Study The Effect Of Rotenone On The Proliferation,Apoptosis And Invasion Of Human Lung Cancer Cell And The Mechanism Between Rotenone And Induction Of Apoptosis

Background and significanceAs one of the most common malignant tumor, lung cancer possess a high incidence and mortality which is increased especially due to grave pollution of environment. By the statistics, lung cancer is increasing by an annual growth rate of 26.9 percent in China with the highest mortality. Treatments on lung cancer contain surgery, radiotherapy, chemotherapy and molecular targeted therapy. Despite of continuous improvement in the therapeutic methods, the prognosis is still poor for the patients of lung cancer, because of late detection and diagnosis, or the insensitivity to the therapy.Cancer cell is charactered by excessive proliferation and block of apoptosis, which potentiate both cancer initiation and progression. It is the principal strategies of treating cancer that suppress proliferation and induce apoptosis. Autophagy is another programmed cell death with complex link with apoptosis. In the normal state, autophagy is crucial for cellular homeostasis. Under oxidative stress, increase of autophagy may reduce the accumulation of injured cell organelle and protein, prevent cell carcinogenesis. However, autophagy would provide nutritional support for the tumor cell during the cancer initiation and progression. Autophagy is dynamic process including formation of phagophores, formation of autophagosomes, fusion between autophagosomes and lysosomes into autolysosomes, degradation in the lysosomes. Autophagic flux is used to describe this dynamic process and reflect the activity of cell autophagy. LC3 protein is the specific marker on the membrane of autophagosomes and autolysosomes. The expression level of LC3 and the ratio of LC3Ⅱ/Ⅰ may reflect the activity of cell autophagy. P62 protein integrated and degraded in lysosome, is one indicator of detecting autophagic flux.Rotenone, which is an inhibitor of the mitochondrial electron transport chain complex I, results in the activation of NOX2 and release of ROS, and has been shown to display anticancer activity through the induction of apoptosis in various cancer cells. The mechanistic link between rotenone-dependent activation of NOX2 and induction of apoptosis is still elusive. In this present study, we used the human lung cancer A549 cells to detect the relationship between rotenone and proliferation, apoptosis and invasion, and investigate the mechanism of rotenone inducing apoptosis of human lung cancer cell.Part I Effect of rotenone on the proliferation, apoptosis and invasion of human lung cancer A549 cellsObjective:Detect the effect of rotenone on the proliferation, apoptosis and invasion of human lung cancer A549 cells, investigate the anticancer activity of rotenone.Methods:1. A549 cells were cultured in DMEM containing 10% FBS, at 37℃ in a humidified atmosphere at 95% air and 5% CO2.2. After overnight attachment, medium was replaced with serum-free DMEM and treated with various doses of rotenone. Cells were harvested 24 hours after the treatment.3. MTT assay was used to detect the activity of A549 cell. The trypan blue exclusion assay and Alamar blue assay were used to determine the cytotoxic effect of rotenone on A549 cell.4. Flow cytometry analysis was used to test cell cycle and apoptosis of A549 cell.5. Western blot was used to test the expression of Bcl-2 and Bax, in order to investigate the induction of apoptosis by rotenone.6. The effect of rotenone on invasive ability of A549 cell was tested by transwell invasion assay.Results:1. MTT assay showed that rotenone caused a dose-dependent reduction in the cell viability of A549 human lung cancer cells.2. Trypan blue exclusion assay and Alamar blue assay showed the cytotoxic effect of rotenone on A549 cells.3. Flow cytometry analysis and western blot showed that rotenone induce the apoptosis of A549 cells.4. Transwell invasion assay showed that the invasive ability of A549 cells was inhibited.Conclusions:1. in vitro, rotenone inhibited the proliferation of A549 cells by reduction in the cell viability and cytotoxic effect.2. Rotenone induce the apoptosis of A549 cells.3. Rotenone inhibited the invasive ability of A549 cells.Part Ⅱ Experimental study on the mechanism of rotenone inducing apoptosis in human lung cancer cellObjective:investigate the mechanism of rotenone inducing apoptosis of human lung cancer cell by activating ROS system and regulating autophagic flux.Methods:1:A549 cells were cultured in DMEM containing 10% FBS, at 37℃ in a humidified atmosphere at 95% air and 5% CO2.2. A549 cells were pre-treated with 5 μmol/L of gp91 ds, an inhibitor of NADPH oxidase assembly, or treated with hydrogen peroxide (H2O2) or Bafilomycin A, which prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes, at the indicated doses and time frame.3. Treat A549 cells with 500 nmol/L rotenone for 6 hours. ROS production was detected using the Total ROS/Superoxide detection kit. Test the expression of LC3 by immunofluorescence detection. Test the expression of p62, LC3 I, LC3Ⅱ, Akt, p-Akt by western blot.4. Treat A549 cells with 500 nmol/L rotenone for 24 hours. ROS production was detected using the Total ROS/Superoxide detection kit. Test the expression of p62, LC3 Ⅰ, LC3Ⅱ by western blot.Results:1:After treatment with 500 nmol/L rotenone for 6 hours:1.1:rotenone treated cells showed an increase in ROS generation compared to untreated cells, which was significantly suppressed in the cells pre-incubated with the NOX2-specific inhibitor gp91 ds.1.2:Immunofluorescence analysis showed an increase in LC3 expression levels compared to untreated cells, which were partially inhibited upon inhibition of Nox2 activity with gp91 ds.1.3:Increase in p62 level upon rotenone treatment and this increment is suppressed by pre-incubation with gp91 ds.1.4:There was no increase in LC3Ⅱ, and p62 level by bafilomycin-A in rotenone treated cells compared to bafilomycin-A alone.1.5:Rotenone treated cells showed a significant increase in PI3K (data not shown) and Akt phosphorylation compared to untreated cells. Pre-treatment with gp91 ds did not inhibit Akt phosphorylation mediated by rotenone2:After treatment with 500 nmol/L rotenone for 6 hours:2.1:Prolonged activation of NOX2-activity further increases ROS generation. Inhibition of Nox2-activity by gp91 ds dramatically decreased the rotenone-dependent increase in ROS generation.2.2:We found a significant increase in LC3Ⅱ level, but no changes in LC3Ⅰ level. Inhibition of NOX2-activity significantly rescued the level LC3Ⅱ.2.3:We observed a significant decrease in p62 level upon prolonged NOX2-activation with low dose of rotenone compared to untreated cells, which was almost completely rescued upon NOX2-inhibition2.4:4 hours of post-treatment with bafilomycin-A in rotenone-treated cells showed a decrease in LC3Ⅱ and p62 levels compared to bafilomycin-A alone treated cellConclusions:1. Mild activation of NOX2 impairs autophagic flux in a PI3K-Akt-mTORC1 dependent pathway.2. Chronic exposure increases NOX2-dependent ROS generation and increases autophagic flux...