Metformin and phenethyl isothiocyanate combined treatment in vitro is cytotoxic to ovarian cancer cultures
© Chan and Miskimins;licensee BioMed Central Ltd. 2012
Received: 4 April 2012
Accepted: 4 June 2012
Published: 10 July 2012
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.
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.
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.
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.
KeywordsMetformin Phenethyl isothiocyanate Ovarian cancer
Ovarian cancer continues to have a disturbing discrepancy in incidence to mortality rate. While ovarian cancer trends as eighth in overall incidence among women in the United States, ovarian cancer mortality is significantly higher and ranks fifth in female cancer deaths. The difference in incidence to mortality rates is a combined result of poor ovarian cancer screening and rapid development of resistance to current chemotherapeutics. Poor ovarian cancer screening is inherent to its non-specific initial symptoms, and CA-125 and transvaginal ultrasound provide no additional benefit in reducing mortality . Expanding available chemotherapies may improve the long term survival of ovarian cancer patients by overcoming the resistance to platinum based therapies and providing alternate mechanisms for cancer cell death. Metformin and PEITC have both been individually identified as potential ovarian cancer therapies [2–5]. This work examines the combination of metformin and PEITC as a novel treatment combination.
The exact mechanisms for the anti-cancer effects of both metformin and PEITC are not entirely clear. However, several groups have found both metformin and PEITC to affect complexes within the mitochondrial electron transport chain [6, 7]. While cancer cells have an altered metabolism that heavily utilizes glycolysis, even in the presence of oxygen, mitochondrial oxidative metabolism is still necessary to generate intermediates needed for cell replication and reducing agents to deal with oxidative stress. This potential inhibition of complexes in the electron transport chain may increase reactive oxygen species (ROS) within the cell, especially in the mitochondria, leading to damage and eventually cancer cell death.
The mechanism of cisplatin resistance in ovarian cancer is not precisely known, but may be due to reduced uptake . Some research suggests cisplatin uptake is dependent on the copper transporter CTR1 . Metformin is highly concentrated in cells, likely through OCT1 , and has not been found to be affected by changes that occur in cisplatin resistant cell lines. PEITC is a lipophilic compound that does not rely on an active transporter and can directly cross the cell membrane. When taken orally in milligram amounts, peak PEITC plasma levels based on HPLC analysis were measured in the low micromolar range .
After being associated with lower cancer incidence in diabetic patients , metformin has been studied in a variety of cancers as a potential therapy. In breast cancer cells metformin has been found to induce cell death and improve the efficacy of existing therapies [13, 14]. Previous research with metformin in ovarian cancer has suggested that mechanisms in addition to AMPK activation are responsible for inhibition of cell growth . Metformin's partial inhibition of complex I, NADH dehydrogenase, has been identified as an effect potentially upstream of AMPK activation . This mechanism is also interesting because it may provide an AMPK independent pathway for metformin to damage cancer cells through ROS production and mitochondrial damage.
PEITC gained interest as a potential anti-cancer agent based on findings of lower cancer incidence in people with diets high in cruciferous vegetables . With PEITC specifically, preclinical animal models also associated the drug with decreased carcinogenesis [16, 17]. Early studies of PEITC in transformed ovarian epithelial cells also found ROS generation to mediate cell death . This ROS generation was later found to be a result of complex III, cytochrome bc1 complex, inhibition in colorectal cancer cells . These drugs which work in alternative pathways from cisplatin may provide additional therapeutic options in ovarian cancer.
Metformin, PEITC, cisplatin, and most other reagents were purchased from Sigma Aldrich (St. Louis, MO). Tissue culture media, DMEM and RPMI were purchased from Thermo-Fisher Scientific (Waltham, MA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Atlanta, GA). Fluorescent dyes: MitoSOX, 2’,7’-dichlorodihydrofluorescein (DCF), dihydroethidine (DHE), dihydrorhodamine 123 (DHR), Sytox green, and MitoTracker Green were all purchased from Invitrogen (Grand Island, NY). Antibodies to detect AMPK, PARP, cleaved PARP were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to detect MnSOD were purchased from BD Pharmingen (San Diego, CA).
Cell lines were incubated in a 37 C, 5% CO2 humidified chamber. OVCAR3, CAOV3, SKOV3, and PA-1 were obtained from ATCC. A2780 and A2780cis were received as a kind gift from Dr. Subhash Chauhan. OVCAR3, CAOV3, SKOV3, and PA-1 were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B. A2780 was maintained in RPMI with 10% FBS, 100 units/mL penicillin/streptomycin, and 250 ng/mL amphotericin B. A2780cis cells were maintained in complete RPMI media supplemented with 1 μM cisplatin every 3rd passage to maintain selection for cisplatin resistance.
Multiple methods were used in cell counting. For cells counted by glass hemacytometer with Trypan blue exclusion, adherent cells were harvested by trypsinization. For live and dead cell counts, media was also collected and added to the cell suspension. Cells were pelleted by centrifugation (200 x g) and each sample was resuspended in an equal volume. Samples were then mixed with an equal volume of 0.4% Trypan blue and counted using a glass hemacytometer. Cells excluding Trypan blue were counted as live, stained cells were counted as dead. Sytox green (Invitrogen) fluorescent dye was also used for dead cell analysis. In a 96-well plate format, cells were plated in phenol red free media 24 hours prior to treatment with drugs of interest. At desired time points, Sytox green dye was added to wells at 1 μM final concentration and incubated for 10 minutes to stain DNA of dead cells. Fluorescence was measured using a Spectramax M5 fluorescence plate reader at excitation 485, emission 530, cutoff 515. Total cell fluorescence was measured by adding Triton X-100 at a final concentration of 0.1% to all wells, incubating for 10 minutes, and remeasuring Sytox fluorescence.
Anchorage dependant colony formation assay
Cells were plated at 300–500 cells per 100 mm dish 24 hours before beginning treatment conditions. Cells were allowed to grow for 7–10 days before fixation. Colonies were fixed using 70% ethanol, and stained using 1% crystal violet dissolved in 10% ethanol. Number of colonies was obtained using image analysis software (AlphaInnotech). Colonies were defined as a minimum of 50 cells in a group.
Samples were harvested in an SDS based buffer, sonicated and heated before running. Proteins were separated by SDS-PAGE and transferred to PVDF membrane by semi-dry transfer apparatus. Antibodies for detection of proteins of interest were diluted in blocking buffer, 5% BSA in TBST. Signal was detected using HRP-conjugated secondary antibodies and Amersham ECL prime detection reagent (GE healthcare). Exposures were captured either by photographic film or CCD camera imaging system (UVP).
Flow cytometry for oxidative stress
After varying treatment periods, adherent cell cultures were detached with trypsin and transferred to culture tubes as a cell suspension. Cells were then incubated with fluorescent dyes to determine levels of oxidative stress products. MitoSOX (Invitrogen) was used to measure mitochondrial specific superoxide. Dihydroethidine (DHE) was used to measure total cellular superoxide. Absorbance was measured with the FL2 channel (Ex / Em) of an Accuri C6 flow cytometer. Data was collected and analyzed using Cflow Plus software (BD-Accuri).
Live cell confocal microscopy
Cells were cultured on a chambered coverglass (Thermo scientific) in phenol red free DMEM with 10% FBS. After completing desired drug treatment courses, cells were stained with dyes of interest in phenol red free DMEM with 5% FBS. MitoSOX was used at 5 μM final concentration and MitoTracker green at 100 nM final concentration. Cells were incubated with dye for 15 minutes at 37 °C, and then washed twice with PBS. Phenol free DMEM with 5% FBS was used during live cell imaging. An Olympus FV1000 imaging system on an Olympus IX81 inverted microscope with heated 5% CO2 stage chamber was used for image capture. Image processing and analysis was completed using Olympus FluoView software.
Error bars displayed are standard deviations. Comparison of two groups was done using a two-tailed student’s t-test. Analysis of multiple groups was performed by ANOVA or Chi-squared test using R, the statistical computing program.
CalcuSyn (BioSoft) was used to calculate combination index values for drug combinations. Drug combinations were given at constant ratios and effect measured was either cell death or cell growth inhibition.
Metformin is cytostatic, PEITC is cytotoxic in multiple ovarian cancer lines
ROS is increased with both metformin and PEITC
Metformin and PEITC combined are effective in a cisplatin resistant cell line
Expanding ovarian cancer therapies to include compounds such as metformin and PEITC is attractive for multiple reasons. Metformin has a broad safety profile, and while the mechanism is not exactly known, a tremendous amount of patient dosage data exists. The risk for lactic acidosis in metformin is minimal, with cases only commonly occurring with coexisting kidney dysfunction . Similarly, PEITC's dosage safety can be partially inferred from cruciferous vegetable intake. A study of a defined watercress diet showed significant uptake of PEITC in humans . A current phase I trial of PEITC in leukemia patients will further clarify safety and dosing.
Metformin's complex mechanisms of action may provide many difficulties to cancer cells attempting to counter its actions. Because metformin likely acts through multiple pathways, a cancer cell may be able to respond to the stress of one change, but still be affected by other alterations. Metformin's resistance to a variety of ROS scavengers, propyl gallate, butylated hydroxyanisole, Mito-TEMPO (data not shown) shows that metformin is not reliant on ROS production to inhibit cancer cell growth despite its actions on complex I. Still, complex I inhibition partially contributes to the cell inhibition as shown through the methyl succinate supplementation experiment. The methyl succinate prevention of metformin’s effect was diminished as metformin dose increased, suggesting metformin acts through multiple pathways as it accumulates within the cell. Higher doses of metformin may act through AMPK pathway activation through a still unknown exact mechanism. Likely, metformin's cellular effects are a combination of AMPK pathway activation with subsequent mTOR inhibition and complex I partial inhibition increasing ROS.
Relevant dosing for metformin in vitro remains unclear. Most groups work in the low millimolar range, from 1–20 mM [3, 22, 23]. While this seems initially unattainable in vivo, metformin is not metabolized and has been found to be highly concentrated with the cells, especially within mitochondria . Moreover, these concentrations are necessary for in vitro work to mimic the effects observed with in vivo studies where approved diabetic metformin doses have been used. Thus, in order to achieve the concentrated cellular metformin levels and study the potential changes that occur in vivo over longer periods of treatment, high doses over short time courses must be used in vitro.
While PEITC does not have the long historical use in humans that metformin has, its general safety can be inferred from diets high in cruciferous vegetables. Also, similar to metformin, PEITC has been associated with cancer prevention in several cohort studies [15, 24].
Although PEITC is more effective at inducing cancer apoptosis, it is also more simply reversed by increasing ROS scavengers. Metformin's enhancement of PEITC could be due in part to depletion of reducing agents needed to handle the ROS stress induced by PEITC. Additionally, metformin may synergize with PEITC through inhibition of complex I to provide extra mitochondrial oxidative stress, shifting the ROS balance towards cell death.
Additional therapies that utilize different mechanisms than cisplatin are especially attractive for ovarian cancer. The combination therapy of metformin and PEITC is interesting because of its effectiveness despite cisplatin resistance. Additional work is warranted to determine if this synergy is maintained with in vivo models. Current data suggests metformin and PEITC as a combination treatment have potential as a novel ovarian cancer therapy.
Complex I inhibition is partially involved in metformin's growth inhibition of ovarian cancer, possibly by increasing ROS and sensitizing cancer to additional oxidative stress. PEITC induces ovarian cancer cell death by increasing ROS. When given together, metformin and PEITC show a synergistic increase in cell death in several ovarian cancer cell lines, including cisplatin resistant A2780cis cell line. This novel combination may be useful in combating the high mortality of cisplatin resistant ovarian cancers.
Reactive oxygen species
Electron transport chain
The technical assistance of Allison Haugrud and Kelly Graber was appreciated. We also thank Yongxian Zhuang for insight on experimental design. This work was supported in part through grant KG100497 (W.K. Miskimins) from Susan G. Komen for the Cure, and a graduate student research grant (DKC) from the University of South Dakota. Flow Cytometry and Imaging Cores were supported by NIH CoBRE grants 5P20RR024219-02 and 5P20RR017662-09.
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