Activated P53Signaling Pathway Via ROS To Induce Apoptosis Of Non Small Cell Lung Cancer Cell A549by Gambogic Acid

Background and PurposesGamboge is the dry resin secreted from Garcinia hanburyi Hook.F.G.. Gambogic acid (GA) is the main active component of Gamboge, which has been confirmed with the broad-spectrum antineoplastic activity by modern pharmacology study. The proliferation of cancer cells including carcinoma of lung, liver, breast, colon etc. can be inhibited effectively by GA. GA educes anti-tumor activity by promoting the apoptosis of tumor cells, inhibiting the telomerase activity, angiogenesis as well as the expression of nuclear pore protein, inducing the G2/M phase arrest of the tumor cells, and activating T lymphocytes to kill tumor cells etc.. While the molecular mechanism of which that inhibits the proliferation of lung carcinoma has not yet to be illuminated.-We used p53wild-type non-small cell lung cancer(NSGLG) cell line A549for the research to observe the inhibitory effect of GA on cell proliferation, analyze the effect of GA on the Reactive oxygen species (ROS) levels in NSCLC cells, the mitochondrial membrane potential (△ψM) as well as the apoptosis rate, detect the expression of p53and its downstream target genes Bax, Bik and PUMA which are pro-apoptotic, and approach the molecular mechanism of the apoptosis of NSCLC cells induced by GA. Furthermore, we used the antioxidant N-acetylcysteine (NAc), to pretreat the A549cells to analyze the effect of removing the endogenous ROS on GA-induced A549cells apoptosis and the expression of p53as well as the apoptosis-related proteins. So we can research the role of ROS in the GA regulated p53expression to induce the apoptosis of A549cells.MethodsThe p53wild-type NSCLC cells A549were cultured in vitro. The effect of cell proliferation by GA in different concentrations for24,48and72hours was analyzed by MTT assay. The apoptosis rate of A549cells by GA after24hours use was detected by Annexin V-FITC/PI double staining. The effect of ROS in A549cells by GA was measured by DCFH-DA. The changes of△ψM in A549cells after16hours use of GA were analyzed by JC-1probe method. The expression of p53and its downstream target genes Bax, Bik and PUMA as well as the anti-apoptotic proteins Bcl-2and caspase cascade reactive proteins were detected by Western blot.We further pretreat the A549cells with NAc for2hours to remove endogenous ROS, then analyze the effect of GA on the A549cells apoptosis and the expression of p53as well as the apoptosis-related proteins. ResultsThe cell proliferation of NSCLC cells was inhibited by GA in a dose-dependent manner. The survival rates were98.3%±4.8%,92.7%±4.0%,73.1%±5.2%,42.6%±3.7%, and21.5%±3.8%after cells were treated by GA (0.5,1,2.5,5, and10μM) for24hours.96.2%±5.4%,84.5%±4.6%,62.6%±4.8%,21.8%±5.0%, and12.7%±3.6%after cells were treated for48hours.95.4%±6.1%,80.2%±5.7%,43.5%±4.3%,18.6%±4.8%, and10.3%±2.9%after treated for72hours. The apoptosis rates were22.4%±5.7%,40.0%±6.2%, and66.6%±5.2%after A549cells were treated by GA (2.5,5, and10μM) for24hours. The expression of cleaved caspase9, cleaved caspase3and cleaved PARP were significantly higher in a dose-dependent manner. Compared with control group, the ROS levels were significantly increased after treated by GA for2hours. After treated by GA for16hours, the mitochondrial membrane potential detection found that in experimental groups were decreased significantly compared with the control group. The use of GA for24hours increased the expression of p53and its downstream target pro-apoptotic genes Bax, Bik and PUMA and decreased the expression of pro-survival protein Bcl-2compared with the control group.We also acquired the conclusion that A549cells pretreated by5mM NAc can efficiently inhibit the GA induced (10μM) cell apoptosis. The expression of p53in GA+NAc experimental group had no significant disparity with the control group. Also, the caspase3and PARP were not activated.ConclusionGA could induce the apoptosis of the p53wild-type NSCLC cells through up-regulating the intracellular level of ROS to activate the expression of p53and its downstream target pro-apoptotic genes Bax, Bik and PUMA, which triggers loss of△ψM and activation of mitochondria apoptosis pathways to induce the apoptosis of human lung cancer cells.