Anticancer Effect Of Plumbagin On Human Gastric Cancer Cells Through Inhibiting Nf-κB Signaling Pathway

Aim:To investigate the effects of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on proliferation, apoptosis, migration and invasion of human gastric cancer SGC-7901 cells as well as the underlying mechanisms.Methods:Human gastric cancer cell lines SGC-7901 was used. Cell viability was examined using MTT viability assay. Cell morphology was observed by DAPI staining and optical microscopy. Apoptosis was further detected via Annexin V/PI double-labeled flow cytometry. The effects of combination of plumbagin with the chemotherapeutic agent on cell viability were assayed by CCK-8. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. The cell cycle progression was detected by PI-labeled flow cytometry. Wound healing and Boyden chamber assays were used to explore the migration and invasion abilities of cell. The location of NF-κB p65 was detected by confocal microscopy. Western blotting was used to assess the expression of Bax, Bcl-2, caspase3, NF-KB-regulated gene products and TNF-a-induced activation of p65, IκBα, and IKK.Results:We demonstrated that plumbagin concentration-dependently caused cellular apoptosis. The IC50 value of plumbagin in gastric cancer cells was 19.12μmol/L. With the increased concentration of plumbagin, more cells showed typical apoptotic morphological changes. The compound (0-20μmol/L) concentration-dependently induced apoptosis rate of SGC-7901 cells was 1.77%±0.31,8.00%±1.67,30.57%±1.25,35.33%±1.31, respectively. The results showed an increase in the cytotoxic effect induced by TNF-a and cisplatin in the presence of plumbagin. After being treated by plumbagin, the number of EdU+cells and cell clone formation were dramatic decline. Treatment with plumbagin increased the percentage of cells in S and G2/M phase in a dose-dependent manner, as compared to control. Plumbagin caused inhibition of cell growth by inducing cancer cells to undergo S-G2/M phase arrest in gastric cancer cells. The concentration 0-20μmol/L of plumbagin had showed an inhibitory role on the migration and invasion abilities. Plumbagin inhibited TNF-a-induced nuclear translocation of NF-κB p65. Meanwhile, plumbagin up-regulated the expression of Bax, cleaved-caspase3, and also downregulated the expression of pro-caspase3, NF-KB-regulated gene products involving in antiapoptosis (IAP1, XIAP, Bcl-2, and Bcl-xL), invasion (TF), and angiogenesis (VEGF). In addition to inhibition of NF-κB p65 nuclear translocation, plumbagin also suppressed TNF-a-induced phosphorylation of p65 and IKK, and degradation of IκBα.Conclusion:Here we presented that plumbagin caused apoptosis in gastric cancer cells, induced the S-G2/M phase arrest, and inhibited cell proliferation, migration and invasion abilities in cells in vitro. Plumbagin also potentiated the apoptotic effects of TNF-a and cisplatin. Meanwhile, plumbagin downregulated the expression of NF-κB-regulated gene products and the activation of NF-κB, which plays a key role in the anticancer activity of plumbagin.