Role of Protein Kinase‐C and Rho Kinase in the Cytotoxic Effects of Bitter Melon Extract on Metastatic Breast Cancer Cells
Authors: Choi Heeyun , Bhetwal Bhupal P. 
Published: June 2021

IntroductionBitter melon is a common vegetable in the kitchens of most of Asian countries. Mitter melon Extract (BME) is known to inhibit proliferation of cancer cells including MCF‐7 cells which are metastatic breast cancer cells. The PKC and ROK play critical roles in cell division, migration, and survival. However, the roles of protein kinase‐c (PKC) and rho kinase (ROK) inhibition on MCF‐7 cells have not been established. Moreover, whether potential effects of BME's effects on MCF‐7 cells are mediated by PKC and ROK are unknown.ObjectiveWe aimed to investigate if BME exerts cytotoxic effects on breast cancer cells (MCF‐7 cells) and if PKC and ROK mediate BME's effects.HypothesisBME inhibits proliferation of MCF‐7 cells by decreasing PKC activity and increasing ROK activity.MethodsFresh bitter melons were purchased from an Asian grocery store and the extract (BME) was extracted, centrifuged, and filter sterilized. Equal number of MCF‐7 cells was plated in culture flasks containing DMEM medium and increasing amount of BME [0%, 0.5%, 1%, 2%, 5%, and 10% of BME (v/v)] was added to the culture flasks. To study whether PKC or ROK play any roles in mediating BME's effects, MCF‐7 cells were cultured in the following conditions; 0% BME, 1% BME, PKC inhibitor (GF109203x; 0.5μM)/ROK inhibitor (H‐1152; 1 μM), and inhibitor combined with 1% BME. After culturing cells for 4 days (for the dose‐response study) and 2 days (for the inhibitor studies), pictures of cultures were taken, and cell viability was determined using Trypan blue dye and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. After culturing cells for the required number of days, culture was centrifuged and cells were collected, protein was extracted using RIPA buffer and proteins candidates of the ROK pathways were detected by western blotting using HRP‐conjugated chemiluminescence system. Multiple comparison ANOVA was used to determine statistical significance.Results and ConclusionsBME dose‐dependently inhibited viability of MCF‐cells (N=3) starting form 1% (v/v), which was measured by two independent viability assay methods. Also, BME dose‐dependently decreased expression of MYPT1 protein, which is a target of ROK (N=1; duplicate) but the beta‐actin expression (N=1, duplicate) was not altered. Neither PKC inhibitor (N=5) nor ROK inhibitor (N=6) alone had significant effects on cell viability. However, when these inhibitors were combined with 1% BME, the effect on cell viability was of the same magnitude that the 1% BME alone had. In the future, we plan to measure phosphorylation of ROK's and PKC's target proteins by western blotting and also test if the BME exerts any cytotoxic effects on non‐cancerous breast epithelial cells.Support or Funding InformationBiomedical science master's thesis funding, Marian University College of Osteopathic Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.