Effect Of GAPDH Gene Knockdown On Apoptosis Of Human Lung Squamous Cell Carcinoma Lines NCI-H520

Objective:Cancer cells and normal cells have different energy metabolism phenotype,Cancer cells use glycolysis to produce energy,therefore,relationship between glycolytic enzymes and tumors has becoming a hot topic.Glyceraldehyde 3-phosphate dehydrogenase(GAPDH)is significantly increased in lung squamous cell carcinoma.It is screened based on 2D-DIGE combined with high-throughput screening technology mass spectrometry by our laboratory.Initiation and progression of cancer are a very complicated process regulated by a variety of signal-networks,and mitochondria might play an important role in this process.Mitochondria balanced a variety of metabolic processes in tumor cells including pro-apoptotic and anti-apoptotic,glycolysis and oxidative phosphorylation etc.The intact mitochondria is very important to cell survival and energy metabolism.The change of mitochondria-related protein and cytochrome C is a sign of the mitochondrial membrane structure was destroyed.Bcl-2 protein family is one of the important indicators of cell apoptosis and is also closely related to the mitochondrial membrane.Therefore,this study aims to explore whether the GAPDH gene knockdown has an effect on apoptosis of human lung squamous cell carcinoma lines NCI-H520 by affecting the mitochondria.We expect to break the intrinsic thought that regard GAPDH as "Housekeeping genes",and explore the functions except glycolytic enzymes.Methods:1.In our previous study,the empty vector shRNA-NC and the shRNA-GAPDH were stably transfected into the lung squamous cancer cell lines NCI-H520 and establish stable cell lines—NCI-H520-shRNA-GAPHD-6,NCI-H520-shRNA-GAPHD-24 and the negative control NCI-H520-shRNA-NC.Logarithmic phase cell lines were taken separately to detect the expression of GAPDH and verify the efficiency of GAPDH gene knockdown.2.Fluorescence detection was used to detect the relative quantity of the ATP in the four cell lines.3.CDDP was used to establish the apoptosis cell model.Then we use a series of apoptosis methods,such as flow cytometry analysis(FCM),DAPI staining and transmission electron microscopy(TEM)to investigate the apoptosis of NCI-H520,NCI-H520-shRNA-NC,NCI-H520-shRNA-GAPHD-6 and NCI-H520-shRNA-GAPHD-24.4.We use Western-blot assay to detect the expression change of apoptosis-associated protein,such as Caspase-3,PARP,Bcl-xL,Bcl-2 and Bax in NCI-H520,NCI-H520-shRNA-NC,NCI-H520-shRNA-GAPHD-6 and NCI-H520-shRNA-GAPHD-24 after treated with CDDP.5.Western-blot assay was used to detect the expression of autophagy protein Beeline 1 after treated with CDDP.Results:1.Fluorescence test results showed no significant differences in the relative quantity of ATP in the four cell lines.2.After treated with CDDP,the result of Flow cytometry analysis(FCM)and DAPI staining showed that the apoptosis rate of the NCI-H520-shRNA-GAPHD-6 and NCI-H520-shRNA-GAPHD-24 cell lines was significantly higher than the negative control NCI-H520-shRNA-NC(*p<0.05),while NCI-H520-shRNA-NC and wild type NCI-H520 was no obvious difference.After treated with CDDP,typical morphological features of apoptotic cells were detected by transmission electron microscopy:apoptotic cell morphology:cells shrank and turned smaller,the microvilli disappear,the nucleus fragmentation,budded around the cell membrane.A large number of autophagic bodies in the cell of GAPDH gene knockdown was also observed.3.By Western Blot assay,we found that the expression of apoptosis-related proteins Caepase-3,Bcl-xL,Bcl-2 in NCI-H520-shRNA-GAPHD-6 and NCI-H520-shRNA-GAPHD-24 cell lines has decreased significantly compared to the negative control NCI-H520-shRNA-NC(*p<0.05),the expression of Cleaved-caspase-3,Cleaved-PARP and BAX protein increased obviously(*p<0.05),the ratio of Bcl-2 and Bax reduces the six times(*p<0.05).4.After the cisplatin-induced apoptosis,the result of immunofluorescence showd that the release of cytochrome C from mitochondria to the cytosol has significant increase in NCI-H520-shRNA-GAPDH6 and NCI-H520-shRNA-GAPDH24 cell lines compared to the negative control NCI-H520-shRNA-NC(*p<0.05).5.The expression of autophagy protein Becline 1 in the NCI-H520-shRNA-GAPHD-6 and NCI-H520-shRNA-GAPHD-24 cell lines increased obviously when compared to the negative control group<0.05).Conclusions:1 GAPDH gene knockdown does not affect the relative quantity of ATP in the four cell lines.2.GAPDH gene knockdown promoted cisplatin-induced apoptosis of human lung cancer cell line NCI-H520.3.GAPDH gene knockdown promoted the inactivation of PARP and activation of Caspase-3,meanwhile,promoted the expression of Bax and suppressed the expression of Bcl-xL and Bcl-2.4.GAPDH gene knockdown promoted the release of cytochrome C from mitochondria to the cytosol.