Metformin and phenethyl isothiocyanate combined treatment in vitro is cytotoxic to ovarian cancer cultures

Abstract

Background: High mortality rates in ovarian cancer are largely a result of resistance to currently used chemotherapies. Expanding therapies with a variety of drugs has the potential to reduce this high mortality rate. Metformin and phenethyl isothiocyanate (PEITC) are both potentially useful in ovarian cancer, and they are particularly attractive because of their safety.

Methods: Cell proliferation of each drug and drug combination was evaluated by hemacytometry with Trypan blue exclusion or Sytox green staining for cell death. Levels of total and cleaved PARP were measured by Western blot. General cellular and mitochondrial reactive oxygen species were measured by flow cytometry and live cell confocal microscopy with the fluorescent dyes dihydroethidine and MitoSOX.

Results: Individually, metformin and PEITC each show inhibition of cell growth in multiple ovarian cancer cell lines. Alone, PEITC was also able to induce apoptosis, whereas metformin was primarily growth inhibitory. Both total cellular and mitochondrial reactive oxygen species were increased when treated with either metformin or PEITC. The growth inhibitory effects of metformin were reversed by methyl succinate supplementation, suggesting complex I plays a role in metformin's anti-cancer mechanism. PEITC's anti-cancer effect was reversed by N-acetyl-cysteine supplementation, suggesting PEITC relies on reactive oxygen species generation to induce apoptosis. Metformin and PEITC together showed a synergistic effect on ovarian cancer cell lines, including the cisplatin resistant A2780cis.

Conclusions: Here we show that when used in combination, these drugs are effective in both slowing cancer cell growth and killing ovarian cancer cells in vitro. Furthermore, the combination of these drugs remains effective in cisplatin resistant cell lines. Novel combinations such as metformin and PEITC show promise in expanding ovarian cancer therapies and overcoming the high incidence of cisplatin resistant cancers.

Figure 1

Individual effects of metformin and PEITC on ovarian cancer linesin vitro.A. Metformin growth inhibition of multiple human ovarian cancer cell lines after 48 hour treatment with control (H₂O vehicle) 8 mM metformin. B. PEITC growth inhibition of multiple human ovarian cancer cell lines after 24 hour treatment with control (DMSO vehicle) or 5 μM PEITC. Both metformin and PEITC induce a significant decrease in cell proliferation across all tested cell lines. Cell proliferation and growth inhibition was measured using hemacytometry with Trypan blue exclusion. C. Anchorage dependant colony formation assay on cisplatin resistant SKOV3 line shows significantly fewer colonies in metformin [5 mM] compared to cisplatin [5uM] treatment. D. In CAOV3, metformin is just as effective as cisplatin in reducing colony formation. * p < 0.01; # p < 0.05; n.s. = not significant.

Figure 2

Individual cell death effects of metformin and PEITC.A. Metformin treatment [8 mM] on multiple human ovarian cancer cell lines after 48 hours shows minimal cell death induction. B. PEITC [5 μM] induces significant cell death of multiple human ovarian cancer cell lines after a 24 hour treatment. Cell death percentage was determined using hemacytometry with Trypan blue exclusion to count both live and dead cells. * p < 0.01.

Figure 3

Cause of cell death for PEITC treated cells. PARP cleavage is increased by treatment with PEITC in multiple ovarian cancer cell lines, indicating death by apoptosis. Western blot for full length and cleaved PARP after 48 hours of treatment is displayed. Blots are representative images of band intensity. Bar graph represents cleaved PARP band intensity normalized to β-actin. * p < 0.01, # p < 0.05, n.s. = not significant.

Figure 4

Mitochondrial reactive oxygen species (ROS) increased with metformin and PEITC treatment. A. Live cell confocal image of SKOV3 cells treated for 24 hours. MitoSOX, a mitochondrial specific superoxide detector, exhibits increased fluorescence in metformin and PEITC treated cells comparison to control treated cells. B. Mean fluorescence intensity from dihydroethidium (DHE) used to detect total cellular ROS. Cell lines were treated for 24 hours with or without metformin [8 mM] and DHE intensity was measured by flow cytometry.

Figure 5

NAC supplementation reverses PEITC cell death.A. The effect of NAC supplementation on control (DMSO vehicle), metformin [5 mM], and PEITC [5 μM] growth inhibitory effects on PA-1 cell line after 24 hours. Metformin is relatively unaffected by the addition of NAC, while PEITC's growth inhibition is significantly reduced. B. The dead cell percent of PA-1 cells treated with control (DMSO vehicle), metformin, or PEITC treatment with and without NAC pre treatment. Metformin alone does not induce high amounts of cell death with or without NAC. PEITC's cell death is strongly reduced to levels similar to control when pretreated with NAC to act as an ROS scavenger. * p < 0.01; n.s. = not significant.

Figure 6

Methyl succinate partially reverses metformin's effects. When 12.5 mM methyl succinate is added to complete growth media, the 48 hour growth inhibition effects of metformin on PA-1 are reduced. Methyl succinate serves as an alternate energy source to glucose that bypasses complex I in the electron transport chain. Metformin at higher doses can overcome the reversal of growth inhibition provided by methyl succinate. * p < 0.01.

Figure 7

Combination of metformin and PEITC.A. Combination of low doses of metformin and PEITC on CAOV3 show an improved effect on cell death after 72 hours compared to either drug individually. Combination can reduce doses necessary to achieve high levels of cell death. B. In the cisplatin resistant ovarian cancer line A2780cis, metformin and PEITC work synergistically and are more effective than cisplatin. Cell death is significantly increased when metformin and PEITC are given together. Cisplatin, even at higher doses, is unable to produce similar cell death. C. Combination index (CI) values for A2780cis calculated using CalcuSyn software. CI values below 1 are considered synergistic combinations, with greater synergy as the value approaches zero. Metformin and PEITC are synergistic at equal ratio effective doses (ED). * p < 0.01 when compared to control treatment.