Activated JNK Signaling Pathway Via ROS To Induce Apoptosis Of Non Small Cell Lung Cancer Cells By Gambogic Acid

[Background and Objective] Gamboge 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, while the molecular mechanism of which that inhibits the proliferation of lung carcinoma has not yet illuminated.We used non-small cell lung cancer(NSCLC) cell lines for 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 activity of JNK signaling pathway, the mitochondrial membrane potential (MMP) as well as the expression of apoptosis-associated proteins, and approach the molecular mechanism of the apoptosis of NSCLC cells induced by GA.[Methods] The NSCLC cells H1975, H292and H460were cultured in vitro. The effect of cell proliferation by GA in different concentrations for24,48and72hours was analyzed by MTT assay. The apoptosis rate of H1975cells by GA after24hours use was detected by Annexin V-FITC/PI double staining. The effect of ROS in H1975 cells by GA was measured by DCFH-DA. The changes of MMP in H1975cells after16hours use of GA were analyzed by JC-1probe method. The activity of JNK signaling pathway and the expression of apoptosis-associated proteins were detected by Western blot.[Results] The cell proliferation of NSCLC cells was inhibited by GA in a dose-dependent manner, in which H1975cells were relatively sensitive. Compared with the control group cells, the survival rates were decreased(91.8%±5.1%,63.7%±3.8%,31.5%±4.8%,10.6%±2.7%) after cells were treated by GA (0.5、1、2.5and5μM) for24hours,89.1%±5.4%,41.5%±4.1%,12.2%±3.2%,8.9%±2.1%after cells were treated for48hours, and84.1%±6.4%,18.5%t3.2%,7.2%±2.0%,6.8%t1.3%after treated for72hours. The apoptosis rates were25.2%,51.8%and75.1%after H1975cells were treated by GA (1、2.5and5μM) for24hours. The expression of cleaved caspase9, cleaved caspase3and cleaved PARP were significantly higher than the control group in a dose-dependent manner. Compared with control group, the ROS levels were significantly increased after treated by GA for2hours, and the expression of phospho-JNK (p-JNK) were up-regulated. 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 pro-apoptotic protein Bax、Bak、Bik and decreased the expression of pro-survival protein Bcl-2.[Conclusion] GA could induce the apoptosis of the NSCLC cells through up-regulating the intracellular level of ROS to activate JNK signaling pathway, which triggers loss of MMP and activation of mitochondria apoptosis pathways to induce the apoptosis of human lung cancer cells.