Figure 1: BITC inhibits breast tumor growth in nude mice.

Human phospho-antibody array analyses reveal BITC-induced increased phosphorylation of p53 and ERK and BITC induces p53-phosphorylation in an ERK-dependent manner. (A) MCF7 cells derived tumors were developed in nude mice and treated with vehicle or BITC. Tumor growth was monitored by measuring the tumor volume for 6 weeks. (n = 8 mice/group); (P < 0.001). (B) Tumors from vehicle (V) and BITC-treated mice were subjected to immunohistochemical (IHC) analysis using Ki67 antibodies. Bar diagrams show quantitation of IHC-analysis. Columns, mean (n = 8); bar, SD. *significantly different (P < 0.005) compared with control. (C) TUNEL-positive cells in tumor sections were counted. Each bar represents the mean (n = 6–8). *P < 0.01, compared with controls. (D,E) MCF7 breast cancer cells were treated with 2.5 μM BITC and subjected to Human phospho-antibody array analyses. Relative levels of protein phosphorylation (normalized intensity for each antibody) were calculated for each untreated and treated sample. *P < 0.001, compared with controls. (F) Immunoblot analysis of phosphorylated-p53-Ser15 (p-p53), total p53, p21 and BAX in breast cancer cells treated with 2.5 μM BITC as indicated. (G) Breast cancer cells were treated with 2.5 μM BITC and subjected to immunofluorescence analysis of p53. (H) Breast cancer cells were treated with 2.5 μM BITC for various time-intervals as indicated followed by nuclear-cytoplasmic fractionation. Nuclear and cytoplasmic lysates were examined for p53. Lamin B and actin were included as controls. (I) Breast cancer cells were treated as in F, total lysates were immunoblotted for pERK and total ERK expression. (J) MCF7 cells were pretreated with 10 μM U0126 for 2 hours followed by treatment with 2.5 μM BITC. Total lysates were immunoblotted for pERK, total ERK, p-p53-Ser15 and total p53 expression. (K) MCF7 cells were transiently transfected with siERK-siRNAs for 48 h and subjected to colony-formation assay in the presence or absence of 2.5 μM BITC. Cells overexpressing ERK-CA are included as ‘gain-of-function’ controls. Histogram represents average number of colonies counted (in six micro-fields). *P < 0.001, compared with vehicle controls (C); **P < 0.005, compared with BITC-treated cells; ***P < 0.05, compared with BITC + ERKsi cells.