Rescue effects of insulin on the cytotoxic effects of bitter melon extract in the human breast cancer cells

Abstract

Bitter melon extract (BME) is known to inhibit breast cancer cells proliferation. Due to the high metabolic rate of cancer cells, we hypothesized that low glucose levels are detrimental to cell survivability and that BME exerts a cytotoxic effect by altering the ability of a cell to utilize glucose. Through this study, we seek to investigate if insulin is capable of mitigating the cytotoxic effects of BME and provide a “rescue effect”.Fresh bitter melons were purchased from an Asian grocery store and the extract (BME) was prepared, centrifuged, and filter sterilized. The MCF‐7 cells were cultured in the DMEM medium (normal or low glucose) with either 1% or 2 % BME (v/v) and in the presence or absence of insulin at various doses [50ng/mL, 100ng/mL, 200ng/mL]. After culturing cells for 24 hours in standard DMEM complete medium, BME and insulin were added to corresponding wells and pictures of cultures were taken every 24 hours for 2 days. After the 2 days, cell viability was then assessed using an MTT colorimetric assay at 620 nm.BME dose‐dependently inhibited MCF‐7 cell viability. Insulin demonstrated a rescue effect and increased the cell viability despite the addition of BME. Insulin also dose‐dependently enhanced MCF‐7 cell viability to an extent when added alone. Following were the % absorbance values with increasing doses of insulin. All values are relative to control (without any addition of BME or Insulin) considered as 100% and corrected for absorbance of DMEM alone; [DMEM only – 38.33%, 1% BME – 34.33%, 0% BME + 200ng Insulin – 49.87%, 1% BME + 50ng Insulin – 39.53%, 1% BME + 100ng Insulin – 39.97%, 1% BME + 200ng Insulin – 41.70%]. 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. The same trial was repeated with 2% BME in order to better visualize the cytotoxic effects of BME and further demonstrate the subsequent rebound in cell viability following addition of insulin. All values are relative to control (without any addition of BME or Insulin) considered as 100% and corrected for absorbance of DMEM alone; [DMEM only – 58.87%, 2% BME – 17.07%, 0% BME + 200ng Insulin – 63.97%, 2% BME + 50ng Insulin – 55.70%, 2% BME + 100ng Insulin – 55.43%, 2% BME + 200ng Insulin – 59.30%].When we treated cells with increasing doses of insulin, an enhanced rescue effect was seen as reflected by the increasing absorbance values. We further tested the effects of BME and insulin by culturing the cells in low glucose DMEM. The raw data is included in the abstract due to space limitation.Our data indicates the addition of insulin is capable of rescuing cells from the cytotoxic effects of BME. Insulin seems to be capable of mitigating the cytotoxic effects of BME and, in some cases, even enhancing the viability of cells despite the prior treatment with BME. The relatively diminished rescue effect with 200ng of insulin and 2% BME may be due to the inherent strain the cells were already experiencing with BME. The high dose of insulin, coupled with the strain of attempting to replicate in sub‐optimal conditions, may have led to cells that were incapable of meeting the metabolic demand placed by the large dose of insulin and subsequent influx of glucose. Further study may be warranted to discover the threshold of benefit that insulin can provide and amount of glucose necessary for, or detrimental to, cell survival.