The July 2010 blog entry HSP70 to the rescue describes how heat shock protein 70 (HSP70) works to promote survival of cells under stress and provides examples of the positive hormetic effects of this chaperone protein. For example HSP70 is neuroprotection in case of cerebral ischemia. HSP-70 is also protective of cancer cells which gives it its good-guy – bad-guy characteristics. This blog entry briefly reviews the actions of HSP70 again, its role in cancer cells, and research efforts aimed at turning it off in cancer cells.
Roles of HSP70 in cells
One of HSP70’s key job is to act as a cell’s protein-folding officer, detecting unfolded or improperly folded proteins, refolding them properly if possible and, if a protein can’t be folded properly, signaling the cell to commit apoptosis. Unfolded or improperly folded proteins can lead to dysfunctional cells and a number of disorders. In my treatise I have discussed incorrect protein folding as a theory of aging. To protect themselves against folded proteins, cells have evolved what is known as the unfolded protein response (UPR). The UPR process is described in the review paper Signal integration in the endoplasmic reticulum unfolded protein response. “The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways — cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.”
“Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. — Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100(ref).”
So, HSP70 keep cells alive by refolding badly-folded proteins and preventing excess aggregation of proteins in cells. And it helps transportation of proteins to their proper intracellular locations. HSP70 also keeps cells alive by other means. “In addition to the well-studied role of Hsp70 as a molecular chaperone assisting in correct protein folding, several new mechanisms by which Hsp70 can prevent cell death have been described. Hsp70 is now known to regulate apoptotic cell death both directly by interfering with the function of several proteins that induce apoptotic cell death as well as indirectly by increasing levels of the anti-death protein bcl-2(ref).”
HSP70 family members and other heat shock proteins are thought to play a role in delaying aging. Widespread protein aggregation in cells is thought to play a prominent role in aging, at least in lower species and quite possibly in humans(ref). The 2009 publication The shock of aging: molecular chaperones and the heat shock response in longevity and aging–a mini-review concludes “Molecular chaperones play an important role in the deterrence of protein damage during aging and their expression is required for longevity. Chemical stimulation of HSP synthesis might therefore be a significant strategy in future design of antiaging pharmaceuticals.” The previous blog post Valproic acid – The phoenix drug arises again pointed out that valproic acid appears to induce HSP70 in cortical neurons and this may be responsible for some of the neuroprotective and therapeutic effects of valproic acid.
A more complete discussion of HSP70’s protective roles can be found in the blog entry HSP70 to the rescue.
HSP70 in cancer cells
Recently, awareness has been developing that the same mechanisms that make HSP70 protective of normal stressed cells makes it protective of cancer cells.
For some time it has been known that HSP70 is highly expressed in certain cancer cells, although its exact role was unknown. The 2000 paper Hsp70 plasma membrane expression on primary tumor biopsy material and bone marrow of leukemic patients stated “With the exception of mammary carcinomas, an Hsp70 plasma membrane expression was found on freshly isolated human biopsy material of colorectal, lung, neuronal, and pancreas carcinomas, liver metastases, and leukemic blasts of patients with acute myelogenous leukemia. Since normal tissues and bone marrow of healthy human individuals do not express Hsp70 on the cell surface, Hsp70 can be considered as a tumor-selective structure in vivo.”
The 2004 article Heat shock protein 70 promotes cancer cell viability by safeguarding lysosomal integrity started to zoom in on the cancer-specific cell-protective effects of HSP70. “The major heat-inducible Hsp70 is a potent survival protein that confers cytoprotection against numerous death-inducing stimuli and increases the tumorigenicity of rodent cells. The depletion of Hsp70 by adenovirus-mediated transfer of antisense cDNA induces caspase-independent death of tumorigenic cells while non-tumorigenic cells are unaffected, suggesting that Hsp70 has cancer-specific function(s). We have recently demonstrated that the depletion of Hsp70 in cancer cells results in a cysteine cathepsin-dependent death, which is preceded by lysosomal destabilization and release of lysosomal constituents to the cytosol. In line with this, Hsp70 localizes to the membranes of lysosomes in human colon carcinoma cells and immortalized murine embryonic fibroblasts (MEFs) and prevents lysosomal membrane permeabilization and cell death induced by tumor necrosis factor (TNF), etoposide and H2O2. These findings identify Hsp70 as the first survival protein that functions by stabilizing the lysosomal membrane.”
It is increasingly clear that mobilization of HSP70 as well as other heat shock proteins is a survival strategy employed in several cancer lines. The 2010 publication Heat shock proteins in breast cancer progression – A suitable case for treatment? reports “Heat shock proteins (HSP) and heat shock factor 1 (HSF1), key factors in the heat shock response (HSR) have been implicated in the etiology of breast cancer. At least two members of the HSP family, Hsp27 and Hsp70 undergo significant increases in cellular concentration during the transformation of mammary cells. These changes result in HSP-mediated inhibition of tumour cell inactivation through blockade of the apoptosis and replicative senescence pathways. The increases in HSP thus mediate two of the common hallmarks of cancer and favour cell birth over cell death.”
“The cancer microenvironment exposes malignant cells to a variety of stressful conditions that promote protein misfolding. HSP70 helps cancer cells deal with this stress. Unlike normal cells, which typically express little, if any, of HSP70, cancer cells contain high levels of this protein all of the time. Indeed, HSP70 has been termed a cancer-critical survival factor, since cancer cells probably require the actions of this protein to survive the protein-altering adverse conditions(ref).”
A July 2010 report in Science Daily states “Professor Afshin Samali, lead author of the study and head of the Department of Biochemistry at NUI Galway, and his team have discovered that under cell stress conditions Hsp70 interacts with a receptor in the cell, activating survival mechanisms and preventing apoptosis, the normal cell death mechanism. — Professor Samali states: “Our results have identified a novel protein-protein interaction that helps cancer cells to survive stressful growth conditions. By interfering with this interaction we hope to develop a new class of anticancer drugs.” The source publication for the study mentioned in this quote deals with the mechanism of action of HSP72: “Here, we report that Hsp72, a stress-inducible cytosolic molecular chaperone, can bind to and enhance the RNase activity of IRE1Î±, providing an important molecular link between the heat shock response and the ER stress response. Importantly, increased production of active XBP-1 was necessary for Hsp72 to exert its prosurvival effect under conditions of ER stress. Our results suggest a mechanism whereby Hsp72 overexpression helps cells adapt to long-term ER stress in vivo by enhancing the pro-survival effects of the IRE1Î±/XBP1 branch of the UPR.” HSP72 is the same as HSP70 as related to the HSPA1A gene.
Inhibiting HSP70 in cancer cells
The growing awareness of the importance of HSP70 as a survival factor in cancers has motivated a search for new molecules that can inhibit the expression of HSP70.
HSP70 inhibition via anti-sense Hsp70 cDNA
A page from the Sloan-Kettering Institute web site discusses inhibitors of HSP70 expression. One of those is anti-sense Hsp70 cDNA. “It is documented that inhibition of Hsp70 expression by anti-sense Hsp70 cDNA resulted in inhibition of tumor cell proliferation and induction of apoptosis. Depletion of Hsp70 by Ad.asHsp70 led to massive cell death of all tumorigenic cell lines tested (carcinomas of breast, colon, prostate and liver as well as glioblastoma). In spite of an effective depletion of Hsp70, Ad.asHsp70 had no effect on the survival or growth of fetal fibroblasts or non-tumorigenic epithelial cells of breast or prostate.”
HSP70 inhibition via quercetin
The same Sloan-Kettering web page mentions use of quercetin as a HSP70 inhibitor. “Inhibition of hsp70 gene expression has been documented after pharmacological intervention with the flavanoid quercetin. The agent induced apoptosis in several tumor cell lines. In addition, inhibition of hsp70 accumulation by quercetin made cells more susceptible to apoptotic inducers. Quercetin also sensitized cells to hyperthermia, chemotherapy and radiation. Inhibition of hsp70 synthesis as well as induction of apoptosis by treatment with quercetin combined with hyperthermia was reported to be confined to leukemic cells, and not to normal hematopoietic progenitor cells.” The ability of quercetin to kill tumor cells via a mechanism involving HSP70 has been known for some time. The 1994 paper Induction of Apoptosis by Quercetin: Involvement of Heat Shock Protein stated “Quercetin, a widely distributed bioflavonoid, inhibits the growth of tumor cells. The present study was designed to investigate the possible involvement of apoptosis and heat shock protein in the antitumor activity of quercetin. — These results suggest that quercetin displays antitumor activity by triggering apoptosis and that HSP70 may affect quercetin-induced apoptosis.”
The same Sloan-Kettering web page goes on to say “In spite of its evident utility in cancer treatment, quercetin is not potent enough to grant its clinical use. Since the introduction of anti-sense mRNA or siRNAs into humans will be problematic because the extent of inhibition cannot be modulated, and the effects of quercetin are likely pleiotropic, small molecules that directly compromise but not completely inhibit the activities of Hsp70 chaperones will prove clinically valuable to combat cancer. In addition, the above data suggest that an Hsp70 inhibitor concentration can be identified that will not be toxic to healthy cells. To date, however, Hsp70 inhibitors have not been tested in cell or animal cancer models, and very few Hsp70 inhibitors have been identified. — We are interested in identifying both inhibitors of Hsp70 activity and expression and efforts in this regard are currently underway.”
HSP70 inhibition via PES
Late last year there was a report of a possibly promising substance, PES. The October 2009 publication A Small Molecule Inhibitor of Inducible Heat Shock Protein 70 reports “The multifunctional, stress-inducible molecular chaperone HSP70 has important roles in aiding protein folding and maintaining protein homeostasis. HSP70 expression is elevated in many cancers, contributing to tumor cell survival and resistance to therapy. We have determined that a small molecule called 2-phenylethynesulfonamide (PES) interacts selectively with HSP70 and leads to a disruption of the association between HSP70 and several of its cochaperones and substrate proteins. Treatment of cultured tumor cells with PES promotes cell death that is associated with protein aggregation, impaired autophagy, and inhibition of lysosomal function. Moreover, this small molecule is able to suppress tumor development and enhance survival in a mouse model of Myc-induced lymphomagenesis. The data demonstrate that PES disrupts actions of HSP70 in multiple cell signaling pathways, offering an opportunity to better understand the diverse functions of this molecular chaperone and also to aid in the development of new cancer therapies.”
According to a November 2009 Science Daily article on this research Inhibitor Of Heat Shock Protein Is A Potential Anticancer Drug, Study Finds, “The inhibitor, called PES, interferes with the HSP70 activities that the cancer cell needs to survive, so by targeting HSP70, one can target the cancer cell. — The investigators showed that PES interacts with HSP70 by blocking its stress-relieving functions. It also induces HSP70-dependent cell death by disrupting the cell’s ability to remove damaged components. Paradoxically for a compound first identified for blocking the cell-death pathway of apoptosis, PES does kill cells, but by a different mechanism. — PES seems to be specifically targeting HSP70, a protein that is differentially expressed in normal versus cancerous cells, and “one that the cancer cell seems to require to survive” says George. “It’s still early days — we don’t know what it will do in a human. But, the exciting part is that this is a pathway and a protein target that clearly is important for cancer cells. — Given the extreme heterogeneity of cancer cells, simultaneously disabling networks of signaling pathways may be important. Indeed, PES was more or less equally effective in every type of cancer cell tested, she says, “which is unusual and supports the idea that it is targeting a protein that is required for the functioning of multiple pathways.”
The article goes on “”We found several known HSP70-interacting proteins that were no longer interacting properly when the cells were exposed to the small molecule,” Leu notes. — Among those were proteins that help HSP70 refold misfolded proteins and proteins that abet its protein trafficking functions. — When they then studied the effect that loss of those functions had on the cell, the team discovered that PES blocks the cell’s ability to get rid of the proteins damaged by cellular stress in a process called autophagy, a process in which cells were basically eating themselves to death. In mice, Murphy and her students Julia Pimkina and Amanda Frank found that PES could inhibit tumor formation and significantly extend survival. — “That was one of the highlights from our perspective, because PES has potential to be developed as a therapeutic,” says Murphy.’
Wrapping it up:
· HSP70 assures proper folding of proteins in cells and, via this and other mechanisms, is strongly protective of cells under stress and possibly plays a positive role with respect to longevity.
· HSP70 also plays a major role in protecting cancer cells, setting off a search for substances that can inhibit HSP70 expression in cancer cells.
· Quercetin, a substance in the anti-aging firewalls regimen, inhibits the expression of HSP70 in cancer cells, but its effects may be too weak for its use as a clinical therapy.
· A substance called PES shows promise as a possible drug candidate for inhibiting HSP70 expression in cancer cells.
· The research in this area still involves experimentation with laboratory animals and may or may not lead to clinical trials in humans.