Studies On Promoting Cellular Senescence By Valproic Acid Combined With All-trans Retinoic Acid In Cervical Cancer Cells

[Background] Cervical cancer is the most common malignancy of the female genital tract, and seriously threat to the health of women and its incidence just behind breast cancer. The incidence of cervical cancer has increased significantly in recent years and younger and younger, the incidence of 2% to 3% annual growth rate. Early cervical cancer can be surgery, but mainly in patients with advanced platinum-based chemotherapy, but the toxic side effects, patients tend to develop resistance to them, thus limiting further treatment, the patient survival rate was significantly reduced. Therefore, need an effective and low toxicity of cancer treatment.Conventional wisdom holds that genetic mutations in the process of tumor formation plays a vital role, With the development of molecular biology in recent years, changes in the role of epigenetic tumor development has been recognized, thus breaking the previous "genetic determinism" point of view. Epigenetic phenomenon is not dependent on the genetic DNA sequence, epigenetic abnormalities including abnormal DNA methylation, aberrant regulation of non-coding RNA, histone modifications abnormalities. More and more studies confirm the occurrence and development of epigenetic abnormalities and cervical cancer has a close relationship. VPA(valproate acid, VPA) is a broad spectrum of anti-epileptic drugs, but also a histone deacetylase(histone deacetylase, HDAC) inhibitors. In vitro, VPA has a role in the inhibition of cell proliferation, induction of apoptosis and differentiation in a variety of solid tumors and leukemia, its efficient, safe, low toxicity, oral clinical features make it extremelyprospects. All-trans retinoic acid(all-trans retinoic acid, ATRA) is a biologically active metabolite of vitamin A, binds to its receptor, with the promotion of the role of epithelial cell differentiation and maturation, has been used as a differentiation inducing agent used in clinical cancer treatment. Our previous studies have demonstrated that VPA can make cancer cells combined with ATRA cell cycle arrest and induction of cell differentiation, but no significant increase in apoptosis, On this basis, further analysis of VPA joint anti-cancer effect of ATRA, observe its effect on cellular aging and to explore its possible molecular mechanisms, so as to provide a reference for the search for new treatments for cervical cancer.[Objective] To study valproate acid(VPA) combined with all-trans retinoic acid(ATRA) induced Cervical Cancer cells senescence,and explore its mechanism.[Methods] The experiment is divided into the control group,the VPA group,ATRA and VPA+ATRA groups. Hela and Siha cells were treated with combinated use of 3 mmol/L VPA, 1 μmol/L ATRA or respectively. The control group was treated with vehicle only. Using Cell Titer 96® Aqueous cell proliferation assay to detect the inhibitory rate of proliferation. Using Annexin V-PE/7AAD assay to detect the apoptosis. Apply SA-β-galactosidase staining method to observe the senescence. The m RNA expressions of P16,P63 and h TERT were detected using Q-PCR and its protein expressions were determined using Western blotting. Using inmunohistochemistry detection P16、P63 expression in tumor xenografts. Using flow cytometry detection the cell cycle.[Results](1) The proliferation of He La and Si Ha cells were significantly inhibited by VPA. The effect of VPA combinated with ATRA was stronger than individual treatment(P<0.05).(2) VPA alone or in combination with ATRA after He La, Si Ha increasedapoptosis is not obvious(3) SA-β-galactosidase staining showed that β-galactosidase staining rate is up-regulated after VPA combined with ATRA in cervical cancer He La and Si Ha cells,the difference was significant compared with control group and individual group(P<0.05).(4) Q-PCR and Western blotting showed that VPA enhanced the m RNA and protein expression of P16, P63 and reduce the expression of h TERT. The efficacy was more powerful in combination therapy group.(5) VPA group,the expression of P16, P63 of ATRA group were increased, compared with control group was statistically significant(P <0.05), and after the two-drug combination is more effective(P <0.05).(6) VPA combined ATRA caused by cancer cells in G1 phase arrest.[Conclusion] Valproate acid combined with all-trans retinoic acid can inhibit the growth of cervical cancer cell lines. The effect of inducing cervical cancer cells senescence may be related with up-regulation of P16, P63 and down-regulation of h TERT.