| [Background] Cervical cancer is one of three malignant tumor of the female reproductive system in women and seriously threat to the health of women and its incidence just behind breast cancer. Worldwide, about500,000new cases diagnosed each year and approximately200,000women died of cervical cancer, of which80%occur in developing countries, which may be related to screening and treatment of cervical cancer conditions in developing countries. In our contry approximately130,000new cases diagnosed each year, and the age of onset is becoming younger and younger. More than90%of cervical cancer people associated with high-risk human papillomavirus (HR-HPV) which mainly for HPV16,18subtypes persistent infection. With the popularization and application of universal cervical cytology screening, early diagnosis and treatment of precancerous cervical lesions and cervical cancer has been improved, the morbidity and mortality of cervical cancer have declined. But its pathogenesis is still not entirely clear.With the development of molecular biology, it has proven that due to the changes of genetic and epigenetic makes the activation of oncogene and inactivatio of tumor suppressor genes plays an important role in the development of cervical cancer.Epigenetic defined as genetic functions changed that can be inheritted by the cell mitosis but DNA sequences not involved.Epigenetic abnormalities including abnormal DNA methylation, histone modifications change, deletion of genomic imprinting and abnormal regulation of non-coding RNA.Currently the most studied are DNA methylation and histone acetylation.More and more evidences have shown that abnormal epigenetic change like abnormal tumor suppressor gene promoter methylation or histone modifications is related to the occurrence and development of cervical cancer. Valproic acid is a traditional anti-epileptic drug, and can inhibit histone deacetylase activity and decrease the level of histone acetylation. Valproic acid can inhibit cell proliferation and promote apoptosis and differentiation in a variety of tumors.All-trans retinoic acid (ATRA) is a biologically active metabolite of vitamin A, which combined with its receptor RAR02to promote the differentiation and maturation of epithelial cells. ATRA has been as an induction of differentiation agents for clinical cancer therapy. Studies have shown that, RARβ2expression was reduced in many tumors including cervical cancer. Its expression and regulation are closely related with histone methylation and deacetylation.Our preliminary results confirm that VPA combined with ATRA can improve the degree of cervical cancer cell lines histone acetylation, through epigenetic pathways restart/upregulate the expression of RARβ2. Thus, we speculate that ATRA binding to RARβ2, startup epithelial cell normal differentiation and maturation pathways, increased the expression of E-cadherin and epithelial cell differentiation markers Filaggrin, Involucrin and Loricrin, thereby promoting differentiation and exert its anti-tumor effect.Three-dimensional cell culture (TDCC) refers to a cell culture in a carrier having a three-dimensional structure, the migration and growth of cells in the three dimensional structure of the carrier forming a cell-carrier dimensional complex. TDCC not only simulate the vivo environment greatly, but also show the cell culture conditions intuitive and controllable advantages. TDCC has important applications in drug toxicity and efficacy evaluation trials. In vitro, TDCC builde a cell developmental system that similar to vivo cells’, and provide a good model for the tumor microenvironment drug designe.This experiment will build a three-dimensional culture models of cervical cancer cells, to explore the effect of cervical cancer cells’growth and differentiation status that induced by the combination of VPA and ATRA. Thereby, it will provide a direction for the search of new treatments for cervical cancer.[Objective] To investigate cervical cancer cells differentiation in three-dimensional culture model induced by the combination of valproic acid (VPA) and all-trans retinoic acid (ATRA).[Methods] HeLa and SiHa cells were seeded and cultured in three-dimensional culture scaffolds. The cells were assigned into three groups based on the treatment received: VPA (3mmol/L) and ATRA (1μmol/L) in combination, VPA and ATRA plus PI3K inhibitor LY-294002, and vehicle only. After2-week culture, the scaffolds were harvested, embedded in paraffin and sliced. The expressions of retinoic acid receptorβ2(RARβ2), E-cadherin and epithelial cell differentiation markers filaggrin, involucrin and loricrin were detected by immunofluorescence assay. To verify the role of PI3K/Akt pathway in cell differention induced by the combination of VPA and ATRA, RARβ2/p-AKT, E-cadherin/p-AKT and p-Akt/involucrin were detected by double-labeling immunofluorescence and further analyzed their temporal relationships.[Results](1)VPA combined with ATRA changed the tumor morphology in three-dimensional cell culture models, SiHa and HeLa cells’complex layers significantly reduced compared with control group;(2)Compared with the control group, VPA combined with ATRA increased the expression of RARβ2and E-cadherin (p<0.0\) and induced differention revealed by the expression of epithelial cell differentiation markers filaggrin, involucrin and loricrin (p<0.05) in both HeLa and SiHa cells in three-dimensional culture models.(3)The treatment of the combination of VPA and ATRA plus LY-294002led to little difference in the expression of RARβ2and E-cadherin, but significant decrease in Akt phosphorylation (p<0.01) and the differentiation markers expression (p<0.01) as compared with cells treated with VPA and ATRA.[Conclusion] VPA combined with ATRA induces differentiation of cervical cancer cells in three-dimensional cell culture model. Its potential mechanism may contribute to the activation of PI3K/Akt signaling pathway. |
PDF Full Text Request