OBJECTIVE: To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells. METHODS: Cells culture, flow cytometric an...OBJECTIVE: To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells. METHODS: Cells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used. RESULTS: HSP70-PC immunization rendered protective effect to both naive tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P展开更多
Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes(HSP70-PCs)on HER-3-overexpressing breast cancer.Methods In this study,we first s...Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes(HSP70-PCs)on HER-3-overexpressing breast cancer.Methods In this study,we first studied the expression of HER-3 in breast cancer tissues and its relationship with patient characteristics.We then purified HSP70-PCs from primary breast cancer cells with different HER-2 and HER-3 expression profiles and determined the cytotoxicity of autogenous dendritic cells(DCs)and CD8+T cells induced by these complexes.Third,recombinant human HSP70-HER-3 protein complexes were used to inhibit the autogenous HSP70-PCs purified from HER-3-overexpressing breast cancer cells,and the resulting immunological response was examined.Results The results show that HSP70-PCs can be combined with recombinant HSP70-HER-3 protein complexes to induce stronger immunological responses than autogenous HSP70-PCs alone and that these treatments induce autogenous CD8+T cell killing of HER-3-positive breast cancer cells.Conclusion These findings provide a new direction for HSP70-DC-based immunotherapy for patients with HER-3-overexpressing breast cancer.展开更多
Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complex...Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.展开更多
Background：To develop a vaccine-based immunotherapy for sarcoma,we evaluated a mixture of heat shock proteins （mHSPs） as a vaccine for sarcoma treatment in a mouse model.Heat shock protein/peptides （HSP/Ps） are a...Background：To develop a vaccine-based immunotherapy for sarcoma,we evaluated a mixture of heat shock proteins （mHSPs） as a vaccine for sarcoma treatment in a mouse model.Heat shock protein/peptides （HSP/Ps） are autoimmune factors that can induce both adaptive and innate immune responses;HSP/Ps isolated from tumors can induce antitumor immune activity when used as vaccines.Methods：In this study,we evaluated the effects of mHSP/Ps on prophylactic antitumor immunity.We extracted mHSP/Ps,including HSP60,HSP70,GP96,and HSP l 10,from the mouse sarcoma cell lines S 180 and MCA207 using chromatography.The immunity induced by mHSP/Ps was assessed using flow cytometry,ELISPOT,lactate dehydrogenase release,and enzyme-linked immunosorbent assay.Results：Of S180 sarcoma-beating mice immunized with mHSP/Ps isolated from S180 cells,41.2% showed tumor regression and long-term survival,with a tumor growth inhibition rate of 82.3% at 30 days.Of MCA207 sarcoma-bearing mice immunized with mHSP/Ps isolated from MCA207 cells,50% showed tumor regression and long-term survival with a tumor growth inhibition rate of 79.3%.All control mice died within 40 days.The proportions of natural killer cells,CD8＋,and interferon-γ-secreting cells and tumor-specific cytotoxic T-lymphocyte activity were increased in the immunized group.Conclusions：Vaccination with a polyvalent mHSP/P cancer vaccine can induce an immunological response and a marked antitumor response to autologous tumors.This mHSP/P vaccine exerted greater antitumor effects than did HSPT0,HSP60,or tumor lysates alone.展开更多
The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty heal...The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty healthy 6-week-old male Wulong geese were selected and randomly divided into three groups. The control group was slaughtered without heat treatment. Treatment group 1 was shocked with an acute heat treatment at （42 ± 1）℃ for 5 h before they were immediately slaughtered. Treatment group 2 was kept for 12 h after the heat treatment under normal conditions in order to recover and was then slaughtered. Cardiac tissue was taken in order to make paraffin sections for the immunohistochemistry experiment and the liver tissue was used to purify HSP70. The geese heart HSP70 expression differences in the three groups were determined and at the same time the experiments of HSP70 purification and appraisal in the liver tissue were carried on. HSP70 purification and synthesis of HBV PreS1 multi-peptides unified the complex, which was determined by bi-specific antibody enzyme-linked immune sandwich assay. The results indicated that widespread HSP70 positive pellets in the cardiac muscle were found under hot shock conditions. HSP70 expression in the treatment group 1 was centered in the karyotheca and its periphery, but in treatment group 2, it was centered in the surrounding cell membrane. The HSP70 purification could be obtained through two sets of purification plans; both the synthesis peptide and the HSP70 purification form the complex under certain conditions. The double antibody sandwich ELISA technique was applied to detect if the complex had been formed. Positive results showed that the complex was formed. The specific expression of HSP70 under heat shock shifted with time, suggesting that HSP70 possibly had some function in cell protection. High-purity HSP70 protein can be obtained under low-pressure chromatography conditions, and in comparison with each other, it was better in the flow of the molecular sieve preliminary separation, ConA-agarose chromatography and the ADP-agarose chromatography. Under certain condition in vitro, the synthetic peptide could combine with HSP70 to form the compound, thereby providing a further experimental foundation for the immunity function of the complex.展开更多
Heat shock proteins(Hsps)are a family of abundantly expressed ATP-dependent chaperone proteins.Hsp90 is an eminent member of Hsp family.Thus far,two primary functions have been described for Hsp90:first,as a regulator...Heat shock proteins(Hsps)are a family of abundantly expressed ATP-dependent chaperone proteins.Hsp90 is an eminent member of Hsp family.Thus far,two primary functions have been described for Hsp90:first,as a regulator of conformational change of some protein kinases and nuclear hormone receptors,and the other as an indispensable factor in cellular stress response.Hsp90 has an essential number of interaction proteins since it participates in almost every biological process and its importance is self-evident.Hsp90 has an inextricable relationship in the pathogenesis of cancer,especially in the proliferation and irradiation of cancer cells,thus being a notable cancer target.Since the discovery of geldanamycin,the first inhibitor of Hsp90,from the bacterial species Streptomyces hygroscopicus,even more attention has been focused toward Hsp90.Many structure-based inhibitors of Hsp90 have been designed to develop an innovative method to defeat cancer.However,already designed inhibitors have various deficiencies,such as hepatotoxicity,poor aqueous solubility,instability,and non-ideal oral bioavailability.Based on the aforementioned reasons and to achieve an optimal performance and fewer side effects,we designed a novel inhibitor of Hsp90,called FS5,and resolved the crystal structure of the Hsp90^N-FS5 complex(1.65 A°,PDB code 5XRB).Furthermore,we compared the complexes Hsp90^N,Hsp90^N-GDM,and Hsp90^N-ATP and suggest that the inhibitor FS5 may compete with ATP for binding to Hsp90,which can be regarded as a potential strategy for the development of novel cancer drugs in the future.展开更多
The peptides mixture was prepared from tumor cells by freezing-thawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide...The peptides mixture was prepared from tumor cells by freezing-thawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocytes were T cells which were specifically cytotoxic to tumor cells, the in vivo growth of both ascitic and solid carcinoma could be suppressed by immunization with HSP70-peptides and the lifetime of tumor-bearing mice was prolonged, the in vivo immunization with HSP70-H22-peptides had no impact on the function of mouse liver and kidney, suggesting that there was no occurrence of autoimmunity in vivo after immunization.展开更多
文摘OBJECTIVE: To study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells. METHODS: Cells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used. RESULTS: HSP70-PC immunization rendered protective effect to both naive tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P
基金Supported by a grant from the National Natural Science Foundation of China(No.81260392).
文摘Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes(HSP70-PCs)on HER-3-overexpressing breast cancer.Methods In this study,we first studied the expression of HER-3 in breast cancer tissues and its relationship with patient characteristics.We then purified HSP70-PCs from primary breast cancer cells with different HER-2 and HER-3 expression profiles and determined the cytotoxicity of autogenous dendritic cells(DCs)and CD8+T cells induced by these complexes.Third,recombinant human HSP70-HER-3 protein complexes were used to inhibit the autogenous HSP70-PCs purified from HER-3-overexpressing breast cancer cells,and the resulting immunological response was examined.Results The results show that HSP70-PCs can be combined with recombinant HSP70-HER-3 protein complexes to induce stronger immunological responses than autogenous HSP70-PCs alone and that these treatments induce autogenous CD8+T cell killing of HER-3-positive breast cancer cells.Conclusion These findings provide a new direction for HSP70-DC-based immunotherapy for patients with HER-3-overexpressing breast cancer.
文摘Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.
文摘Background：To develop a vaccine-based immunotherapy for sarcoma,we evaluated a mixture of heat shock proteins （mHSPs） as a vaccine for sarcoma treatment in a mouse model.Heat shock protein/peptides （HSP/Ps） are autoimmune factors that can induce both adaptive and innate immune responses;HSP/Ps isolated from tumors can induce antitumor immune activity when used as vaccines.Methods：In this study,we evaluated the effects of mHSP/Ps on prophylactic antitumor immunity.We extracted mHSP/Ps,including HSP60,HSP70,GP96,and HSP l 10,from the mouse sarcoma cell lines S 180 and MCA207 using chromatography.The immunity induced by mHSP/Ps was assessed using flow cytometry,ELISPOT,lactate dehydrogenase release,and enzyme-linked immunosorbent assay.Results：Of S180 sarcoma-beating mice immunized with mHSP/Ps isolated from S180 cells,41.2% showed tumor regression and long-term survival,with a tumor growth inhibition rate of 82.3% at 30 days.Of MCA207 sarcoma-bearing mice immunized with mHSP/Ps isolated from MCA207 cells,50% showed tumor regression and long-term survival with a tumor growth inhibition rate of 79.3%.All control mice died within 40 days.The proportions of natural killer cells,CD8＋,and interferon-γ-secreting cells and tumor-specific cytotoxic T-lymphocyte activity were increased in the immunized group.Conclusions：Vaccination with a polyvalent mHSP/P cancer vaccine can induce an immunological response and a marked antitumor response to autologous tumors.This mHSP/P vaccine exerted greater antitumor effects than did HSPT0,HSP60,or tumor lysates alone.
文摘The experiment was conducted to study the specific expression of HSP70 caused by heat shock, HSP70 purification and the characteristics of coalescence with antigenic peptide in the formation of the complex. Sixty healthy 6-week-old male Wulong geese were selected and randomly divided into three groups. The control group was slaughtered without heat treatment. Treatment group 1 was shocked with an acute heat treatment at （42 ± 1）℃ for 5 h before they were immediately slaughtered. Treatment group 2 was kept for 12 h after the heat treatment under normal conditions in order to recover and was then slaughtered. Cardiac tissue was taken in order to make paraffin sections for the immunohistochemistry experiment and the liver tissue was used to purify HSP70. The geese heart HSP70 expression differences in the three groups were determined and at the same time the experiments of HSP70 purification and appraisal in the liver tissue were carried on. HSP70 purification and synthesis of HBV PreS1 multi-peptides unified the complex, which was determined by bi-specific antibody enzyme-linked immune sandwich assay. The results indicated that widespread HSP70 positive pellets in the cardiac muscle were found under hot shock conditions. HSP70 expression in the treatment group 1 was centered in the karyotheca and its periphery, but in treatment group 2, it was centered in the surrounding cell membrane. The HSP70 purification could be obtained through two sets of purification plans; both the synthesis peptide and the HSP70 purification form the complex under certain conditions. The double antibody sandwich ELISA technique was applied to detect if the complex had been formed. Positive results showed that the complex was formed. The specific expression of HSP70 under heat shock shifted with time, suggesting that HSP70 possibly had some function in cell protection. High-purity HSP70 protein can be obtained under low-pressure chromatography conditions, and in comparison with each other, it was better in the flow of the molecular sieve preliminary separation, ConA-agarose chromatography and the ADP-agarose chromatography. Under certain condition in vitro, the synthetic peptide could combine with HSP70 to form the compound, thereby providing a further experimental foundation for the immunity function of the complex.
基金supported by the Open Project of Key Laboratory of Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases,Ministry of Education(No.XN201904)Gannan Medical University(No.QD201910)+1 种基金the National Natural Science Foundation of China(Nos.31770795 and 31971043)the Jiangxi Province Natural Science Foundation(No.20181ACB20014)
文摘Heat shock proteins(Hsps)are a family of abundantly expressed ATP-dependent chaperone proteins.Hsp90 is an eminent member of Hsp family.Thus far,two primary functions have been described for Hsp90:first,as a regulator of conformational change of some protein kinases and nuclear hormone receptors,and the other as an indispensable factor in cellular stress response.Hsp90 has an essential number of interaction proteins since it participates in almost every biological process and its importance is self-evident.Hsp90 has an inextricable relationship in the pathogenesis of cancer,especially in the proliferation and irradiation of cancer cells,thus being a notable cancer target.Since the discovery of geldanamycin,the first inhibitor of Hsp90,from the bacterial species Streptomyces hygroscopicus,even more attention has been focused toward Hsp90.Many structure-based inhibitors of Hsp90 have been designed to develop an innovative method to defeat cancer.However,already designed inhibitors have various deficiencies,such as hepatotoxicity,poor aqueous solubility,instability,and non-ideal oral bioavailability.Based on the aforementioned reasons and to achieve an optimal performance and fewer side effects,we designed a novel inhibitor of Hsp90,called FS5,and resolved the crystal structure of the Hsp90^N-FS5 complex(1.65 A°,PDB code 5XRB).Furthermore,we compared the complexes Hsp90^N,Hsp90^N-GDM,and Hsp90^N-ATP and suggest that the inhibitor FS5 may compete with ATP for binding to Hsp90,which can be regarded as a potential strategy for the development of novel cancer drugs in the future.
基金This work was supported by the National Natural Science Foundation of China (Grant No.39970322) Trans-Century Training Programme Foundation for Talent under the supervision of Ministry of Education of China.
文摘The peptides mixture was prepared from tumor cells by freezing-thawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocytes were T cells which were specifically cytotoxic to tumor cells, the in vivo growth of both ascitic and solid carcinoma could be suppressed by immunization with HSP70-peptides and the lifetime of tumor-bearing mice was prolonged, the in vivo immunization with HSP70-H22-peptides had no impact on the function of mouse liver and kidney, suggesting that there was no occurrence of autoimmunity in vivo after immunization.
