Study On Apoptosis Mechanism Of Human Hepatocellular Carcinoma 7721 Cells Induced By Tanshinone Ⅰ

Objective: Liver cancer is the third leading cause of cancer deaths in China.The current treatment options include surgical therapy(liver resection and liver transplantation)and non-surgical therapy(interventional therapy,radiotherapy,chemotherapy,etc.),but studies have shown that most patients with liver cancer will suffer from some other complications such as postoperative recurrence of cancer,insignificant effects of radiotherapy and chemotherapy,prone to multidrug resistance,and severe adverse drug reactions.Therefore,there is an urgent need to find some drugs that have good therapeutic effect and can effectively reduce the adverse reactions in patients.As a large country in the development,research and application of traditional Chinese medicine,China has found that many active extracts of natural medicinal plants have some good anti-tumor efficacy.Among them,Tanshinone I has been pointed out in recent reports that have significant anti-cancer effects and low toxicity.However,the relevant underlying mechanism is yet to be completely clear.The purpose of this experiment is to use 7721 cells as the object of this study to research the related mechanism of tanshinone I to promote cell apoptosis,and to provide preliminary theoretical and experimental basis for tanshinone I as an anti-tumor drug for clinical application.Methods: The cells were stimulated with different concentrations of tanshinone I.Morphological changes of the cells were examined by inverted microscope.The cell viability of each group was detected by CCK8.The cell viability was monitored by RTCA.The cell cycle,apoptosis and changes of reactive oxygen species were detected by flow cytometry,at the same times,the cells were stained with DAPI to detect the apoptosis of hepatocellular carcinoma cells by fluorescence microscopy.WB was used to detect the expression of cell cycle,apoptosis,and autophagy-related proteins.Finally,the cells were pretreated with NAC and 3-MA to detect the viability and apoptosis of 7721 cells.Results: 1.Tanshinone I can significantly inhibit the proliferation of 7721 cells in a timeand dose-dependent manner.Under inverted microscope,the tumor cells became round and the number was dramatically reduced after the drug stimulation.2.Tanshinone I can reduce the expression of Cycline A1 protein in 7721 cells,and increase the expression of P21 protein,which can also promote cell arrest in S phase.3.The tanshinone I can increase the early apoptosis rate of the cells from 0.24% to 8.98%,after the staining with DAPI,the number of fluorescent bright spots increases significantly with the increase of concentration,and the expression of P-ERK protein can be reduced,and the apoptosis marker protein Caspase3 and PRAP can be reduced which suggest that the drug may release the relevant signal factors through the extracellular signal-regulated kinase pathway to induce apoptosis of the cells.4.After tanshinone I stimulated cancer cells,it could induce the increase of intracellular ROS levels.And the cell viability increased significantly after drug stimulation with the treatment of NAC(P<0.001),and the rate of cell apoptosis was significantly reduced(P<0.001).5.Tanshinone I can participate in the induction of autophagy,resulting in increased expression of the autophagy marker proteins LC3-I,LC3-II and Beclin1,and decreased expression of P62 protein at the same time;The cell viability decreased significantly(P<0.01),and the level of apoptosis increased continuously after stimulated by tanshinone I with the treatment of 3-MA,suggesting that tanshinone I may trigger the occurrence of intracellular protective autophagy,and the addition of autophagy inhibitors can inhibit the growth of cancer cells.Conclusions: Tanshinone I has obvious anti-tumor activity against hepatocellular carcinoma and can arrest the cell cycle and induce apoptosis,which is a potential therapeutic drug for liver cancer.The mechanism of tanshinone I-induced apoptosis of hepatoma cells may be to stimulate the cancer cells to effectively inhibit the activation of extracellular signal-regulated kinases by increasing the level of reactive oxygen species in the cells,so as to decrease the expression level of p-ERK protein and activate apoptotic proteins such as caspase3.In addition,tanshinone I can promote the occurrence of protective autophagy in hepatoma cells,inhibit autophagy can enhance the tanshinone I-induced apoptosis,so when using drugs with autophagy inhibitors can effectively improve the anti-tumor activity of drugs,which can improve the clinical efficacy of tanshinone I treatment for hepatocellular carcinoma in the future.