Introduction: Withaferin A (WA) is a natural product withanolide compound with historical anti‐inflammatory activity and recent anticancer activity in vitro. The purpose of this study was to evaluate the mechanism of action of withaferin A in melanoma cancer cells and test its efficacy in a murine orthotopic model of metastatic melanoma.

Methods: Human melanoma cells lines SKMEL28, NPA, DRO and the murine B16F10 cell line were used for in vitro analysis. MTS cell viability assay and cell proliferation analysis was performed to determine the concentration‐dependent effect of withaferin A on melanoma cells. Effects on cell cycle arrest and apoptosis were evaluated by PI/Annexin V staining with flow cytometry and confirmed through Western analysis. MAPkinase and heat shock protein expression after WA treatment was evaluated by Western analysis. Inhibitors of MEK1/2, JNK and p38 MAP kinase were used to examine the relationship between WA‐mediated MAPkinase protein expression and modulation of heat shock proteins. In vivo analysis was performed on BalbC mice injected s.c. with 5 million B16F10 melanoma cells, allowing tumors to grow and metastasize. Treatment was with 5mg/kg/d WA i.p. for 3 weeks. Tumor growth and survival was followed prospectively. In vitro and in vivo variables were statistically evaluated using SPSS 17.0 software and significance was defined for p<0.05.

Results: Withaferin A induces reduction of cell viability and cell proliferation in all melanoma cell lines tested [Range of IC50(B16F10 =209.7±16nM); (SKMEL‐28=650.1 ±25nM)]. Mechanistic studies demonstrate that WA induced a cell cycle shift from G0/G1 arrest to G2/M arrest in all melanoma cells lines tested. Annexin V /PI staining on FC as well as caspase activation and PARP cleavage on Western studies confirm that WA induces apoptosis in melanoma cells. WA reduced p‐Akt levels and total Akt levels in all cell lines tested in a concentration and time‐dependent manner. Withaferin A modulates MAP kinase proteins, inducing activation of p‐ERK1/2 , p38 MAPkinase and SAPK/JNK in all melanoma cell lines. HSP expression analysis yielded strong induction of HSP72 expression in all cell lines, reduction of HSP90 in SKMEL28 cells, and strong reduction of HSF1 cellular levels in a dose dependant manner with almost complete reduction at IC50 concentrations. Confocal microscopy analysis showed that withaferin A‐induced expression of HSP72 was increased in the cytoplasm and that HSP72 was transported to the nucleus upon withaferin A stimulation. In vivo studies demonstrate almost complete tumor regression with CR in 80% of animals treated with 5 mg/kg/day WA compared to controls (p<0.01). Additionally at 5/mg/kg dosing, there was complete reversal of organ and lymphatic metastatic disease by inspection after 3 weeks treatment in the 80% of animals responding without progression once the treatment was discontinued.

Conclusion: Our present results show that withaferin A is a potent therapy against melanoma cells in vitro and in vivo through induction of cell cycle arrest, apoptosis, and modulation of MAPkinase and HSP expression. This is the first report of inhibition of HSF1 expression in cancer cells induced by withaferin A treatment. These results support future proof of concept studies for translation to potential therapies for melanoma patients.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B84.