Effects And Mechanism Of Dynasore On The Proliferation,Apoptosis,Invasion And Migration Of The Non-small-cell Lung Cancer Cell Line A549

Lung cancer is the leading cause of cancer deaths worldwide,most of which are non-small cell lung cancer.Cisplatin is a first-line chemotherapeutic drug that is widely used as a treatment for lung cancer and works by inhibiting cell proliferation,invasion and metastasis.However,since cisplatin often appears resistance in the treatment of tumors,which reduces its curative effect and limits its clinical application,it is urgent to further explore more effective therapeutic drugs.In recent years,studies have found that the occurrence of lung cancer is related disorders of mitochondrial fusion and fission.Dynamin-related protein 1(Drp1)is required for mitochondrial division.Inhibition of Drp1 in lung cancer cells can reduce the proliferation and increase cell apoptosis of lung cancer cells.Dynasore is a small molecule inhibitor of non-selective GTPase activity,both in vivo and in vitro,can inhibit dynamin 1,dynamin 2 and Drp1 GTPase activity.However,the effect of Dynasore on theproliferation,apoptosis,invasion and metastasis of lung cancer A549 cells is not yet clear.Objective:The main purpose of this study is to investigate the effect and mechanism of Dynasore on the proliferation,apoptosis,invasion and metastasis of A549 cells and to detect the anticancer effect of cisplatin or its combination with Dynasore on lung cancer A549 in order to explore a more effective treatment strategy of lung cancer.Methods:The protein expression levels of DRP1 in lung cancer cell lines A549,H1299,H460 and LK2 and human bronchial epithelial cells HBE were detected by western blotting.According to the results,we select the A549 cell line as the key detecting object.A549 lung cancer cells were randomly divided into four groups after culture,(Control group,3% DMSO),cisplatin group(cisplatin 30 ?M),Dynasore group(Dynasore 80 ?M)and Dynasore + cisplatin group(Dynasore 80 ?M,Cisplatin 30 ?M).After intervention of each group,the proliferation of each group was detected by MTT method.The colony formation of each group was detected by colony formation assay.The levels of mitochondrial membrane potentials(MMPs)and ROS were measured by the fluorescence spectrophotometer according to the instructions of JC-1 kit and reactive oxygen species(ROS)kit.The contents of adenosine triphosphate(ATP),malondialdehyde(MDA)and reduced glutathione(GSH)were detected by the spectrophotometer according to the instructions of ATP,MDA and GSH kit.The invasion ability of cells in each group was detected by matrigel invasion assay.Scratch test was used to detect the cell migration ability.The protein expression of mitochondrial fission protein Drp1,Dynamin 2,Proliferating cell nuclear antigen(PCNA),cytochrome C(Cyt C),apoptosis inducing factor(AIF)and Cleaved Caspase-3 protein were detected by Western blotting.The protein expression of Drp1,Dynamin 2,PCNA and Cleaved Caspase-3 were measured by immunofluorescence staining.Results: 1.Compared with HBE cell line,the expressions of Drp1 in A549,H1299,H460 and LK2 cells were significantly increased(P <0.05),and the expression of Drp1 in A549 cell line was significantly higher than that in H1299,H460 and LK2 cells(P<0.05).However,there was no significant difference in Drp1 expression between H1299,H460 and LK2 cell lines.2.Dynasore inhibits the expression of Drp1 and dynamin 2 in lung cancer A549 cells.The results of Western blotting were as follows: The contents of Drp1 and Dynamin2 in Dynasore group were significantly lower than those in Control group,and compared with control group,the levels of Drp1 and Dynamin2 in cisplatin group and Dynasore + cisplatin group were significantly increased(P <0.05).Compared with cisplatin group,The content of Drp1 and Dynamin 2 in Dynasore group and Dynasore +cisplatin group were significantly decreased(P <0.05).Compared with Dynasore group,the content of Drp1 and Dynamin 2 in Dynasore + cisplatin group were significantly increased(P <0.05).We used the average fluorescence intensity to reflect the expression of Drp1 and Dynamin 2 protein in each group.The results of immunofluorescence were consistent with the above Western blotting results(P <0.05).3.Dynasore inhibits the proliferation of lung cancer A549 cells.The results of Western blotting were as follows: PCNA expression in Dynasore group,cisplatin group and Dynasore + cisplatin group were significantly lower than that in Control group(P<0.05);Compared with cisplatin group,the expression level of PCNA in Dynasore group was significantly increased,while the expression level of PCNA in Dynasore + cisplatin group was significantly decreased(P <0.05).Compared with Dynasore group,theexpression level of PCNA in Dynasore + cisplatin group was significantly decreased(P<0.05).We used the average fluorescence intensity to reflect the expression of PCNA protein in each group.The results of immunofluorescence were consistent with the above Western blotting results(P <0.05).MTT experiments and cloning results: Compared with the control group,the cell viability and the proliferative capacity in the Dynasore group,cisplatin group and Dynasore + cisplatin group were significantly decreased(P <0.05).Compared with the cisplatin group,the proliferation of cells in the Dynasore group were significantly increased,while the cell viability and the proliferative capacity in Dynasore+ cisplatin group were significantly decreased(P <0.05).The cell viability and proliferative capacity in Dynasore + cisplatin group were significantly decreased compared with the Dynasore group(P <0.05).These results are consistent with the results of PCNA protein level.4.Dynasore induced mitochondrial dysfunction in lung cancer A549 cells.Compared with the control group,the levels of MMPs,ATP and GSH were significantly decreased,while the ROS and MDA contents were significantly increased in the Dynasore group,the cisplatin group and the Dynasore + cisplatin group(P <0.05).Compared with the cisplatin group,the levels of MMPs,ATP and GSH were significantly increased,and the contents of ROS and MDA were significantly decreased in Dynasore group(P <0.05),while the levels of MMPs,ATP and GSH were significantly decreased,and the contents of ROS and MDA were significantly increased in the Dynasore +cisplatin group(P <0.05).Compared with the Dynasore group,the MMPs,ATP and GSH levels were significantly decreased,and ROS and MDA contents were significantly increased in the Dynasore + cisplatin group(P <0.05).5.Dynasore activates mitochondrial apoptotic pathway in lung cancer A549 cells to induce apoptosis.The results of Western blotting were as follows: compared with Control group,the contents of Cyt C and AIF in mitochondria were significantly decreased,the content of Cyt C and AIF in cytoplasm and the expression of Cleaved Caspase-3 were significantly increased in the Dynasore group,cisplatin group and Dynasore + cisplatin group(P <0.05).Compared with the cisplatin group,the contents of Cyt C and AIF in the mitochondria were significantly increased,while the content of Cyt C and AIF in thecytoplasm and the expression of Cleaved Caspase-3 were significantly decreased in the Dynasore group(P <0.05),while the mitochondrial Cyt C and AIF contents were significantly decreased,the contents of Cyt C in cytoplasm and the AIF the nucleus and Cleaved Caspase-3 expression were significantly increased in the Dynasore + cisplatin group(P <0.05).Compared with Dynasore group,The contents of Cyt C and AIF in the mitochondria were significantly decreased,the content of Cyt C in the cytoplasm and and the AIF the nucleus as well as the expression of Cleaved Caspase-3 were significantly increased in the Dynasore + cisplatin group(P<0.05).We further used immunofluorescence technique to detect the level of cleaved Caspase-3 in each group by using the number of Cleaved Caspase-3 positive cells.The change trend of the number of cleaved Caspase-3 positive cells in each group was consistent with the above-mentioned results of the Western blot(P <0.05).6.Dynasore inhibits the invasion and migration of lung cancer A549 cells.Compared with the control group,the number of invasive cells and the cell metastasis ability were significantly decreased in the Dynasore group,the cisplatin group and the Dynasore + cisplatin group(P <0.05);compared with the cisplatin group,the number of invasive cells and the cell metastasis ability in the Dynasore group were significantly increased,while the number of invasive cells and the cell metastasis ability were significantly decreased in the Dynasore + cisplatin group(P <0.05).Compared with Dynasore group,the number of invasive cells and the cell metastasis ability were significantly decreased in the Dynasore + cisplatin group(P <0.05).Conclusion:Dynasore can inhibit the proliferation,invasion and metastasis and induce apoptosis of lung cancer A549 cells,whose mechanism may be related to the inhibition of mitochondrial division and autophagy.Dynasore can enhance the anticancer effect of cisplatin on lung cancer A549 cells.