Mechanistic Insights on Cytotoxicity of KOLR by targeting Signaling Complexes of phosphodiesterase 3B and Rap guanine nucleotide exchange factor 3

Abstract Background Protein signaling complexes play important roles in prevention of several cancer types and can be used for development of targeted therapy. The roles of signaling complexes of phosphodiesterase 3B (PDE3B) and Rap guanine nucleotide exchange factor 3 (RAPGEF3), which are two important enzymes of cyclic adenosine monophosphate (cAMP) metabolism, in cancer have not been fully explored. Methods The natural product Kaempferol-3-O-(3′′,4′′-di-E-p-coumaroyl)-α-L-rhamnopyranoside designated as KOLR was extracted from Cinnamomum pauciflorum Nees leaves using reverse phase chromatography, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. The antitumor effect of KOLR was analyzed by multiple cell proliferation and metastasis experiments. The PDE3B/RAPGEF3 complex was found to be the target of KOLR through mRNA sequencing, Co-Immunoprecipitation assay, gene knock-down, gene mutation of drug-resistance cell line, and molecular docking. In vivo studies have shown that KOLR has the same antitumor mechanism. Results KOLR exhibited cytotoxic effects against selected cancer cells, except for AsPC-1 pancreatic cancer cell line. KOLR stabilized PDE3B/RAPGEF3 signaling complex thus inhibiting AKT phosphorylation and Rap-1 activation. Notably, mutation of RAPGEF3 G557A inhibited effect of KOLR on stabilizing PDE3B/RAPGEF3 complex in AsPC-1 cells. Furthermore, downregulation of PDE3B expression inhibited cytotoxic effect of KOLR on tumor cells. Downregulation of RAPGEF3 and Rap-1 expression promoted apoptosis of tumor cells and inhibited tumor metastasis. PDE3B inhibits activity of RAPGEF3 and activation of downstream signaling pathway. Conclusion The findings of this study show that KOLR could stabilize PDE3B/RAPGEF3 signaling complex to play an anti-tumor role and the PDE3B/RAPGEF3 complex is a potential therapeutic cancer target.