Cell Cycle Dysregulations And Relevant Anti-Cancer Effect Induced By Toxoplasma Gondii Infection To Mouse Colon Carcinoma CT26 Cells

Background and Objective malignant tumors are harm to human health severely, which is resistance to radiotherapy or chemotherapy and easy to relapse after surgery. Among the malignant tumors, Colon Carcinoma is prominent particularly. The cell cycle specific agents (CCSA) such as etoposide (VP-16) has a stronger effect on tumor cells in S and G2 phases during the cell cycle, the intensity of which are subject to cell cycle phase of one or several cycles of cell number. The role of these drugs on cancer cells is generally weak and needs to take some time to work in vivo. The anticancer effects of these drugs are improved by the adoption of certain factors affecting the distribution of cell cycle to promote S phase and G2 / M phase percentage increases, thereby killing effects of the CCSA drugs on tumor cell are increased.Recent reports suggested that the host cell infected by Toxoplasma gondii tachyzoites could stop replication and block in the G2/M phase. the altered environment was immediately conducive to bradyzoite differentiation. In this study, the effect of T. gondii RH infection on cell cycle distribution and the molecular mechanisms involved in cell cycle progression of mouse colon carcinoma cell line (ct26) were explored. Secondly, the effect of T. gondii infection on anticancer action of etoposide (VP-16) was observed. Finally, the effect on cell cycle dysregulations and other anti-cancer action induced by Toxoplasma gondii infection in vivo were detected by tumor-bearing mice models.Methods1. The comitant culture models of ct26 cells with different amounts of tachyzoites of T. gondii RH strain were established, the multiplicity of infection(moi) of which were 2:1, 4:1, 8:1, 16:1 respectively. The parasitism of Toxoplasma gondii tachyzoites in ct26 cells was observed by Giemsa staining, and flow cytometry (FCM) was adopted to analyze the cell cycle progression in ct26 infected with T. gondii tachyzoites at different multiplicity of infection(moi) and different time points(6h,12h,24h,48h). The mRNA levels of cyclinB1 and cdc2 were detected by Semi-quantitative RT-PCR, and the protein levels of cyclinB1 were detected by Western blot.2. Take the normal ct26 cells as control, ct26 cells infected with T. gondii tachyzoites at the moi of 2:1 had been cultured for 24 hours before they were treated with different concentrations of VP-16, then the growth inhibition rates of ct26 cells were investigated by the CCK-8 stain method.3. Based on the results from study in vitro, the tumor-bearing mice models of normal ct26 cells and T. gondii infected ct26 cells were built respectively, and then etoposide (VP-16) was intraperitoneal injected. The time of tumor development, growth rate of tumor and survival time of mice were measured. Tumors were taken off and weighed firstly for measurement of inhibition rate, then the tumor tissues made into single cell suspensions and the distributions of cell cycle were detected by flow cytometric assay.4. Tumors were taken out for paraffin processing. The pathohistology structure of tumors was observed by HE dyeing, and the microvessel density (MVD) and expressions of VEGF and TSP1 in tumor tissue were evaluated by methods of immuno-histochemistry. CD4⁺ T cell and CD8⁺ T cell differentiation were measured by FACS analysis.Results1. The parasitism and proliferation of Toxoplasma gondii tachyzoites in ct26 cells were observed by Giemsa staining and oil immersion lens. In the flow cytometric analysis, the percentage of G₀/G_l phase of the ct26 cells was decreased, while the percentage of G₂/M cells was increased significantly in the groups infected with T. gondii (P<0.01). The maximum change happened at 24h after infection with T. gondii RH at the moi of 8:1, the percentage of cells in G2/M phase increased by 12.77%, and then the percentage of cells in G2/M phase decreased at 48h. Notably, the model at the moi of 2:1 made the percentage of S and G₂/M phase of the cells increase by 14.92% at 24h, which was suitable for measurements of cytotoxicity of VP-16. There were no significant change of cdc2 mRNA level after infected by T. gondii for 3h-24h, while both mRNA level and protein level showed a slight increase of the cyclinB1 level appeared after 3h of T. gondii infection, followed by a dramatic decrease of cyclinB1 at 6h, 12h, and 24 h. The cyclinB1 mRNA level was just 16.55% of control group at 24h.2. The data of CCK-8 showed that the Proliferation inhibitory rate of VP-16 to ct26 cells infected by T. gondii (moi=2:1, 24h) increased by 10%¹5% in each dose group (P<0.01).3. The time of tumor development of T. gondii infected group (moi=2:1, 24h) delayed 3 days. It was found that the tumor grew slower (P<0.05) and the inhibition ratio reached to 42.57%. The inhibitory effect of VP-16 combined with T. gondii was superior to those of VP-16 or T. gondii alone (P<0.05). T. gondii infection also prolonged life of mice. The average survival time of mice in T. gondii group increased significantly to 29.6±7.02 days, compared with mice in NS control group(18.8±2.39 days), while average survival time of mice between VP-16 group and control group was nonsignificant. In results of FCM, after infection with T. gondii, the percentage of G₀/G₁ phase decreased obviously and the percentage of S phase increased from 10.83 % to 34.13 %, while G2/M phase of ct26 cells increased nonsignificant. At the same time, an apoptosis rate of 20.58% appeared as a notable apoptosis peak in DNAtapes. The results were quite different from results in vitro. 4. It was shown by HE staining that the tumor cells which came from T. gondii infection group showed disarrangement, slice necrosis areas and apoptosis. Results from immunohistochemical staining showed that the microvessel density(MVD) and expressions of VEGF in tumor tissue from T. gondii infection group were lower than normal ct26 cells group, while the expressions of TSP1 were up-regulated(P<0.05). Results from FCM showed that though there were no significant change of the percentage of CD8⁺ T cells, the percentage of CD4⁺T cells increased from normal ct26 cells group's 26.00% to 46.96 %, and the ratio of CD4⁺/CD8⁺ were up-regulated.Conclusions1. T. gondii parasitised in cytoplasm of ct26 cells and induced host cell cycle arrest at the G2/M phase. Missing expression of cyclinB1 could be the main cause of G2/M arrest.2. The G2/M arrest induced by T. gondii potentiated growth-inhibition of VP-16 to ct26 cells in vitro.3. Tumors which came from ct26 cells infected by T. gondii (moi=2:1, 24h) appeared later and grew slower than the mice group which planted by normal ct26 cells. The inhibition ratio of VP-16 increased and the survival time of tumor-bearing mice extended more significantly. The main characteristics of ct26 cell cycle distribution in vivo were S arrest and apoptosis peak, which were different from vitro.4. T. gondii also played other roles in anti-cancer through inducing tumor tissue necrosis and apoptosis, inhibiting angiogenesis of malignant tumor, and correcting the ratio disturbance of T-lymphocyte subsets.