Fig. 7. Cisplatin-mediated apoptosis is regulated by Itch expression and mitochondrial cholesterol.

A Cell viability assay of the Itch KD cell line in response to Cisplatin treatment for 72 h at 0–6 µg/ml. B Apoptosis assay using the Itch KD cell line treated with 0–6 µg/ml Cisplatin for 72 h. Data was expressed as a percentage of control. C Analysis of apoptotic markers in whole cell lysates from cells treated with Cisplatin in U18666A. D Western blot analysis of total expression levels of STARD1 and VDAC2 (dimers) in control and Itch KD OE33 cell lines. Error bars represent the standard error of the mean. Statistical significance was calculated by Student’s t test. E Quantification of STARD1 band intensity normalised to the loading control in three independent experiments. Statistical analysis was performed using Student’s t test, ***p < 0.001. F Immunoprecipitation of VDAC2 from OE33 detergent extract. The samples were incubated overnight with a control IgG and the VDAC2 antibody and washed four times. Co-immunoprecipitated proteins were separated by SDS-PAGE electrophoresis and STARD1 and Itch were detected by Western blotting. G A schematic image illustrating the suggested model for Itch in regulation of extrinsic and intrinsic apoptosis. A reduction in TRAIL-R at the cell surface impairs downstream apoptotic signalling, which moreover is not further amplified at the mitochondria. Loss of Itch expression results in an increase in mitochondrial cholesterol which impairs membrane fluidity, binding of caspase-8, subsequent activation of Bid and Bax. Together, this results in reduced pore formation in the outer mitochondrial membrane and reduced cytochrome c release. Mechanistically loss of Itch expression results in reduced ubiquitination and degradation of the STARD1/VDAC2 lipid transfer complex that mediates cholesterol import to mitochondria. A stabilisation of STARD1/VDAC2 promotes import of mitochondrial cholesterol which is anti-apoptotic. Statistical significance; *p < 0.05, ***p < 0.001.