Sunitinib Combined with Th1 Cytokines Potentiates Apoptosis in Human Breast Cancer Cells and Suppresses Tumor Growth in a Murine Model of HER-2^pos Breast Cancer

Figure 4

Sunitinib and Th1 cytokine treatment maximizes cytochrome C release from mitochondria and degradation of PARP. SKBR-3 cells were seeded at per mL and were left untreated (No Rx) or treated with Th1 cytokines (5 ng/mL each), sunitinib (10 μM), or sunitinib and Th1 cytokines (both) for 72 hours. (a) Cells were harvested, permeabilized, and stained first with anti-cytochrome C antibodies, followed by an APC-labeled secondary antibody. Cells were then analyzed by flow cytometry using a 642 nm excitation laser. Histograms display a single representative experiment of cytochrome C expression in SKBR-3 cell lines out of four total experiments with similar results. (b) Treated cells were harvested and extracted in RIPA buffer in the presence of protease and phosphatase inhibitors. Protein lysate (30 μg/well) was separated on a 4-15% SDS gel and electrotransferred onto nitrocellulose membranes. Blots were then probed with anti-PARP (116 kDa) and anti-actin (loading control; 42 kDa) and bands visualized using chemiluminescence substrate with the Fuji LAS 3000 detection system. Illustrated blot representative of 3 separate experiments with similar results.

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