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.
Highlights
Justicidin B (Figure 1) is a naturally occurring cytotoxic arylnaphtalene lignan endowed with powerful bioactivities [1]
The inhibitory effects on A375 cell proliferation was assessed by the Sulforhodamine B (SRB) method following cell exposure to several concentrations of Justicidin B and its derivatives at different time points 24, 48 and 72h
The GI50 of Justicidin B and Diphyllin methyl ether were found at the low μM range, whilst their glycosylated derivatives at the nM range
Summary
Justicidin B (Figure 1) is a naturally occurring cytotoxic arylnaphtalene lignan endowed with powerful bioactivities [1]. The cytostatic activities of Diphyllin and some of its derivatives were described in 1979 by González et al [4] who adscribed them to their ability to block the DNA synthesis in both normal and malignant cells pointing to an intercalating action in the minor groove. The authors claimed that Diphyllin itself have no value as anti-cancer drug, first because its negative cytotoxic index -high tocixicity on both cancer and human primary cells. Modern studies pointed that its anti-proliferative action on cancer cells may involve the cell cycle arrest in the S-phase and inhibition of protein synthesis [5] and cytotoxic activity towards human monocytes and skin tissues [6] and that it is effluxed by P-glycoprotein (Pgp) [7], limiting its therapeutic potential. Cleistanthin A (diphyllin O-(3,4-Di-O-methyl-D-xylopyranoside) is reported to be more toxic to cancer cells than to normal ones [8, 9]
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