The Effect And Mechanisms Of Atorvastatin On Proliferation,Apoptosis And Migration Of Human Lung Cancer HCC827 Cells

Lung cancer ranks first among male and female cancer patients,and it is a serious threat to human health.Surgery and chemotherapy are two main treatment methods for lung cancer at present,but the treatment results are not satisfactory.Among them,a considerable proportion of patients develop drug resistance.Finding new drug targets is important for reducing lung cancer mortality.The statin is a hydroxymethyl glutaryl coenzyme A(HMG-CoA)reductase inhibitor,which is widely used for prevention and treatment of hyperlipidemia.In recent years,epidemiological studies have shown that long-term use of statins can effectively reduce the risk of cancer.However,its mechanism of action on lung cancer is still unclear.In this study,we studied the effect of atorvastatin on proliferation and apoptosis of human lung cancer cells and further explored its related mechanisms.Tumor metastasis is the leading cause of death in patients with malignant tumors.Epithelial mesenchymal transition(EMT)is an important mechanism for tumor metastasis,which is accomplished by complex multi-gene regulation.EMT can enhance cell migration.TGF-?1 is a key factor in the EMT process.In this experiment,the migration ability of HCC827 cells was observed by wound healing assay and Transwell migration assay,and the in vitro EMT model of HCC827 cells induced by TGF-?1 was established to further detect the effect of atorvastatin on migration.The expression of the marker proteins E-cadherin,N-cadherin and migration-associated protein MMP2 during EMT was also examined.Part 1 Effect of atorvastatin on proliferation and apoptosis of human lung cancer HCC827 cellsObjective: To investigate the effect of atorvastatin(ATO)on proliferation and apoptosis of human lung cancer HCC827 cells and explore its mechanism.Methods: The inhibitory effect of atorvastatin on the proliferation of HCC827 and A549 cells was detected by trypan blue staining.The cell cycle of HCC827 was detected by flow cytometry.Cell morphology was observed under Optical microscope.Hoechst 33342 and TUNEL assay were used to detect apoptosis.The level of reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe;Colorimetric method was used to detecti the content of malondialdehyde(MDA);The expression levels of P-H2 A.X and apoptosis related proteins Cleaved-caspase 3?Bax and Bcl-2 were detected by Western blot.Results:1.The result of trypan blue staining showed that the survival rate of human lung cancer HCC827 cells and A549 cells decreased with the increase of atorvastatin concentration from 0 to 40 ?M.The inhibitory effect of atorvastatin on HCC827 cells was more obvious than that on A549 cells in a dose-and time-dependent manner.2.Flow cytometry results showed that the number of cells in G1 phase significantly increased and the S phase and G2 phase decreased after treatment with different concentrations of atorvastatin(2.5 ?M,10 ?M,40 ?M).3.The morphology of the cells was observed under light microscopy.The cells in the control group grew well.The cells in the atorvastatin group shrank with the volume smaller,and the number of dead cells floating in the culture solution increased significantly.4.Hoechst 33342 staining results showed that the morphology of HCC827 nucleus showed apoptotic features such as chromatin condensation,crescent after atorvastatin treatment.5.The results of TUNEL fluorescence staining showed that the green fluorescence intensity in the atorvastatin group was significantly higher than that in the control group,which indicated that atorvastatin induced DNA fragmentation in HCC827 cells.6.The level of reactive oxygen species(ROS)in HCC827 cells was detected by DCFH-DA fluorescent probe.The results showed that green fluorescence increased significantly with the increase of atorvastatin concentration compared with the control group,indicating a significant increase in intracellular ROS level.7.Colorimetric detection of malondialdehyde(MDA)content showed that the MDA content in HCC827 cells increased with the increase of atorvastatin concentration compared with the control group.8.Atorvastatin up-regulated the expression of P-H2 A.X,Cleaved caspase 3 and Bax protein in HCC827 cells,down-regulated the expression of Bcl-2 protein,and up-regulated the ratio of Bax/Bcl-2.Summarize: Atorvastatin can significantly inhibit the growth of HCC827 and A549 cells,And the inhibition of HCC827 cell growth is more obvious,The growth inhibition may be related to cell cycle arrest;Atorvastatin can induce apoptosis in HCC827 cells,which may be related to oxidative stress and mitochondria-mediated pathways involved in atorvastatin-induced apoptosis in HCC827 cells.Part 2 Effect of atorvastatin on migration of human lung cancer HCC827 cellsObjective: To investigate the effect of atorvastatin on the migration of human lung cancer HCC827 cells and explore its mechanism.Methods: The effect of atorvastatin on the migration of HCC827 cells was detected by wound healing assay and Transwell migration assay.The expression of E-cadherin,N-cadherin and MMP2 protein was detected by western blot.Results:1.The results of wound healing showed that the atorvastatin could significantly inhibit the migration of HCC827 cells,and the TGF-?1 could significantly promote the migration of HCC827 cells.The atorvastatin also significantly inhibited the migration of HCC827 cells promoted byTGF-?1.2.The number of transmembrane cells in each group after 24 h of drug treatment was calculated in vitro transwell migration experiments.Atorvastatin significantly reduced the number of transmembrane cells in HCC827 cells compared with control group,and TGF-?1 significantly increased the number of HCC827 cells.The number of transmembrane cells was significantly lower in the atorvastatin combined with TGF-?1 group than in the TGF-?1 alone group.3.Atorvastatin reduced the expression of N-cadherin and MMP-2 protein in HCC827 cells and had no effect on the expression of E-cadherin protein.TGF-?1 could significantly increase the expression of N-cadherin and MMP-2 proteins in HCC827 cells,and had no effect on the expression of E-cadherin protein;Atorvastatin could also significantly reduce the expression of N-cadherin and MMP-2 protein in HCC827 cells stimulated by TGF-?1,and had no effect on the expression of E-cadherin protein.Summarize: Atorvastatin inhibits the migration of HCC827 cells and also inhibits the migration of TGF-?1 stimulated HCC827 cells.This may be related to the inhibition of EMT process in HCC827 cells and down-regulation of MMP2 protein expression.Conclusions:1.Atorvastatin can inhibit the proliferation of HCC827 cells,which may be related to atorvastatin interfering with HCC827 cell cycle;atorvastatin can induce apoptosis in HCC827 cells,which may be related to oxidative stress-mediated pathway and The regulation of death-related factors is involved in the apoptosis of HCC827 cells induced by atorvastatin.2.TGF-?1 can induce EMT in HCC827 cells.Atorvastatin can inhibit the migration of HCC827 cells and inhibit the migration of HCC827 cells induced by TGF-?1.It may interfere with the EMT process of HCC827 cells and down-regulate MMP2 protein expression.