Background: Resveratrol is known as a natural phytoalexin found in grapes and wine, which has significant antitumor activity under in vitro and in vivo conditions. In recent years, great progress has been made in understanding the underlying mechanisms of resveratrol in inducing cellular apoptosis of melanoma cells. Our previous study has shown that the apoptosis regulation of resveratrol in melanoma cells was independent of activation of classical apoptosis-related protein p53.
Materials and methods: MTT assay and 5-bromo-2'-deoxyuridine staining assay were used to analyze cell viability and proliferation. Immunofluorescence analysis of γ-H2AX was employed to clarify DNA damages. Annexin V–propidine iodide/fluorescein isothiocyanate assay was performed to evaluate the cell apoptosis. The mechanisms underlying the activation of M2-type pyruvate kinase (PKM2) by Erk1/2 to stabilize and maintain Bcl-2 signaling was investigated by subcellular fractionation analyses, immunofluorescence analysis, co-immunoprecipitation assay, ubiquitination assay, and glutathione S-transferase pull-down assay.
Results: In the present study, we found that resveratrol dramatically inhibited melanoma cell proliferation and induced cell apoptosis through upregulation of p53 in a concentration-dependent manner. Conversely, p53 downregulation by short hairpin RNA couldn’t rescue resveratrol-induced cell proliferation inhibition or apoptosis enlargement. Additionally, we found that resveratrol downregulated antiapoptotic protein Bcl-2 and activated Bax in the protein levels by promoting Bcl-2 degradation and cytochrome c release. Moreover, we discovered that PKM2, had a key role in cell apoptosis triggered by resveratrol through interacting with Bcl-2. Based on these results, we overexpressed PKM2 in melanoma cells and found that this prevented resveratrol-induced apoptosis by stabilizing the protein level of Bcl-2.
Conclusion: Taken together, our results provided a novel mechanism accounting for the apoptosis induction of resveratrol in melanoma cells and suggested that downregulating Erk/PKM2/Bcl-2 axis appears to be a new approach for the prevention or treatment of melanoma.
Keywords: antitumor, ubiquitination, cytochrome c, ER stress