Oncotarget

Research Papers:

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

Aljawharah Al-Qathama _, Simon Gibbons and Jose M. Prieto

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Oncotarget. 2017; 8:95999-96012. https://doi.org/10.18632/oncotarget.21625

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Abstract

Aljawharah Al-Qathama1,2, Simon Gibbons1 and Jose M. Prieto1

1Centre for Pharmacognosy and Phytotherapy, University College London School of Pharmacy, London WC1N 1AX, United Kingdom

2Department of Pharmacognosy, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia

Correspondence to:

Aljawharah Al-Qathama, email: aljawharah.alqathama.10@ucl.ac.uk

Simon Gibbons, email: simon.gibbons@ucl.ac.uk

Jose M. Prieto, email: j.prieto@ucl.ac.uk

Keywords: apoptosis; caspases; Diphyllin; melanoma; cell cycle

Received: February 21, 2017     Accepted: August 26, 2017     Published: October 06, 2017

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.


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