The Preliminary Investigation On The Effects Of ALDOA On Apoptosis In Lung Cancer And Its Mechanism

Objective:Fructose-bisphosphate aldolase A(ALDOA)has been initially identified from lung squamous cell carcinoma as a key regulator in tumorigenesis.In our previous study,we demonstrated that ALDOA can promote cell proliferation and migration in lung squamous cell carcinoma.However,the direct evidence for a relationship between ALDOA and lung cancer apoptosis has not been found,The aim of this study is to explore the function of ALDOA to regulate the cell apoptosis in lung cancer.We could figure out the molecular mechanism of apoptosis related to ALDOA from a signal pathway standpoint.Method:1.In our previous studies,we successfully established ALDOA stably knocked-down cell lines in NCI-H520 with interference RNAi technique.At first,the expression of ALDOA was detected by Western blotting.2.Use Western blotting to detect the expressions of ALDOA in 5 kinds of lung cancer cell lines:HBE,NCI-H520,A549,973,H157 and futher screen the cell line with lower expression of ALDOA.Then we transfect pcDNA4.0-ALDOA and pcDNA4.0-NC plasmids respectively to the cancer cell line with lower expression of ALDOA.Western blot analysis were used to confirm if ALDOA was successfully transfected.3.Cisplatin(CDDP),by simulating chemotherapy to induce tumor cell apoptosis.4.Then we use a series of apoptosis detection methods,such as DAPI staining and flow cytometry(flow cytometry analysis,FCM)to detect apoptosis rate of each cell line.5.Western blotting was used to detect the the expression of the protein related with apoptosis,such as Caspase-3,Cleaved-Caspase-3,Cleaved-PARP,Bax,Bcl-2.6.Furthermore,we detected the key proteins of extrinsic and intrisic pathway,Cleaved-Caspase-8 and Cleaved-Caspase-9 by Western blotting.7.The release of cytochrome C from mitochondria into cytosol was detected by immunofluorescence staining.Results:1.Western blot analysis demonstrated that the expression level of ALDOA remarkably decreased in knockdown ALDOA NCI-520 cells,indicating that the expression of ALDOA was successfully knocked down in NCI-520 cells(*p<0.05).2.Then,the protein expression levels of ALDOA in various lung cancer cell lines(HBE,NCI-H520,A549,973 and H157)were determined.Western blot analysis displayed the low expression of ALDOA in the H157 cell line.Therefore,pcDNA4.0-ALDOA and pcDNA4.0-NC plasmids were transfected into H157 cells,respectively.There was a significant increase in ALDOA expression after the transfection of pcDNA4.0-ALDOA.3.DAPI staining and flow cytometry analysis results revealed that apoptotic activity was enhanced in the knockdown of ALDOA cell lines(*p<0.05).This result was also reversely confirmed in ALDOA-overexpression cell line.4.Further investigation revealed that the occurrence of apoptosis was associated with the activation of caspases-3 and cleavage of PARP(*p<0.05).In addition,we also found that ALDOA knockdown resulted in the upregulation of Bax,but downregulation of Bcl-2(*p<0.05).At the same time these results were reversely confirmed in ALDOA overexpressing cell line.5.Western blotting indicated that both of the key protein involved in the intrinsic and extrinsic apoptosis pathway were increased in ALDOA knockdown cell lines(*p<0.05).6.The release of cytochrome C from mitochondria into cytosol was markedly increased in ALDO A knockdown cell lines.Conclusion:1.Taken together,our results provide evidence that the depletion of ALDOA can enhance cisplatin-induced apoptosis.2.The overexpression of ALDOA may prevent cisplatin-induced cell apoptosis in NSCLC cells.3.ALDOA suppressed apoptosis of lung cancer cells and this effect was associated with multiple apoptosis related proteins,including Caspase-3,Cleaved-Caspase-3,Cleaved-PARP,Bax,Bcl-2.4.Knockdown of ALDOA promotes lung cancer cell apoptosis through both the extrinsic and intrisic apoptosis pathways.4.Furthermore,we also found that the role of ALDOA on cell apoptosis was associated with the release of cytochrome C from mitochondria to cytoplasm and then the mitochondrial downstream signals and onset of apoptosis may be activated.