The Growth Inhibition And The Apoptosis Of HeLa Cells With Chimonanthus Salicifolius Extract

Background: Chimonanthus salicifolius is one of the most widespread used Chinese herbal drug in the She ethnic group. It is usually used for the treatment of gastro-intestinal discomfort and other gastrointestinal diseases. So far, it has been developed into Shanlameiye Keli, Piweishu capsule and healthy tea. Piweishu capsule has shown an evident effect in Adjuvant treatment of cancer chemotherapy. Therefore, Chimonanthus salicifolius may play a role as antitumor agent.Objective: The aim of this study was to investigate the growth inhibitory effect of Chimonanthus salicifolius extract on HeLa cells, the induction of cell cycle arrest and apoptosis in HeLa cells, and to explore the possible mechanism.Methods: The antiproliferative effect and apoptosis effect of Chimonanthus salicifolius extract on HeLa cells have been studied by using MTT assay, FCM analysis, Transmission electron microscopy assay, Hoechst33258/PI staining, DNA laddering assay, RT-PCR and Western Blot analysis.Results: (1) The results showed: a) Tween-80 had little influence on HeLa cells, and there was no significant difference in HeLa cells between Tween-80 and control groups, b) The results showed that proliferation of HeLa cells could be inhibited by Chimonanthus salicifolius extract, and have a time and dose dependent manner, c) Chimonanthus salicifolius extract had low Cytotoxicity on HeLa cells, and there were significant differences between HeLa cells and FL cells, d) The rabbit serum containing Chimonanthus salicifolius had cytotoxic effect on the HeLa cells. (2) Flow cytometric analysis suggest that Chimonanthus salicifolius extract could induce cell cycle G₂/M arrest on HeLa cells in vitro. (3) Transmission electron microscopy showed that cells treated with Chimonanthus salicifolius extract appeared chromatic margination, vacuoles, Karyorrhexis and apoptosis body forming. (4) Hochest 33258/PI staining showed that cell nucleus concentrated, and showed no red fluorescence. (5) Agarose gel electrophoresis showed characteristic "DNA ladder". (6) RT-PCR analysis showed the intensity of the p21 mRNA band and the ratio of optical density integral of P21 mRNA/GAPDH were increased with time dependent. (7) Western blot analysis showed the expression of P21 was up-regulated.Conclusion: The results in our study suggested that Chimonanthus salicifolius extract could inhibit the proliferation, induce apoptosis and G₂/M arrest of HeLa cells in vitro. In our prestent study, we have confirmed the antitumor activities of Chimonanthus salicifolius, and this provides an experimental basis for the further analysis of Chimonanthus salicifolius.