Figure 6

Calcarea carbonica triggers T cell-mediated tumor killing via p53-Bax-caspase-3 cascade. (A) EAC, MCF-7 and MDA-MB-231 cells were co-cultured with untreated-/placebo-/calcarea carbonica-primed T cells and subjected to Western blot/RT-PCR analysis to determine the expression profile of p53/Bax/Bcl-2 at protein and Bax/Bcl-2 at mRNA levels (left panels). Right panels represent quantitative data for Western blot. (B) Graphical representation of Bcl-2/Bax protein ratio in tumor cells co-cultured with calcarea carbonica-primed T cells. (C) Wild-type p53-expressing cells were transfected with p53-siRNA (inset) and scored for percent apoptosis when co-cultured with calcarea carbonica-primed T cells. (D) Bax and cytochrome c levels were determined in cytosolic and mitochondrial fractions of tumor cells co-cultured with placebo-/calcarea carbonica-primed T cells by Western blot analysis (left panels). Middle panels represent quantitative data. α-Actin and MnSOD were used as internal protein markers (right panels). (E) Graphical representation of mitochondrial trans-membrane potential of tumor cells co-cultured with calcarea carbonica-primed T cells pre-treated with cyclosporine-A. (F) Expression profiles of pro-/active- forms of caspase-3 in EAC and HBL-100 cells and pro-/active caspase-9 in caspase-3-null MCF-7 cells co-cultured with calcarea carbonica-primed T cells. Right panels represent quantitative data. (G) Expression profiles of active caspase-3 in EAC and caspase-9 in MCF-7 cells co-cultured with calcarea carbonica-primed T cells pre-treated with cyclosporine-A (left panel). Middle panel represent quantitative data. In parallel set, cells were scored for percentage apoptosis (right panel). (H) Percent apoptosis of EAC and HBL-100 cells co-cultured with calcarea carbonica-primed T cells in the presence of caspase-3 inhibitor (Z-DEVD-FMK) or transfected with caspase-3-siRNA and percent apoptosis of MCF-7 cells co-cultured with calcarea carbonica-primed T cells in the presence of caspase-9 inhibitor (Z-LEHD-FMK). Values are mean ±SEM of five independent experiments. *p < 0.05 and **p < 0.001 when compared with respective placebo/drug-treated sets.