The cytotoxic role of the inhibition of different steps in the PI-3-kinase pathway and acto-myosin cross bridge formation in human metastatic breast cancer cells
Bhupal P. Bhetwal Ph.D
Medicine and Health Sciences
Introduction: Externally 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 Phospha-tidyl-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 human metastatic breast cancer cell line (MCF-7) survival. Hypothesis: We hypothesized that all three drugs would inhibit cell proliferation. Aims: Our 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. Methods: PI-3-kianse inhibitor (Wortmannin); MLCK inhibitor (ML-7), and lastly, a 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. Results and Discussion: All three drugs inhibited cell growth in a dose-dependent manner. The observable differences between the different drugs were the concentrations at which they were effective. Images of each plate were taken and correlated with the MTT assay graphs. Acknowledgement: The research was partially supported by the Research and Honors and Awards Committees at MU-COM
Copyright 2019 all authors
Thirumeni, Rajashree OMS-2 and Bhetwal, Bhupal P. Ph.D, "The cytotoxic role of the inhibition of different steps in the PI-3-kinase pathway and acto-myosin cross bridge formation in human metastatic breast cancer cells" (2019). MU-COM Research Day. 163.