Investigation Of The Apoptosis And Autophagy Induction Effects Of 7-O-geranylquercetin On Human Lung Cancer Cells In Vitro

Objective:To investigate the apoptosis and autophagy induction effects of 7-O-geranylquercetin on human lung cancer cell lines A549 and NCI-H1975 in vitro.Methods:(1)In the study of apoptosis induction effect of 7-O-geranylquercetin on human lung cancer cells,MTT assay was used to detect the cell viability of lung cancer cells A549 and NCI-H1975 after exposed to various concentrations of 7-O-geranylquercetin(0,15,25 and 35 ?M)at different time(12,24 and 48 h);DAPI,AO/EB staining and transmission electron microscopy(TEM)were used to observe the morphology of the cells;Flow cytometry was used to detect the cell cycle distribution;AV-PI double-staining was used to detect influence of 7-O-geranylquercetin on cell apoptosis;Western blotting assay was used to detect the expression of apoptosis-related proteins,such as Bax,Bcl-2,pro-caspase 3 and cleaved-PARP;Besides,Z-VAD-FMK,a caspase inhibitor,was utilized to verify whether 7-O-geranylquercetin-induced apoptosis depends on caspase or not;Real-time PCR assay was used to detect the influence of 7-O-geranylquercetin on the expression of hTERT gene in lung cancer cells.(2)In the study of autophagy induction effect of 7-O-geranylquercetin on human lung cancer cells,TEM was used to observe autophagosome and western blotting assay was used to detect the expression of autophagy-related proteins,such as LC3-?,Beclinl,ATG5 and p62.(3)In the study of relationship between 7-O-geranylquercetin-induced apoptosis and autophagy,autophagy inhibitor CQ and Beclinl siRNA were respectively used to inhibit autophagy to further investigate the influence of 7-O-geranylquercetin-induced autophagy on apoptosis.Furthermore,flowcytometry was used to detect ROS generation in lung cancer cells exposed to 7-O-geranylquercetin.And NAC,a ROS inhibitor.was used to study the role of ROS in 7-O-geranylquercetin-induced apoptosis and autophagy.Results:(1)In the study of apoptosis induction effect of 7-O-geranylquercetin on human lung cancer cells,MTT assay revealed that 7-O-geranylquercetin could inhibit cell viability of the two lung cancer cell lines obviously in a dose-and time-dependent manner,and the effect was stronger than that of quercetin.The cytotoxicity of 7-O-geranylquercetin to lung cancer cells A549 and NCI-H1975 was much stronger than that to normal lung cells HLF.Morphological results showed that cell shrinkage,the nucleus changes,cell number decrease and cell apoptosis number increase occurred after the lung cancer cells were exposed to 7-O-geranylquercetin.Cell cycle result revealed that 7-O-geranylquercetin could arrest the lung cancer cells at G0/G1.AV-PI double-staining showed that 7-O-geranylquercetin induced apoptosis in a dose-dependent manner.Western blotting assay indicated that 7-O-geranylquercetin induced up-regulation of Bax and cleaved-PARP,down-regulation of pro-caspase 3 and Bcl-2 with dose-dependence.The apoptosis mechanism study showed that 7-O-geranylquercetin induced caspase-dependent apoptosis.Real-time PCR result showed that 7-O-geranylquercetin down-regulated the expression of hTERT mRNA.(2)In the study of autophagy induction effect of 7-O-geranylquercetin on human lung cancer cells,TEM results showed that autophagosome appeared obviously in 7-O-geranylquercetin treated cells;Western blotting assay revealed that the expression of autophagy-related proteins LC3-? and Beclinl up-regulated and that of p62 down-regulated in a dose-dependent manner.while the expression of ATG5 changed little.(3)In the study of relationship between 7-O-geranylquercetin-induced apoptosis and autophagy,7-O-geranylquercetin-induced apoptosis weakened significantly after the lung cancer cells were treated with CQ and Beclin 1 siRNA,indicating that autophagy may promote the apoptosis.The preliminary research of the mechanism revealed that 7-O-geranylquercetin could increase the ROS generation,and 7-O-geranylquercetin-induced apoptosis and autophagy were inhibited by the ROS inhibitor NAC.Conclusion:This study found that 7-O-geranylquercetin possessed good anti-lung cancer effects in vitro.The effects were mainly manifested in induction of apoptosis and apoptosis-promoting autophagy on lung cancer cells A549 and NCI-H1975.and might be related with excessive ROS generation.7-O-geranylquercetin is expected to be a new agent against lung cancer,and has great research and development value.