Figure 6.

Sorafenib but not CCCP induces Parkin-dependent suppression of Mcl-1 and activates apoptotic initiator caspases. A and B, HeLa Parkin–WT or Parkin–T240R cells were exposed to 20 μm sorafenib (A) or 10 μm CCCP in a 6-h time course. Sorafenib induces robust PARP cleavage and activation of initiator caspase-3 characterized by reduction of the pro-caspase signal. The effect of CCCP on PARP or pro-caspase-3 cleavage is modest. The effect of sorafenib was not observed in the mutant Parkin cell line. C and D, effect of sorafenib on anti-apoptotic Bcl-2 family of proteins was monitored by immunoblotting (IB). Sorafenib causes a depletion of Mcl-1 but not Bcl-xL or Bcl-2 in a Parkin-dependent manner. E, sorafenib and CCCP have similar effects on PINK1 stabilization and Parkin activation but differ in Mcl-1 suppression. F, suppression of Mcl-1 by sorafenib can be reversed by proteasome inhibitor MG132. G, immunoblotting showed the suppression of endogenous Mcl-1 expression by Mcl-1 shRNA. HeLa cells expressing Venus–Parkin and RFP–Smac were infected with shRNA for Mcl-1. H, CCCP induces apoptosis in HeLa cells with reduced Mcl-1 expression. Sorafenib induced stronger apoptosis in Mcl-1 knockdown cells. Apoptotic cell death was quantified by counting cells positive for RFP–Smac release. I and J, overexpression of CFP-tagged mouse Bcl-2 in HeLa Parkin cells suppresses sorafenib induced apoptosis. Immunoblotting confirms the ectopic expression of CFP-mBcl-2. Quantification of apoptotic percentages of HeLa Parkin cells and HeLa Parkin mBcl-2 cells upon incubation with 20 μm sorafenib.