Silibinin-induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells

We reported previously that higher doses (150-250 μM) of silibinin enhanced fission and inhibited fusion of mitochondria, accompanying Apoptosis of double-positive breast Cancer cell line MCF-7 cells and triple-negative breast Cancer cell line MDA-MB-231 cells. We report here three important questions yet unclarified in the previous study; 1) Whether enhanced fission of mitochondria by the treatment of silibinin leads to Mitophagy, 2) Whether Mitophagy positively contributes to Apoptosis and 3) Whether estrogen receptor-positive (ER⁺) MCF-7 cells and estrogen receptor-negative (ER^-) MDA-MB-231 cells are affected in a different way by silibinin treatment, since silibinin often works through ERs signaling pathway. Mitophagy driven by Pink1/Parkin signaling, plays an important role in eliminating damaged mitochondria. Indeed, increased expression of Pink1 and the recruitment of Parkin and LC3-II to mitochondria by the treatment with silibinin account for silibinin induction of Mitophagy. In this study, the effects of mitochondrial division inhibitor 1 (mdivi-1) and small interfering RNA targeting dynamin-related protein 1 (DRP1) were examined to reveal the effect of mitochondrial fission on Mitophagy. As expected, mdivi-1 or siRNA targeting DRP1 reversed silibinin-induced mitochondrial fission due to down-regulation in the expression of DRP1. Inhibition of mitochondrial fission by mdivi-1 prevented induction of Mitophagy as well as Autophagy in both MCF-7 and MDA-MB-231 cells, indicating that silibinin-induced mitochondrial fission leads to Mitophagy. Inhibition of mitochondrial fission efficiently prevented silibinin-induced Apoptosis in MCF-7 and MDA-MB-231 cells in our previous work, and the second point of the present study, inhibition of Mitophagy by Pink1 or Parkin knockdown increased silibinin-induced Apoptosis of these cells, respectively, suggesting that the Mitophagy induced by silibinin treatment serves as a cytoprotective effect, resulting in reduction of Apoptosis of Cancer cells in both cells. In the third point, we studied whether estrogen receptors (ERs) played a role in silibinin-induced Mitophagy and Apoptosis in MCF-7 and MDA-MB-231 cells. ERα and ERβ are not involved in silibinin-induced mitophagic process in MCF-7 and MDA-MB-231 cells. These findings demonstrated that silibinin induced mitochondria fission leads to Mitophagy, which attenuates silibinin-induced Apoptosis not through ERs-Pink1 or -Parkin pathway in MCF-7 and MDA-MB-231.