Mechanism Of Gamabufotalin Suppressing COX-2 Expression In Lung Cancer Cells

Objective Gamabufotalin(CS-6), a major bufadienolide of Chansu, has been used for cancer therapy due to its desirable metabolic stability and less adverse effect. However, the underlying mechanism of CS-6 involved in anti-tumor activity remains poorly understood. In this study, we aim to study the cytotoxic effect of CS-6 on human lung cancer A549 cells and then to investigate the possible mechanism on inhibiting tumor growth.Methods In the present study, human lung cancer A549, H1299 and H322 cells were treated by different concentrations of gamabufotalin(CS-6), and then the cell viabilities were detected by MTT assay. At the same time, the cell viability of human normal lung cell line HLF cells was also evaluated at the same dose. The biological functions of CS-6 were investigated by clonogenic cell survival,wound-healing and apoptosis assays in NSCLC cells. In mechanism investigation, the nuclear localization and interaction between transcriptional co-activator p300 and NF-κB p50/p65 and their binding to COX-2 promoter were analyzed by confocal immunofluorescence, DNA-protein binding by streptavidin-agarose pulldown assay and chromatin immunoprecipitationassays, after treatment with CS-6. In order to find the target for CS-6, molecular docking study was used to simulate the interaction of CS-6 with IKKβ. The in vivo anti-tumor efficacy of CS-6 was also analyzed in xenografts nude mice. Western blot was used to detect the protein expression level.Results Gamabufotalin(CS-6) strongly suppressed COX-2 expression by inhibiting the phosphorylation of IKKβ via targeting the ATP-bindingsite, thereby abrogating NF-κB binding and p300 recruitment to COX-2 promoter, then affecting transcription and DNA replication. In addition, CS-6 induced apoptosis by activating the cytochrome c and caspase-dependent apoptotic pathway. Moreover, CS-6 markedly down-regulated the protein levels of COX-2 and phosphorylated p65 NF-κB in tumor tissues of the xenograft mice, and inhibited tumor weight and size.Conclusions Our study found that gamabufotalin(CS-6) showed significantly inhibition on COX-2 expression, and the efficacy may be through targeting IKKβ/NF-κB signaling pathway. The present work provides pharmacological evidence that CS-6 exhibits potential use and may be developed to be a new drug in the treatment of COX-2-mediated diseases such as lung cancer.