The Antitumor Effect And Mechanism Of Marine Sponge-derived Stellettin B

Objective:This study aims to explore the anti-tumor effects and molecular mechanisms of Stel B on human non-small cell lung cancer A549 cells and investigate the autophagy induced by Stel B on human glioblastoma cancer SF295 cells based on the previous research, which provide a preliminary theory evidence and experimental foundation for the development of these compounds as anti-tumor drugs.Methods:1. WST-8 assay was used to determine the inhibitory effect of Stel B on the proliferation of A549 cells and SF295 cells.2. The effect of Stel B on cell cycle distribution was analyzed by flow cytometer;Western blot was used to analyze the expression or phosphorylation level of cell cycle-related proteins such as cyclin D1, Rb and p27.3. To investigate whether Stel B induced apoptosis in A549 cells, flow cytometry and DAPI stainingwere carried out;we examined the effect of Stel B on expression level of cleaved PARP in A549 cells by Western blot and the changes in ROS level were detected by using flow cytometry.4. To examine whether Stel B induced autophagy in A549 cells, plenty of analytical methods such as MDC staining, transmission electron microscopy, Western blot assay and m RFP-GFP-LC3 plasmid transfection method were employed.5. We examined the activity of Stel B on the representative signal proteins in PI3K/Akt pathway in A549 cells and SF295 cells by Western blot.6. WST-8 assay was used to detect anti-tumor activity of Stel B after blocking autophagy by autophagy inhibitor chloroquine.Results:1. Stel B inhibited proliferation of A549 cells in a dose-dependent manner, with IC50 value as 0.022 ?M.2. Stel B induced cell cycle arrest at G1 phase in A549 cells. Treatment by Stel B caused reduction in expression of cyclin D1 and phosphorylation of p Rb, and enhancement in p27 expression.3. Stel B promoted ROS generationand induced late-stage apoptosis in A549 cells,cytoplasmic shrinkage and nuclear fragmentation were observed under inverted phase contrast microscope after treatment with Stel B in A549 cells. The apoptosis level increased in a concentration-dependent manner following Stel B treatment; the amount of the cleaved PARP was increased in Stel B treated cells.4. MDC staining showed that the amount of the bright green fluorescence near the nuclearwas increased in Stel B treated A549 cells.; the expression of autophagy marker proteins including LC3 BII increased; As shown in the electron micrographs, a plenty of autophagic vacuoles which contain subcellular materials,were observed in Stel B-treated cells; Confocal microscopy showed that after treatment by Stel B, from the process of forming autophagosome to the degradation of lysosome was smooth.The results proved that Stel B chould induce autophagy in A549 cells.5. Stel B inhibited proliferation of SF295 cells in a dose-dependent manner, with IC50 value as 0.026 ?M. The results of MDC staining, Western-Blotandlaser confocal microscope showed that Stel B could induce autophagy in SF295 cells(The effects of Stel B on cell cycle and apoptosisin SF295 cells were reported before).6. After treatment by Stel B, the expression level of PI3K-p110 and phosphorylation of Akt, m TOR and p70S6 K were downregulated in A549 cells and the expression level of PI3K-p110 and phosphorylation of Akt were also blocked in SF295 cells.7. After blocking autophagy by autophagy inhibitor CQ, the inhibition of Stel B on A549 cells and SF295 cells increased.Conclusions:1. Stel B showed potent activities on A549 cells and SF295 cells at low n M doses,which showed potent antitumor effects in vitro.2. Stel B treatment induced G1 arrest, apoptosis and autophagy in A549 cells. The G1 arrest induced by Stel B might be attributed to the expression of cyclin D1 and phosphorylation of p RB decreased and the expression of p27 increased.Promotion of ROS generation and induce the PARP cleaved by Stel B lead to apoptosis cell death in A549 cells. Studies on molecular mechanisms elucidated that Stel Binduced G1 arrest and apoptosis in A549 cells via blocking PI3K/Akt Pathway.3. Stel B induces autophagy in SF295 cells, and the degree of autophagy was higher in A549 cells than in SF295 cells. After blocking autophagy by autophagy inhibitor CQ, the inhibition of Stel B in A549 cells and SF295 cells increased,which indicate that Stel B-induced autophagy had protective effects on A549 cells and SF295 cells.4. Similarly, Stel B induced autophagy via blocking PI3K/Akt Pathway. And the relationship among its effects on cell cycle arrest, apoptosis, and autophagy is expecting for further research.