Study The Effect And Mechanism Of Sanguinarine On Paclitaxel Resistant Ovarian Cancer Cells

[Objective]To investigate the effect of sanguinarine on Taxol resistant ovarian cancer growth and chemosensitivity of A2780/Taxol cells and the mechanism, provide the basis for the treatment of paclitaxel resistant ovarian cancer.[Method]Cultured A2780/Taxol cells to paclitaxel resistant ovarian cancer cell line, MTT assay of sanguinarine, paclitaxel, the inhibition rate of A2780/Taxol cells, determine the sanguinarine and paclitaxel and the optimal concentration and time. A2780/Taxol cells were divided into control group, sanguinarine group, paclitaxel group and sanguinarine combined with paclitaxel group, control group neither sanguinarine nor paclitaxel , sanguinarine group mixed 2.0μ mol/L sanguinarine, paclitaxel group mixed 2μ g/mL paclitaxel, sanguinarine combined with paclitaxel group mixed 2.0μmol/L sanguinarine and2μ g/mL paclitaxel. After 48h culture, MTT assay, cell clone formation assay, flow cytometry and Western blot assay of sanguinarine on A2780/Taxol cell proliferation, colony formation, apoptosis, cell cycle and the effect of Bax, Caspase-3 and beta -tubulin III and TGF- beta /Smad signal pathway.[Results]1. Sanguinarine could significantly inhibit the proliferation of A2780/Taxol cells in a dose and time dependent. According to the relationship of sanguinarine dose, time,determination of sanguinarine concentration of 2 mol/L, the reaction time was 48h, to the following experiment. The working concentration of paclitaxel was 2 g/mL, and the drug resistance coefficient (IR) of paclitaxel was (A2780/Taxol).2. The cell proliferation assays demonstrated that compared with the control group,paclitaxel group, sanguinarine group and sanguinarine combined with paclitaxel could significantly inhibit the proliferation of A2780/Taxol cells, sanguinarine combined with paclitaxel group the highest inhibition rate, the difference was statistically significant (P<0.05).3. The cloning results showed that compared with the control group, paclitaxel group,sanguinarine group and sanguinarine combined with paclitaxel group clone formation rate were significantly decreased, sanguinarine combined with paclitaxel group were significantly reduced, the difference was statistically significant (P<0.05).4. Flow cytometric detection of apoptosis showed that compared with the control group, paclitaxel group, sanguinarine group and sanguinarine plus paclitaxel group significantly increased apoptosis rate and apoptosis of paclitaxel combined with sanguinarine group cell rate increased significantly, the difference was significant(P<0.05).5. Results of the 5 flow cytometry cell cycle showed that compared with the control group, sanguinarine group cell cycle percentage was no difference (P>0.05);paclitaxel group G0/G1, the percentage of S phase cells decreased, the percentage of G2/M phase cells increased significantly,the differences were statistically significant(P<0.05),reduce the percentage; sanguinarine combined with paclitaxel group G0/G1 phase cell percentage of G2/M phase cells increased, there was significant difference(P<0.05); sanguinarine combined with paclitaxel group and the paclitaxel group comparison showed that the percentage of G2/M cells decreased significantly, while G0/G1 phase cell percentage increased, the difference was statistically significant(P<0.05).6. Western blot results showed that compared with the control group, paclitaxel group,sanguinarine group, sanguinarine combined with paclitaxel group Caspase-3, Bax and Smad7 protein expression were significantly increased, sanguinarine combined with paclitaxel group the highest expression level, the difference was statistically significant (P<0.05); compared with the control group, the expression of paclitaxel group, sanguinarine group, sanguinarine combined paclitaxel group TGF- beta 1, beta Smad3, -tubulin Ⅲ were significantly reduced, sanguinarine combined with paclitaxel group expression was the lowest, the difference was statistically significant (P<0.05).[Conclusion]1. Sanguinarine can inhibit the growth of paclitaxel resistant ovarian cancer cell A2780/Taxol has anti-tumor effect. Sanguinarine combined effect of paclitaxel on apoptosis rate increased significantly, increased the inhibitory effect of paclitaxel on the proliferation of A2780/Taxol cells, increase cell sensitivity to taxol.2. Sanguinarine can upregulate the expression of paclitaxel resistant ovarian cancer cell line A2780/Taxol apoptosis related protein Bax and Caspase-3,promote the apoptosis of A2780/Taxol cells.3. Sanguinarine inhibits TGF- /Smad signaling pathway, reduce the expression of beta-tubulin Ⅲ, reversal of paclitaxel resistant ovarian cancer.