PMC-A induces dose-dependent cell death by activating intrinsic apoptotic pathway.

<p>HCT116 wt, p53−/− and Bax −/− cells were collected and analyzed by flow cytometry following 24 h treatment with indicated doses of PMC-A (A). Difference of mean values between treated and untreated cells of the same cell line were tested using unpaired student’s t-test. PMC-A caused significant dose-dependent cell death in all cell lines. Mean values of PMC-A-treated p53−/− or Bax −/− cells against those of PMC-A-treated wt cells were tested using paired student’s t-test; <b>*</b>P<0.05, <b>**</b>P<0.01. Lack of Bax expression significantly protected the cells from PMC-A induced cell death at doses of 50, 75, and 100 µM. Wild type and p53−/− HCT116 cells were treated with 50 µM PMC-A and were harvested at indicated time points, lysed and immunoblotted with anti-cleaved caspase-3, anti-caspase-9, and anti-β-actin antibodies (B). Mitochondria mediated (intrinsic) apoptotic pathway was activated as shown by cleavage of caspase-9 and caspase-3 in both cell types following the treatment. HCT116 wt cells that were pretreated with specific caspase inhibitors Z-LEHD-FMK, Z-DEVD-FMK, or Z-VAD-FMK were collected and analyzed by flow cytometry following 24 h 50 µM PMC-A treatment as well as unpretreated/untreated cells (C). Difference of mean values between pretreated and unpretreated cells were tested using unpaired student’s t-test; <b>**</b>P<0.01. Pharmacological inhibition of caspase-9, -3 and all caspases at once resulted in protection from PMC-A induced cell death. The results from at least 3 independent experiments were shown as means ± SD for Panel A and C. Western results from one of three independent experiments are shown. β-actin was used as loading control. Control cells were treated with solvent only.