Differential modulation of Bax/Bcl-2 ratio and onset of caspase-3/7 activation induced by derivatives of Justicidin B in human melanoma cells A375.

Abstract

Diphyllin and its derivatives are well known cytotoxic natural products structurally related to the anti-cancer drug podophyllotoxin. We here study their structure-activity relationship upon human melanoma cells for first time. To this end, human melanoma A375 cells were incubated with Justicidin B and its 4-methoxylated or 4-glycosylated derivatives to evaluate their selective cytotoxicity and study their effects on cell cycle distribution, caspase activation, apoptosis induction using Annexin V-FITC/PI staining, cell morphology and western blot analysis. Diphyllin methyl ether (GI50 = 3.66 μM) and Justicidin B (GI50 = 1.70 μM) caused an elevation of both early and late apoptosis populations whereas Diphyllin apioside (GI50 = 0.84 μM) and its acetate (GI50= 0.39 μM) enhanced late apoptosis population only (Annexin V-positive/PI-positive). All induced cell cycle arrest at S phase and classic morphological indicators of apoptosis (blebbing, apoptotic bodies, and nuclear fragmentation) accompanied with an elevation of cells with low DNA content (sub-G1). All compounds increased the Bax/Bcl-2 ratio by enhancing Bax expression which evidences the involvement of the mitochondria (intrinsic pathway) in the apoptotic process. These caspase-3/7 results evidence that 4-methoxylation or 4-O-glycosylation of Justicidin B -a caspase independent mitochondrial apoptosis-inducer- triggers caspase-3/7 activation at different times (24h vs. 48h, respectively). Interestingly, the methoxylation causes attenuation of Bcl-2 protein expression contrarily to Diphyllin methyl ether or the O-glycosylated derivatives. Finally, the compounds exhibited significantly less toxicity when tested in adult human dermal fibroblasts and their GI50 in melanoma Sk-Mel-5 cells was not influenced by MDR1/Pgp inhibitors. This study may inform the synthesis of future Diphyllin derivatives with different apoptosis mechanism of action towards human melanoma cells.

• Authors: Aljawharah Al-Qathama, Simon Gibbons, Jose M Prieto
• Journal: Oncotarget (Oncotarget) Vol. 8 Issue 56 Pg. 95999-96012 (Nov 10 2017) ISSN: 1949-2553 [Electronic] United States
• PMID: 29221182 (Publication Type: Journal Article)