Discovery of a novel protein kinase C activator from Croton tiglium for inhibition of non-small cell lung cancer

Abstract

BackgroundThe incidence of non-small cell lung cancer (NSCLC) accounts for approximately 85–90% of lung cancer, which has been shown to be challenging for treatment owing to poorly understanding of pathological mechanisms. Natural products serve as a source of almost all pharmaceutical preparations or offer guidance for those chemicals that have entered clinical trials, especially in NSCLC. PurposeWe investigated the effect of B10G5, a natural products isolated from the Croton tiglium, in human non-small cell lung canceras as a protein kinase C (PKC) activator. MethodsThe cell viability assay was evaluated by the MTT assay. The apoptosis and cell cycle distribution were assessed by flow cytometry. Reactive oxygen species (ROS) production was determined by using the fluorescent probe DCFDA. Cell migration ability of H1975 cells was analyzed by using the wound healing assay. The inhibiting effect of B10G5 against the phosphorylation level of the substrate by PKCs was assessed by using homogeneous time-resolved fluorescence (HTRF) technology. The correlation between PKCs and overall survival (OS) of Lung Adenocarcinoma (LUAD) patients was analysis by TCGA portal. The binding mode between B10G5 and the PKC isoforms was explored by molecular docking. Protein expression was detected by western blotting analysis. ResultsB10G5 suppressed cell proliferation and colony formation, as well as migration ability of NSCLC cells, without significant toxic effect on normal lung cells. B10G5 induced the cell apoptosis through the development of PARP cleavage, which is evidenced by means of the production of mitochondrial ROS. In addition, the B10G5 inhibitory effect was also related to the cell cycle arrest at G2/M phase. Mechanistically, molecular modelling technology suggested that the potential target of B10G5 was associated with PKC family. In vitro PKC kinase assay indicated that B10G5 effectively activated the PKC activity. Western blotting data revealed that B10G5 upregulated PKC to activate PKC-mediated RAF/MEK/ERK pathway. ConclusionOur results showed that B10G5, a naturally occurring phorbol ester, considered to be a potential and a valuable therapeutic chemical in the treatment of NSCLC.