Effect Of Arsenic Trioxide And Quercetin On Cervical Cancer Hela Cell Line Immunosuppressive Action

Objective: Chinese herbs are the predominant medicine resource in our country. Cervical cancer is one of serious diseases threatening women severely in the world. second only breast carcinoma, nowadays the incidence tend to youth. There is close correlation between the growth and development of cervical cancer and HPV infecton. Obviously, without effective resistance and escaping mechanisms, few mutant cells could develop tumor. There are many possible ways for tumor to escape immune surveillance, among them, tumor immunosuppression should play an important role. Our previous studies showed that supernatants of some tumor cells could inhibit immune functions and some anti-tumor herbs could reverse this immunosuppression. CD4⁺CD25⁺ regulatary T cells possess immune nullipotency and immunosuppression, and play a role in immunities of tumor, it is one of several mechanisms to escape immune surveillance. The literature reported that the content of CD4⁺CD25⁺ Treg in the cervical cancer of peripheral blood increase highly. we used cervical cancer Hela cell line, whose immunosuppression had been observed in pre-experiment, and two ingredients of Chinese herbs (arsenic trioxide As₂O₃) and (quercetin Que) , whose anti-tumor activities had been reported but not involved their anti-imunosuppressive effects. This study aims to offer experimental evidence to the new method of selecting anti-tumor Chinese herbs and studying their new anti-tumor mechanism.Methods:1. The preparation of various kinds of immune supernatants We collected supernatants of cervical cancer Hela cells after treated by arsenic trioxide (As₂O₃)and quercetin(Que). After treated with As₂O₃ and Que for 48 hours respectively, cervical cancer Hela cells were washed and re-cultured with new complete medium for 48 hours: The supernatants were collected and named as As-S (Supernatant of Hela cells aftert reated by As₂O₃), Qu-S (supernatant of Hela cells after treated by quercetin) and control-S( supernatant of Hela cells after treated by only complete medium).2. The effects of different supernatants on peripheral blood lymphocyte proliferation induced by PHA and CTL cytotoxicity were detected By MTT3. Subpopulation phenotype of peripheral blood mononuclear cells (PBMC) were detected by Flow Cytometry (FCM) Assay.4. The concentrations of five immunosuppressive molecules (including TGF-β1, VEGF, IL-4, L-6 and IL-10) in different supernatants were measured by quantitative ELISA5. To analysis the relations between five immuno- suppressive molecules and The effects of different supernatants downregulated by Arsenic trioxide and quercetin.6. Statistics Methods: Statistic analysis was performed with SPSS 13.0. All experiment data were indicated Mean±STD.Results:1. The effects of different supernatants of Hela cells treaded by Arsenic trioxide on peripheral blood lymphocyte immune function1.1 For supernatants of Hela cells treated with As₂O₃, peripheral blood lymphocyte proliferation induced by PHA,CTL cytotoxicity and inhibition of CD4⁺CD25- T and CD4⁺CD25⁺ T decreased significantly, compared with supernatants of Hela treated without As₂O₃ , while inhibition of CD4⁺CD25⁺ T increased significantly.1.2 For supernatants of Hela treated with As₂O₃, the concentrations of TGF-β1,IL-4 and IL-10 decreased greatly, while concentrations of VEGF and IL-6 have no significant change1.3 The analysis of correlation Indicated the positive correlations existed between TGF-β1 and the inhibition of peripheral blood lymphocyte proliferation induced by PHA,CD25⁺ expression, and also positive correlations with CD4⁺CD25⁺T expression; the positive correlations also existed between IL-4 and CD4⁺CD25- expression,the inhibition of CTL cytotoxicity; meanwhile positive correlations existed between VEGF and inhibition of CTL cytotoxicity.2. The effects of different supernatants of Hela cells treaded by quercetin on peripheral blood lymphocyte immune function2.1 For supernatants of Hela treated with quercetin, peripheral blood lymphoidcell proliferation induced by PHA,CTL cytotoxicity and inhibition of CD4⁺CD25- T decreased significantly, compared with supernatants of Hela treated without quercetin, while inhibition of CD4⁺CD25⁺ T increased significantly.2.2 For supernatants of Hela treated with quercetin, the concentrations of TGF-β1,IL-6 and IL-10 decreased greatly ,while concentrations of VEGF and IL-4 have no significant change .2.3 The analysis of correlation Indicated the positive correlations existed between TGF-β1 and the inhibition of T lymphocyte proliferation induced by PHA or CD25⁺ expression; CTL cytotoxicity exist positive correlations with VEGF and IL-4 ; the positive correlations also existed between IL-10 and CD4⁺CD25- expression. but have no correlations between CD4⁺CD25⁺ T expression and five immunosuppressive molecules.Conclusion:1. There were obvious immunosuppression effects of supernatants of Hela on immune functions of peripheral blood mononuclear cells (PBMC),It may be the cervical cancer Hela cells secret immunosuppressive molecules, such as TGF-β1 and IL-10, the increase of CD4⁺CD25⁺ regulator T cells may be involved in tumor immunty.2. Arsenic trioxide and quercetin can reverse immunosuppressive function of Hela cells by downregulating the secretion of immunosuppressive molecules, such as TGF-β1 and IL-10, which is one of their anti-tumor mechanisms.3. Arsenic trioxide and quercetin can down-regulate the expression of CD4⁺CD25⁺Treg, recover the functions of CD4⁺T and activities of CTL cells, to achieve the effect of anti-tumor growth ,this effect should be one of their anti-tumor mechanisms.4. Detecting effects on tumor immunosuppression could be apractical new method to select anti-tumor Chinese herbs and to study their anti-tumor mechanism.