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Activation of ERK1/2 is required for the antimitotic activity of the resveratrol analogue 3,4,5,4′‐tetramethoxystilbene (DMU‐212) in human melanoma cells

Background Human melanoma is a malignant tumor that originates from abnormal growth of melanocytes. It is the most lethal form of skin cancer and its incidence is rising rapidly worldwide. In Europe, approximately 100 000 new cases of melanoma skin cancer will develop every year, whereas approximately 20 000 cases lead to fatalities (1). Due to the increasing incidence of melanoma worldwide, the development of novel non-cytotoxic chemotherapeutic agents with improved efficacy is imperative (2,3). Resveratrol (Fig. S1) is a naturally occurring phytoalexin found in grapes, cranberries and peanuts generated in response to pathogenic attack that possesses a wide diversity of anticancer effects such as inhibition of kinase enzyme activity and induction of apoptosis (4–8). DMU-212 or 3,4,5,40-tetramethoxystilbene (Fig. S1) is a resveratrol analogue containing methoxy groups in place of the hydroxyl groups of the stilbene moiety that has shown promising metabolic stability and bioavailability in in vivo studies, compared to resveratrol as well as higher potency than resveratrol in inhibiting proliferation of cancer cells in vitro (9,10). In our previous study, we demonstrated the ability of DMU-212 to inhibit proliferation of MCF-7 breast adenocarcinoma, and HepG2 liver hepatoma cells at submicromolar toxicities (11). Question addressed In the present study, we sought to investigate the antiproliferative effect and further mechanism of action of DMU-212 in human melanoma cells. Experimental design The effects of DMU-212 in A375, MeWo, M5 and Bro cells were investigated by cell viability studies, cell cycle analysis, Western blotting and confocal immunofluorescence. Cell viability was estimated using MTT, and cell cycle analysis was performed using flow cytometry with propidium iodide staining. Western blotting was employed to investigate the expression levels of p21, p53, cyclin B1, cyclin A2, chk2, p-chk2, ERK1/2, p-ERK1/2, Bax, Bcl-2, caspase 3 and 9 proteins, whereas confocal microscopy was used for staining of the mitotic spindle and detection of p-ERK (Full methods in data S1). Results The results of our investigation indicated inhibition of cellular proliferation of A375, MeWo, M5 and Bro human melanoma cells at submicromolar or micromolar concentrations (IC50 = 0.5 lM for A375 and Bro and IC50 = 1.25 lM for MeWo and M5 cells) occurring after treatment with DMU-212 for 96 h (Fig. 1a, Fig. S2). DMU-212 was superior to resveratrol (IC50 = 30 lM) in inhibiting cell growth of human melanoma cells (Fig. 1a, Fig. S2). Cell cycle analysis demonstrated that A375 cells pretreated with 24 h with DMU-212 were arrested at the G2/M phase of the cell cycle (85 3.5% vs 20 2.5% for control), while a subG1 peak corresponding to 4.5 0.4% of cells (vs 0.5 0.1% for control) was observed at 20-lM treatment of DMU-212 which is consistent with late apoptosis (Fig. 1b, Fig. S3). Similar results were obtained for MeWo and M5 melanoma cells (Fig. S4). Nuclear staining with TOPRO-3 revealed a large increase in mitotic index (22 2.5% vs 3.7 0.4% for control) after incubation of A375 cells with DMU212 for 24 h that was similar in ratio with the G2/M blockage noted in the flow cytometry experiments, whereas visualization of A375 nuclei indicated blockage of the cells mitotic division at the prometaphase stage (Fig. 1e, c). Staining with b-tubulin demonstrated