The Effect Of Tanshinone On The Apoptosis And The Expression Of Regulated Proteins In Lung Cancer Cell Line

Objective:This study aims to?1?determine the influence of dihydrotanshinone?DTS?and tanshinone I?Tan I?on the apoptosis of lung carcinoma cells and the mechanisms by which these compounds regulate protein expression;?2?determine,through experiments,the inhibitory effect of DTS on human lung adenocarcinoma GLC-82 cell proliferation and the influence of DTS on cell cycles and apoptosis;and?3?confirm the significant change of the protein expression in preliminary research and explore the mechanism of protein expression.An animal model was established to investigate the tumor suppression effect and mechanism of Tan I combined with radiotherapy on Lewis lung carcinoma-bearing mice.Methods:Human lung adenocarcinoma GLC-82 cells cultured in vitro were divided into blank control group and DTS group.The inhibitory effects of different concentrations(5,10,20,40,80,and 100?g·mL^-1)of DTS on the proliferation of GLC-82 cell were examined through MTT assay to confirm if the inhibitory effect of DTS was time-and dose-dependent,and the suppression ratio was calculated.Different time gradients of DTS administration were set.The proportion of different GLC-82 cell cycle phases was examined through flow cytometry and single propidium iodide?PI?staining.The effects of different action time of DTS on GLC-82 cell apoptosis were examined using annexin V/PI double staining.Expression of Bcl-2,Bax,and Caspase-3 proteins were detected through Western blot.A cancer xenograft model of Lewis lung in C57BL/6mice was established by inoculating Lewis lung cancer cells and randomly dividing into model control group,5-FU+radiotherapy control group?postive control group?,and Tan I(10,20,and40mg·kg^-1)+radiotherapy group.Dietary status of tumor-bearing mice was observed,and the relative volume of tumor and tumor growth delay time were calculated.The tumors were isolated and weighed to calculate the tumor inhibition rate.Cell apoptosis was analyzed through TUNEL.The expression of Bcl-2 and Bax proteins were analyzed through Western blot.Results:MTT assay showed a significant,time-dependent inhibitory effect of different concentrations of DTS on GLC-82 cell growth.With the increase of the DTS concentration,the inhibition rate of cell proliferation significantly increased compared with that of the blank control group?P<0.05 or P<0.01?.DTS could induce GLC-82 cell apoptosis,and the effect increased with increased administration time.A significant deference was observed between treatment groups and blank control group?P<0.05 or P<0.01?.Western blot indicated that increased DTS action time did not significantly change the expression level of Bax protein,whereas the expression of Bcl-2 protein decreased gradually.The caspase family proteins were activated,and the expression of caspase-3 protein was upregulated,whereas that of Bcl-2 protein was downregulated.Different doses of Tan I combined with radiotherapy had a certain inhibitory effect on the growth of Lewis lung cancer xenograftin mice.The inhibitory effect of high-dose(40 mg·kg^-1)Tan I combined with radiotherapy was stronger,whereas moderate-(20 mg·kg^-1)and high-dose(40 mg·kg^-1)Tan I alone had a relatively strong and positively dose-dependent radiosensitization effect.The inhibitory rates were 27.14%,38.46%,and 59.78%,respectively,which were significantly higher than that of the model control group?P<0.01?.In high-Tan-I-dose group,cell shrinkage was observed with an apoptosis index of 51.3%,which is significantly higher than that of the model control group?P<0.05?.Additionally,high-dose Tan I downregulated Bcl-2 protein expression but significantly upregulated Bax protein expression to induce cell apoptosis.Moreover,high-dose Tan I(40mg·kg^-1)+radiotherapy and 5-FU+radiotherapy presented a similar radiosensitization effect,and both could induce cell apoptosis.Conclusions:DTS has a potent cytotoxity against lung adenocarcinoma GLC-82cells and could induce cell apoptosis.The mechanism of action may be related to the downregulation of Bcl-2 protein expression and activation of Caspase-3 protein to induce apoptosis.Tan I can effectively suppress the growth of cancer xenograft of Lewis lung in mice and has a certain tumor inhibition effect.High Tan I dose causes cell shrinkage possibly by downregulating Bcl-2 protein expression and upregulating Bax protein expression to induce cell apoptosis and then exerting a radiosensitization effect.