Effects Of Tanshinone ?A On E6, E6TP1 And RET Expression-associated PNI In Cervical Cancer Cells

Background and objectives:Cervical cancer incidence rate occupies first place in malignant tumors of female reproductive system. It is a serious problem that threatens women's life and health. In many malignant tumors, including cervical cancer, Perineural invasion (PNI) is considered to be a bad prognostic factor, but the mechanism of PNI is not completely clear. Current studies suggest that glialcellline-derived neurotrophic factor (GDNF) plays an important role in the process of PNI, and it has biological effects by activating receptor RET. There is a close correlation between the occurrence and development of E6 protein and cervical cancer. E6TP1 is a tumor suppressor protein can interact with E6. It is through the ubiquitin proteasome pathway, and by the activation of Rap 1, promote the occurrence and development of cervical cancer.Tanshinone ?A is one of the most abundant effective components in Radix Salviae miltiorrhizae. It has anti-inflammatory, antioxidant, and cytotoxic activities. It was found that Tanshinone ?A could inhibit the growth of cervical cancer cells by down regulating the expression of E6 E7 and HPV oncogenes.This experiment through the study of tanshinone ?A on cervical cancer CaSki and HeLa and C33A cell survival rate and migration effects, and detection of cervical carcinoma perineural invasion related E6, E6TP1, RET, P-RET protein expression is to explore the related mechanism of the occurrence and development of PNI in cervical cancer; the effect of Tan ?A on the inhibition of the growth of cervical cancer cells and occurrence and development of PNI in cervical cancer.Methods:Cervical cancer CaSki, HeLa and C33A cells cultured in vitro. The growth inhibition of CaSki, HeLa and C33A cells of cervical cancer by Tan ?A in different concentration were determined by crystal violet method. The Western Blot was used to detect the effects of Tan ?A on E6?E6TP1 and RET expression-associated PNI in cervical cancer cells. The effect of Tan ?A on cell migration ability of CaSki, HeLa and C33A cells was detected by cell scratch test.Results:1?Tan ?A induce the apoptosis of CaSki, HeLa and C33A cells in dose-dependent manner(P>0.05),. The inhibitory effect of Tan ?A on the growth of HeLa cells was significantly different than that of C33A and CaSki, the difference was statistically significant (P<0.05), and there was no significant difference between CaSki and C33A cells (P>0.05).2?In CaSki cells, after the effection of different concentrations of Tan ?A on the action of 24h, compared with the control group, the expression of E6 protein was down regulated, the expression of E6TP1 protein was up regulated, the expression of RET and P-RET protein were reduced, and the difference was statistically significant (P<0.05); Besides with the increase of the concentration of the E6TP1 protein expression had no statistical difference (P>0.05), the expression of E6, RET and P-RET protein decreased gradually with the increase of drug concentration, and the difference was statistically significant (P<0.05). In HeLa cells, after the effection of different concentrations of Tan ?A on the action of 6h, compared with the control group, the expression of E6 protein was down regulated, the expression of E6TP1 protein was up regulated, the expression of RET and P-RET protein were reduced, and the difference was statistically significant (P<0.05); Besides with the increase of the concentration of the E6TP1 protein expression had no statistical difference (P>0.05), the expression of E6, RET and P-RET protein decreased gradually with the increase of drug concentration, and the difference was statistically significant (P<0.05). After C33A cells were treated with Tan ?A, compared with the control group, the expression of E6TP1 protein was down regulated, and the expression of P-RET and RET were down regulated, and the difference was statistically significant (P<0.05).3?Tan ?A can inhibit invasion and migration ability of CaSki, C33A cells. In the experimental group, the difference of migration distance was significant between the 48h and the 0h; Compared with the control group, the migration distance of CaSki and C33A cells in the experimental group after 48h was reduced, and the difference was statistically significant (P<0.05); In the experimental group 12h and 24h, the difference of their migration distance compared with the control group,was not statistically significant (P>0.05); In HeLa cells, the differences of the migration distance in the experimental group in 12h,24h,48h, compared with the control group, were not statistically significant (P>0.05).Conclusions:After Tan ?A treatment of HPV positive cell line CaSki and HeLa, the expression of E6 protein was down regulated, and it had a certain anti HPV virus.After Tan ?A treatment of HPV 16 positive cell line CaSki, by down regulating the expression of E6 protein, the expression of E6TP1 protein was up-regulated, and the expression of RET and P-RET protein was down regulated,the cell migration ability was inhibited. It is suggested that Tan ?A may block the activation of downstream signal pathway of RET to inhibit the occurrence and development of cervical cancer PNI by regulating the expression of E6?E6TP1 and RET. This has laid a certain experimental foundation for studying the mechanism of cervical cancer PNI.After Tan ?A treatment of HPV negative cell line C33A, the expression of E6TP1 protein was down regulated, but the expression of RET and P-RET protein was also down regulated, and cell migration was inhibited. This suggests that Tan ?A may by regulating other cervical cancer PNI related protein expression or directly down regulate the expression of RET and P-RET protein, block activation of RET downstream signaling pathways, inhibit the occurrence and development of cervical cancer PNI.