Elena Y. Komarova

Downstream caspases are novel targets for the antiapoptotic activity of the molecular chaperone Hsp70

Abstract The response of cancer cells to apoptosis-inducing agents can be characterized by 2 opposing factors, the proapoptotic caspase cascade and the antiapoptotic stress protein Hsp70. We show here that these factors interact in U-937 leukemia cells induced to apoptosis with anticancer drugs, etoposide and adriamycin (ADR). The protective effect of Hsp70 was verified using 2 approaches: mild heat stress and transfection-mediated overexpression of the Hsp70 gene. The increase in Hsp70 levels attained by these 2 methods was found to postpone caspase activation for 12–18 hours. An in vitro assay was developed using mouse myeloma NS0/1 cells, which lack the expression of Hsp70. Measurement of DEVD-ase activity in extracts of apoptotic NS0/1 cells incubated with purified Hsp70 showed that Hsp70 reduced caspase activity by up to 50% of its control value in a dose-dependent manner. The hypothesis that the inhibitory effect of Hsp70 on caspase-3/7 activity related to a direct interaction between Hsp70 and the caspases was tested by reciprocal immunoprecipitations and Far-western analyses. These tests were performed with extracts of Hsp70-overexpressing, control, and ADR-treated U-937 cells and using anti–caspase-3, caspase-7, and anti-Hsp70 antibodies, and the data clearly showed that Hsp70 was able to interact with the proforms of these caspases in cell lysates and with reconstituted purified proteins but did not bind the activated forms of either caspase-3 or -7. This association was also corroborated by a novel, enzyme-linked immunosorbent assay–like assay, protein interaction assay, that combined the advantages of immunoprecipitation and immunoblotting in a 96-well microplate–based assay. Thus, Hsp70 may act to suppress caspase-dependent apoptotic signaling through binding the precursor forms of both caspase-3 and caspase-7 and preventing their maturation.

Drug-induced Myc-mediated apoptosis of cancer cells is inhibited by stress protein Hsp70

The Myc oncoprotein serves a dual function by stimulating cells both towards growth and apoptosis. The latter functions are often abrogated during tumor development. The Hsp70 stress protein is a potent anti‐apoptotic molecule, but its potential role in protecting cells from Myc‐mediated apoptosis has not been investigated. Our results show that activated Myc potentiated apoptosis induced by the cancer drugs etoposide (ETO) and camptothecin (CAMP) in v‐Myc‐expressing human U‐937 monoblastic cells and in Rat1 cells containing a conditionally active Myc/estrogen receptor (MycER) fusion protein. However, both heat shock and ectopic Hsp70 expression protected the cells from Myc‐mediated apoptosis after drug treatment in both systems. The increased susceptibility to the anti‐tumor drugs by activated Myc was enhanced by siRNA‐mediated knockdown of Hsp70 expression in U‐937 cells. Addressing the mechanisms by which Myc and Hsp70 promotes and inhibits drug‐induced apoptosis, respectively, we found that v‐Myc stimulated cytochrome c release and activation of effector caspase‐9, ‐3 and ‐7, but not of initiator caspase‐8. Inhibition of caspase‐9 specifically reduced v‐Myc‐stimulated apoptosis, whereas inhibition of caspase‐8 and ‐3/7 reduced apoptosis both in v‐myc‐expressing and parental ETO‐treated U‐937 cells. Interestingly, Myc‐stimulated activation of effector caspases was inhibited, but cytochrome c release was not affected by Hsp70 expression, suggesting that Hsp70 interferes with the proapoptotic function of Myc downstream of mitochondria, at the level of caspase‐9 and downstream caspases. In conclusion, Hsp70 seems to have key function in inhibition of apoptosis mediated by Myc and may therefore play an important role in Myc‐driven oncogenesis.

Effective immunotherapy of rat glioblastoma with prolonged intratumoral delivery of exogenous heat shock protein Hsp70

Chaperone Hsp70 can activate adaptive immunity suggesting its possible application as an antitumor vaccine. To assess the therapeutic capacity of Hsp70 we administered purified chaperone into a C6 glioblastoma brain tumor and explored the viability and tumor size as well as interferon gamma (IFNγ) production and cytotoxicity of lymphocytes in the treated animals. Targeted intratumoral injection of Hsp70 resulted in its distribution within the area of glioblastoma, and caused significant inhibition of tumor progression as confirmed by magnetic resonance imaging. The delay in tumor growth corresponded to the prolonged survival of tumor‐bearing animals of up to 31 days versus 20 days in control. Continuous administration of Hsp70 with an osmotic pump increased survival even further (39 days). Therapeutic efficacy was associated with infiltration to glioblastoma of NK cells (Ly‐6c+) and T lymphocytes (CD3+, CD4+ and CD8+) as well as with an increase in the activity of NK cells (granzyme B production) and CD8+ T lymphocytes as shown by IFNγ ELISPOT assay. Furthermore, we found that Hsp70 treatment caused concomitantly, with a tenfold elevated IFNγ production, an increase in anti‐C6 tumor cytotoxicity of lymphocytes. In conclusion, continuous intratumoral delivery of Hsp70 demonstrates high therapeutic potential and therefore could be applied in the treatment of glioblastoma.

The discovery of Hsp70 domain with cell-penetrating activity

Chaperone Hsp70 can cross the plasma membrane of living cells using mechanisms that so far have not received much research attention. Searching the part of the molecule that is responsible for transport ability of Hsp70, we found a cationic sequence composed of 20 amino acid residues on its surface, KST peptide, which was used in further experiments. We showed that KST peptide enters living cells of various origins with the same efficiency as the full-length chaperone. KST peptide is capable of carrying cargo with a molecular weight 30 times greater than its own into cells. When we compared the membrane-crossing activity of KST peptide in complex with Avidin (KST–Av complex) with that of similarly linked canonical TAT peptide, we found that TAT peptide penetrated SK-N-SH human neuroblastoma cells at a similar rate and efficiency as the KST peptide. Furthermore, KST peptide can carry protein complexes consisting of a specific antibody coupled to the peptide through the Avidin bridge. An antibody to Hsp70 delivered to SK-N-SH cells with high expression level of Hsp70 reduced the protective power of the chaperone and sensitized the cells to the pro-apoptotic effect of staurosporine. We studied the mechanisms of penetration of KST–Av and full-length Hsp70 inside human neuroblastoma SK-N-SH and human erythroleukemia K-562 cells and found that both used an active intracellular transport mechanism that included vesicular structures and negatively charged lipid membrane domains. Competition analysis of intracellular transport showed that the chaperone reduced intracellular penetration of KST peptide and conversely KST peptide prevented Hsp70 transport in a dose-dependent manner.

Discovery and optimization of cardenolides inhibiting HSF1 activation in human colon HCT-116 cancer cells

Combinational anticancer therapy demonstrates increased efficiency, as it targets different cell-survival mechanisms and allows the decrease of drug dosages that are often toxic to normal cells. Inhibitors of the heat shock response (HSR) are known to reduce the efficiency of proteostasis mechanisms in many cancerous cells, and therefore, may be employed as anti-tumor drug complements. However, the application of HSR inhibitors is limited by their cytotoxicity, and we suggested that milder inhibitors may be employed to sensitize cancer cells to a certain drug. We used a heat-shock element-luciferase reporter system and discovered a compound, CL-43, that inhibited the levels of heat shock proteins 40, 70 (Hsp70), and 90 kDa in HCT-116 cells and was not toxic for cells of several lines, including normal human fibroblasts. Consequently, CL-43 was found to reduce colony formation and motility of HCT-116 in the appropriate assays suggesting its possible application in the exploration of biology of metastasizing tumors. Importantly, CL-43 elevated the growth-inhibitory and cytotoxic activity of etoposide, cisplatin, and doxorubicin suggesting that the pro-drug has broad prospect for application in a variety of anti-tumor therapy schedules.

Knock-down of Hdj2/DNAJA1 co-chaperone results in an unexpected burst of tumorigenicity of C6 glioblastoma cells.

The chaperone system based on Hsp70 and proteins of the DnaJ family is known to protect tumor cells from a variety of cytotoxic factors, including anti-tumor therapy. To analyze whether this also functions in a highly malignant brain tumor, we knocked down the expression of Hsp70 (HSPA1A) and its two most abundant co-chaperones, Hdj1 (DNAJB1) and Hdj2 (DNAJA1) in a C6 rat glioblastoma cell line. As expected, tumor depletion of Hsp70 caused a substantial reduction in its growth rate and increased the survival of tumor-bearing animals, whereas the reduction of Hdj1 expression had no effect. Unexpectedly, a reduction in the expression of Hdj2 led to the enhanced aggressiveness of the C6 tumor, demonstrated by its rapid growth, metastasis formation and a 1.5-fold reduction in the lifespan of tumor-bearing animals. The in vitro reduction of Hdj2 expression reduced spheroid density and simultaneously enhanced the migration and invasion of C6 cells. At the molecular level, a knock-down of Hdj2 led to the relocation of N-cadherin and the enhanced activity of metalloproteinases 1, 2, 8 and 9, which are markers of highly malignant cancer cells. The changes in the actin cytoskeleton in Hdj2-depleted cells indicate that the protein is also important for prevention of the amoeboid-like transition of tumor cells. The results of this study uncover a completely new role for the Hdj2 co-chaperone in tumorigenicity and suggest that the protein is a potential drug target.

Sensitizing tumor cells to conventional drugs: HSP70 chaperone inhibitors, their selection and application in cancer models

Hsp70 chaperone controls proteostasis and anti-stress responses in rapidly renewing cancer cells, making it an important target for therapeutic compounds. To date several Hsp70 inhibitors are presented with remarkable anticancer activity, however their clinical application is limited by the high toxicity towards normal cells. This study aimed to develop assays to search for the substances that reduce the chaperone activity of Hsp70 and diminish its protective function in cancer cells. On our mind the resulting compounds alone should be safe and function in combination with drugs widely employed in oncology. We constructed systems for the analysis of substrate-binding and refolding activity of Hsp70 and to validate the assays screened the substances representing most diverse groups of chemicals of InterBioScreen library. One of the inhibitors was AEAC, an N-amino-ethylamino derivative of colchicine, which toxicity was two-orders lower than that of parent compound. In contrast to colchicine, AEAC inhibited substrate-binding and refolding functions of Hsp70 chaperones. The results of a drug affinity responsive target stability assay, microscale thermophoresis and molecular docking show that AEAC binds Hsp70 with nanomolar affinity. AEAC was found to penetrate C6 rat glioblastoma and B16 mouse melanoma cells and reduce there the function of the Hsp70-mediated refolding system. Although the cytotoxic and growth inhibitory activities of AEAC were minimal, the compound was shown to increase the antitumor efficiency of doxorubicin in tumor cells of both types. When the tumors were grown in animals, AEAC administration in combination with doxorubicin exerted maximal therapeutic effect prolonging animal survival by 10–15 days and reducing tumor growth rate by 60%. To our knowledge, this is the first time that this approach to the high-throughput analysis of chaperone inhibitors has been applied, and it can be useful in the search for drug combinations that are effective in the treatment of highly resistant tumors.

Etoposide-Induced Apoptosis in Cancer Cells Can Be Reinforced by an Uncoupled Link between Hsp70 and Caspase-3

The Hsp70 chaperone binds and inhibits proteins implicated in apoptotic signaling including Caspase-3. Induction of apoptosis is an important mechanism of anti-cancer drugs, therefore Hsp70 can act as a protective system in tumor cells against therapeutic agents. In this study we present an assessment of candidate compounds that are able to dissociate the complex of Hsp70 with Caspase-3, and thus sensitize cells to drug-induced apoptosis. Using the PASS program for prediction of biological activity we selected a derivative of benzodioxol (BT44) that is known to affect molecular chaperones and caspases. Drug affinity responsive target stability and microscale thermophoresis assays indicated that BT44 bound to Hsp70 and reduced the chaperone activity. When etoposide was administered, heat shock accompanied with an accumulation of Hsp70 led to an inhibition of etoposide-induced apoptosis. The number of apoptotic cells increased following BT44 administration, and forced Caspase-3 processing. Competitive protein–protein interaction and immunoprecipitation assays showed that BT44 caused dissociation of the Hsp70–Caspase-3 complex, thus augmenting the anti-tumor activity of etoposide and highlighting the potential role of molecular separators in cancer therapy.

Hydrocortisone 21-hemisuccinate did not prevent exogenous GAPDH-induced apoptosis in human neuroblastoma cells

These data are related to our paper “GAPDH-targeted therapy – a new approach for secondary damage after traumatic brain injury on rats” (Lazarev et al., In press), in which we explore the role of exogenous GAPDH in traumatic brain injury-induced neuron death, and the therapeutic application of small molecules that bind to the enzyme. The current article demonstrates the induction of apoptosis by exogenous GAPDH and the effectiveness of the hydrocortisone derivative for suppressing the pathogenic action of the enzyme.

GAPDH-targeted therapy – A new approach for secondary damage after traumatic brain injury on rats

Massive neuronal death caused by a neurodegenerative pathology or damage due to ischaemia or traumatic brain injury leads to the appearance of cytosolic proteins in the extracellular space. We found that one of the most abundant cellular polypeptides, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), appearing in the medium of dying cells or body fluids is able to form aggregates that are cytotoxic to adjacent cells. Since we previously showed that the hydrocortisone derivative RX624 can inhibit the ability of GAPDH to transport the enzyme complex with polyglutamine and reduce the cytotoxicity of the complex, we explored the effects of GAPDH on SH-SY5Y neuroblastoma cells. We found that the latter treated with particular forms of GAPDH molecules die with a high efficiency, suggesting that the exogenous enzyme does kill adjacent cells. RX624 prevented the interaction of exogenous GAPDH with the cell membrane and reduced the level of death by more than 10%. We also demonstrated the efficiency of RX624 treatment in a rat model of traumatic brain injury. The chemical blocked the formation of GAPDH aggregates in the brain, inhibited the cytotoxic effects of cerebrospinal fluid and rescued the motor function of injured rats. Importantly, RX624 treatment of rats had a similar effect as the intracranial injection of anti-GAPDH antibodies.