Multiple Mechanisms Of Novel Therapeutic Alliance Of Non-invasive Pelvic Cavity Regional Moderate Hyperthermia Combine With HSP70-PC Derived From Autologous Tumor Against Orthotopic Rat Invasion Bladder Tumor

Multiple mechanisms of novel therapeutic alliance of non-invasive pelvic cavity regional moderate hyperthermia combine with HSP70-PC derived from autologous tumor against orthotopic rat invasion bladder tumorTo study the mechanism of non-invasive pelvic cavity regional moderate hyperthermia (MHT) against to rats containing orthotopic invasion bladder tumors and the mechanism of HSP70-peptides complex (HSP70-PC) against to rats containing orthotopic invasion bladder tumors, to further learn the mechanism of novel therapeutic alliance of non-invasive pelvic cavity regional moderate hyperthermia combine with HSP70-PC against to rats containing orthotopic invasion bladder tumor, we observed the effectiveness of solo therapy and therapeutic alliance respectively. The studies contain six parts:â‘  The study of HSP70 of orthotopic rat invasion bladder tumor provoked and up-regulated by non-invasive pelvic cavity regional moderate hyperthermia: Invasion bladder tumor model of inbred line F344 rat was erected by MNU. The pelvic cavity of rat was heated to 41 ℃ by Double RF Tumor Hyperthermia System. The proteinaceous expression regularity of HSP70 in bladder tumor cells heated by different MHT programs was detected by immune electron microscopy, immunohistochemistry and SELDI-TOF-MS respectively.â‘¡ Purification of HSP70-PC from orthotopic rat invasion bladder tumor heated bynon-invasive pelvic cavity regional moderate hyperthermia: High purity HSP70-PCwas purified from rat invasion bladder tumor by our improved methods of liquidchromatography of three steps.â‘¢ The pathologic and immunologic changes of orthotopic rat invasion bladder tumor were induced by HSP70-PC derived from autologous tumor: We applied HSP70-PC to rats containing orthotopic invasion bladder tumors. Then we observed the effects of the therapy. This is the first experiment of therapy of HSP70-PC against to rat invasion bladder tumor in vivo.â‘£The morphologic changes of orthotopic rat invasion bladder tumor induced by non-invasive pelvic cavity regional moderate hyperthermia: The pathologic changes of tumor heated by different MHT programs were detected by electron microscopy and light microscope respectively.⑤The immunologic changes of rat containing orthotopic invasion bladder tumor induced by non-invasive pelvic cavity regional moderate hyperthermia: The changes of immunocytes in tumor, lymphonode of draining domain around bladder and spleen in rat in different MHT programs were detected by electron microscopy and light microscope respectively. The quantity of Rat IFN-γ was detected by ELISA. â‘¥Multiple mechanisms of novel therapeutic alliance of non-invasive pelvic cavity regional moderate hyperthermia combine with HSP70-PC against orthotopic rat invasion bladder tumor: After a month of sequential therapy of non-invasive pelvic cavity regional MHT and HSP70-PC subcutaneous injection, we studied the pathologic changes of bladder tumor, lymphonode of draining domain around bladder and spleen. The quantity of Rat IFN-γ was detected by ELISA. Part IThe study of HSP70 of orthotopic rat invasion bladder tumor provoked and up-regulated by non-invasive pelvic cavity regional moderate hyperthermiaObjective To explore whether heat shock protein 70 of orthotopic rat bladder tumor can be provoked and up-regulated by non-invasive pelvic cavity regional moderate hyperthermia, we studied proteinaceous expressive regulation of HSP70 in rat invasion bladder tumor cell. Methods Six-week-old female inbred line F344 rats were infused methyl-nitroso-urea transurethrally (MNU) transurethrally once every other week for 6 times. Then the rats were bred 4 weeks continuously. Thus rat invasion bladder tumor model had been erected. NRL-00II Double RF Tumor Hyperthermia System was applied to every rat. One rat was laid down in the lucite bracket and its pelvic was put in heating field. The rat pelvic was heated to 41 ℃ and persisted an hour. Groups: Each group contained 5 rats. Group A was control. Group B was heated once. Group C was heated twice interval 24 hours. Group D was heated twice interval 72 hours. The localization of HSP70 of the bladder tumor cells were detected by protein A-10nm colloidal gold labeled immune electron microscopy. The positive cell rates of HSP70 were detected by immunohistochemistry to paraffin embedded bladder tumor. Quantity of HSP70 (HSP70 Intensity and HSP70 MZ Area) from supernatant of tumor lysate of every specimen was detected by SELDI-TOF-MS. Results Immune electron microscopy of 10nm colloidal gold labeled indicated that HSP70 of group A was located in cytoplasm without heat stress. HSP70 of group B, C and D were located in tumor cell nuclear, mitochondrion and cytosol after heat stress. Immunohistochemistry showed that the positive cell rate of HSP70 of group A was significant less than that of group B, C and D (P<0.01). Nevertheless there were non-significant difference among group B, C and D (P>0.01). SELDI-TOF-MS indicated that quantity of HSP70 were group C, D, B and A in descending (P<0.01). Conclusions HSP70 can be provoked and up-regulated by non-invasive pelvic cavity regional moderate hyperthermia in orthotopic rat invasion bladder tumor. Non-invasive pelvic cavity regional moderate hyperthermia was a mild thermotherapy. Not all tumor cells of HSP70 could be provoked and up-regulated by it. The positive cell rate of HSP70 was not increasing with heat times. But the quantity of HSP70 was increasing with heating times. The quantity of HSP70 of group of heated twice interval 24 hours was the highest. Part IIPurification of HSP70-PC from orthotopic rat invasion bladder tumor heated by non-invasive pelvic cavity regional moderate hyperthermiaObjective To obtain purified HSP70-PC, we isolated and purified the protein from orthotopic rat invasion bladder tumor heated by non- invasive pelvic cavity regional moderate hyperthermia by liquid chromatography. Methods Fifteen rat models of invasion bladder tumor had been erected previously. Every rat pelvic was heated to 41 ℃ by NRL-00II Double RF Tumor Hyperthermia System in a lucite bracket and persisted an hour internal. Bladder tumor cell lysate supernatant was injected to Mono Q column, Con-A Sepharose column and 2'5'ADP-Sepharose 4B column in order. HSP70-PC was identified by SDS-PAGE, Western blotting and SELDI-TOF-MS respectively. Results We obtained high purity HSP70-PC detected by SDS-PAGE, Western blotting and SELDI-TOF-MS respectively. Molecular weight of peptides binding with HSP70 identified by SELDI-TOF-MS ranged from 500 to 2500 Dalton. Conclusions High purity HSP70-PC can be purified from rat invasion bladder tumor heated by non-invasive pelvic cavity regional moderate hyperthermia by our improved methods of liquid chromatography of three steps. Part IIIThe pathologic and immunologic changes of orthotopic rat invasion bladder tumor were induced by HSP70-PC derived from autologous tumorObjective To explore the effectiveness and mechanism of treatment of HSP70-PC derived from autologous tumor after heated by non-invasive pelvic cavity regional moderate hyperthermia. Methods 1. Establishment of orthotopic rat bladder tumor model: Six-week-old female inbred line F344 rats of 20 totals were infused MNU transurethrally once every other week for 6 times. Then 5 rats were continuous bred 4 weeks, and the other 15 rats were continuous bred 8 weeks respectively. 2. HSP70-PC has been purified from rat bladder tumor heated by non-invasive pelvic cavity regional moderate hyperthermia by our improved methods in our previous study. 3. Therapy to orthotopic rat bladder tumor: The 15 rats were separated randomly to group A (control), group B (HSP70-PC therapy) and group C (tumor lysate therapy). 4. The wet weight of bladder containing tumors was weighed after 4 weeks. The pathologic grade and stage of tumor were detected. The positive cells of S-100 Protein and CD8 immerged in tumor, the positive cells of S-100 Protein and CD8 in lymphonode of draining domain around bladder, the positive cells of S-100 Protein and the positive cells of CD8 in spleen and PCNA of tumor cells were detected by immunohistochemistry. Apoptosis of tumor cell were detected by TUNEL. The quantity of Rat IFN-γ in serum was detected by ELISA. Results The weight of bladder containing tumors of group B (HSP70-PC therapy) were significant lower than that of group A (control) and C (tumor lysate therapy) (P<0.01). But there was non-significant difference between group A and C (P =0.610). The stages of tumor of group B were significant lower than that of group A and C (P <0.01). But there were non-significant of stages difference between group A and C (P =1). There were non-significant differences of grades of tumor among groups A, B and C (P =1). The ratio of apoptosis index (AI) of tumor cells, the ratio of CD₈ positive cells in tumor and the quantity of Rat IFN-γ in serum of group B were significant higher than that of group A and C (P <0.01). But there were non-significant difference of them between group A and C (P =0.870, 0.597, 0.979) respectively. The descending ranks of the ratio of S-100 protein positive cells and the ratio of CD₈ positive cells in spleen were group B, C and A (P <0.01). There were non-significant differences among three groups of PCNA labeled indexes (PCNA LI) of tumor cells, the ratio of S-100 protein positive cells immerged in tumor and in lymphonode of draining domain around bladder (P =0.808, 0.718, 0.847) respectively. Conclusions HSP70-PC can improve both cellular immunity of rat containing orthotopic bladder tumor and tumor cell apoptosis. Nevertheless it could not affect proliferation of tumor cells. Part IVThe morphologic changes of orthotopic rat invasion bladder tumor induced by non-invasive pelvic cavity regional moderate hyperthermiaObjective To explore what effectiveness on orthotopic rat invasion bladder tumor treated by different non-invasive pelvic cavity regional MHT programs, we studied morphologic changes of the tumor. Methods F344 rat models of invasion bladder tumor had been erected previously. Every rat pelvic was heated to 41 ℃ and persisted an hour by NRL-00II Double RF Tumor Hyperthermia System in different MHT programs. Group of short duration contained group A (tumor control), B (heated once), C (heated twice interval 24 hours) and D (heated twice interval 72 hours). Group of long duration contained group E (heated twice interval 24 hours) and F (heated twice interval 72 hours) obtained 4 times heat totally in 4 weeks. The pathologic changes of tumor were detected by electron microscopy and light microscope respectively. Results The descending ranks of wet weight of bladder containing tumors were group A, D and C (P<0.01). There were non-significant difference between group A and B (P=0.737). The descending ranks of wet weight of bladder containing tumors were group E, G and F (P<0.01). There were non-significant differences of stage and grade of tumor in group of short duration and in group of long duration CP>0.05). Electron microscopy indicated that there were some viable apoptotic cells in group of short duration. Apoptotic cells and apoptotic bodies were seen frequently in group of long duration. The ratio of HSP70 positive cells of group B, C and D was significant higher than that of group A (P<0.01). Nevertheless there were non-significant differences in group B, C and D. The ratio of HSP70 positive cells were group F, G and E in descending (P<0.01). In group of short duration, the descending ranks of PCNA LI of tumor cells were group A, B, D and C (P<0.01). AI of tumor cells were group C, D, B and A in descending. (P<0.01). In group of long duration, the descending ranks of PCNA LI of tumor cells were group E, G and F in descending (P<0.01). AI of tumor cells were group F, G and E in descending. (P<0.01). Conclusions Non-invasive pelvic cavity regional moderate hyperthermia could inhibit the proliferation of tumor cells and induce apoptosis of tumor cells. Thermotolerance of tumor cells were not emerged in heated groups of long duration. Part VThe immunologic changes of rat containing orthotopic invasion bladder tumor induced by non-invasive pelvic cavity regional moderate hyperthermia Objective To explore immunologic changes of rat containing orthotopic invasion bladder tumor induced by different non-invasive pelvic cavity regional MHT programs. Methods F344 rat models of invasion bladder tumor had been erected previously. Every rat pelvic was heated to 41℃ and persisted an hour by NRL-00II Double RF Tumor Hyperthermia System in different MHT programs. Group of short duration contained group A (tumor control), B (heated once), C (heated twice interval 24 hours) and D (heated twice interval 72 hours). Group of long duration contained group E (heated twice interval 24 hours) and F (heated twice interval 72 hours) obtained 4 times heat totally in 4 weeks. The changes of immunocytes in tumor, lymphonode of draining domain around bladder and spleen in rat in different MHT programs were detected by electron microscopy and light microscope respectively. The quantity of Rat IFN-γ was detected by ELISA. Results Electron microscopy indicated that there were some neutrophile granulocytes and macrophages emerged in tumor of group B, C and D in group of short duration. The aggregation of neutrophile granulocytes and macrophages containing apoptosis bodies were observed in group F and G in group of long duration. The descending ranks of positive cells of S-100 protein in tumor, lymphonode and spleen in group of short duration were group C, D, B and A (P<0.01). The positive cells of CD₈ in tumor, the positive cells of CD₈ in spleen and the quantity of Rat IFN-γ in serum in group of short duration were non-significant difference among of group A, B, C and D respectively (P>0.05). All of the detected indexes in group of long duration were group F, G and E in descending. (P<0.01). Conclusions The redistribution of immunocytes of rats containing orthotopic invasion bladder tumor was followed with the increasing of frequency of non-invasive pelvic cavity regional MHT. These changes could profit for tumor antigen presentation and effective immunocytes attack to tumor cells. Part VIMultiple mechanisms of novel therapeutic alliance of non-invasive pelvic cavity regional moderate hyperthermia combine with HSP70-PC against orthotopic rat invasion bladder tumorObjective To learn effectiveness and mechanism of novel therapeutic alliance of non-invasive pelvic cavity regional MHT combine with HSP70-PC subcutaneous injection. Methods F344 rat models of invasion bladder tumor had been erected previously. Pelvic cavity of rat was heated to 41 ℃ by Double RF Tumor Hyperthermia System. HSP70-PC had been purified from F344 rat bladder tumor by our improved methods in previous study. Rats were separated randomly to group A (control), B (heat therapy), C (HSP70-PC therapy) and D (therapeutic alliance). After a month of sequential therapy of non-invasive pelvic cavity regional MHT and HSP70-PC subcutaneous injection, we studied the pathologic changes of bladder tumor, lymphonode of draining domain around bladder and spleen. The quantity of Rat IFN-γ was detected by ELISA. Results Apoptosis cells and bodies in bladder tumor in group D were more than that of other groups observed by transmission electron microscope. Moreover, more lymphocytes, neutrophile granulocytes and macrophages containing apoptotic bodies were emerged in tumor in group D. The wet weight of bladder containing tumors, the stages of tumor and PCNA LI of group D were significant lower than that of group A, B and C (P<0.01). There were non-significant differences of grade of tumor among groups A, B, C and D (P=1). The ratio of HSP70 positive cells of tumor and the ratio of S-100 protein positive cells in tumor of group D were significant higher than that of group C (P<0.01). But there were non-significant difference of the two indexes between group B and D. (P=0.825, 0.686). The AI of tumor, the ratio of CD₈ protein positive cells in tumor, the ratio of positive cells of S-100 protein in lymphonode, the ratio of positive cells of CD₈, the ratio of positive cells of S-100 protein in spleen and the quantity of Rat IFN-γ in serum of group D were significant higher than that of group A, B and C (P<0.01). Conclusions The roles of promotion apoptosis of tumor cells, inhibition to proliferation of tumor cells and up-regulation immunity of rat containing orthotopic invasion bladder tumor evoked by non-invasive pelvic cavity regional MHT were in coordination and complementation with the effectiveness of antitumor immunity evoked by HSP70-PC. The effectiveness of therapeutic alliance to rats containing orthotopic invasion bladder tumor was the best in the three therapies.