The Effect And Mechanism Of ROS In The Inhibition Of Proliferation Of Raddeanin A On Ovarian Cancer Cell Skov3

BackgroundOvarian cancer is one of the most often diagnosed therioma of the female reproductive system, and the mortality of ovarian cancer has stayed high in the list. The major treatment to ovarian cancer at present is surgical operation with a postoperative chemotherapy. Though the operation chemotherapy and radiotherapy has been improved greatly these years, the long-time survival fails to gain an obvious rise to match, in which the side effects of chemotherapy drugs and the formation of drug resistance might be involved. Therefore, seeking for new drug treatment and clarifying its mechanism are of great significance and application value. Researches and applications of Chinese herbs and medicines enjoyed a long history, and a great amount of traditional medicines have been proved of anti-tumor activity and light toxic side effects, thus becoming a research focus. Raddeanin A is a well-famed Chinese herbs and also known as an effective anti-tumor drugs, yet the underlying mechanism of its anti-tumorr effects needs further explored. Reactive oxygen species (ROS) are important cellular redox signaling molecules that regulates proliferation and apoptosis, and various medicines perform its therapeutic effects by altering the level of ROS in tumor cells.PurposeIn this study, ovarian cancer cells (Skov3) are treated with raddeanin A to verify its role in cell proliferation and explore the underlying mechanism.Materials and methods1. MTT was employed to detect the proliferation inhibition in Skov3cells treated with different concentrations of raddeanin A.2. Flow cytometry was employed to detect the ROS levels in Skov3cells treated with raddeanin A.3. MTT was employed to detect the proliferation inhibition in Skov3cells, which were treated with NAC before the use of raddeanin A.4. Cellular morphology of Skov3cells is observed under phase contrast microscope and the cells were treated with NAC before the use of raddeanin A.5. Flow cytometry and DHE fluorescence staining were employed to detect intracellular ROS level and the cells were treated with NAC before the use of raddeanin A.6. Flow cytometry was employed to detect the cell apoptosis and cell cycle of Skov3cells, which were treated with NAC before the use of raddeanin A. 7. Western Blot was employed to assess the expression of P53, CDK4, cyclinB1, Chkl, P21, ATR in Skov3cells, which were treated with NAC before the use of raddeanin A.8. Acridine orange staining was employed to assess autophagy lysosome in cells, which were treated with NAC before the use of raddeanin A.9. Western Blot was employed to assess the expression of LC3and the cells were treated with NAC before the use of raddeanin A.10. MTT was employed to assess the proliferation of Skov3cells, which were treated with3-MA before the use of raddeanin A.Results1. Raddeanin A can inhibit Skov3proliferation and this inhibition is dose-dependent.2. Raddeanin A can upregulate the intracellular ROS level, this induction can be inhibited by a combined use of NAC, which promote cell inhibition rate. This effect has nothing to do with apoptosis, a rise in G2/M stay was detected and the expression of ATR, Chkl and cyclinB1proteins were downregulated, and the expression of P21and P53proteins are upregulated.3. Raddeanin A can induce autophagy of Skov3cells and upregulate the expression of LC3protein. The pretreatment with NAC can inhibit the autophagy and downregulate the expression of LC3protein.4.Raddeanin A with3-MA can promote cell inhibition rate after the cell was pretreatmented by3-MA.Conclusions1. Raddeanin A can inhibit the proliferation of Skov3cells, and this inhibition is dose-dependent.2. Raddeanin A has an evident anti-tumor activity and it can inhibit its own activity by producing a low level of ROS.3. After a removal of ROS, the anti-tumor activity of raddeanin A was increased, but this effect has nothing to do with cell apoptosis. On the one hand, ATM/ATR-Chkl/Chk2-Cdc25pathway is regulated, therefore the cell cycle was stopped at G2phase, and on the other hand, cellular autophagy was inhibited, thus the self-protection of tumor cells was weakened.