A Study On Anti-tumor Immunological Effect Of HSP70-PC Derived From Lung Carcinoma And Its Correlation With Heat Syndrome Of TCM

Syndrome is an important theory of science of TCM which guides people to know the occurrence, development and manifestations of diseases. It is a main evidence for the clinicians of TCM to recognize and treat diseases according to the results of differentiating syndrome and determinating treatment of diseases. A study on syndrome of TCM is a long-term topic on basic research of TCM. Molecular biology as an advanced subject offers good condition for studying on the mechanism of syndrome of TCM. Exploring the mechanism of syndrome from the level of gene and protein has already been the focus of attention.Known commonly as molecular chaperones for proteins, heat shock proteins (HSPs) play essential roles in assisting protein synthesis, folding, assembly, transportation, metabolism and maintaining their normal structure and function of cells; as factors protecting cells from heat, HSPs can prevent cells from being hurt and improve the ability of tolerance to stress, especially the ability of thermoresistance. They are also correlated with many kinds of diseases and play important roles in tumor and anti-infection immune. Among these HSPs, molecular weight with 70kD (heat shock protein70, HSP70) was studied most profoundly. It is clear that HSP70 is expressed in minor quantities under normal physiological conditions and its expression can be powerfully induced to much higher levels as a result of heat shock or other forms of stress. The research shows that HSP70 overexpresses not only instantly when the cells are stressed, but also increases its expression in the course of many diseases such as tumor, inflammation, trauma, aging, cardiovascular disease and immunologic disease. Especially the important roles of HSP70 in tumor genesis and development have been uncovered. It is reported that HSP70 expresses highly in many kinds of tumors, which could be a marker of tumor progress. Furthermore, it can participate in the active immunity of tumor too.In the 1990s, it was first reported that the HSPs purified from a given cancer were observed to elicit protective immunity specific to that particular cancer but HSPs derived from normal tissues did not elicit protective immunity to any cancers tested which was paid attention to by some foreign researchers. Considering that HSPs are conservative and HSP cDNAs would not differ between cancers and normal tissues, they think that there should not be HSP70 molecule per se to elicit specific anti-tumor immunity. So they began to search the material basis from HSP70 preparation and they found that some antigenic peptides were responsible for the specificity of immunogenicity of HSP70 preparation. The dissociation of peptides from HSP70 preparation resulted in abrogation of the immunogenicity, and that the HSP70 was not immunogenic of itself. Researchers have already found in animal models HSP molecules derived from a given cancer including HSP70, HSP90, gp96 are peptide-binding proteins. They can chaperone, present, process antigenic peptides and activate antigen presenting cells (APC). They are associated with peptides inside of cells to generate heat shock protein-peptide complexes (HSP-PC) which were observed to elicit strong protective immunity specific to that particular cancer. At present, the research on the anti-tumor efficacy of HSP70—PC is focused on the animal models. It is unclear whether HSP70 purified from human lung carcinoma tissue has anti-tumor immunity and the mechanism of HSP70 in tumor immune is unclear either. Recently, some clinical trials on HSP-PC derived from human as tumor vaccine are being carried out at abroad. But it is limited to the study of liver cancer, renal carcinoma and malignant melanoma etc. and the efficient rate of HSP-PC immunotherapy is low. There are of great difference in the therapeutic effect among cancer patients which confines it widely applied. Considering that heat stress is a primary factor of inducing HSP gene expression and heat shock response (HSR) is similar to heat syndrome of TCM, we carry out some research on the correlation of HSP family with heat syndrome when we explore the mechanism of syndrome with biological technology from the level of molecule.It has already been proved that the immune effect induced by HSP70 derived from cancers and that from normal tissue is different because they chaperone different antigenic peptides. Furthermore, we have already verified from previous study that HSP70 shows a high expression in tumor such as lung cancer, cancer of colon, gastric cancer etc.. Its level of expression is related to the state of the patients be in, especially in the patients of heat syndrome. The results reveals that HSP70 expression has the specificity of syndrome of TCM. Therefore, we think that HSP70 purifiedfrom tumor with patients of different syndrome may be complexed with different antigenic peptides in quality. We think that the difference in the therapeutic effect among cancer patients suffering from the same tumor in clinical application may be related with patients of different syndrome and the group of heat syndrome may be better than that of non-heat syndrome.Thus, we carry out some research including:First, we chose patients suffering from non-small cell lung carcinoma according to the diagnostic criteria of the disease and the syndrome of TCM and differentiated heat syndrome and non-heat syndrome. Tumor and corresponding non-tumor lung samples were obtained from these patients at the time of open thoracotomy for lung carcinoma.Second, the purification and identification of HSP70-PC: we want to establish the method of HSP70-PC purified from human lung tissue, we purify HSP70-PC from tumor and corresponding non-tumor lung tissue obtained from patients with heat syndrome and non-heat syndrome by the method of cell lysis, ADP affinity chromatography, DEAE ion exchange chromatography, SDS-PAGE, Western-blot etc.. HSP70-PC would be identified by the technology of Western—blot and quantitated by Bradford assay with ultraviolet spectrophotometer.Third, the observation of HSP70-PC purified from tumorous tissues and distal tumor-free lung tissues effecting on human lung cancer cells: peripheral blood mononuclear cells (PBMC) were isolated from blood samples of healthy people by using the method of density gradient centrifugation. We used the technology of cell culture, the MTT cell proliferation assay to measure cell proliferation rate and the reduction in cell viability and investigated whether HSP70-PC purified from human lung carcinoma is able to induce T cell proliferation and tumor cytotoxic reactivity to A-549 cells in vitro.Based on the above-mentioned research, the results are summarized as follows:First, we collected proteins eluate purified by ADP affinity chromatography and DEAE ion exchange chromatography and they were resolved on 12% SDS-PAGE. After electrophoresis, the gel was coomassie stained and 70kD molecules were identified. Then the proteins of the eluate were characterized by SDS-PAGE and transferred onto PVDF membrane immunoblotted with anti-HSP70 antibody. We could see a clear brown protein lane in the PVDF membrane which was coloured by DAB colouration assay and the proteins of the eluate were confirmed to be HSP70. After that, they were quantitated by Bradford assay with ultraviolet spectro-photometer and about 75-100ug HSP70—PC could be obtained from lg tissue.Then, when we used HSP70—PC as immunogen to sensitize human lymphocyte in vitro, we found that they were sensitized efficiently by HSP70 preparation purified from lung cancer tissue. Significant stimulation was observed with HSP70—PC purified from lung cancer of heat syndrome, compared with the control group(P<0.01) and there was also different with HSP70—PC purified from lung cancer of non-heat syndrome(P<0.05). When human lung cancer A-549 cells were co-cultured with lymphocytes pulsed by HSP70—PC, they showed a definite cytotoxicity to A-549 cells at effectontarget ratio of 20:1. The cytotoxicity was significantly more in comparison with the control group( P<0.01). It is worthy of being noticed that we could see the trend of increasing in the heat syndrome group. Under the same condition, HSP70-PC purified from distal tumor-free lung tissue didn't show any proliferation and cytotoxic reactivity.In conclusion, we obtained very pure HSP70 preparations through the method of chromatographies from human tissue, which is shown to be associated with antigenic peptides, that is HSP70-PC. It was indicated that HSP70-PC purified from tumor tissues of heat syndrome and non-heat syndrome patients chaperoned tumorous peptides which can induce CTL immunity. We can see that the immunological effect elicited by HSP70-PC purified from lung carcinoma may well be correlated with the heat syndrome. There was the trend of increasing in the heat syndrome group and it was expected to enlarge samples to study, further. Our research results allows a subsequent study of anti-tumor immunity of HSP70-PC derived from lung tumor of different syndromes. It provides an essential material basis for researchers to analyze the antigenic peptides. Furthermore, it also implied that HSP70-PC derived from lung carcinoma could be a reliable source of tumor-specific antigens for clinical application and permits an expeditious application of HSP70-PC to the immunotherapy of cancer.New ideas of the study merit to be specific mentioned:First, on the aspect of methodology, we purify HSP70-PC from tumorous tissues and corresponding distal tumor-free lung tissues and establish the method of HSP70-PC purified from human lung tissue.Second, the research on the anti-tumor efficacy of HSP70—PC was focused on the animal models and the kind of cancers being studied were limited. It is unclear whether HSP70 purified from human lung carcinoma tissue has anti-tumor immunity and the mechanism of HSP70 in tumor immune is unclear either. We observe the...