The Expression Of Heat Shock Protein 70, 90α In Gastric Cancer And The Experimental Study On Antitumor Effect Of Heat Shock Protein 70, 90α Vaccines

ObjectivePrevious studies found that all organisms would have a similar response by elevated temperatures, it was called heat shock response initially, the typical manifestation was the expression of a group of protein increased rapidly, so it was denominated as heat-shock protein. Subsequently, people found heat shock response also could be induced by other factors, such as amino-acid analog, heavy metal, metabolized poison, ischemia, anoxia and injury. At present, it is thought that heat shock response induced by various stress factors should be called stress response accurately, heat shock proteins be called stress proteins.According to regulating mode, HSP are divided into two major groups: HSP and glucose regulated protein. they also can be divided into several major families, HSP8,10,28,32,47,60,70,90,110kDa based on their size and induced pattern. Now they are usually classified into four families, HSP90, HSP70, HSP60 and small HSPs, besides high molecular weight HSP (100-110KD). They are highly conserved and abundant stress proteins in both eukaryotes and prokaryotes. They widely participate in various physiological process including protein folding, transfer, body immunity and cell apoptosis.The role of HSP participating in physiological process in vivo can be described as follows:1. Molecular chaperones2. Regulate the function of hormone receptor and some protein kinase3. Increase the thermotolerance of the cell4. Participate in cell proliferation regulating5. It is a mark of cell injuryBesides above physiological functions, the expression of heat shock proteins in tumors and its values in tumor immunotherapy deserves more and more atten-tion.An important cause of tumorigenesis is low tumor immunogenicity, escaping the immune monitoring of the body. It is known that T cell mediated tumor immune reaction plays an important role in antitumor response, whereas, the activation of T cell need recognize two signals: (T)antigenic peptide MHC molecular compound (2)co-stimulatory. Many tumors cannot successfully presenting antigens or there was down regulation of the immune-related molecular on cell surface , which lead to low tumor immunogenicity. Recent years, it was found that HSP participate in the specific tumor immune response when studying the immu-nostimulatory principles of tumors. The expression of HSP closely related with tumor immunogenicity, it suggested that HSP has important role in the immuno-therapy of tumor.Some authors found that some antigens extracting from tumors that had specific tumor immunostimulatory function related with HSP. HSPs have been highly conserved throughout evolution. The possibility of HSPs as a changeable tumor antigen is small. Further study showed that these HSPs are HSP-polypeptide compound, which combined with polymorphism polypeptide, the specificity of their induced immunity are determined by the specificity of the polypeptide. The tumor growth inhibiting function of HSPs was first found by Srivastava in 1986. they isolated soluble HSPs from many animal tumors, because HSPs carried specific tumor antigen, HSP peptide vaccines are performed. Immunization animals with HSP peptide vaccines before inoculating tumors to the animals confirmed that they inhibit tumor growth and metastasis. Thereafter, various animal experiments by different laboratory achieve remarkable effect using HSP ( include HSP70 and gp96) on tumors (include melanoma, sarcoma, Colon cancer, prostate cancer, live cancer, leukemia and lung cancer). HSPs could slow tumor growth, cause tumor regress, reduce metastasis lesions, prevent metastasis or elongate survival period. Moreover, their function were specific, they only protected tumor from which HSP extracted, they had no protective immunity to other syngeneic or different tumors. Meanwhile, extracting HSP carried large a-mount of protein peptides, probably include antigen peptide library of this individual tumor, and become a polypeptide vaccine. Its function greatly exceedsthat of vaccine prepared with one antigen. Studies demonstrate that tumor cell not only express individual antigens, but also express common antigens, peptide carried by HSP probably include tumor individual peptide, common peptide and normal cell peptide. But normal cell peptide is in the immune tolerance status, so it will not cause immune response.At present, further researches on antitumor immunity of HSP polypeptide vaccines are done all over the world, and have gained some success. We aim at experimental study of HSP on gastric cancer to find a new way for gastric cancer therapy.Materials and methods1. SpecimensAll specimens were obtained from 41 patients (29 males and 12 females) with gastric cancer who underwent operation in Liaoning Tumor Hospital between June 2001 and June 2002, aged 40 to 78 years ( median, 64. 7years). The normal-appearing tissues beside the tumor were obtained as the normal control. The pathological materials were obtained according to the criterion of Japan gastric cancer stipulation and TNM staging.Animals;Kunming mice were purchased from animal center of Chinese Medical University (4-6 weeks old, body weight, 20-24g). S180 cell line was kindly provided by the first laboratory of tumor institute of china medical university.2. ReagentsTrizol( America Invitrogen)RT-PCR kits were bought from Dalian Bao engineering limited company Taq enzyme (Japan TaKaRa)Mouse anti-HSP70 monoclonal antibody ( Wuhan Boster Company) Rabbit anti-HSP90ct polyclonal antibody (American NeoMakers Company) SABC Immunohistochemistry kit were bought from Wuhan Boster Company Primers of HSP70>HSP90a andj3-actin were synthesized by Beijing Aoke Biology Engineering Limited CompanyAmpholytes(pH 4-6) were bought from Military Medical Science InstituteWestern blot kit were bought from Wuhan Boster CompanyAnnexin V apoptosis detecting kit were bought from German Bendermedsys-tems companyAnti-mouse T lymphocyte double fluorescent direct labeling kit were bought from Xiehe gem cell gene engineering limited company3. Experimental methods1) Immunohistochemical labeling: sections were deparaffinized serially with xylene. Endogenous peroxidase activity was quenched with 3% H2O2 Sections were placed in the citrate buffer for microwave oven treated for 10 min. Sections were then incubated in the goat serum for 20 min to eliminate non-specific staining. The following primary antibodies (1;200) were used in the room temperature for 90 min and then washed in PBS. Avidin-goat-anti-mouse IgG incubation for 20 min at room temperature followed by the addition of SABC was carried out in succession. 3,3' -diaminbezidine tetrahydrochloride ( DAB) was used as a chromogen substrate and sections were then counterstained with hematoxylene for 10 min.2)RT-PCRTotal RNA was extracted by One-step method. The purification was determined by the spectrophotometry. The purification of RNA = OD260/OD280( > 1.6). The RNA was reverse transcripted to cDNA for PCR amplification. The amplified products were electrophoresed on 2% agarose gel. Gels were stained with ethidium bromide and photographed. All signals were normalized to the mRNA levels of p-actin, and expressed as a ratio. 3) Preparation of polypeptide vaccinesSI80 cell line was inoculated to the abdomen cavity of Kunming mice (2 x 10 cells per mouse). Two weeks later, the peritoneal fluid was collected. Ly-sate buffer was added to the peritoneal fluid. The homogenate was centrifuged at high speed for 90 min. The supernatant was isoelectric focused for 4h at 12W twice. After the second isoelectric focusing, the samples were collected in 20 pipes by vacuum pump. The separated fractions were resolved on SDS-PAGE. After electrophoresis, the proteins were stained with cosmic S-250 and thentransferred to the nitrate cellulose membrane. The site of the protein was determined by Western blotting with HSP70 and 90a antibodies. The pipes were grouped according to the results of Western blotting. The samples with same proteins were mixed and frozed for using.4) Animal vaccinationFourty kumning mice were randomized into 4 groups, with 10 mice per group. The prepared HSP-70 polypeptide vaccines were injected to the peritoneal cavity of mice in group 1, the prepared HSP-90a were injected to the peritoneal cavity of mice in group 2, the prepared HSP-70 + 90a were injected to the peritoneal cavity of mice in group 3, the group 4 were treated as the control. Afterwards, additional injection was given every 3 days. Twenty-four hours after first injection, approximately 2 x 106 cells of S180 cell line were injected into the peritoneal cavity of every mouse. Peritoneal fluid growth and survival period of the mice were observed.5) Collection and detection of peritoneal fluid and blood of the mice: After a period of time, the mice were weighted. Peritoneal fluid and bloodwere collected. The animals were sacrificed by pull-off the neck.(DCareful monitor the development of peritoneal fluid. The fluid were double stained with Annexin V -FITC and PI. DNA ploidy and apoptosis were detected by flow cytometry.(5)The blood was collected into two pipes. Forty micro liters of PE labeled anti-mouse CD8 monoclonal antibody and FITC labeled anti-mouse CD3 monoclonal antibody were added to the blood in one pipe. Forty micro liter of PE labeled anti-mouse CD4 monoclonal antibody and FITC labeled anti-mouse CD3 monoclonal antibody was added to the other pipe beforehand. Double parameters were detected by flow cytometry to determine the content of CD4 + T lymphocyte and CD8 + T lymphocyte.6) Statistical analysisThe significance of differences was assessed by SPSS11.0 software.Results1. Immunohistochemical resultsThe expression of HSP70 and 90a was high in the gastric cancer than that in the normal control. The expression in gastric cancer was positive, even strong positive in some cases. The positive rate was 75. 6% and 80. 5%. Whereas, it was negative or weak positive in the normal control group, the positive rate was only 29. 3% and 41. 5%. there was significant difference between gastric cancer group and normal group( P < 0. 05 ).The expression of HSP70 and 90 a was high in undifferentiated type than in differentiated type. It was high in group with lymph node metastasis than without lymph node metastasis. The expression of HSP70 and 90a was different in different TNM staging, it was the highest in stageIVcases, obviously high than in the other stages.2. RT-PCR resultsThe ratio of OD260 to OD280 of total RNA was greater than 1.8, it showed that the purification of total RNA was good.RT-PCR amplication of HSP70 mRNA in the gastric cancer showed positive or strong positive bands, whereas in the normal group, it showed weak bands or negative results. In the cases with more than 5 lymph node metastasis or with distal metastasis, the expression of HSP70 and 90a was obviously higher than that in the cases without metastasis or less lymph node metastasis. The expression of HSP70 and 90 a was obviously different in undifferentiated and differentiated cancer. It was strong positive in undifferentiated cases. The expression of HSP70 and 90a was different in different staging too. The expression increased with the progression of the staging.3. Animal experimentThe survival period of the mice injected with SI80 cell line after immunizing with HSP70, 90a and 70 + 90a polypeptide vaccines was 27. 4 ± 8. 6d, 28.9 ±9. 2d and 28.4 ±8.9d respectively, it was obviously longer than that of control group(20. 3 ±9.7d). On the same time, the average weight of mice inthe group with immunizing with HSP70, 90a and 70 +90a vaccines was 50.6 ± 13.6g, 51. 2 ±9.9g and 50.9 ± 12. lg respectively,it was obviously lower than that in the control group(1.4 ± 12. 6g). there was no significant difference of the survival period and growth rate of peritoneal fluid between HSF70 and 90a groups.After immunizing with HSP70, 90a and 70 + 90a polypeptide vaccines, the apoptosis rate in the peritoneal fluid of mice were 12. 73% ±5. 34% , 7. 21% ±2. 83% and 8. 89% ±3. 35% respectively, there were no significant differences when compared with control group. The CD4 + T and CD8 + T lymphocytes in the blood increased obviously in the group with immunization, there was significant difference when compared with control group.Conclusions1. The expression of HSP70 and 90a was higher in the gastric cancer than that in the control group. It related to the pathological factors of gastric cancer, with the progression of the cancer, the expression increased.2. Isoelectric focusing technique can purify and prepare HSP70 and 90a polypeptide vaccines, and with this technique high purification HSP70 and 90a polypeptide vaccines can be obtained.3. Animal experiment demonstrated that tumor-derived HSP70 and 90a polypeptide vaccines have obvious antitumor effect, this kind of vaccines can stimulate the proliferation of CD4 + T and CD8 + T lymphocytes which will kill tumor cell directly.4. Heat-shock protein polypeptide vaccines can provide a new method for tumor immunotherapy.