p53-p66shc/miR-21-Sod2 signaling is critical for the inhibitory effect of betulinic acid on hepatocellular carcinoma

• BA inhibited tumor growth and induced apoptosis in mice treated by DEN plus CCl4.
• BA inhibited proliferation and induced apoptosis in hepatocellular carcinoma cells.
• Increase of p53 was responsible for the pro-apoptotic and anti-tumor effect of BA.
• Increase of p66shc and miR-21 and reduction of Sod2 was involved in anti-tumor effect of BA.
• p53-p66shc/miR-21-Sod2 signaling is critical for the anti-tumor effect of BA.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third cause of cancer-related death. Betulinic acid (BA) is a pentacyclic triterpene, possessing potential pro-apoptotic activities. The present study was designed to evaluate the effect of BA on tumor growth in mice and HCC cell proliferation in vitro. We found that BA dose-dependently inhibited tumor growth in mice induced by DEN plus CCl4 (D/C) and suppressed cell viability and proliferation in several HCC cell lines. In addition, BA increased mitochondrial ROS generation and mitochondrial dysfunction, activating molecular apoptotic events and leading to apoptotic cell death. p53 was increased by BA in D/C-treated mice and HCC cells and inhibition of p53 significantly suppressed the pro-apoptotic and anti-tumor effect of BA. Increase of p66shc was involved in the pro-apoptotic and anti-tumor effect of BA. Moreover, BA-induced increase of p66shc was dependent upon p53. Sod2 expression was reduced by BA treatment, and a Sod2 mimic (MnTBAP) significantly blocked the pro-apoptotic and anti-tumor effect of BA. Furthermore, miR-21 was increased by BA in D/C-treated mice and HCC cells and inhibition of miR-21 significantly suppressed the pro-apoptotic effect of BA. miR-21 inhibitor attenuated BA-induced decrease of Sod2 and p53 inhibitor blocked BA-induced increase of miR-21. These results demonstrated that p53 is responsible for the anti-tumor effect of BA through up-regulation of p66shc and miR-21 and down-regulation of Sod2 expression, leading to mitochondrial ROS accumulation and apoptosis. The p53-p66shc/miR-21-Sod2 signaling is critical for BA-inhibited tumor growth and cancer cell proliferation.

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