The cytotoxic role of the inhibition of PI‐3‐kinase, and acto‐myosin cross‐bridge formation in human metastatic breast cancer cells.

Abstract

IntroductionExternally added survival/growth factors are essential for the survival of healthy cells, however cancer cells are believed to be self‐sufficient in supporting their auto‐proliferation without the need of exogenously added growth factors. The Phosphatidyl‐Inositol‐3‐kinase (PI‐3‐kinase) pathway is crucial in cell survival. Also, cancer cells exhibit enhanced potential to metastasize via increased acto‐myosin cross bridges formation which requires increased Ca2+ influx via cell surface voltage‐gated Ca2+ channels leading to activation of myosin light chain kinase (MLCK). The MLCK then phosphorylates myosin and forms acto‐myosin cross‐bridges. The role of MLCK, PI‐3‐kinase, and calcium channel in cancer cell growth has not been extensively tested. In this study, we tested the individual effects of blocking cell survival pathway (PI‐3 kinase), voltage‐gated Ca2+ channels and MLCK on MCF‐7 cell (a human metastatic breast cancer cell) survival using pharmacological inhibitors.HypothesisWe hypothesized that all three drugs would inhibit cell proliferation.AimsOur aim was to firstly test if these drugs had any effect at all on cancer cell growth. Secondly, it was to test each drug at different concentrations and see which concentration had the most effect on cell death.MethodsPI‐3‐kianse inhibitor (Wortmannin); MLCK inhibitor (ML‐7), and lastly, a voltage‐gated calcium channel inhibitor (nifedipine) were used. 1% Bitter melon extract (BME) was also tested as this was found to inhibit MCF‐cell growth in our lab by earlier investigators. Cells were thawed, mixed with a medium that contained all essential nutrients for cell growth (DMEM + fetal bovine serum), and then were added onto 96‐well plates and allowed to grow for 48 hours. We incorporated control plates (no drug), plates with drugs at different concentrations, a plate with the compound the drug was originally dissolved in (DMSO or ethanol), and a plate with 1% BME (bitter melon extract). The plates were analyzed under a microscope and cell viability was measured using MTT assays. Images of each plate were taken and correlated with the MTT assay results. Each experiment was repeated at least 4 times in duplicate or triplicate to plot graphs and draw statistical inferences.Results and DiscussionAll three drugs independently inhibited cell growth in a dose‐dependent manner. The observable differences between the different drugs were the concentrations at which they were effective. The results imply that Ca2+ influx and its downstream pathway leading to acto‐myosin interaction is essential for the MCF‐7 cell attachment to the plate and hence its survival.Support or Funding InformationSummer research stipend to medical students at MUCOMDepartmental faculty research support to faculties at MUCOM