AGINGSCIENCES™ – Anti-Aging Firewalls™

As a kid in a traditional Italian family, I was raised on olive oil.  And I now consume generous quantities of extra-virgin olive oil (EVOO) just about every day.  For one thing, I love its taste.  I am so hooked on EVOO that I shudder when a friend serves me a wonderful meal with a great salad, but sets out bottles of commercial salad dressing instead of dressing it with EVOO.  I am particularly bothered by bottled dressings with big labels saying MADE WITH REAL ITALIAN OLIVE OIL and showing ladies in colorful dresses carrying baskets of olives while the very fine print on the back of the bottle lists the amount of olive oil as only 15%, and it’s not even extra-virgin oil.  I have always known that olive oil is good for me.  But for most of my life I could not respond intelligently if somebody asked me “why?”  I thought here, as a break from heavy stuff in molecular biology, I would addresses that question and review some of the research on olive oil.  I particularly focus on EVOO. 

Olive oil and the Mediterranean Diet

There is a two-part generic argument often heard for olive oil.  The first part is that a Mediterranean Diet contributes significantly to health and longevity.  The second part is that olive oil is an essential component of a Mediterranean diet and therefore must be one of the key “good for you” components.  There seems to be good research evidence for the first-part argument as outlined in my August 2009 blog post Recent research on the Mediterranean diet.  The second part of the argument by itself does not meet the “beyond a reasonable doubt” test needed to convict somebody in a court trial.  What if something else in the Mediterranean Diet is providing most of the benefits, like the tomatoes?  The rest of this blog entry will establish the value of EVOO beyond a reasonable doubt.

Olive oil and heart disease risk

A good place to start is with a carefully controlled and fairly-large international 2006 study that  directly relates phenolic content of olive oils to familiar lipid levels like HDL and triglycerides:  The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial “BACKGROUND: Virgin olive oils are richer in phenolic content than refined olive oil. — OBJECTIVE: To evaluate whether the phenolic content of olive oil further benefits plasma lipid levels and lipid oxidative damage compared with monounsaturated acid content. DESIGN: Randomized, crossover, controlled trial. SETTING: 6 research centers from 5 European countries. PARTICIPANTS: 200 healthy male volunteers. MEASUREMENTS: Glucose levels, plasma lipid levels, oxidative damage to lipid levels, and endogenous and exogenous antioxidants at baseline and before and after each intervention. INTERVENTION: In a crossover study, participants were randomly assigned to 3 sequences of daily administration of 25 mL of 3 olive oils. Olive oils had low (2.7 mg/kg of olive oil), medium (164 mg/kg), or high (366 mg/kg) phenolic content but were otherwise similar. Intervention periods were 3 weeks preceded by 2-week washout periods. RESULTS: A linear increase in high-density lipoprotein (HDL) cholesterol levels was observed for low-, medium-, and high-polyphenol olive oil: mean change, 0.025 mmol/L (95% CI, 0.003 to 0.05 mmol/L), 0.032 mmol/L (CI, 0.005 to 0.05 mmol/L), and 0.045 mmol/L (CI, 0.02 to 0.06 mmol/L), respectively. Total cholesterol-HDL cholesterol ratio decreased linearly with the phenolic content of the olive oil. Triglyceride levels decreased by an average of 0.05 mmol/L for all olive oils. Oxidative stress markers decreased linearly with increasing phenolic content. Mean changes for oxidized low-density lipoprotein levels were 1.21 U/L (CI, -0.8 to 3.6 U/L), -1.48 U/L (-3.6 to 0.6 U/L), and -3.21 U/L (-5.1 to -0.8 U/L) for the low-, medium-, and high-polyphenol olive oil, respectively. LIMITATIONS: The olive oil may have interacted with other dietary components, participants’ dietary intake was self-reported, and the intervention periods were short. CONCLUSIONS: Olive oil is more than a monounsaturated fat. Its phenolic content can also provide benefits for plasma lipid levels and oxidative damage. International Standard Randomised Controlled Trial number: ISRCTN09220811”.  So, in only three weeks cholesterol and triglyceride scores improved in the olive oil takers and the scores increased most markedly in those taking the olive oil with the most phenolic content, i.e., the extra-virgin olive oil. And the result is not just because the olive oil is a “good fat.”

The 2007 publication Changes in the phenolic content of low density lipoprotein after olive oil consumption in men. A randomized crossover controlled trial reports on a trial cohort of 30 men, and my impression is that this cohort may have been part of the larger cohort of the first study mentioned above.  The writeup has a somewhat different focus, however, focusing on the antioxidant properties of virgin olive oil. “Olive oil decreases the risk of CVD (cardiovascular disease). This effect may be due to the fatty acid profile of the oil, but it may also be due to its antioxidant content which differs depending on the type of olive oil. In this study, the concentrations of oleic acid and antioxidants (phenolic compounds and vitamin E) in plasma and LDL were compared after consumption of three similar olive oils, but with differences in their phenolic content. Thirty healthy volunteers participated in a placebo-controlled, double-blind, crossover, randomized supplementation trial. Virgin, common, and refined olive oils were administered during three periods of 3 weeks separated by a 2-week washout period. Participants were requested to ingest a daily dose of 25 ml raw olive oil, distributed over the three meals of the day, during intervention periods. All three olive oils caused an increase in plasma and LDL oleic acid (P < 0.05) content. Olive oils rich in phenolic compounds led to an increase in phenolic compounds in LDL (P < 0.005). The concentration of phenolic compounds in LDL was directly correlated with the phenolic concentration in the olive oils. The increase in the phenolic content of LDL could account for the increase of the resistance of LDL to oxidation, and the decrease of the in vivo oxidized LDL, observed in the frame of this trial. Our results support the hypothesis that a daily intake of virgin olive oil promotes protective LDL changes ahead of its oxidation.”

Coming back to the Mediterranean Diet, the 2005 review article The phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health relates the phenolic content of olive oil to the diet’s salutary benefits. “The Mediterranean diet is rich in vegetables, cereals, fruit, fish, milk, wine and olive oil and has salutary biological functions. Epidemiological studies have shown a lower incidence of atherosclerosis, cardiovascular diseases and certain kinds of cancer in the Mediterranean area. Olive oil is the main source of fat, and the Mediterranean diet’s healthy effects can in particular be attributed not only to the high relationship between unsaturated and saturated fatty acids in olive oil but also to the antioxidant property of its phenolic compounds. The main phenolic compounds, hydroxytyrosol and oleuropein, which give extra-virgin olive oil its bitter, pungent taste, have powerful antioxidant activity both in vivo and in vitro.”

Again, the messages appear to be that it is the phenolic ingredients in olive oil that are important, that their antioxidant activities are important and that the most healthful olive oil is the one with the most concentration of the polyphenols, namely first-press extra-virgin olive oil (EVOO). 

Further, though olive oil seems to be a simple substance the biochemical activities of olive oil polyphenols is not simple, as discussed in the 2008 paper Nutritional benefit of olive oil: the biological effects of hydroxytyrosol and its arylating quinone adducts.  “A unique characteristic of olive oil is its enrichment in oleuropein, a member of the secoiridoid family, which hydrolyzes to the catechol hydroxytyrosol and functions as a hydrophilic phenolic antioxidant that is oxidized to its catechol quinone during redox cycling. Little effort has been spent on exploring the biological properties of the catechol hydroxytyrosol quinone, a strong arylating electrophile that forms Michael adducts with thiol nucleophiles in glutathione and proteins. This study compares the chemical and biological characteristics of hydroxytyrosol with those of the tocopherol family in which Michael adducts of arylating desmethyltocopherol quinones have been identified and correlated with biologic properties including cytotoxicity and induction of endoplasmic reticulum stress. It is noted that hydroxytyrosol and desmethyltocopherols share many similarities, suggesting that Michael adduct formation by an arylating quinone electrophile may contribute to the biological properties of both families, including the unique nutritional benefit of olive oil.” 

The 2007 document The olive oil antioxidant hydroxytyrosol efficiently protects against the oxidative stress-induced impairment of the NObullet response of isolated rat aorta reports “Moreover, hydroxytyrosol was found to be a potent OH(*) scavenger, which can be attributed to its catechol moiety. Because of its amphiphilic characteristics (octanol-water partitioning coefficient = 1.1), hydroxytyrosol will readily cross membranes and provide protection in the cytosol and membranes, including the water-lipid interface. The present study provides a molecular basis for the contribution of hydroxytyrosol to the benefits of the Mediterranean diet.”

A number of other published studies confirm these messages like the 2004 publication  Effects of differing phenolic content in dietary olive oils on lipids and LDL oxidation–a randomized controlled trial and the 2010 publication Biological activities of phenolic compounds present in virgin olive oil, the 2005 report International conference on the healthy effect of virgin olive oil, the 2009 report Chemistry and health of olive oil phenolics, and a number of others. 

Olive oil and cancers

A number of studies relate the effects of the active polyphenols in olive oil to killing (induction of apoptosis in) cancer cells.  For example the 2009 study Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells reports “Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 mug/mL of oleuropein or 50 mug/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G(1) to S phase transition manifested by the increase of cell number in G(0)/G(1) phase.”

The 2009 study Extra-virgin olive oil polyphenols inhibit HER2 (erbB-2)-induced malignant transformation in human breast epithelial cells: relationship between the chemical structures of extra-virgin olive oil secoiridoids and lignans and their inhibitory activities on the tyrosine kinase activity of HER2 reports “Extra-virgin olive oil (EVOO – the juice of the olive obtained solely by pressing and consumed without any further refining process) is unique among other vegetable oils because of the high level of naturally occurring phenolic compounds. We explored the ability of EVOO polyphenols to modulate HER2 tyrosine kinase receptor-induced in vitro transformed phenotype in human breast epithelial cells. — EVOO polyphenols induced strong tumoricidal effects by selectively triggering high levels of apoptotic cell death in HER2-positive MCF10A/HER2 cells but not in MCF10A/pBABE matched control cells. EVOO lignans and secoiridoids prevented HER2-induced in vitro transformed phenotype as they inhibited colony formation of MCF10A/HER2 cells in soft-agar. Our current findings not only molecularly support recent epidemiological evidence revealing that EVOO-related anti-breast cancer effects primarily affect the occurrence of breast tumors over-expressing the type I receptor tyrosine kinase HER2 but further suggest that the stereochemistry of EVOO-derived lignans and secoiridoids might provide an excellent and safe platform for the design of new HER2 targeted anti-breast cancer drugs.”

The 2008 paper Analyzing effects of extra-virgin olive oil polyphenols on breast cancer-associated fatty acid synthase protein expression using reverse-phase protein microarrays is another of several more relating EVOO to breast cancer. “These findings reveal for the first time that phenolic fractions, directly extracted from EVOO, may induce anti-cancer effects by suppressing the expression of the lipogenic enzyme FASN in HER2-overexpressing breast carcinoma cells, thus offering a previously unrecognized mechanism for EVOO-related cancer preventive effects.”

The 2008 document tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO) relates to the same theme.  “Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion. CONCLUSION: The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.”   

It is interesting to me that the emphasis in the last-mentioned study and in several other studies seems to be not on promoting the use of EVOO as an anti-cancer health measure but rather on identifying biochemical pathways on which to base new drug developments.  Perhaps this is because the point of departure of most of these studies is acknowledging the health benefits of a Mediterranean diet and consuming lots of olive oil.  And, perhaps cynically, I wonder if it reflects research funding sources for whom finding a new blockbuster drug may be more important than public health. 

A 2010 study publication Extra-virgin olive oil-enriched diet modulates DSS-colitis-associated colon carcinogenesis in mice reports “RESULTS: Disease activity index (DAI) was significantly higher on SFO (sunflower oil) vs. EVOO diet at the end of the experimental period. EVOO-fed mice showed less incidence and multiplicity of tumors than in those SFO-fed mice. beta-catenin immunostaining was limited to cell membranes in control groups, whereas translocation from the cell membrane to the cytoplasm and/or nucleus was showed in DSS-treated groups and its expression was higher in SFO-fed animals. Cytokine production was significantly enhanced in SFO-fed mice, while this increase was not significant in EVOO-fed mice. Conversely, cyclooxigenase-2 (COX-2) and inducible nitric oxidase synthase (iNOS) expression were significantly lower in the animal group fed with EVOO than in the SFO group. CONCLUSIONS: These results confirm that EVOO diet has protective/preventive effect in the UC-associated CRC. This beneficial effect was correlated with a better DAI, a minor number of dysplastic lesions, a lower beta-catenin immunoreactivity, a proinflammatory cytokine levels reduction, a non modification of p53 expression and, COX-2 and iNOS reduction in the colonic tissue.”   

Olive oil and inflammation

A study in done back 2001 Protective effects upon experimental inflammation models of a polyphenol-supplemented virgin olive oil diet again demonstrated a dose-dependent effect of olive oil polyphenols in protecting rats from induced inflammation damage. “CONCLUSIONS: This study demonstrates that virgin olive oil with a higher content of polyphenolic compounds, similar to that of extra virgin olive oil, shows protective effects in both models of inflammation and improves the disease associated loss of weight. This supplementation also augmented the effects of drug therapy.”   

Synergy of olive oil with other supplements  

A 2006 study Intestinal anti-inflammatory activity of combined quercitrin and dietary olive oil supplemented with fish oil, rich in EPA and DHA (n-3) polyunsaturated fatty acids, in rats with DSS-induced colitis found synergy between administration of fish oil, quercetin and olive oil in a rat model of colitis.  “In addition, a complete restoration of colonic glutathione content, which was depleted as a consequence of the colonic insult, was obtained in rats treated with QR plus FO diet; this content was even higher than that obtained when colitic rats were treated with FO diet alone. When compared with the control colitic group, the combined treatment was also associated with a lower colonic nitric oxide synthase and cyclooxygenase-2 expression as well as with a significant reduction in different colonic proinflammatory mediators assayed, i.e. leukotriene B(4), tumor necrosis factor alpha and interleukin 1beta, showing a significantly greater inhibitory effect of the latter in comparison with rats receiving FO diet without the flavonoids (quercetin). CONCLUSIONS: These results support the potential synergism between the administration of the flavonoid and the incorporation of olive oil and n-3 PUFA to the diet for the treatment of these intestinal inflammatory disorders.”

EVOO and gene expression

Finally, I want to mention that new studies are starting to look at the effects of EVOO on gene expression.  For example, the 2010 publication Gene expression changes in mononuclear cells from patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil.  “BACKGROUND: Previous studies have shown that acute intake of high-phenol virgin olive oil reduces pro-inflammatory, pro-oxidant and pro-thrombotic markers compared with low phenols virgin olive oil, but it remains unclear if the effects attributed to its phenolic fraction are exerted at the transcriptional level in vivo. To achieve this goal, we aimed at identifying in humans those genes which undergo expression changes mediated by virgin olive oil phenolic compounds. RESULTS: Postprandial gene expression microarray analysis was performed on peripheral blood mononuclear cells at the postprandial period. Two virgin olive oil-based breakfasts with high (398 ppm) and low (70 ppm) content of phenolic compounds were administered to 20 patients with metabolic syndrome following a double-blinded random crossover design. To eliminate the potential effect that might exist in their usual dietary habits, all subjects followed a similar low-fat, carbohydrate rich diet during the study period. Microarray analysis identified 98 differentially expressed genes (79 underexpressed and 19 overexpressed) when comparing the intake of phenol-rich olive oil with the low-phenol olive oil. Many of those genes are linked to obesity, dyslipemia and type 2 diabetes mellitus. Among these, several genes are involved in inflammatory processes mediated by transcription factor NF-kappa B, activator protein-1 transcription factor complex AP-1, cytokines, mitogen-activated protein kinases MAPKs or arachidonic acid pathways. CONCLUSION: This study shows that intake of a breakfast based in virgin olive oil rich in phenol compounds is able to repress the in vivo expression of several pro-inflammatory genes, thereby switching the activity of peripheral blood mononuclear cells to a less deleterious inflammatory profile. These results provide at least a partial molecular basis for the reduced risk of cardiovascular disease observed in Mediterranean countries, where virgin olive oil represents the main source of dietary fat.”   

I could continue this blog entry citing more and more studies but it should be clear by now that the old Italian folklore about the health value of olive oil is right-on and the pungent extra-virgin variety is by far the best.  What I have to figure out now based on the final study quoted is how can I work extra virgin olive oil into my regular daily breakfast?  I want to do that without adding carbs and confounding tastes like blueberries and EVOO.  I will need to do some experimenting.  Perhaps they will go together fine.

Please see the medical disclaimer for this blog.

In the recent blog post Harnessing the engines of finance and commerce for life-extension, I characterized a new approach to health, medicine and  longevity called Personalized Predictive Preventative Participatory Medicine (PPPPM). I also promised to describe specific examples in subsequent blog entries.  This posting is concerned with a Canadian initiative on the way to becoming a full PPPPM.  The initiative is called the PROOF Centre of Excellence, where PROOF stands for prevention of organ failure.  A presentation on this topic was made at the Bio-IT World Conference & Expo last week by Raymond Ng.  His talk was on Developing Combinatorial Biomarker Panels for End-stage Organ Failures.  PROOF represents a medium-scale PPPPM in early-stage development, where focus is presently on identification and development of biomarkers. 

As stated on the PROOF website “The Centre of Excellence for the Prevention of Organ Failure (PROOF Centre) discovers, develops, commercializes and implements bio-molecular markers (biomarkers) to prevent, predict, diagnose and better treat heart, lung and kidney failure. The PROOF Centre is a cross-disciplinary engine of devoted partners including industry, academia, health care, government, patients and the public focused on reducing the enormous socioeconomic burdens of heart, lung and kidney failure and on improving health.”

I will review elements of PROOF paying particular attention to the criteria by which I characterized PPPPM.

1.      PROOF addresses an important category of disease processes. Organ failure is a condition where an organ does not perform its expected function.  There are many possible types of organ failure and multiple possible causes.  “Epidemics of inactivity, dietary imbalance, hypertension, obesity, diabetes, air pollution and tobacco use set the stage for accelerated risk for, and occurrence of, vital organ failure. One in four Canadians are believed to be at risk of organ failure; similar to the incidence worldwide. Current methods of detecting organ failure are frequently ineffectual, often costly, at times invasive and generally unsuitable for early diagnosis(ref).” In Canada alone, the personal, societal and economic consequences of vital organ failure (heart, lung and kidney) has a cost of more than $35 billion a year. 

2.     Availability of reliable predictive biomarkers for various kinds of organ failure could make a big difference. “Precise and accurate recognition of a patient’s risk of organ failure has the potential to dramatically improve preventive care, treatment decisions and clinical outcomes, while lowering both social and economic costs(ref).”  This web page illustrates how PROOF views the importance of biomarkers and articulates the core strategy of a PPPPM initiative. “Current treatment strategies are reactive in that, for the most part, interventions are initiated after significant pathological changes have occurred and are often irreversible, which greatly increases the management costs, disease burden, and results in poorer outcomes. The typical current intervention occurs after the disease is irreversible and costly –. However, a new treatment strategy that makes use of predictive, diagnostic, or prognostic biomarker information may help to guide earlier effective interventions when the disease process is still modifiable. As such, overall treatment costs may be reduced and outcomes can be improved.”  

3.     PROOF is a highly collaborative activity. Proof is a not-for-profit center established in 2008, funded initially by the Canadian Networks for Centres of Excellence for Commercialization and Research, hosted by the University of British Columbia and based at the St. Paul hospital in Vancouver BC.   It defines itself as the hub of a multi-disciplinary group of partners drawn from industry, academia, government, health care and the public.

Among the PROOF Centre partners are Luminex, Pfizer of Canada, Genome BC, Astellas Pharma Canada and UVic Genome BC Proteomics Centre.   “The PROOF Centre has partnered with Genome British Columbia to bring new blood tests into clinical practice for heart and kidney transplant patients.  IO Informatics have partnered with the PROOF Centre to provide a data integration and knowledge explorer infrastructure. This will allow investigators involved in the Centre to access data from all biomarker programs within the PROOF Centre(ref).” 

Among the PROOF Centre partners are:

• “clinical and academic leaders to define decision-points in patient management when/where a biomarker panel could change care and then implement observational biomarker discovery studies,

• technology leaders in the public and private sectors to pilot or augment technology platforms for multiplexed analysis,

• leaders of established patient cohorts at the “right” stage of disease to assess the power of biomarker panels in changing care,

• health economics leaders to evaluate clinical settings and patient groups in which biomarker solutions would change healthcare costs and/or lead to a wealth creation opportunity, and

• “front-line” health care and laboratory professionals who understand the harsh practicalities of bringing new tests based on biomarkers into care systems(ref).”

4.     PROOF envisages the following stages of evolution which are characteristic of a PPPPM:

a.     “Biomarker discovery: cohort – single site,  high performance platforms – genomics, proteomics, metabolomics, and computation & analysis to develop sets of biomarkers,

b.     Biomarker development:  multi-site biomarker trial, high performance platforms/assay validation, computation & analysis to validate sets of biomarker signals,

c.      Clinical drug development:  develop assays and platforms that can move into the clinical laboratory, assay development partnerships,

d.     Regulatory filing:  voluntary exploratory data submission (VXDS). FDA and Health Canada submissions

e.     Clinical implication and experience feedback(ref).”

5.     PROOF is already a going concern.  Representative of PROOF’s initial impacts:

·        “Through its flagship program, “Biomarkers in Transplantation”, the PROOF Centre team has discovered levels of genes and proteins in the blood that allow the diagnosis or prediction of acute rejection in heart and kidney(ref).”  Put differently: “The PROOF Centre has discovered and internally validated blood-based proteomic and genomic biomarker tests to diagnose and predict allograft immune rejection in heart and kidney transplantation(ref).”

·        A Canada-Wide Biomarker Trial is underway and will test this new method to provide better care for heart and kidney transplant patients in preparation for a submission to regulatory agencies(ref)” 

My impression is that PROOF is somewhere in the middle of the scale in size and reach as far as existing PPPPMs go.   I suspect PROOF will grow in scale, in sophistication of techniques and in partnering activities as time progresses.  Dimensions of expansion will probably include more “omics” screening, enhanced collaborative computer networking among its partners and active relationships with care agencies with active feedback from clinical experience. 

PROOF may seem small-scale given US standards and appears tucked up in the Canadian NorthWest.  But its biomarkers, once validated and in widespread use, may be of immense help to everybody.

On several occasions both in this blog and in my treatise I have pointed out the need for integrating the various disparate theories of aging into an overall systems framework.  Following is the abstract for a presentation I will be offering at the American Aging Association’s 39th Annual Meeting and 24th Annual Meeting of the American College of Clinical Gerontology in Portland Oregon June 4-7, 2010:

Towards a systems view of aging

A comprehensive systems theory of aging must embrace the validated teachings of multiple existing special theories of aging, theories that range from oxidative damage to loss of mitochondrial function to neurological degeneration to telomere shortening and cell senescence to decline in hormone levels.  The author has characterized 14 such major theories and an additional 7 candidate ones.   While each such theory is correct in its own framework of reference, on the surface most seem to be largely independent of the others.  However, on the levels of molecular biology and genomics a rich network of links exists among these theories.  The author suggests two overarching frameworks for integrating and clarifying existing understandings from the diverse theories of aging.  One framework is lifelong programmed changes in global gene expression due to DNA methylation, histone acetylation and other epigenomic modifications.  For example, aging-related decline of efficacy of DNA repair machinery might possibly result from promoter methylation of the Mms22 gene, resulting in increasing susceptibility to oxidative damage with age.  Promoter methylation of the P21 and P53 apoptosis genes can result in increased susceptibility to cancers.  The second framework sees aging as decline in functioning of the stem cell supply chain, the chain where adult stem and progenitor cells progressively differentiate as-needed into other cells of increased specificity and decreased pluripotency, resulting in lifelong renewal of somatic cell types.  As the supplies of multipotent mesenchymal and haemopoietic stem cells available in their niches for differentiation decline because of their replicative senescence, for example, fewer progenitor and somatic cells are available to replace ones that have died or become senescent.  The paper will embody insights developed over a multi-year period and described in the author’s online treatise ANTI-AGING FIREWALLS – THE SCIENCE AND TECHNOLOGY OF LONGEVITY and in the hundreds of postings in the author’s blog www.anti-agingfirewalls.com.

In the course of the next couple of weeks I expect to produce a more detailed version of that presentation for this blog.

Suppose I reported that billions of dollars will be spent on life-extension research next year and that soon that number will reach tens of billions?  The first reaction of aging-science researchers would be “no way.”  They would point to the pittance allocated to longevity research by the NIH.   Yet, I am about to assert here that investing in life-extension is going big time. This is the first of a series of related blog posts on an emerging paradigm in medicine and its implications for longevity.  The series is inspired by what I learned at the Bio-IT World Conference & Expo last week from listening to many speakers and from wandering through the exhibit floor and talking with many people.    

Aging science vs. life-extension engineering 

I drew an important distinction in the blog entry What are aging, life-extension and anti-aging?  I pointed out that there are a lot of life extension approaches, ones that increase the life expectancy of a defined population, that are of an engineering or social nature.  These approaches may or may not have anything to do with postponing aging from a biochemical viewpoint.  Example approaches that have worked are better sanitation systems, clearing out water and air pollution, eradicating parasite populations, systematic inoculation programs, building safer highways, and implementing speed limits and seatbelt laws.  Indeed, the fact that the average American lives twice as long as our predecessors did 150 years ago has mostly to do with these kinds of life extension measures.  While the results of these engineering and social approaches have been life extension, they are invariably thought of in other terms such as “public health,” “cleaner environment,” and “food safety.”  A powerful new engineering approach in medicine with life extension consequences is gearing up right now.

In contrast, in my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY treatise and in this blog I have largely come at aging through the lenses of aging science, identifying 14 of the leading theories of what causes aging and another 7 candidate theories of aging and then writing about developments relating to them.  So I have dealt with topics relating to aging theories  ranging from oxidative damage  and chronic inflammation at the “classical” end of the spectrum to telomere shortening and damage and increasing mTOR signaling at the “new science” end of the spectrum.  I will doubtlessly continue to do the same.

What I am going to discuss in this particular series of blog entries is a different engineering and social approach to life extension that, in my opinion at least, will also end up telling us many of the things we need to know about the science of aging. 

Personalized Predictive Preventative Participatory Medicine (PPPPM)

The new approach is directed to health and medicine in the coming decades, although I believe it will have major implications for life extension and perhaps even for slowing biological aging.  I will call it Personalized Predictive Preventative Participatory Medicine (PPPPM).  The dimensions of PPPPM are only now becoming clear and I will only generally characterize it here, saving detailed characterizations and discussions of examples for subsequent blog entries. 

In a nutshell, the essence of PPPPM is:

1.     The objective of PPPPM is not so much to cure diseases as it is to detect and predict disease susceptibilities before a disease starts or at very early stages of disease progression and initiate personalized interventions to prevent the progression of the disease before it becomes symptomatic or does damage. 

2.     PPPPM is participatory in the sense that the collaborative participation of large numbers of health research institutions and care agencies is involved in doing the research and creating the infrastructure to make it workable.  It involves a tight and continuing linkup loop of researchers, practitioners, patients and healthy people who want to stay healthy.

3.     The predictions of PPPPM are based on the identification of sets of biomarkers that are predictive of disease susceptibilities and stages of disease progression for particular diseases.  The biomarkers can consist of known gene mutations, SNPs, copy number variations and the like, and other “omics” markers (proteomic, transcriptomic, metabolomic, epigenomic etc.) as well as the results of all kinds of existing clinical tests and clinical data.

4.     The biomarkers will be arrived at through massive correlation analyses and pattern matching between public data bases of the kinds of data involved and association studies of many different kinds.  See the blog entry Genome-wide association studies for examples.  The biomarkers will be continuously refined through feedback from personal histories, of course with numerous layers of personal privacy protection.

5.     Identifying PPPM biomarkers will proceed one disease at a time, the challenges including creation of massive public data bases of “omics” information and performing multivariate association studies.  It is a task that requires mobilization of incredible networked computer power and human analytics.  

6.     Identifications of disease-prevention interventions including drug candidates will proceed along the same lines using the same kinds of tools and analytics, where interventions will be determined on the basis of the known biomarker patterns as well as individual patient or well-persons’ patterns of “omic” markers.

7.     Fully implementing PPPM will require genetic, genomic and other “omic” profiling on the part of increasing numbers of people, something that should become commonplace in 10-15 years. 

Here is a simplified example of how the approach could work.  Say a healthy person discovers early in life that he has a susceptibility to Alzheimer’s disease.  For example, a current-generation genetic test shows he possesses the APOE4 gene allele, a predictor of late-onset AD.  Starting at around age 50, he would periodically have his other Alzheimer’s-predictive biomarkers checked.  These should show early signs of the actual disease before any dementia is detected.  This might, for example, require a combination of epigenomic scans and lab tests.  If and when very-early disease signs are detected, strong preventative measures are immediately initiated.  These might consist of drugs, supplements or lifestyle changes. 

The basic concept is that it should be a lot easier to stop a disease in its very early stages from progressing or reverse its progress than waiting for symptoms to show up and damage is done, and then trying to “cure” it.  All too many diseases like Alzheimer’s have shown themselves to be remarkably resistant to being cured because by the time they are symptomatic, it is too late to stop them.

PPPPM may sound like a grand concept for the future, and it is.  However it is already being actively pursued for several disease conditions by large research consortia.  Many large relevant databases already exist and many important biomarkers are already known.  One of several existing examples of PPPPM is the cancer biomedical informatics grid, “a virtual network of interconnected data, individuals and individuals that redefines how research is conducted, care is provided, and patients/participants interact with the biomedical research enterprise.”

The current energy and vitality of PPPPM follows from the fact that it is not just a research approach but also heavily involves finance, entrepreneurship and industry participation.  Large pharmaceutical companies who badly need new drug candidates to replace those going off-patent are using the PPPPM approach to discover them.  A number of large government agencies like the National Cancer Institute are involved.  And dozens of new companies and players are lining up to do the information processing and collaborative networking required.  Players with names like Microsoft and IBM are involved.  There is the smell of billions of dollars involved.   But I will leave details and descriptions of concrete examples to subsequent blog entries.

And what happens when diseases of aging are effectively postponed?  It is a no-brainer that on the average people live longer, that is life extension. And I have no doubt that as effective PPPPM approaches gear up and more and more biomarker-disease correlations and prevention-treatment disease correlation are learned, we will be learning more and more about how to postpone biological aging too.

Life-extension lives!

Just as a post-script, the basic principles of computer science were already well-established in 1950, but computers were doing very little to help people then.  It took years of commercial activities, engineering and technology innovations, inventions and new approaches in manufacturing, software and marketing to get where we are now where computers empower all aspects of our lives.  This was a social-engineering-industrial-marketing feedback process involving institutions, government (for early Internet development), businesses, entrepreneurial activities and intense involvement of business of all kinds and now, everybody.  It was tumultuous with many failures and incredible successes, and continues to be. The same kind of process has already started related to health.  We are already deeply engaged in a similar tumultuous process of life extension and it will be a wonderful crazy ride.  Real practical life extension is no fantasy.

I am out attending the Bio-IT World Conference & Expo in Boston a good part of this week, paying particular attention to the track on Systems and Predictive Technology.  The conference is focused on a fundamental shift happening in biological and medical research, drug discovery, disease prevention, and the practice of medicine – with far-reaching implications for longevity.  Being at the conference is providing me with important contacts, new materials and exciting ideas for future blog posts.  However, these few days there is no time left over for me to do my usual job of researching and writing blog posts.  I will be back at it again soon, probably starting on the weekend.

Don’t let the fancy name scare you off.  The underlying concepts are fairly simple.  The basic idea is to go after cancer cells with viruses that kill them.  To help the viruses escape the immune system, they are packaged in stem cells that are expected to snuggle up to the cancer cells.  It is hoped that the approach will go after cancer stem cells as well as mature cancer cells and therefore possibly provide a basic cure for the cancer concerned.  The broader area, oncolytic virotherapy is an approach to curing cancers that has been intensely researched for a number of years.  What is new is using stem cells or other human cells for safely getting the viruses to and into the target cancer cells. 

Some background 

The 10th theory of aging in my treatise is Susceptibility to Cancers.  The probability of incidences of cancer rises rapidly with advanced aging.  Cancer is second major cause of age-associated mortality and cancer in turn often induces other age-related maladies.  Finally, cancer therapies like radiation and some forms of chemotherapy can accelerate the aging process. The War on Cancer going back to 1971 has produced marginal results for the hundreds of billions of dollars spent on research.  In 2008 the NCI spent $4.83 billion on 5,380 research grants.  The relative lack of progress for the amount of effort invested is probably due to the fact that basic scientific knowledge related to the biomolecular, cell-cycle, signaling pathways, genetic and epigenetic processes involved in cancer did not exist until recent years.  And this knowledge could not exist until extremely powerful computers capable of analyzing molecular, genomic and proteomic data became available.  So, the early approaches to curing cancer tended to be trial-and-error approaches based on screening chemical compounds which mostly ended in failure.  Many current approaches are much more sophisticated, however, and stem cell oncolytic virotherapy is one of them. 

On oncolytic virotherapy

The idea of plain-old oncolytic virotherapy is more than 50 years old though most real progress has been made in the last dozen years.  See the 2007 publication Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress.  “Therapeutic oncolytic viruses (virotherapeutics) constitute a novel class of targeted anticancer agents that have unique mechanisms of action compared with other cancer therapeutics. The development of virotherapeutics has evolved from the use of in vitro-passaged strains (first generation), to genetically engineered selectivity-enhanced viruses (second generation) and finally to genetically engineered transgene-expressing ‘armed’ oncolytic viruses (third generation). Descriptions of cancer remissions following virus infections date back to a century ago. Initial patient treatment publications, written up to 50 years ago, consisted of case reports or case series of treatment with first-generation, non-engineered viruses. Over the past decade, hundreds of patients with cancer have been treated on prospectively designed clinical trials (including phase III), evaluating over 10 different agents, including engineered second-generation and third-generation viruses. This Review summarizes and interprets the data from clinical reports over the last century, including safety, efficacy and biological end points (viral and immunologic).”

By 2008 much progress had been made as indicated in the publication Oncolytic virotherapy: molecular targets in tumor-selective replication and carrier cell-mediated delivery of oncolytic viruses.  “Tremendous advances have been made in developing oncolytic viruses (OVs) in the last few years. By taking advantage of current knowledge in cancer biology and virology, specific OVs have been genetically engineered to target specific molecules or signal transduction pathways in cancer cells in order to achieve efficient and selective replication. The viral infection and amplification eventually induce cancer cells into cell death pathways and elicit host antitumor immune responses to further help eliminate cancer cells. Specifically targeted molecules or signaling pathways (such as RB/E2F/p16, p53, IFN, PKR, EGFR, Ras, Wnt, anti-apoptosis or hypoxia) in cancer cells or tumor microenvironment have been studied and dissected with a variety of OVs such as adenovirus, herpes simplex virus, poxvirus, vesicular stomatitis virus, measles virus, Newcastle disease virus, influenza virus and reovirus, setting the molecular basis for further improvements in the near future. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells (or cellular vehicles) to deliver OVs to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules.”

So, with all that great history and the discovery of many powerful anti-cancer viruses, why are oncolytic virotherapies not now in widespread use?   A large part of the answer appears to be that usually a) there is a problem getting the virus specifically to the cancer cells and b) the human immune system detects and wipes out the virus before it can get to the cancer cells and do its job.  The immune system in such a case is just doing its job.  The 2008 publication Cell carriers to deliver oncolytic viruses to sites of myeloma tumor growth reports “Several studies have illustrated the potential of utilizing oncolytic viruses (measles, vaccinia, Vesicular Stomatitis Virus and coxsackievirus A21) for the treatment of MM (multiple myeloma), but there are significant barriers that prevent the viruses from reaching sites of myeloma tumor growth after intravenous delivery. The most important barriers are failure to extravasate from tumor blood vessels, mislocalization of the viruses in liver and spleen and neutralization by antiviral antibodies.”  These problems have led to approaches using “Trojan horse” cells that hide the viruses from the immune system and that can home-in to the cancer cells.

Cell-based oncolytic virotherapy

The March 2010 publication Crossing the boundaries: stem cells and gene therapy provides an overview of the current situation.  “Oncolytic virotherapy is an emerging therapeutic modality for the treatment of cancer. It entails construction of viruses with the ability to selectively target and lyse tumor cells. This branch of therapy has significantly advanced in the past decade, heralded by the development of several novel viruses. Despite the initial success of oncolytic virotherapy in the preclinical setting, however, this treatment modality remains hindered by several obstacles. First, failure to achieve effective viral delivery to targeted tumor beds is a well known limitation. Second, the virus-neutralizing mechanisms of the host immune system, which are in place to protect from viral pathogens, may also hinder the therapeutic potential of virotherapy. One approach to tackling these shortcomings is the use of cell-based carriers to both help with delivery of the virus and shield it from immunosurveillance. Stem cells have recently surfaced as a potential cell-based candidate for delivery of virotherapy. Their unique migratory and immunosuppressive qualities have made them an exciting area of investigation. The focus of this review is to discuss the benefits of stem-cell-based delivery of oncolytic virotherapy and its role in cancer treatment.”

The idea of using cells to deliver oncolytic viruses in cells goes back a few years.  The 2007 publication Cell-based delivery of oncolytic viruses: a new strategic alliance for a biological strike against cancer outlined the strategy of cell-based delivery but at that time did not emphasize the use of stem cells as delivery vehicles.  The 2009 report Cell carriers for oncolytic viruses: Fed Ex for cancer therapy amplifies on this theme. “Oncolytic viruses delivered directly into the circulation face many hazards that impede their localization to, and infection of, metastatic tumors. Such barriers to systemic delivery could be overcome if couriers, which confer both protection, and tumor localization, to their viral cargoes, could be found. Several preclincal studies have shown that viruses can be loaded into, or onto, different types of cells without losing the biological activity of either virus or cell carrier. Importantly, such loading can significantly protect the viruses from immune-mediated virus-neutralizing activities, including antiviral antibody. Moreover, an impressive portfolio of cellular vehicles, which have some degree of tropism for tumor cells themselves, or for the biological properties associated with the tumor stroma, is already available.” 

Stem cells appear to be excellent candidates for the Trojan horse role. 

The 2010 publication Treatment of metastatic neuroblastoma with systemic oncolytic virotherapy delivered by autologous mesenchymal stem cells: an exploratory study reports on a small-scale human trial: “The tumor stroma engrafting property of intravenously injected mesenchymal stem cells (MSCs) may allow the use of MSCs as cellular vehicles for targeted delivery. In this work, we study the safety and the efficacy of infusing autologous MSCs infected with ICOVIR-5, a new oncolytic adenovirus, for treating metastatic neuroblastoma. Four children with metastatic neuroblastoma refractory to front-line therapies received several doses of autologous MSCs carrying ICOVIR-5, under an approved preliminary study. The tolerance to the treatment was excellent. A complete clinical response was documented in one case, and the child is in complete remission 3 years after this therapy. We postulate that MSCs can deliver oncolytic adenoviruses to metastatic tumors with very low systemic toxicity and with beneficial antitumor effects.”

A May 2009 blog entry Trojan-horse stem cells might offer an important new cancer therapy discusses an approach to killing cancer stem cells that is very akin to the one described here.  Instead of the death payload delivered to cancer cells being an oncolytic adenovirus as discussed here, the payload in the case of that blog entry is a molecule called TRAIL (A TNF-related apoptosis-inducing ligand in case you wanted to know).  “TRAIL induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells(ref).”  

Lots of research action

There is much more going on relating to oncolytic virotherapy, way more than I can start to cover here.  Here, for example, is a starting list of relevant publications produced in 2010 alone:

·         A High-throughput Pharmacoviral Approach Identifies Novel Oncolytic Virus Sensitizers.

·         Regression of human prostate tumors and metastases in nude mice following treatment with the recombinant oncolytic vaccinia virus GLV-1h68.

·         Oncolytic measles viruses encoding interferon beta and the thyroidal sodium iodide symporter gene for mesothelioma virotherapy.

·         Oncolytic herpes simplex virus vectors and chemotherapy: are combinatorial strategies more effective for cancer? 

·         Adenovirus retargeting to surface expressed antigens on oral mucosa.

·         Antiangiogenic cancer therapy combined with oncolytic virotherapy leads to regression of established tumors in mice. 

·         Crossing the boundaries: stem cells and gene therapy.

·         Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1 in rat models.

·         Cancer Stem Cells: The Final Frontier for Glioma Virotherapy. 

·         Noninvasive monitoring of mRFP1- and mCherry-labeled oncolytic adenoviruses in an orthotopic breast cancer model by spectral imaging.

·         United virus: The oncolytic tag-team against cancer!

·         Combining oncolytic virotherapy and tumour vaccination.

·         Clinical trials with oncolytic reovirus: Moving beyond phase I into combinations with standard therapeutics. 

·         Oncolytic herpes simplex virus armed with xenogeneic homologue of prostatic acid phosphatase enhances antitumor efficacy in prostate cancer.

·         Oncolytic parvoviruses as cancer therapeutics.

·         Evaluation of continuous low dose rate versus acute single high dose rate radiation combined with oncolytic viral therapy for prostate

Treatment of metastatic neuroblastoma with systemic oncolytic virotherapy delivered by autologous mesenchymal stem cells: an exploratory study.

·         Double-regulated oncolytic adenovirus-mediated IL-24 overexpression exhibits potent antitumor activity on gastric adenocarcinoma.

·         Oncolysis using herpes simplex virus type 1 engineered to express cytosine deaminase and a fusogenic glycoprotein for head and neck squamous cell carcinoma.

·         Type I interferon-sensitive recombinant newcastle disease virus for oncolytic virotherapy.

·         Oncolysis of prostate cancers induced by vesicular stomatitis virus in PTEN knockout mice.

·         Oncolytic (replication-competent) adenoviruses as anticancer agents. 

·         Combination gene therapy of lung cancer with conditionally replicating adenovirus and adenovirus-herpes simplex virus thymidine kinase.

·         Phase I trial of intraperitoneal administration of an oncolytic measles virus strain engineered to express carcinoembryonic antigen for recurrent ovarian cancer.

·         Parvovirus H1 selectively induces cytotoxic effects on human neuroblastoma cells.

·         Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo. 

·         Vesicular stomatitis virus oncolysis is potentiated by impairing mTORC1-dependent type I IFN production.

·         Myxoma virus virotherapy for glioma in immunocompetent animal models: optimizing administration routes and synergy with rapamycin. 

·         Improved potency and selectivity of an oncolytic E1ACR2 and E1B19K deleted adenoviral mutant in prostate and pancreatic cancers.

·         hTERT-promoter-dependent oncolytic adenovirus enhances the transduction and therapeutic efficacy of replication-defective adenovirus vectors in pancreatic cancer cells.

·         Antitumor effects of telomelysin in combination with paclitaxel or cisplatin on head and neck squamous cell carcinoma. 

·         Intelligent design: combination therapy with oncolytic viruses.

·         International Society for Cell and Gene Therapy of Cancer 2009 Annual Meeting held in Cork, Ireland.

·         Single-cycle viral gene expression, rather than progressive replication and oncolysis, is required for VSV therapy of B16 melanoma. 

·         Multimodal approach using oncolytic adenovirus, cetuximab, chemotherapy and radiotherapy in HNSCC low passage tumour cell cultures.

·         Adenovirus-mediated cancer gene therapy and virotherapy (Review). 

Despite all this history, the use of stem cells as delivery vectors for oncolytic viruses is fairly new and this aspect of oncolytic virotherapy is still largely in the preclinical stage. 

The bottom line? 

Do I think cell-delivered oncolytic virotherapy will be an effective approach to wiping out cancers?  My guess is that the answer depends on whether the particular virotherapy concerned infects and wipes out cancer stem cells as well as cancer cells.  If the answer is “no” then the cell-delivered oncolytic virotherapy will be just another in a long list of ways to send the cancer into temporary remission with a high probability of it returning.  Paying lots of attention to the virus delivery vehicle and little attention to whether the delivered virus will wipe out cancer stem cells is not likely to get us very far. 

The 2008 publication Virotherapy as An Approach Against Cancer Stem Cells is optimistic in this respect. “Targeting of cancer stem cells might be key for improving survival and producing cures in patients with metastatic tumors. Viruses enter cells though infection and might therefore not be sensitive to stem cell resistance mechanisms. During the last decades, oncolytic adenoviruses have been shown to effectively kill cancer cells, by seizing control of their DNA replication machinery and utilizing it for the production of new virions, ultimately resulting in the rupture of the cell.”  The 2007 publication Targeting the Untargetable: Oncolytic Virotherapy for the Cancer Stem Cell appears also to be tentatively optimistic.  “Oncolytic viruses may represent an effective therapeutic approach to target cancer stem cells.^6,7 ” 

If the authors are right and the virotherapy kills cancer stem cells, then at last we might be on the way to real cancer cures.  Finally!

Perhaps the two most comprehensive theories explaining aging in my treatise are Programmed Epigenomic Changes and Stem Cell Supply Chain Breakdown.  Recent research related to the epigenetics of stem cells deals with the profound underlying relationships between those two theories.  The research relates to questions such as “What causes a stem cell to proliferate (e.g. reproduction through mitosis making more stem cells of the same kind), and what causes a stem cell to differentiate (e.g. generate more specific progenitor or somatic cells)?  The subject has been called Epigenetic alchemy for cell fate conversion.   I review some of that current research here. The key players I am going to focus on here are DNA methyltransferases and their key regulatory roles.

Background on DNA methylation

I have discussed DNA methylation and its role in aging in a number of my earlier blog entries.  See for example Epigenetics, epigenomics and aging, DNA methylation, personalized medicine and longevity and Histone acetylase and deacetylase inhibitors, DNA demethylation – a new way of coming at cancers,  and Epigenetics going mainstream.

“DNA methylation, particularly when applied to CG-rich promoter sequences, has been shown to silence gene expression in a heritable manner. DNA methylation is therefore a form of cellular memory. Because DNA methylation is not encoded in the DNA sequence itself, it is called an epigenetic modification (“epi”, Greek origin: “above” or “upon”). The transcriptional silencing associated with 5-methylcytosine is required for fundamental biological processes such as embryonic development, protection against intragenomic parasites, X-inactivation(ref).”,

It has long been known that “DNA methylation is impacted by aging and impacts on aging(ref).  Methylation in the promoter region of genes is thought generally to be associated with gene silencing.  Longevity-related and health-promoting genes may be turned off in the process of aging due to progressive methylation(ref).”  I remind my readers that the 13^th theory of aging  covered in my treatise, Programmed Epigenomic Changes, envisages aging as a systematically articulated set of epigenomic changes including  changes in DNA methylation in cells accumulated with aging. One researcher goes so far as to assert that DNA methylation is the cause of aging.  See my blog entry Homicide by DNA methylation.

Much of the new research relates to the life-and-death roles of DNA methyltransferases in adult stem cells and what causes stability in embryonic stem cells.  “– the DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions.  All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor(ref).” As mentioned, a methyl group transferred to  a GpC site in the promoter region(ref) of a gene generally serves to silence that gene.  “CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. “CpG” is shorthand for “—C—phosphate—G—“, that is, cytosine and guanine separated by a phosphate(ref).”  

The new research relates to How DNA methyltransferases 1.  initiate and maintain methyl marks, 2. are involved in self-renewal of  embryonic stem (ES) cells, and 3. act in somatic (adult) stem cells including: hematopoietic, epithelial, neural and muscle cells.  It also relates to the molecular factors that keep embryonic stem cells from differentiating and the role of methyltransferases once those cells start differentiating.  I start out with research on adult stem cells. 

DNA methyltransferases and  adult stem cells

The April 2010 review publication DNA methylation in adult stem cells: New insights into self-renewal summarizes the important role of methyltransferases in preserving adult stem cell lineages. “Methylation of cytosine residues in the context of CpG dinucleotides within mammalian DNA is an epigenetic modification with profound effects on transcriptional regulation. A group of enzymes, the DNA methyltransferases (DNMTs) tightly regulate both the initiation and maintenance of these methyl marks. Loss of critical components of this enzymatic machinery results in growth, viability and differentiation defects in both mice and humans, supporting the notion that this epigenetic modification is essential for proper development. Beyond this, DNA methylation also provides a potent epigenetic mechanism for cellular memory needed to silence repetitive elements and preserve lineage specificity over repeated cell divisions throughout adulthood. Recent work highlighting the specialized roles of DNA methylation and methyltransferases in maintaining adult somatic stem cell function suggests that further dissection of these mechanisms will shed new light on the complex nature of self-renewal.” 

The 2010 study DNMT1 maintains progenitor function in self-renewing somatic tissue gets down to more specifics.  “Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation.  — These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.”

The 2009 publication DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells establishes a similar critical role for DNMT1  in hematopoietic stem and progenitor cells. “DNA methylation is essential for development and in diverse biological processes. The DNA methyltransferase Dnmt1 maintains parental cell methylation patterns on daughter DNA strands in mitotic cells; however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we conditionally disrupted Dnmt1 in the hematopoietic system. Defects were observed in Dnmt1-deficient HSC self-renewal, niche retention, and in the ability of Dnmt1-deficient HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had specific impact on myeloid progenitor cells, causing enhanced cell cycling and inappropriate expression of mature lineage genes. Dnmt1 regulates distinct patterns of methylation and expression of discrete gene families in long-term HSCs and multipotent and lineage-restricted progenitors, suggesting that Dnmt1 differentially controls these populations. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment.”

The methyltransferases are also important in maintaining genomic stability of neural stem cells. Then 2009 study Cellular epigenetic modifications of neural stem cell differentiation reports : “Emerging information indicates that epigenetic modification (i.e., histone code and DNA methylation) may be integral to the maintenance and differentiation of neural stem cells (NSCs), but their actual involvement has not yet been illustrated. In this study, we demonstrated the dynamic nature of epigenetic marks during the differentiation of quiescent adult rat NSCs in neurospheres. A subpopulation of OCT4(+) NSCs in the neurosphere contained histone marks, trimethylated histone 3 on lysine 27 (3me-H3K27), 2me-H3K4, and acetylated H4 (Ac-H4). A major decrease of these marks was found prior to or during differentiation, and was further diminished or reprogrammed in diverse subpopulations of migrated NSCs expressing nestin or beta-III-tubulin. –. Furthermore, we found an outward translocation of DNA methylation marker 5-MeC, DNMT1, DNMT3a, and MBD1 in NSCs as differentiation began and proceeded; 5-MeC from homogeneous nucleus to peripheral nucleus, and DMNT1a and 3a from nuclear to cytoplasm, indicating chromatin remodeling. —  These results indicate that chromatin is dynamically remodeled when NSCs transform from the quiescent state to active growth, and that DNA methylation modification is essential for neural stem cell differentiation.”

Embryonic and induced pluripotent stem cells and maintenance of pluripotency

The methyltransferases play somewhat of a different role when it comes to fully pluripotent cells – embryonic stem cells and, most likely, induced pluripotent stem cells.  Philosophically, I like the position taken in the 2008 paper Capturing pluripotency.  “In this Essay, we argue that pluripotent epiblast founder cells in the embryo and embryonic stem (ES) cells in culture represent the ground state for a mammalian cell, signified by freedom from developmental specification or epigenetic restriction and capacity for autonomous self-replication. We speculate that cell-to-cell variation may be integral to the ES cell condition, safe-guarding self-renewal while continually presenting opportunities for lineage specification.”

A key question is “When does a pluripotent stem cell like an Esc or iPSC stay pluripotent and when does it differentiate into a less-pluripotent state?  Addressing this question is the September 2009 publication Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. “Coordinated transcription factor networks have emerged as the master regulatory mechanisms of stem cell pluripotency and differentiation. Many stem cell-specific transcription factors, including the pluripotency transcription factors, OCT4, NANOG, and SOX2 function in combinatorial complexes to regulate the expression of loci, which are involved in embryonic stem (ES) cell pluripotency and cellular differentiation. This review will address how these pathways form a reciprocal regulatory circuit whereby the equilibrium between stem cell self-renewal, proliferation, and differentiation is in perpetual balance. We will discuss how distinct epigenetic repressive pathways involving polycomb complexes, DNA methylation, and microRNAs cooperate to reduce transcriptional noise and to prevent stochastic and aberrant induction of differentiation. We will provide a brief overview of how these networks cooperate to modulate differentiation along hematopoietic and neuronal lineages.”

Also addressing the same question is the 2008 publication Esrrb activates Oct4 transcription and sustains self-renewal and pluripotency in embryonic stem cells.  “The genetic program of embryonic stem (ES) cells is orchestrated by a core of transcription factors that has OCT4, SOX2, and NANOG as master regulators. Protein levels of these core factors are tightly controlled by autoregulatory and feed-forward transcriptional mechanisms in order to prevent early differentiation. Recent studies have shown that knockdown of Esrrb (estrogen-related-receptor beta), a member of the nuclear orphan receptor family, induces differentiation of mouse ES cells cultured in the presence of leukemia inhibitory factor. – Supporting all of these data, stable transfection of Esrrb in ES cell lines proved sufficient to sustain their characteristics in the absence of leukemia-inhibitory factor. In summary, our experiments help to understand how Esrrb coordinates with Nanog and Oct4 to activate the internal machinery of ES cells.”

These two studies suggest that, unlike the case for adult stem cells, other factors like Esrrb are important in maintaining the undifferentiated status of fully pluripotent stem cells.  This result was also observed in a 2006 mouse study which concluded “Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA.”  The 2010 publication Polycomb complexes act redundantly to repress genomic repeats and genes also suggest other mechanisms that inhibit differentiation in ESCs.

What causes embryonic stem cells to differentiate? The 2009 report Cdk2ap1 is required for epigenetic silencing of Oct4 during murine embryonic stem cell differentiation indicates “Here, we show that Cdk2ap1, a negative regulator of Cdk2 function and cell cycle, promotes Oct4 promoter methylation during murine embryonic stem cell differentiation to down-regulate Oct4 expression.”

When ESCs do differentiate, then promoter methylation comes into play as indicated in the April 2010 publication Targeting of de novo DNA methylation throughout the Oct-4 gene regulatory region in differentiating embryonic stem cells. “Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyze the progression of DNA methylation of the approximately 2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a.”

Summarizing the situation:

·        The pluripotency and differentiation of ESCs and iPSCs are regulated by complex networks which maintain dominance of cell ground-state pluripotency transcription factors like OCT4, SOX2 and NANOG until differentiation is triggered.  Apparently, the methyltransferases do not play a dominant role in that process though they may be involved.

·        When ESCs start to differentiate, silencing of the OCT4 gene seems to take place via promoter methylation of this gene.  At that point methyltransferases become important for maintaining lineages of adult stem cells

.·        Adult stem cells, including neural progenitor cells and hematopoietic stem cells depend on DNA methylation for their survival in undifferentiated state.  This methylation in turn depends critically on the actions of DNA methyltransferases.  In plain language, the methyltransferases keep lineages of adult stem cells continuing in their niches throughout life instead of having all the adult cells differentiating early in life leaving no reserves of such cells.

So, DNA promoter regulation via methylation in stem cells is an important mechanism for the operation of the stem cell supply chain. 

There is also a growing number of publications on DNA methylation and the role of methyltransferases in cancer stem cells, and I will probably take that topic up in a later blog post.

DNA repair is a major defense against the second cause of aging described in my treatise Cell DNA Damage.  Such repair is absolutely necessary. Damage can be caused by oxidative processes, radiation exposure, and exposure to environmental toxins, cigarette smoke and some antibiotics, and anti-inflammatory drugs(ref). Even without extraordinary exposure, in the course of a normal good day a person may have a million or more events of DNA damage occur in his or her body.  Further, the kinds of DNA damage can be of multiple types(ref).  Failure to repair damage can lead to cell death, cancer, a number of diseases and premature aging.   

In response to this challenge, cells have evolved numerous repair strategies.  Some are very clever and still being discovered. I discussed one such line of defense against an important form of DNA damage, double-strand breaks, in my March 2010 blog entry DNA repair cleanup failure – a root cause for cancers?  I concluded that entry by saying “There is a lot more interesting research related to DNA repair beyond the thread covered here and I will probably come back to that topic again before too long.”  That time is now. I review several additional natural DNA repair strategies together with news of recent discoveries.   

Ku and making ends meet 

In the earlier blog entry and with respect to the substance Ku that I am concerned with here, the focus is on one particular important kind of breaks, double-strand breaks (DSBs), breaks that can occur naturally in cell differentiation or that are created by radiation and certain chemicals.    A double-strand break results in a broken chromosome, and this kind of DNA damage is particularly difficult to repair.  Because these breaks completely threaten genomic integrity, evolution has provided us with a number of sophisticated approaches for automatic DNA repair. Non-homologous DNA end-joining (NHEJ) is the main pathway for repairing double-stranded DNA breaks.  It functions throughout the cell cycle to repair such lesions. “NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately.^[1][2][3][4] Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells(ref).^[5] ” 

A protein called Ku has been known for some time to be involved in NHEJ(ref)(ref).  A colorful animation of the role of Ku in NHEJ can be found here.  Previously, it was thought that Ku worked simply by recognizing  broken ends and then recruiting other factors that cleaned up the ends and then joining them again.  The ends-cleanup processing is necessary because the strand breaks are often associated with nucleotide damage so that simply connecting ends would result in mutated chromosomes. The April 2010 online publication Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends says that Ku does the ends-cleanup job itself. “Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well.”  As reported in Science Daily quoting Dale Ramsden one of the authors and investigators, “Ku is a very exciting protein because it employs a unique mechanism to repair a particularly drastic form of DNA damage.  — Damage to DNA in the form of a broken chromosome, or double strand break, can be very difficult to repair — it is not a clean break and areas along the strand may be damaged at the level of the fundamental building blocks of DNA — called nucleotides — It has been assumed in the past that double strand breaks are the most difficult class of DNA damage to repair and it is often presumed that they simply cannot be repaired accurately.”  Part of the importance of this new research is showing that repairs of double-strand breaks can be more accurate than previously thought.  Ku-based healing is not only at the chromosome level but also at the nucleotide level. 

DNA unwrapping/wrapping in repairing single-strand breaks. 

My power boat ties up to the dock using heavy 3-strand polyvinyl rope.  If a single strand breaks I can fuse it together with heat from a small blowtorch.  But first I must unwrap the strands some to identify the break and make room for the repair.  It turns out that the DNA repair machinery does something very similar.  The 2009 publication DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway reports on such unwrapping.  As explained simply in a Science Daily article: “They found that the proteins that initially recognize the damage amplify the distortion of the DNA around the damaged site by bending the DNA and separating the strands of the double helix. This makes it easier for the next protein to recognize and cut out the damaged portion of the DNA. The cells then patch up the empty space using the healthy half of the DNA as a model to repair the cell to its original state. — The study was conducted using a DNA repair system operated in E. coli, but the findings are applicable to other cells because they adopt similar systems.”  The following item relates to the repair process that takes place after the DNA strands unwind. 

A shuttlebus first-responder repair protein, SSB 

For some time, the protein SSB has been known to play a role in excision repair(ref) of DNA single-strand breaks.  The 2009 publication SSB protein diffusion on single-stranded DNA stimulates RecA filament formation lends insight into the ways in which SSB works. “Single-stranded DNA generated in the cell during DNA metabolism is stabilized and protected by binding of ssDNA-binding (SSB) proteins. Escherichia coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high-affinity protein–DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. — tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.”   

A press release from the University of Illinois  explains the actions of SSB in simpler terms.  “– a single-stranded DNA-binding protein (SSB), once thought to be a static player among the many molecules that interact with DNA, actually moves back and forth along single-stranded DNA, gradually allowing other proteins to repair, recombine or replicate the strands.”  SSB is a first responder. Think of it as a crew first sent out on a small railway shuttlebus car when there is trouble with the tracks.  The crew includes the supervisor who will oversee emergency measures and the repairs.  “Whenever the double helix of DNA unravels, exposing each strand to the harsh environment of the cell, SSB is usually first on the scene, said University of Illinois physics professor and Howard Hughes Medical Institute investigator Taekjip Ha, who led the study. — Although DNA unwinding is necessary for replication or recombination, it is normally a transient process, he said. Exposed single-stranded DNA (ssDNA) can be damaged or degraded by enzymes in the cell. Damaged DNA may also come unwound, and ssDNA can bond to itself, forming hairpin loops and other problematic structures. — “If you have lots of single-stranded DNA in the cell, basically it’s a sign of trouble,” Ha said. “SSB needs to come and bind to it to protect it from degradation and to control what kind of proteins have access to the single-stranded DNA.” Although other proteins are known to travel along double-stranded DNA, this is the first study to find a protein that migrates back and forth randomly on single-stranded DNA, Ha said.” 

“– the researchers showed that SSB diffuses randomly back and forth along single-stranded DNA, and that this movement is independent of the sequence of nucleotides that make up the DNA. They also found that an important DNA repair protein in E. coli, RecA, grows along the ssDNA in tandem with the movement of SSB. As the RecA protein extends along the DNA strand it prevents the backward movement of the SSB. — The researchers also found that SSB can “melt” small hairpin loops that appear in single-stranded DNA, straightening them so that the RecA protein can bind to and repair them. In this way SSB modulates the activity of RecA and other proteins that are involved in DNA repair, recombination and replication. — “SSB may be a master coordinator of all these important processes,” Ha said(ref).” 

The role of HMGB1 

No, HMGB1 is not an agency in the Russian secret service.  HMGB1 stands for high mobility group box protein 1.   It is a protein that pays an important role in DNA repair, though what to do about it is controversial. HMGB1 is an Alarmin. “Alarmins are a newly described and still emerging group of structurally diverse, but functionally related, molecules that include defensins, cathelicidins, eosinophil-derived neurotoxin, and HMGB1 — All are released in response to infection and tissue damage, and mediate innate immunity and tissue repair(ref).”  Let’s start with the bad reputation for HMGB1, which is that it causes inflammation and plays a role in creating epileptic episodes and is implicit in the progress of many cancers.  For example the medical news report Salute: epilessia? è colpa della molecola HMGB1 translate into Health:  epilepsy?  It the fault of the molecule HMGB1.  Many publications suggest that targeting HMGB1 could provide effective cancer therapies(ref).  The 2003 publication Dealing with death: HMGB1 as a novel target for cancer therapy suggests the development of anti-cancer drugs that work by inhibiting HMG1. HMGB1 is thought to play a key role in chronic inflammatory autoimmune disease and as well as in severe, acute systemic inflammatory disease(ref). ”Because HMGB1 plays a key role in inflammation, it’s also being targeted in drugs under development for rheumatoid arthritis and sepsis. “   A quite different view of HMGB1 is suggested in the 2008 medical news report Suspect protein HMGB1 found to promote DNA repair, prevent cancer.  “An abundant chromosomal protein that binds to damaged DNA prevents cancer development by enhancing DNA repair”–  “Long known to attach to sites of damaged DNA, the protein was suspected of preventing repair. “That did not make sense to us, because HMGB1 is a chromosomal protein that’s so abundant that it would be hard to imagine cell repair happening at all if that were the case,” Vasquez (senior researcher in the study) said.  In a series of experiments — Vasquez and first author Sabine Lange, — tracked the protein’s impact on all three steps of DNA restoration: access to damage, repair and repackaging of the original structure, a combination of DNA and histone proteins called chromatin. — First, they knocked out the gene mouse embryonic cells and then exposed cells to two types of DNA-damaging agents. One was UV light, the other a chemotherapy called psoralen that’s activated by exposure to darker, low frequency light known as UVA. In both cases, the cells survived at a steeply lower rate after DNA damage than did normal cells. — Next they exposed HMGB1 knockout cells and normal cells to psoralen and assessed the rate of genetic mutation. The knockout cells had a mutation frequency more than double that of normal cells, however, there was no effect on the types of mutation that occurred. –Knock out and normal cells were then exposed to UV light and suffered the same amount of damage. However, those with HMGB1 had two to three times the repair as those without. Evidence suggests that HMGB1 works by summoning other DNA repair factors to the damaged site, Vasquez said.”

Going back to the issue discussed above of DNA unwrapping/wrapping in repairing single-strand breaks, HMGB1 apparently plays an important role in that process.  “Lange and Vasquez hypothesize that HMGB1 normally binds to the entrance and exit of DNA nucleosomes, so is nearby when DNA damage occurs. It then binds to and bends the damaged site at a 90-degree angle, a distortion that may help DNA repair factors recognize and repair the damage. After repair it facilitates restructuring of the chromatin(ref).”

“Pinpointing HMGB1’s role in repair raises a fundamental question about drugs under development to block the protein, Vasquez said.” “Our findings suggest that depleting this protein may leave patients more vulnerable to developing cancer.”  I wonder if this view is giving any pause to those who are out  pushing the development of HMGB1 inhibitors as drug candidates?  I think it should.

I expect I will be coming back to DNA repair yet-again before too long.

Up until a couple of months ago, the answer seemed very clear to me.  Resveratrol offers a number of powerful health-promoting effects.  Also, it turns on the SIRT1 gene activating the same evolutionary-conserved pathway that is known to confer longevity in case of calorie restriction.  But a pair of recent publications cast doubt on this picture.  This blog entry reviews research on the beneficial health effects of resveratrol and whether or not it indeed activates the SIRT1 gene.  I also touch on other substances being developed to turn on the SIRT1 gene and how certain big pharma and biotechcompanies appear to be lining up taking conflicting viewpoints.  Large stakes are involved.   

Resveratrol  (trans-resveratrol, 3,4′,5-trihydroxystilbene) is a substance occurring naturally in several plants in response to stress, attack by pathogens such as bacteria or fungi, or ultraviolet  radiation.  Discovered to be in red wine in 1992, the substance has been extensively studied for its medicinal and possible anti-aging properties.  The resveratrol site of the Linus Pauling Institute, is a good source of information on all aspects of the substance with 109 literature citations but appears not to be up-to-date since no citations subsequent to 2006 are listed. The Wikipedia article on resveratrol is also a good general information source but appears also not to be completely current. 

Bioavailability of resveratrol is low because it is rapidly metabolized and eliminated.  “–much of the basic research on resveratrol has been conducted in cultured cells exposed to unmetabolized resveratrol at concentrations that are often 10-100 times greater than peak concentrations observed in human plasma after oral consumption (ref)(ref)(ref).”   Therefore, conclusions of such studies may not be applicable even for people who take large amounts of supplementary resveratrol. New micronized forms of resveratrol and sublingual tablets may increase bioavailability(ref)(ref).  Resveratrol appears to exercise few side effects even at large dose levels(ref)(ref).  Since effects may vary widely by individual depending on genetic makeup, however, it is appropriate to exercise caution with large doses. 

Example health benefits of resveratrol 

·        Studies have included looking at resveratrol’s actions in leukemia and in colon, stomach, pancreatic, esophageal, intestinal, and breast cancers, both from preventative and therapeutic viewpoints.  “Although resveratrol can inhibit the growth of cancer cells in culture and in some animal models, it is not known whether high intakes of resveratrol can prevent cancer in humans(ref).” See the discussion and citations here. Multiple forms of biological activity against cancers can be involved. For example, resveratrol might prevent cancer by inhibiting  the expression of certain cytochrome p450 enzymes(ref).   To find recent and current publications related to resveratrol and cancer, I suggest doing a search on “cancer resveratrol” in PubMed.org.

·        “Resveratrol has been found to exert a number of potentially cardioprotective effects in vitro, including inhibition of platelet aggregation (47, 48, 68), promotion of vasodilation by enhancing the production of NO (46, 69) and inhibition of inflammatory enzymes (34, 70, 71). However, the concentrations of resveratrol required to produce these effects are often higher than those that have been measured in human plasma after oral consumption of resveratrol (7)(ref).”  There is an impressive number of current and recent publications related to resveratrol and cardiovascular issues as can be found by doing a search on “cardiovascular resveratrol” in PubMed.org. 

·        Numerous studies indicate that resveratrol might be be useful in control of obesity and diabetes.  A March 2010 publication Resveratrol, obesity and diabetes states “It is well established that resveratrol exerts beneficial effects in rodents fed a high-calorie diet. In some studies, resveratrol was reported to reduce body weight and adiposity in obese animals. The action of this compound involves favourable changes in gene expressions and in enzyme activities. The accumulating evidence also indicates the benefits of resveratrol in diabetes and diabetic complications. It is known that resveratrol affects insulin secretion and blood insulin concentration. In animals with hyperinsulinemia, resveratrol was found to reduce blood insulin. Moreover, numerous data indicate that in diabetic rats, resveratrol is able to reduce hyperglycemia.”

·        “In November 2008, researchers at the Weill Medical College of Cornell University reported that dietary supplementation with resveratrol significantly reduced plaque formation in animal brains, a component of Alzheimer and other Neurodegenerative diseases.^[23] In mice, oral resveratrol produced large reductions in brain plaque in the hypothalamus (-90%), striatum (-89%), and medial cortex (-48%) sections of the brain. In humans it is theorized that oral doses of resveratrol may reduce beta amyloid plaque associated with aging changes in the brain(ref).”

·        Re. resveratrol and bone loss see these articles.  Re. resveratrol and prostate function see these articles.  Re. resveratrol and stroke see these articles.  Re. resveratrol and cataracts see these articles.  Re resveratrol and its anti-viral activities see these articles. 

The main messages up to this point are: 

·        Resveratrol appears to offer a wide range of potential health benefits based on biochemical, cell-level and in some cases, small-animal studies.  Except for bioavailability, it appears to be an amazing supplement.

·        It is unclear as to whether taking the usual doses of commercial resveratrol supplements lead to any of these benefits, however, first because of absence of any clinical studies on humans, and second because of resveratrol’s low bioavailability profile. 

Resveratrol activating SIRT1 

SIRT1, you will recall is the evolutionary-conserved gene activated by calorie restriction that, when activated, provides health and life extension in a number of lower species.  The SIRT1 protein is the most interesting member of the sirtuins, protein deacetylases. I have discussed aspects of SIRT1 in several blog posts and cited this current review of 10 years of research in sirtuins by Leonard Guarante and a colleague. 

In the course of these discussions, I have in keeping with the literature repeatedly asserted that resveratrol activates SIRT1.  I have also provided multiple literature citations to back up this assertion.   The blog post SIRT1, mTOR, NF-kappaB and resveratrol and this 2010 publication, for example, indicates how resveratrol inhibits mTOR signaling while activating SIRT1 showing a linkup between the mTOR “shortivity” pathway and the SIRT1 “longevity” pathway.  This article suggests a linkup between TXNIP, another “shortivity” pathway,  and the SIRT1 “longevity” pathway.   

I repeat a couple of paragraphs here from my blog post Visit with Leonard Guarante which are directly relevant to the present discussion.  “The work of Guarante and his colleagues has led to the identification of  resveratrol as an activator of SIRT1 and later to the establishment of Sirtris Pharmaceuticals, a company devoted to the discovery of small-molecule activators of sirtuins that could address diseases of aging.   Reported in the Sirtris web site, “A long-term study of middle-aged mice shows resveratrol improves health and mimics some benefits of dietary restriction(ref).”

“David Sinclair, a key player in sirtuins research at Harvard and founder of Sirtris, originally came from Australia to work at MIT in Guarante’s lab.   Leonard is on the Board of Sirtris which has been acquired by GlaxoSmith Kline (GSK).  The 2007 publication Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes by Sinclair and other Sirtris-affiliated  authors is one of a number of publications relating SIRT1 to type 2 diabetes.  “Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival^10–14   Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme—peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes(ref).” 

Sirtris currently has four SIRT1 activator substances in Phase IIa clinical trials(ref).  Phase I safety and dose trials of SRT2104 are complete.  The substances are identified as SRT2104, SRT2379 and SRT501.  Trials relate to metabolic disease (Type 2 Diabetes), inflammation, cardiovascular disease and oncology.”

The April 2009 paper Sirtuin activators summarized the most-accepted view of the situation at the time of its publication “CONCLUSIONS: To date, resveratrol is the most potent natural compound able to activate SIRT1, mimicking the positive effect of calorie restriction. Resveratrol might help in the treatment or prevention of obesity and in preventing the aging-related decline in heart function and neuronal loss. As resveratrol has low bioavailability and interacts with multiple molecular targets, the development of new molecules with better bioavailability and targeting sirtuin at lower concentrations is a promising field of the medicinal chemistry. New SIRT1 activators that are up to 1000 times more effective than resveratrol have recently been identified. These improve the response to insulin and increase the number and activity of mitochondria in obese mice. Human trials with a formulation of resveratrol with improved bioavailability and with a synthetic SIRT1 activator are in progress.”  The new “1000 times more effective” SIRT1 activators referred to here are the molecules developed by and being tested by Sirtris.As written in a February 2010 Special Report in Gen Sirtuins: Antiaging Medicines or Marketing? – “A large body of basic research does indeed support the nomination of SIRT1 activators as antiaging drugs. SIRT1 activation has profound metabolic effects: It regulates glucose or lipid metabolism through its deacetylase activity for over two dozen known substrates and has a positive role in the metabolic pathway through its direct or indirect involvement in insulin signaling. It also stimulates glucose-dependent insulin secretion from pancreatic β cells and directly stimulates insulin-signaling pathways in insulin-sensitive organs. SIRT1 also reportedly influences adiponectin secretion, inflammatory responses, gluconeogenesis, and levels of reactive oxygen species, which together contribute to the development of insulin resistance.” 

The dissenting publications 

Two dissenting publications appeared recently, ones that challenge whether resveratrol or any of the Sirtris small-molecule activators actually activate SIR1 Also indirectly challenged is whether Sirtris has a sound platform of drug candidates with which to succeed.   

The October 2009 paper Resveratrol is Not a Direct Activator of SIRT1 Enzyme Activity in the journal Chemical Biology and Drug Design  is by a group of authors associated with Amgen Inc.  The researchers claimed that the activation of SIRT1 observed by Sinclair and his colleagues and reported in the publication Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes was not real but was an artifact of the use of a high-throughput in vitro fluorescence polarization assay.  The Amgen authors write “Here, we show that: (i) the Fluor de Lys-SIRT1 peptide is an artificial SIRT1 substrate because in the absence of the covalently linked fluorophore the peptide itself is not a substrate of the enzyme, (ii) resveratrol does not activate SIRT1 in vitro in the presence of either a p53-derived peptide substrate or acetylated PGC-1alpha isolated from cells, and (iii) although SIRT1 deacetylates PGC-1alpha in both in vitro and cell-based assays, resveratrol did not activate SIRT1 under these conditions.” 

The second dissenting paper SRT1720, SRT2183, SRT1460, AND RESVERATROL ARE NOT DIRECT ACTIVATORS OF SIRT1 was published in January 2010 in the Journal of Biological Chemistry.  Several of the co-authors are associated with Pfizer.  SRT1720, SRT2183 and SRT1460 were earlier reported by Sirtris to be SIRT1 activators.  This is a highly technical paper where the researchers developed their own assay approach.  “These data demonstrate that neither the Sirtris series nor resveratrol would serve as useful  pharmacological tools due to their highly promiscuous profiles. – Our results show that the Sirtris series of compounds and resveratrol have little or no effect on SIRT1 activity even with these two full length protein substrates. — In summary, our detailed assessment of the Sirtris series and resveratrol involving several biochemical assays with native substrates and biophysical studies employing NMR, SPR, and ITC demonstrate that these compounds are not direct SIRT1 activators. We also demonstrated that SRT1720 does not show beneficial effects in a rodent diabetes model, which is in contrast to that previously reported (26). The broad selectivity assessment against over 100 targets including receptors, enzymes, ion channels, and transporters show that the Sirtris series and resveratrol are highly promiscuous and would not serve as useful pharmacological tools for studying SIRT1 pathways. In the literature, resveratrol has been widely referred to as a “SIRT1 activator” (For selected recent references, see 40-44) and routinely used to “activate” SIRT1 in variouscellular assays, with only a few questioning the original study that reported its ability to activate SIRT1 in an artificial substrate-based fluorescent assay (28,29,39,45). Likewise, the Sirtris compounds have been referred to as “SIRT1 activators” in recent publications (46-48). Our present data are significant for the field as we provided strong evidence that neither the Sirtris series nor resveratrol are direct SIRT1 activators.” 

Where am I in all this? 

First of all, I am skeptical of the skeptics.  This paper by the Pfizer people appears to condemn resveratrol along with the Sirtris small-molecule compounds for two reasons: 1.  It does not really directly activate SIRT1, and 2.  It is biochemically highly “promiscuous,”  making it a kind of biochemical whore.  Even if point 1. Were correct, what about the very large number of publications (hundreds) from researchers around the world written over nearly two decades documenting the positive health benefits of resveratrol quite independently of whether it has any SIRT1 action?  Did all of those researchers miss seeing or overlook the promiscuity?  Were they carried away by resveratrol’s sexiness?  Perhaps, but I have trouble believing that.   And, if resveratrol were indeed so promiscuous, why does it seem to have so few negative side effects and so many positive effects?   

And as far as resveratrol and SIRT1 are concerned, I would ask the question “If resveratrol does not activate SIRT1, then why does it seem to provide so many of the same health benefits that would be expected from activating SIRT1?” 

Second, the stakes involved are big.  If the Sirtris small molecules turn out to be worthless, then just what will have GSK gotten for the $720 million they paid for Sirtris?  If the original studies done at MIT and Harvard and Sirtris on SIRT1 activation are based on faulty science, what will be the result on the reputations of the key scientists and laboratories involved?  

Third, the results of the Sirtris clinical trials should tell a lot.  If one of the substances turns out to be a blockbuster, this should send the SIRT1 activator skeptics scampering.  If all the Sirtris substances fizzle, then perhaps the rival skeptics are in part right.  But even if all the proprietary Sirtris SIRT1 activators fizzle as drugs, that still won’t explain all the positive research results on health benefits of good old resveratrol. 

Finally, being just an individual, I have no personal desire to get anywhere near the middle of a knife fight between pharmaceutical and biotech giants like Pfizer, GSK and Amgen and research institutions like Harvard and MIT. 

Please see the medical disclaimer for this blog.

Astragalus-based dietary supplements that are known to activate the expression of telomerase have been on the market for several years now.  However, there appears to be a significant difference between what these supplements are widely publicized to do and what published scientific research says they actually do.  Specifically, the promotion and press coverage often implies that such supplements will extend the lengths of telomeres in people who take them and thus confer longevity benefits.  However, there appears to be virtually no clinical research evidence to support such claims.  On the other hand, research does suggest that at least one of the supplements can provide several important health benefits.  In this blog post I seek to penetrate through the thick layers of commercial and PR fog about such supplements and get down to what is actually known about their actions. 

History 

During the 1990s and early 2000s, Geron, a small biotech company, was a leader in research relating to telomeres and telomerase.  Few scientists and no other significant biotech or pharmaceutical company paid much attention to telomeres or telomerase back then.  Based on its research, Geron applied for 279 patents related to telomeres or telomerase.  One of the major areas of research concern to Geron then was telomere activation as an approach to disease prevention and longevity.  The company discovered that certain extracts of the astragalus plant had a capability to activate the expression of telomerase in certain cell types, at least under test-tube conditions.   

From the Geron web site: “Geron, in collaboration with the Biotechnology Research Institute (BRC), a company established by the Hong Kong University of Science and Technology (HKUST), began screening for telomerase activators in early 2000. The source of material for the screen was natural product extracts from traditional Chinese medicines. In the course of the screening, several extracts were discovered that reproducibly up-regulated the low, basal level of telomerase in human skin cells. With analysis of the extract and further testing, one compound in the extracts was identified as a key telomerase activator. It was capable of activating telomerase in other human cells types (e.g., lymphocyte immune cells) at very low concentrations. Another compound, a derivative of the first, was also present in the extract but at lower concentrations and was also found to possess similar telomerase activating properties. These molecules are currently under development for the treatment of degenerative diseases. Other small molecule activators discovered during the course of the research may also be developed for certain disease indications.”

The research resulted in Geron applying for a patent on telomerase activators which was finally issued just a few months ago.  Filed 06/23/2004, the patent is called Compositions and Methods for Increasing Telomerase Activity.  Since publication 05/15/2008, the patent application can be read by anyone and the descriptions found there still provide much  of the scientific rationale for people taking astragalus-based telomerase-activator supplements.  The patent application introduction states “The present invention relates to methods and compositions for increasing telomerase activity in cells. Such compositions include pharmaceutical, including topical, and nutraceutical formulations. The methods and compositions are useful for treating diseases subject to treatment by an increase in telomerase activity in cells or tissue of a patient, such as, for example, HIV infection, various degenerative diseases, and acute or chronic skin aliments. They are also useful for enhancing replicative capacity of cells in culture, as in ex vivo cell therapy and proliferation of stem cells.” 

Geron subsequently shifted its focus to other areas of research including embryonic stem cell therapies and developing drugs that turn telomerase off in cancer cells.  As far as I can tell, Geron is currently pursuing telomere activation mainly via a subsidiary and marketing licensing agreements.  Geron is the majority owner of TA Therapeutics, a Hong Kong subsidiary which is focusing on telomerase activation for organ renewal and prolonging the lives of AIDS patients.  A US company, TA Sciences, has licensed one telomerase-activator extract from Geron called TA-65 in 2002, a nutraceutical it has been marketing it to the public for over three years now. 

Of the Geron-researched telomerase-activating products, two in particular have received the most attention: TA-65 being marketed to the public by TA Sciences and TAT2 under investigation as part of drug development by TA Therapeutics.  Both formulations are carefully guarded proprietary secrets of the companies involved.  I suspect the two substances are either highly related or identical.  There has been much speculation as to what TA-65 consists of, particularly in online longevity-related forums(ref)(ref).  Based on reading the Geron patent, it appears that a number of astragalus membranaceus extracts exhibit varying degrees of capability to promote the expression of telomerase(ref).  One extract mentioned in the patent is astragaloside IV, and another extract with roughly ten times the activation potency is cycloastragenol, and there are others as well.  Based on careful reading of the patent and the dosage originally suggested by TA sciences the best informed guess is that TA-65 and TAT2 are cycloastragenol, but this is only a guess.   

TA Sciences is an active marketing company and any search on Google related to telomerase will often produce prominent advertising related to TA-65 and its health and longevity benefits.  TA-65 does not come cheap.  When the company first started marketing it, it was available only as part of a “Patton Protocol” package with cost of $25,000 for the first year.  (Noel Patton is the founder of TA Sciences.)  Now, the Patton Protocol is offered in either an a-la-cart mode or as a full package.  The cost of six months of the protocol including the TA-65, some other supplements, a visit to a doctor and a number of diagnostic tests is $6,725.  Cost of a six-month supply of TA-65 alone is $4,000.  It is interesting that when it was originally marketed the daily dosage of TA-65 was 5 mg and the daily dosage has been increased now to 100mg, by a factor of 20.  This has led to speculation that the substance may not be pure cycloastragenol which is very expensive to produce. 

Besides TA-65 available from TA sciences, based on the information in the Geron patent other companies have started to market both astragaloside IV and cycloastragenol as telomerase activator supplements(ref)(rev).  These supplements are being sold considerably cheaper than TA-65 with cost of a 30-day supply typically running up to $80.  One such company, Revgenetics, decided to discontinue its cycloastragenol product line when the Geron patent was finally issued and sell of its existing stock at discount, charging $25 for a bottle which contains 30 5mg pills. 

The concept of telomerase activation 

A responsible formulation of the telomerase activation hypothesis is that through systemic intermittent activation of telomerase, specifically in stem and progenitor cells, it may be possible to delay shortening of telomeres and therefore delay the onset of multiple disease and degenerative processes associated with cell senescence.  A very informative 2007 PowerPoint Presentation by Joseph M. Raffaele MD (an affiliate of TA Sciences) states the scientific rationale for telomerase activation and lays out results of a small clinical trial of TA-65.  There is general consensus that too-short telomeres lead to cell senescence leading to the diseases and symptoms of aging.  However, it must be pointed out that many factors affect telomere length, that many complex factors both known and yet-unknown promote or delay the onset of cell senescence, and that telomerase activation does more than affect the lengths of telomeres.  For example, the protein TAp63 strongly affects senescence of stem cells(ref).  I return to this important point later. 

Research on telomerase activators 

So, what research exists on the effects of telomerase activators beyond that which went into the patent?  I will review research here that involves any of the four activator substances mentioned (TA-65, TAT2, Astragaloside IV, Cycloastragenol) recognizing that what is true for one activator may not be valid for another.  With one exception, I will confine myself to publications in established journals or reputable online research publishers and will avoid ungrounded assertions in press releases or opinions stated in blogs.  

·        A small human trial was conducted in 2005 of TA-41, a precursor of TA-65, I believe sponsored by TA Sciences.  This trial is described in a page on the TA Sciences web site and in the aforementioned PowerPoint Presentation.  The trial was a 24-week double-blind, placebo-controlled study involving 36 male subjects between 60 and 85 years of age, a relatively short trial with scale far smaller than typical Phase III FDA-approved trials.  TA-65 is the presumed major metabolite of TA-41. “ — subjects consumed 2 or 4 tablets daily of a placebo control substance (placebo groups) for 12 weeks or 2 or 4 tablets daily of a TA-65 precursor molecule (TA-41) for 12 weeks (product groups). The product tablets each contained 10 mg of TA-41 (an Astragalus extract) along with other botanical extracts and excipients. — The 12 week placebo or product use period was followed by a further 12 week follow-up period.”  My impression is that the experimental design of the study and the treatment of statistical measures were handled quite responsibly.  Nonetheless, because of the small sample size, statistical significance of the results is relatively crude.  The study treated .2 as the p-value for statistical significance though in larger studies .05 or even .01 are typical values.  The major benefits observed among those taking the products were “1.  Apparent improvement in certain immune system measures, 2. Apparent improvement in eye sight, 3.  Apparent improvement in certain sexual function measures, and 4. Apparent improvement in certain skin properties(ref).”  No significant adverse events were identified.  Detailed discussion and diagrams of results can be found on the TA Sciences web page for the study and in the PowerPoint presentation.  To my knowledge the results of this 2005 study have never been published in an established scientific journal.  Nonetheless the study seems to have been well done and I tend to take it seriously.  

·        Dr. Raffaele reports in his 2007 PowerPoint Presentation that “preliminary results of 16 patients o TA-65 for 3 months show an increase of mean lymphocyte telomere length.”  I have seen no further or subsequent details.

·        To my knowledge, there have been no further studies relating actual user experience of those taking TA-65 though by this time there should be considerable experience to report.  Those taking TA-65 as part of the Patton Protocol have had extensive measurements of aging-related biomarkers and their telomere lengths.  I would love to see the data derived from this user cohort laid out.

·        The 2008 study report Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes looked at exposing lymphocyte cells from HIV-infected donors to TAT2. “ — , during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8(+) CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8(+) T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity.  The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation.”  The study suggests a possible health benefit for HIV-infected individuals, individuals who experience an extraordinary high rate of telomere shortening in immune cells due to the disease.  This benefit would have to be verified in clinical tests.  This study says nothing about telomere lengthening.  This study was co-authored by Rita Effros, a leading researcher in the role of telomeres in HIV infections.  This study, by the way, referred to the experimental substance both as TAT2 and as cycloastragenol.

·        A 2005 study Telomerase Therapeutics for Degenerative Diseases describes possible benefits of telomerase activation but provides no experimental results.  There are numerous studies pointing to telomere shortening as an important process contributing to the advance of HIV and studies like this 2010 one looking at telomerase activity and replicative senescence in human CD8 T lymphocytes, but none of those studies are directly concerned with telomerase activation. 

·        The 2009 publication Cycloastragenol extends T cell proliferation by increasing telomerase activity covers another in-vitro study reporting “Naturally, there is a great deal of interest in finding inducers of telomerase that may help delay the onset of cellular aging. There are various nutraceuticals that claim to both increase the health of individuals and delay the onset of cellular aging. We tested the nutraceuticals resveratrol and cycloastragenol for their ability to enhance T cell functions in vitro. In this study we evaluated the effect of these compounds on cellular proliferative capacity, levels of telomerase activity, surface markers and cytokine secretion of human CD4 and CD8 T cells. Our results show that cycloastragenol moderately increase telomerase activity and proliferative capacity of both CD4 and CD8 T cells. These preliminary results suggest that nutraceuticals inhibit the onset of CD4 and CD8 cellular senescence.”  Like in the previously-discussed study the effect was moderate, outside the body, and the study said nothing about extending telomeres. 

Of the telomerase activators mentioned, perhaps the one best covered in the research literature is astragaloside IV.  As I state in my treatise; “Astragaloside IV has been systematically studied for its medicinal properties only recently, mostly in Chinese and European research centers. It is an antiinflammatory, antifibrotic and antioxidant. It is known to have vasodilation and cardioprotective properties. It is neuroprotective and can protect the myocardium against ischemia/reperfusion injury. There are no reported negative side effects. Yet, my impression is that much is yet to be learned about this substance. Specifically, there appears to be little if any research available in the public domain relating astragaloside IV’s medicinal properties to its ability to induce telomerase expression.”  

Research publications related to Astragaloside IV include the 2002 publication Effects of astragaloside IV on myocardial calcium transport and cardiac function in ischemic rats, the 2004 publication Astragaloside IV protects against ischemic brain injury in a murine model of transient focal ischemia, the 2009 publication Effects of Astragaloside IV on heart failure in rats, the 2009 publication Astragaloside IV attenuates cerebral ischemia–reperfusion-induced increase in permeability of the blood-brain barrier in rats, the 2008 report Astragaloside IV inhibits spontaneous synaptic transmission and synchronized Ca2+ oscillations on hippocampal neurons, the 2006 report Effects of astragaloside IV on pathogenesis of metabolic syndrome in vitro, and Effect of astragaloside IV on hepatic glucose-regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin, and the 2006 publication Astragaloside IV from Astragalus membranaceus Shows Cardioprotection during Myocardial Ischemia in vivo and in vitro.  While these and many other research publications relate to potentially beneficial effects of Astragaloside IV, none relate to or even mentions the substance’s role as a telomerase activator.  Of course, some or all of the reported benefits could ultimately be due to telomerase activation. 

Other than the study cited above, the only discussions of cycloastragenol health activities seem to be in longevity blogs chewing over the same material covered here.  It is a relatively unfamiliar substance.  As for astragaloside IV, cycloastragenol suppliers appear to be in China.  Purchasing either of these supplements from a US company, it is good to be on a lookout for independent laboratory verification of contents and purity.  

Observation 1: When it comes to telomerase activation, the contrast between what is reported as “research” in the general and commercial literature and what is reported in the filtered scientific research literature is singularly stark.  Pubmed.org is the definitive National Library of Medicine database of medical and related scientific research, containing millions of literature abstracts covering virtually every article in every research publications worldwide.  The following lists the number of items retrieved using Google and using Pubmed in response to the given query. 

Query                                      Found in Google     Found in Pubmed

TA-65 + telomerase                     21,100                7 (all irrelevant)

TAT2 + telomerase                          4,510                1 (cited here)         

astragaloside + telomerase          7,200                0

cycloastragenol + telomerase      1,820                1 (cited here) 

Observation 2: Published studies suggests that telomerase activation may have a positive effect on the immune function, though this conjecture based on lab cell-level studies must be confirmed via large-scale human studies.  How much affect using what activator and under what conditions are as yet not established.   There is also research strongly suggesting important potential health benefits from taking astragaloside IV in particular, and possibly also from taking TAT2 (likely to be the same thing).  How telomerase activation relates to the beneficial effects of these substances, however, remains mostly unstudied. 

Observation 3:  The case for specifically taking TA-65 is mainly based on propriety information provided by TA sciences and doctors offering TA-65 as a treatment, and by the original research done by Geron. The most compelling positive information is that derived from the 2005 human trial sponsored by TA Sciences.  While TA-65 has an immense standing in the popular literature I have had trouble finding any mention of it in the published scientific literature.  In fact, if a query about TA-65 is made in PubMed, part of the reply is “The following term was not found in PubMed: TA-65.”   

Observation 4:  I remind readers that there are research studies establishing that there are other interventions that result in longer telomeres besides taking the telomerase activators discussed here.  See the January 2010 blog entry Vitamins, supplements and telomerase – upregulation or downregulation?  And also see my blog entries Exercise, telomerase and telomeres, Timely telomerase tidbits, Breakthrough telomere research finding, and Telomere and telomerase writings. 

Observation 5: In the scientific literature I have found no published research whatsoever that establishes that any of the telomerase activators mentioned actually extends telomeres.  The closest the literature comes are statements like “moderately increases telomerase activity, ” “modestly retards telomere shortening,”  and “inhibit the onset of CD4 and CD8 cellular senescence.” And these statements are based on cell-level studies with results that may or may not be applicable in live humans.  It is interesting that the TA Sciences web site does not now make the claim that TA-65 actually extends telomeres.  Unfortunately, however, the claim keeps popping up in news stories and some blog postings about the activator substances. 

Observation 6:  What telomerase activators actually do in humans remains a mystery as far as the published scientific literature is concerned, and what the two proprietary activators consist of still remains a mystery as well. I keep awaiting more trustworthy published information. 

Back to the science of telomeres and telomerase 

For those familiar with the great complexities of telomere biology and pathways affecting telomere length management, it should not be surprising that is not so simple as “take a telomerase activator and get longer telomeres.”  Whether telomeres get longer or shorter or stay the same is determined not only by the presence of telomerase but also by interactions involving many signaling paths and activation cofactors.  “Telomere transcription is regulated by several mechanisms: developmental status, telomere length, cellular stress, tumour stage and chromatin structure(ref).”  Presence of a telomerase activator is only one factor in driving telomere lengths. The literature related to telomerase and telomeres is extremely extensive and I have barely touched on it here.  What is largely missing is literature specifically related to the astragalus-based telomerase activators.  

Further, telomerase has other activities besides telomere length maintenance.  Activating the TERT telomerase component may have other positive effects without making telomeres longer.  These positive effects can include promoting cellular and organismal survival(ref) and increasing the rate of differentiation of quiescent adult stem cells(ref).  So, in principle at least, a telomerase activator could convey health benefits independently of affecting telomere lengths.  We just don’t know the extent to which this happens in humans in response to the astragalus-based supplements.   

Personal experience 

My personal experience with telomerase activators may be untypical.  I started taking a large dose of astragalus extract in July 2007 with the intention of telomerase activation, switched to astragaloside IV 50mg a day in August 2008.  As of mid-December 2009, I switched to taking a 5mg cycloastragenol capsule together with a simple astragalus-extract pill which may possibly increase bioavailability.   On February 14 2010, I upped the daily cycloastragenol dose to 10mg. So I have been on some kind of telomerase activator or the other for close to three years now.  What is the effect?  I don’t know, especially because of all the other anti-aging supplements I have been taking and anti-aging lifestyle patterns I have been observing.  If I use the criteria mentioned in the TA Sciences trial, I can personally comment that now at the age of 80: 

1.      Immune system measures:  I seem to have as good or possibly better resistance to infections or viruses as ever.

2.     Eye sight: Distance vision acuity in both eyes is excellent though I require corrective lenses for reading and close-up vision.  My last eye exam showed no progression of druzen or signs of macular degeneration.

3.      Sexual function measures:   No decline in desire, perhaps even a bothersome increase.  Performance and satisfaction enhanced by sildenafil seems OK.

4.     Certain skin properties:  Quality and texture of skin for my age are excellent.

5.     Hair:  And I add one other thing I have been monitoring, which is hair.  About a year ago I wrote in my treatise “I have noticed a few small effects so far. The light patina of grey hairs on my mostly-bald scalp seems to me to be a bit thicker with a few black hairs as well. I have been nearly bald for over 30 years. It is known that in animal models at least, conditional telomerase induction causes proliferation of hair follicle stem cells (ref). It remains to be seen whether I will see more or darker hair as I continue with telomerase activation. Also there seems to be some increase in my sexual libido but this may be a subjective impression. I also do not know if my daily schedule of alternating taking the telomerase activator with the other supplements with a few hours of separation is effective or whether I would be better off alternating every other day or even every other week.”  Since then there are many more grey hairs but not black ones.  I am no longer absolutely bald.  The grey hairs seem to keep coming back but very slowly.   

I am planning to stay with the cycloastragenol supplements until my supply runs out in 3 months or so.  I am not sure what I am going to do for telomerase activation after then. I keep waiting for the “shoe to drop” with more definitive research results becoming available as I have been waiting for three years now.  I think it is ridiculous that we are still relying on 2005 test data from 36 people who were on an activator for only two weeks when now many people have been on such activators for three years or more and have been subjected to systematic age-biomarker and telomere length testing.