Dual role played by Ca 2+ in the cytotoxicity of human metastatic breast cancer cells

Abstract

Introduction 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 (VGCC) leading to activation of myosin light chain kinase (MLCK). The MLCK then phosphorylates myosin and forms acto-myosin cross-bridges. The role of MLCK, and calcium channel in cancer cell growth has not been extensively tested. Objective In this study, we tested the individual effects of blocking VGCC and MLCK on MCF-7 cell (human metastatic breast cancer cell) survival. We also treated the cells with high KCl to induce increased Ca2+ influx and test if the effects of KCl will be opposite to that of VGCC blocker. Methods The experimental design of this study is primarily focused on the administration of specific inhibitors to MCF-7 cells with an interest in the dose-dependent response of the cells to varying concentrations of the treatments as well as a thorough comparison of the cell morphology, viability, and proliferation to the effects of the 1% BME control. The three drugs that have been investigated so far are Nifedipine, a voltage-gated calcium blocker, Nifedipine in the presence of KCl [in order to artificially depolarize the cell membrane to open the VGCC], and ML-7, a myosin light chain kinase inhibitor. The cell viability was then assessed through an MTT colorimetric assay at 562 nm in order to visually and quantitatively assess the results. Additionally, transfection studies are planned to be carried out to substantiate pharmacological findings of inhibitors. Results & Conclusion Nifedipine dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of nifedipine. All values are relative to control without nifediine considered as 100%; [0.625 um Nif – 43.05%, 1.25 um Nif – 42.19%, 2.5 um Nif – 40.15%, 5 um Nif -39.53%, 10 um Nif – 38.19%, 20 um Nif – 25.1%, 40 um Nif – 22.96%, 1% BME –39.26%]. The 1% BME (bitter melon extract) is the positive control in our studies as our lab have found BME to be toxic to MCF-7 cells. ML-7 also dose-dependently inhibited MCF-7 cell viability. Following were the % absorbance values with increasing doses of ML-7. All values are relative to control without ML-7 considered as 100%; (0.5 um ML7- 70.01%, 1 um ML7 – 65.74%, 2 um ML7 – 57.71%, 4 um ML7 – 49.36%, 8 um ML7 – 48.28%, 16 um ML7 – 34.13%, 1% BME – 36.17%]. When we stimulated cells with increasing doses of KCl, increasing toxicity was seen as reflected by the decreasing absorbance values. We tested the effects of KCl in the presence or absence of nifedipine. Following were the % absorbance values with increasing doses of KCl and/or nifedipine. All values are relative to control (neither KCl nor nifedipine) considered as 100%; [40 mm KCl – 29.55%, 20 mm KCl – 44.024%, 10 mm KCl –56.51%, 40 um Nif – 22.81%, 40 mm KCl + 40 um Nif – 13.69%, 20 mm KCl + 40 um Nif –15.87%, 10 mm KCl + 40 um Nif – 30.91%, EtOH – 86.85%, 1% BME – 35.27%.]. Thus, either inhibiting Ca2+ influx with nifedipine or opening VGCC with high KCl both exerted cytotoxic effects on MCF-7 cells. This suggests that Ca2+ is critical for the viability of these cells but at the same time too much Ca2+ is toxic to the cells.