The role of heat shock proteins Hsp70 and Hsp27 in cellular protection of the central nervous system

Heat shock proteins (Hsps) are highly conserved and under physiological conditions act as molecular chaperones and/or have anti-apoptotic activities. Expression in the brain of two heat shock proteins, the70?kDa Hsp (Hsp70) and the 27?kDa Hsp (Hsp27), is notable because both proteins are highly inducible in glial cells and neurons following a wide range of noxious stimuli including ischemia, epileptic seizure and hyperthermia. In the central nervous system, constitutive expression of Hsp27 is limited to many (but not all) sensory and motor neurons of the brain stem and spinal cord, while there is little or no constitutive expression of Hsp70. However, inducible expression of both Hsp70 and Hsp27 is present in many areas of the brain and retina and is associated with cellular resistance to a variety of insults. The potential for manipulating the expression levels of Hsps for therapeutic advantage in neurodegenerative diseases such as Alzheimer's disease, stroke and glaucoma will be explored.     

The role of heat shock proteins Hsp70 and Hsp27 in cellular protection of the central nervous system.

Heat shock proteins (Hsps) are highly conserved and under physiological conditions act as molecular chaperones and/or have anti-apoptotic activities. Expression in the brain of two heat shock proteins, the70 kDa Hsp (Hsp70) and the 27 kDa Hsp (Hsp27), is notable because both proteins are highly inducible in glial cells and neurons following a wide range of noxious stimuli including ischemia, epileptic seizure and hyperthermia. In the central nervous system, constitutive expression of Hsp27 is limited to many (but not all) sensory and motor neurons of the brain stem and spinal cord, while there is little or no constitutive expression of Hsp70. However, inducible expression of both Hsp70 and Hsp27 is present in many areas of the brain and retina and is associated with cellular resistance to a variety of insults. The potential for manipulating the expression levels of Hsps for therapeutic advantage in neurodegenerative diseases such as Alzheimer's disease, stroke and glaucoma will be explored.     

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp.     

DNA damage and repair in peripheral blood lymphocytes from healthy individuals and cancer patients: a pilot study on the implications in the clinical response to chemotherapy

Drug resistance is considered the main impediment to successful cancer chemotherapy. The quest for a method useful to predict individual responses to chemotherapy prior to treatment is highly desired. This study was designed to determine the individual influences of doxorubicin and cisplatin on the degree of DNA damage, DNA repair and hMSH2 and the hMLH1 protein expression in peripheral blood lymphocytes (PBL) and their correlations with the clinical response. PBL were obtained from 25 cancer patients (pre- and post-chemotherapy) and from 10 healthy persons, cultured and exposed to doxorubicin or cisplatin. Cells were collected at T0 (immediately after drug treatment) and 24h after damage (T24). The alkaline comet assay was employed to assess the DNA damage and repair function, and immunocytochemistry to study hMLH1 and hMSH2 expression. Clinical response was evaluated after three cycles of chemotherapy. Pre-chemotherapy PBL from cancer patients showed significantly higher levels of basal DNA damage than healthy persons, with appreciable interindividual variations between them. The in vivo administration of antineoplasic drugs was accompanied by significant DNA damage, and an increased in the number of apoptotic cells. Cancer patients with complete response showed a high number of apoptotic cells. The DNA migration increased at T0 and at T24 in cisplatin-treated patients, reflecting a decreased rate of cisplatin adducts repair than that observed in healthy individuals. The ability to repair DNA lesions in doxorubicin-damaged cells was very similar between healthy individuals and cancer patients. Cisplatin-treated patients that died by the disease showed lower DNA migration than the mean value. The expression of hMLH1 and hMSH2 was practically identical between healthy individuals and cancer patients. Nevertheless, chemotherapy induced a depletion mostly of hMLH1. In 83% of cisplatin-treated patients with CR the hMLH1 and hMSH2 expression at T24 was higher than the mean. In this pilot study the alkaline comet assay offered information about the amount of DNA damage and the DNA repair status in PBL from individual patients and this seems useful in predicting the response to chemotherapy.     

Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy

Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (>10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (>66%) of Hsp27 had shorter DFS.<0B> <0R>The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies. Int. J. Cancer (Pred. Oncol.) 79:468–475, 1998.© 1998 Wiley-Liss, Inc.     

Analysis of MLH1 and MSH2 expression in ovarian cancer before and after platinum drug-based chemotherapy.

Preclinical studies have demonstrated a relationship between DNA mismatch repair (MMR) status and sensitivity to cisplatin and carboplatin. MMR-deficient cells are resistant to both drugs, and selection for cisplatin resistance in vitro is sometimes accompanied by loss of MMR protein expression. We used immunohistochemical staining techniques to investigate hMLH1 and hMSH2 expression in paired ovarian tumor sections from 54 ovarian cancer patients before and after platinum-based therapy. We sought associations between hMLH1 and hMSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. hMLH1 and hMSH2 staining decreased significantly after platinum-based therapy. The percent of malignant cells that stained positive correlated with the intensity of nuclear staining for both proteins; staining for hMLH1 correlated well with staining for hMSH2. Unexpectedly, expression of nuclear hMLH1 correlated negatively with response to treatment. Expression of nuclear hMLH1 and hMSH2 was positively correlated with pretreatment CA125 level, and expression of nuclear hMSH2 was positively correlated with change in CA125 level after treatment. Tumor stage was associated with expression of nuclear hMSH2, and tumor histological subtype was associated with both hMLH1 and hMSH2 staining. No association was found between expression of either protein and overall survival. These results indicate that the tumor is biologically altered after chemotherapy consistent with treatment-induced selection for cells expressing lower hMLH1 and hMSH2 levels. However, immunohistochemical staining for either hMLH1 or hMSH2 was not highly predictive of drug sensitivity as measured by response or survival.     

Hsp60 and Hsp10 down-regulation predicts bronchial epithelial carcinogenesis in smokers with chronic obstructive pulmonary disease

BACKGROUND: The relation between smoking, chronic obstructive pulmonary disease (COPD), and lung cancer (LC) is an open field of investigation. A higher frequency of adenocarcinoma has been reported in patients with COPD. Heat shock proteins (Hsps) are implicated in tumoral cell growth and differentiation. The aim of the present study was to investigate the expression of Hsp60 and Hsp10 in bronchial biopsies from smokers with COPD and in 10 lung cancer patients and to evaluate the association between Hsps expression and carcinogenetic steps of LC. METHOD: An immunohistochemical study was performed for Hsp60 and Hsp10 in bronchial biopsies from 35 COPD (postbronchodilator forced expiratory volume in 1 second [FEV(1)]: 53 +/- 19% [mean +/- SD]) patients with a history of smoking (53 +/- 34 pack/years) and in 10 patients with adenocarcinoma or adenosquamous carcinoma (ASC). Immunopositivity was quantified in the bronchial epithelium and in specimens with ASC. RESULTS.: In smokers with COPD, 10 out of 35 patients had a normal bronchial epithelium (NBE), 12 showed basal cell hyperplasia (BCH), 5 squamous metaplasia (SM), and 8 dysplasia (Dy). It was found that 58 +/- 23% and 54 +/- 23% of NBE and 48 +/- 29% and 52 +/- 26% of BCH expressed Hsp60 and Hsp10, respectively; in contrast, only 3 +/- 3% and 3.6 +/- 2% of SM, 1.9 +/- 4% and 1.1 +/- 2% of Dy expressed Hsp60 and Hsp10, respectively. ASC specimens were negative for Hsps proteins. Interestingly, NBE also present at the edges of ASC specimens was negative for Hsps proteins. CONCLUSIONS: The loss of Hsp60 and Hsp10 immunopositivity is related to the development and progression of bronchial cancer in smokers with COPD.     

Loss of expression of DNA repair enzymes MGMT,hMLH1, and hMSH2 during tumor progression in gastric cancer

BACKGROUND: Disorders of the DNA repair system that protects against alkylating mutagens are known to play an important role in carcinogenesis. METHODS: We investigated the expression of the DNA repair enzyme that protects against alkylating mutagens, O(6)-methylguanine DNA methyltransferase (MGMT), and the mismatch repair (MMR) enzymes, hMLH1 and hMSH2, in 135 gastric cancer specimens by immunohistochemical means. RESULTS: The immunoreactivity of MGMT and MMR proteins correlated significantly with several clinicopathologic factors. The survival curve in 116 patients showed that a loss of MGMT or hMLH1, but not of hMSH2, correlated with a poor prognosis. Combined evaluation of MGMT and hMLH1 revealed that the survival of patients with negative status for both MGMT and hMLH1 was shortest. However, this significant association between patient survival and MGMT or hMLH1 expression disappeared when early and advanced cancers were separately analyzed, indicating that synchronous losses of MGMT and hMLH1 increase during tumor progression and stage. Further evaluation according to histologic type revealed that loss of MGMT, hMLH1, and hMSH2 expression significantly differed between early and advanced cancer in differentiated-type cancers. In contrast, in undifferentiated-type cancer, loss of MGMT and MMR expression was frequently found even in intramucosal (m) cancer, and no significant difference was found in loss of hMLH1 and hMSH2 between early and advanced cancer. CONCLUSION: These findings demonstrate that the reduced expression of MGMT, hMLH1, and hMSH2 in differentiated-type cancer may play an important role during tumor progression between the early and advanced stage. On the other hand, in undifferentiated-type cancer, loss of MGMT and the MMR proteins appears to be an important event at carcinogenesis or at an earlier step of tumor progression.     

Tumor prevention and antitumor immunity with heat shock protein 70 induced by 15-deoxy-delta12,14-prostaglandin J2 in transgenic adenocarcinoma of mouse prostate cells

The biological modifier delta12-prostaglandin J2 and related prostaglandins have been reported to have significant growth-inhibitory activity with induction of heat shock proteins (Hsps). Tumor-derived Hsps have been shown previously to elicit specific immunity to tumors from which they are isolated. In this study, 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2)-induced Hsp70 was purified from transgenic adenocarcinoma mouse prostate cells (TRAMP-C2). It was then tested for its ability to activate specific CTLs and induce protective immunity against prostate cancer in C57BL/6 mice. Treatment of cells with 8.0 microM 15d-PGJ2 for 24 h caused significant induction of Hsp70 expression. The yield of Hsp70 purified from 15d-PGJ2-treated cells was 4-5-fold higher when compared with untreated TRAMP-C2 cells. Vaccination of mice with Hsps isolated from TRAMP-C2 cells elicited tumor-specific CTLs and prevented the growth of TRAMP-C2 tumors. These results indicate that the induced heat shock proteins may have promising applications for antitumor, T-cell immunotherapy. In particular, these findings have important implications for the development of novel anticancer therapies aimed at promoting an immune response to prostate tumors.     

Hsp27 modulates p53 signaling and suppresses cellular senescence

The small heat shock protein Hsp27 is expressed at high levels in many tumors and provides protection against anticancer drugs. Here, we show that expression of recombinant Hsp27 at elevated levels leads to protection of MCF10A human mammary epithelial cells from doxorubicin. The protection was associated with suppression of the doxorubicin-induced senescence, where Hsp27 inhibited p53-mediated induction of p21, the major regulator of the senescence program. Similarly, Hsp27 inhibited accumulation of p21 and suppressed senescence in response to the p53 activator nutlin-3, indicating that Hsp27 has a general effect on the p53 pathway. In line with these findings, down-regulation of Hsp27 in HCT116 human colon carcinoma cells that express this heat shock protein at high levels caused senescence in a population of cells and sensitized the rest of the cells to doxorubicin-induced senescence (at low doses) or apoptosis (at high doses of doxorubicin). Induction of senescence by Hsp27 down-regulation associated with activation of the p53 pathway and induction of p21. Interestingly, depletion of Hsp27 caused neither significant proteotoxic nor genotoxic stress, and therefore this heat shock protein seems to have a specific effect on the p53 signaling. Indeed, Hsp27 down-regulation was associated with destabilization of HDM2 and stabilization of p53. These data suggest that Hsp27 may play a general role in regulation of cellular senescence by modulating the p53 pathway.     

Thermotolerance and sensitivity of human cancer cells to cisplatin and doxorubicin

Testis tumour cells are more sensitive than most other types of cancer cell to heat, radiation and chemotherapeutic drugs, both in the clinic and in vitro. Since heat shock proteins (HSP) can protect cells from the cytotoxic effects of stress, we studied their role in the sensitivity of testis tumour cells to the frequently used cancer chemotherapeutic drugs, cisplatin and doxorubicin. Clonogenic assays of 3 testis tumour cell lines (833K, GCT27, GH) and 3 bladder cancer cell lines (RT112, HT1376, MGH-U1) following a 1 h exposure to the two drugs confirmed that the testis tumour cell lines were inherently more sensitive. Having shown previously that heat shock proteins were upregulated in both cell types following a heat shock, in this study no induction of HSP was seen following treatment for 1 h with cisplatin or doxorubicin (at concentrations reducing colony forming ability by 50%) in either cell type. In contrast, chronic exposure to cisplatin (at concentrations on the threshold of cytotoxicity), but not doxorubicin, resulted in upregulation of HSP 27, but not HSP73/72, in both cell types. Testis and bladder cancer cells with heat-induced increases in HSP were thermotolerant, and this was associated with increased resistance to doxorubicin, but not cisplatin.     

Loss of hMLH1 expression correlates with improved survival in stage III-IV ovarian cancer patients

Pre-clinical data suggest a relationship between DNA MisMatch Repair (MMR) system failure, particularly the inactivation of genes hMLH1 and hMSH2, and resistance to drugs like cisplatin and carboplatin. We studied the correlation between loss of hMLH1 expression in tumour cells and clinical outcome in 38 patients with ovarian cancer, who underwent cisplatin-based chemotherapy. 19 patients (56%) showed loss of hMLH1 expression (Group A) while 15 patients (44%) showed normal hMLH1 expression (Group B). 4 patients were not evaluable for hMLH1 expression. The 2 groups of patients were similar for clinical characteristics, response to chemotherapy and time to progression. Group A patients showed a median survival of 55 months whereas Group B patients had a median survival of 12 months (P=0.014). Loss of hMLH1 expression was the only independent predictor of survival in the multivariate analysis. Our observations suggest a relationship between loss of hMLH1 and improved survival in advanced ovarian cancer.     

Human colon cancer cells surviving high doses of cisplatin or oxaliplatin in vitro are not defective in DNA mismatch repair proteins

PURPOSE: Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin. The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system. METHODS: Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml. The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied. RESULTS: Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines. Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene. No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells. In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells. CONCLUSIONS: These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins.     

Allelic losses and DNA methylation at DNA mismatch repair loci in sporadic colorectal cancer

Normal and tumor DNA samples of 35 patients with sporadic colorectal carcinoma were analyzed for microsatellite alterations at 12 markers linked to mismatch repair loci: hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2. Remarkably, no correlation was observed between the replication error phenotype (RER+) and allelic losses at these loci. Hemizygous deletions, seen in 6/35 (17%) informative cases at hMLH1, 4/27 (15%) at hMSH2/hMSH6 and 6/34 (18%) at hMSH3, were rarely found in RER+ tumors. Since mismatch repair protein components act in molecular complexes of defined stoichiometry we propose that hemizygous deletion of the corresponding loci may be involved in colorectal tumorigenesis through defects in cellular functions other than replication error correction. The analysis of the methylation status of the promoter region of hMLH1 revealed that methylation might be an important mechanism of this locus inactivation in RER+ sporadic colorectal cancer.      

Deregulation of miR-34a and Its Chaperon Hsp70 in Hepatitis C virus-Induced Liver Cirrhosis and Hepatocellular Carcinoma Patients

Background: MicroRNA deregulation may occur during hepatocellular carcinoma (HCC) genesis and progression stages. MicroRNA-34a (miR-34a) functions as a tumor suppressor and is down-regulated or silenced in a variety of human cancers, while heat shock proteins (Hsps) play important roles in assisting protein folding and preventing both protein aggregation and transport across membranes. The present study aimed to evaluating serum expression of miR-34a and its target Hsp70 for early detection of HCC in patients with liver cirrhosis (LC), focusing on correlations with clinicopathological features. Methods: A total of 180 patients were included: 120 with HCC on top of LC (60 with either early or late HCC) and 60 patients with HCV-related LC. In addition, 60 healthy individuals were considered as controls. Real-time polymerase chain reactions were performed for expression profiling of serum miR-34a and Hsp70 and for allelic discrimination of the promotor variant (rs2763979, C/T). In addition, in silico analysis was carried out. Results: All participants were heterozygote for the promotor polymorphism. miR-34a serum levels were significantly under-expressed in LC and especially HCC patients as compared to controls. Associations with a high Child-Turcotte- Pugh (CTP) score, advanced cancer stage, and number of masses were noted. In contrast the target Hsp70 was significantly overexpressed in cancer patients but not in LC group and inversely correlated with miR-34a levels. Conclusion: Utility of circulating miRNAs as biomarkers for early detection of HCC was raised. Future large-scale studies are warranted to confirm the current findings.     

Expression of heat shock protein (Hsp) 70 and Hsp 40 in colorectal cancer

Heat shock protein (Hsp) 70 and Hsp 40 are stress proteins that cooperate as chaperones in mammalian cells. The present study was designed to determine the expression levels of Hsp 70 and Hsp 40 in colorectal cancer by immunohistochemistry and Western blot analysis. Among 50 colorectal cancer tissues studied, 80% and 14% of tumors showed specific immunoreactivity to Hsp 70 and Hsp 40, respectively. Hsp 70 and Hsp 40 were overexpressed in cancer tissue samples compared with normal tissues, on both analytic sets. However, there were no significant correlations between their expression and various clinicopathological parameters of colorectal cancer. Hsp 70 and Hsp 40 may be tumor markers for colorectal cancer.     

hMLH1 and hMSH2 mutations in families with familial clustering of gastric cancer and hereditary non-polyposis colorectal cancer

The pattern of hMLHI and hMSH2 mutations was assessed to identify the genetic correlation between hereditary gastric and colorectal cancers. Four disease groups and their healthy family members were assembled according to the presentation of gastric cancer: FG, familial clustering of gastric cancer (n = 32); CG, family with one or more colorectal and gastric cancers in first-degree relatives (n = 22); HS, seven HNPCC families corresponding to the Amsterdam criteria (AMS+) and 12 suspected HNPCC families which did not satisfy one of the criteria (AMS-), but no gastric cancer among first- and second-degree relatives (n = 19); and SG, sporadic gastric cancer (n = 33). In the CG group, three were included in AMS + and six in AMS- criteria. Peripheral blood was obtained from them to detect hMLHI and hMLH2 mutations using PCR-SSCP analysis and direct sequencing. The incidence of mutations was 9.4% in the FG group, 54.5% in the CG group, 31.6% in the HS group, and none in the SG group. The incidence, type, and number of the mutation were not different between the CG and HS groups. Thirty-four different mutations included 19 in hMLH1 and 15 in hMSH2. Gastric cancer was the most common extracolonic malignancy in HNPCC and suspected HNPCC families (9/28, 32.1%). The hMLH1 or hMSH2 mutation occurred in seven of 10 families with AMS+, whereas it occurred in four of 18 with AMS- (70% vs. 22.2%, P = .013). Five mutations in the hMLH1 and six mutations in the hMSH2 were exclusively found in families with gastric cancer. All three mutations in the FG group were in hMLHI and there was no mutation in their healthy family members. This study demonstrates that some familial clustering type of gastric cancer appears to be associated with hMLHI mutations thereby indicating a difference from the hereditary gastric cancer studies previously reported. In addition, hMLHI and hMSH2 mutations may impact the gastric cancer carcinogenesis in HNPCC or suspected HNPCC.     

MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models

The ideal cancer vaccine should work regardless of MHC types but currently the barrier generated by MHC specificity hampers the development of human cancer vaccines, requesting to identify strong immunogenic molecules that can induce anti-cancer immune responses without being affected by MHC polymorphism. Tumor-derived exosomes are small membrane vesicles containing tumor antigens as well as other immunologically important molecules such as MHC molecules and heat shock proteins (HSPs). Because of their potential immunogenicity, the plausible utility of tumor-derived exosomes as an MHC independent cancer vaccine was proposed. Here, we investigated whether Hsp70-enriched tumor exosomes can induce stronger immunogenicity as compared to normal tumor-derived exosomes in autologous as well as allogeneic murine models in vitro and in vivo. Western blotting showed that the exosomes of heat-treated tumor cells (HS Exo) contained higher amounts of Hsp70 than the exosomes of untreated cells (CNTL Exo). In both MHC type-identical and -irrelevant antigen-presenting cell models in vitro, HS Exo triggered the increased expressions of MHC class II molecules. Crucially, HS Exo performed greater therapeutic capability in regressing pre-established MHC type-identical and -irrelevant tumors than CNTL Exo in vivo. The analyses of anti-tumor function in allogeneic mouse model demonstrated that HS Exo elicited Th1-polarized immune responses defined by the increased productions of IgG2a and IFN-γ. In summary, the Hsp70-enriched exosomes extracted from heat-treated tumors induced strong Th1 immune responses, resulting in eliminating cancer cells in allogeneic hosts in vivo. These results indicate that HS Exo is a potent MHC independent cell-free cancer therapeutic agent that can be developed for clinical trials.