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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Cancer Res. 2014 Jun 26;74(17):4822–4835. doi: 10.1158/0008-5472.CAN-14-0584

Figure 6.

Figure 6

Dual-drug combinations of PKC-α, MET, and CDK6 inhibitors synergistically suppressed clonogenic and anchorage-independent growth in vitro and in an animal model.(A) The number of colony-forming cells was determined by clonogenic assay in BT549 and MDA-MB-231cells. Cells were treated with safingol (SA; 0.75 μM), crizotinib (Cr; 0.75 μM), and PD0332991 (PD; 0.75 μM), separately or in combination, for 10 days. (B) The number of colony-forming cells was determined by anchorage-independent growth analysis in MDA-MB-231 and BT549 cells. Cells were treated with safingol (0.5 μM), crizotinib (0.5 μM), and PD0332991 (0.2 μM), separately or in combination, for 21 days. Representative images from the clonogenic and anchorage-independent growth assays (carried out as described in Methods) are shown. The data on the right are means with standard deviations for three independent experiments. *, p < 0.05 for differences between combination and single-drug treatments.(C) Representative images of immunohistochemical staining of PKC-α expression in human TNBC tissues (left panels). TNBC tissue array analysis of overall survival in PKC-α-overexpressing patients. To ensure objectivity, the ACIS III automated cellular imaging system (Dako) was used for tissue scoring and quantification. All correlations between kinase expression levels were analyzed using SPSS version 15.0 statistical software (right panel). The overall survival durations after surgery were analyzed using a Kaplan-Meier plot. The Cox proportional hazards regression model was used to assess prognostic factors for overall survival. Cum, cumulative.(D) Seven days after transplantation of MDA-MB-231 cells expressing luciferase and green fluorescent protein, mice were treated with safingol at 20 mg/kg intravenously twice a week and/or crizotinib at 8 mg/kg orally every day. Tumor volume (V) was measured on days 8, 12, 15, 19, and 21 and was normalized to the tumor volume in untreated mice (V0). Error bars indicate standard deviations (n = 5). CTL, control; Inh., inhibition. (E) Body weight of mice described in (D). Data represent mean ± SD. n = 5. *p < 0.05 compared with the control group.