Anti-tumor Effect Of TCS And Its Immunological Mechanisms

Trichosanthin (TCS), a protein purified from the plant Trichosanthes Kirilowii Maxim, is a type I ribosome-inactivating protein (RIP) and can be a potential therapy in treating cancer. Trichosanthin induced apoptosis in RM21 cells by regulating gene Bax. CREB is a possible upstream regulator of Bcl-2 in apoptotic HeLa cells induced by TCS. To immune system, TCS shown the immune tolerance by elevating the percentage of CD4+ cells and cutting down the CD8+ cells'. On the other hand, TCS promoted the IL-2 production of PMBC to against the tumor. Therefore, how did TCS, as a potential anti-tumor medicine, affect the immune system of tumor-bearing bodies? Did TCS induce the anti-tumor immune response or inhibite the anti-tumor immunity? These questions were very important to answer for developping TCS as a clinical medicine.Based on the above hypothesis, we first observed the effctor of TCS on 3LL tumor cell lines and 3LL tumor-bearing mice. If it works, T cell subsets in immune pattern and its immune function was analysised and the immune mechanisms of TCS anti-tumor immune response were explored to providing theoretical and experimental basis for TCS as an anti-tumor drug used in tumor therapy.Part I The anti-tumor effect of trichosanthinIn order to verify whether the trichosanthin has the ability in tumor growth inhibition,3LL cells were studied in this research. The results shown that TCS inhibited the cell proliferation and promoted the apoptosis of 3LL cells, and the IC50 was 50μg/ml.3LL tumor cells were inoculated in nude mice which lack T cells. When the tumor size reached 1mm×1mm,0.4mg/kg TCS was injected beside tumor site each two days for 5 times. We found that TCS slightly inhibited the tumor growth in nude mice. The tumor grown slowly than controls'. These resluts indicated that TCS had the ability to inibite the tumor growth in vitro and in vivo.Recent studies have indicated that TCS inhibited the immune response in normal body. To understand whether this property of TCS would influce its anti-tumor potency, we inoculated 3LL tumor cells in C57BL/6 mice and found that TCS treated mice could be completely inhibited tumor growth in vivo, and mice survival rate was 100%. Tumor growth curves and survival rate were significantly different from the control group and nude mice. These results indicated that the anti-tumor effect of TCS both by acting on tumor cells directly and also through the host's immune system.Part II Trichosanthin induced specific anti-tumor immune response in lewis lung cancer model. In vitro and in vivo experiments of the first part confirmed that 3LL tumor cells proliferation was directely repressed by TCS and the immune system maybe involved in inhibiting the tumor growth in vivo. But some groups have reported that TCS could inhibite the immune response in normal mice. In this section, we first studied TCS impaction on the immune system in normal mice with the therapeutic dose using in this study. We extracted the lymphocytes from spleen and lymph nodes and found that TCS major changed the T cell subsets, specially the CD8+T cells. Next, we observed the impact of TCS on T cells function by FACS. That both IFN-y and IL-4 secretion level were decreased indicated that the therapeutic dose of TCS slightly inhibited the immune system in normal mice. As the dose of TCS and the program we used were different from other groups, the inhibition on the immune system in normal mice was much weaker than theirs.TCS repressed multiplication and advanced apoptosis of tumor cells. The above experimental results in C57BL/6 tumor-bearing mice indicated that immune response involved the anti-tumor effect in vivo though it had been proved that TCS could inhibite the normal immune system. Further, we asked the influence of TCS on lymphocytes proportion in tumor-bearing mice. Lymphocytes from spleen and lymph nodes were acquired and we found that T subsets ratio upregulated significantly in TCS treated tumor-bearing mice, both in CD4+T cells and CD8+T cells. Then we detected the cytokine production T cells from TCS treated tumor-bearing mice. Its IFN-y secretion level was markedly improved and IL-4 production was decreased significantly. These datas suggested that TCS induced a Thl-type pattern of cell-mediated immune response in tumor-bearing mice, which was benifit to the anti-tumor immunity. The above results were different from the datas in normal mice. We hypothesised that it was caused by the changes of tumor enviroment in tumor-bearing mice treated with TCS.Finally, we chose the mice that had TCS treated and survived over 100 days.3LL tumor cells were inoculated into the left groin of mice again, while unrelated tumor cells FBL3 were inoculated in the right inguinal. Normal mice were chose as control. The results showed that TCS treated mice could completely reject the second attack of 3LL tumor cells, and 3LL tumor persistently grown in normal mice. However, FBL3 tumor continued growning in both groups of mice. There were no significant differences between two groups. The proportion of memory T cells (CD44+CD62L-T cells) in TCS treated group was absolutely elevated. Those results suggested that TCS treated tumor-bearing mice generated a special immune memory for 3LL tumors.To sum up, TCS enhanced anti-tumor immunity and increased memory T cells through changing the tumor enviroment in tumor-bearing mice.PartⅢRudiment mechanisms of trichosanthin inducing anti-tumor immune responseTCS inhibited the 3LL tumor growth in vitro and in vivo and repressed immune response in normal mice. But it enhanced anti-tumor immune response and immune memory in tumor-bearing mice. We conjectrued that TCS maybe indirectly enhanced immune system in tumor-bearing mice. We studied the rudiment mechanisms of trichosanthin in inducing anti-tumor immune response.To verify the above hypothesis, splenocytes from tumor-bearing mice were marked by CFSE and cultured with TCS treated inactivated 3LL tumor cells. The proliferation capacity and Thl type cytokines secretion of T cells improved much more than the control group. On the contrary, Th2 type cytokoines, such as IL-4 and IL-10, were significantly reduced.Next, we deeply studied the changes of moleculars on 3LL tumor cells treated with TCS and found that MHC class I and co-simulation moleculars was obviously raised. Furthermore, we discovered that the expression of tumor suppressor protein TSLC1 was significantly up-regulated, which has a ligand, CRTAM, was reported expressed on activitied T cells. The interaction between TSLC1 and CRTAM could promote the proliferation and IFN-y secreton of activitied T cells and enhance the anti-tumor effect of T cells.In summary, trichosanthin induced anti-tumor immune response and elevated survival ratio by significantly enhancing proliferation of CD3+ T cells and inducing Th1-type cytokine production.