Early upregulation of Bim is important to mediate PMC-A induced apoptosis.

<p>Wild type HCT116 cells were transfected with empty, human Bim gene carrying expression plasmids. Transfected and untransfected cells were treated with or without 50 µM PMC-A and harvested 24 h following treatment and analyzed by flow cytometry. The results from at least 3 independent experiments are shown as means ± SD. <b>**</b>P<0.01, mean value of PMC-A-treated mock-tansfected cells against that of untreated mock-transfected was tested using unpaired student’s t-test. <b>*</b>P<0.05, mean values of PMC-A-treated Bim-transfected cells against that of PMC-A-treated mock-transfected were tested using paired student’s t-test. Apoptotic induction by PMC-A treatment was significantly increased in cells overexpressing Bim compared to the cells that possess endogenous Bim expression. Cells transfected with mock vector or Bim were collected, lysed and immunoblotted with anti-Bim antibody to confirm transfection.</p

Synthesis of PMC analogs.

<p>Synthesis of PMC analogs.</p

Transcriptional downregulation of Bcl-2 is critical to mediate PMC-A induced apoptosis.

<p>Wild type HCT116 cells were treated with indicated concentrations of PMC-A and harvested at 24 h post-exposure, lysed and immunoblotted with anti-Bcl-2 and anti-β-actin antibodies (A). β-actin was used as loading control. Downregulation of cellular Bcl-2 was amplified by increasing PMC-A dose. HCT116 wt cells were treated with indicated PMC-A concentrations, harvested after 24 h for total RNA extraction, cDNA synthesis and qRT-PCR analysis (B). A marginal (∼5-fold) decrease in Bcl-2 transcription was evident upon PMC-A treatment (p<0,01). HCT116 wt cells were transiently transfected with expression plasmids either carrying human Bcl-2 gene or mock vector only (C). Transfected cells were treated with 50 µM PMC-A before collection and flow cytometry analysis. The results from at least 3 independent experiments were shown as means ± SD. Difference of mean values between mock and Bcl-2-transfected cells were tested using paired student’s t-test; <b>**</b>P<0.01. Analysis indicates that ectopic Bcl-2 expression renders the cells more resistant against PMC-A induced apoptosis. Bcl-2 expression statuses of mock- and Bcl-2-transfected cells prior to PMC-A treatment are displayed as immunoblotting results obtained from cells that were harvested, lysed and immunoblotted with anti-Bcl-2 antibody (C). Results from one of three independent experiments are shown in immunoblotting results.</p

Chemical structures of PMC analogs.

<p>Chemical structures of PMC analogs.</p

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.</p

PMC-A is the most cytotoxic compound among PMC analogs.

<p>Viabilities of HCT116 cells were analyzed by MTT assay following 24 h treatment with 100 µM of each PMC analog. Wild-type HCT116 cells were assayed colorimetrically following 24 h treatment with PMC analogs. Average absorption values relative to untreated control are displayed after multiplication with 100. The results from at least 3 independent experiments were shown as means ± SD. Difference of mean values between treated and untreated samples were tested using unpaired student’s t-test; <b>**</b>P<0.01. Control cells were treated with solvent only. All tested analogs except the precursor PMC led to significant loss of viability/proliferation while PMC-A and -F proved most cytotoxic to cells.</p