Anti-diabetic Biguanide Drugs Research Articles

Decavanadate and metformin-decavanadate effects in human melanoma cells

Decavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive–non-competitive inhibition with an IC50 value about 6 times higher (87 μM) than the previously described for decavanadate (15 μM). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways.

Abstract 361: NT1195, a novel biguanide, exhibits superior uterine penetration and anti-tumorigenic efficacy as compared to metformin/phenformin in endometrial cancer

Abstract Objectives: Obesity and diabetes are associated with increased risk and worse outcomes in endometrial cancer (EC). Anti-diabetic biguanide drugs such as metformin/phenformin may have anti-tumorigenic effects by behaving as AMPK activators. Metformin requires organic cation transporters (OCTs) for entry into cells whereas phenformin is not as reliant on OCTs for cell entry. NT1195 (NovaTarg) is a novel biguanide designed to have greater affinity for the OCT1/3, PMAT and MATE1 transporters. Thus, we compared head-to-head the pharmacokinetic properties and anti-tumorigenic potential of metformin vs phenformin vs NT1195 in a genetically engineered mouse model of endometrioid EC (LKB1fl/flp53fl/fl). Methods: Pharmacokinetic parameters, including half-life, AUC, bioavailability and uterine concentration were measured in female control mice after oral dosing of metformin, phenformin and NT1195 (5 mg/kg oral for all drugs). AdCre was injected at 6 wks of age to induce invasive EC in LKB1fl/flp53fl/fl mice. Following tumor onset, mice were treated with placebo vs metformin vs phenformin vs NT1195 for 4 wks (50 mg/kg, oral for all drugs). Results: NT1195 (> 24 hrs) had a longer half-life when compared to metformin (3.7 hrs) and phenformin (7.2 hrs). Drug bioavailability was heightened in the NT1195-treated mice when compared to metformin and phenformin (149 vs 83.5 vs 8.31%, respectively). The AUC (h*kg*ng/ml/mg) for NT1195 was significantly higher at 1143 than that of metformin (26) and phenformin (184). Drug accumulation in uterine tissue at 24 hrs post dose demonstrated markedly increased presence of NT1195 (45.4 ng/g), compared to metformin and phenformin, which yielded levels below the limits of quantification. NT1195 had increased anti-tumorigenic efficacy in LKB1fl/flp53fl/fl mice, inhibiting tumor growth by 88% compared to 78% for metformin and 80% for phenformin (p<0.01). Conclusions: When compared to metformin/phenformin, NT1196 had superior bioavailability, a longer half-life and greater uterine tissue penetration, culminating in improved efficacy in an EC mouse model. Thus, NT1195 may be a promising novel biguanide therapy in EC, particularly in women who want to avoid hysterectomy and preserve fertility or are poor surgical candidates. Citation Format: Katherine Tucker, Allison Staley, Yali Fan, Xiaoling Zhao, Yajie Yin, Ziwei Fang, Wenchuan Sun, Kenneth Batchelor, Nick Livingston, Chunziao Zhou, Victoria Bae-Jump. NT1195, a novel biguanide, exhibits superior uterine penetration and anti-tumorigenic efficacy as compared to metformin/phenformin in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 361.

Antidiabetic Biguanides Radiosensitize Hypoxic Colorectal Cancer Cells Through a Decrease in Oxygen Consumption.

Background and Purpose: The anti-diabetic biguanide drugs metformin and phenformin exhibit antitumor activity in various models. However, their radiomodulatory effect under hypoxic conditions, particularly for phenformin, is largely unknown. This study therefore examines whether metformin and phenformin as mitochondrial complex I blockades could overcome hypoxic radioresistance through inhibition of oxygen consumption.Materials and Methods: A panel of colorectal cancer cells (HCT116, DLD-1, HT29, SW480, and CT26) was exposed to metformin or phenformin for 16 h at indicated concentrations. Afterward, cell viability was measured by MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) was examined by western blot. Mitochondria complexes activity and oxygen consumption rate (OCR) were measured by seahorse analyzer. The radiosensitivity of tumor cells was assessed by colony formation assay under aerobic and hypoxic conditions. The in vitro findings were further validated in colorectal CT26 tumor model.Results: Metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced OCR in a dose-dependent manner starting at 3 mM and 30 μM, respectively. As a result, the hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin with an enhancement ratio about 2 at 9 mM and 100 μM, respectively. Regarding intrinsic radioresistance, both of them did not exhibit any effect although there was an increase of phosphorylation of AMPK and ROS production. In tumor-bearing mice, metformin or phenformin alone did not show any anti-tumor effect. While in combination with radiation, both of them substantially delayed tumor growth and enhanced radioresponse, respectively, by 1.3 and 1.5-fold.Conclusion: Our results demonstrate that metformin and phenformin overcome hypoxic radioresistance through inhibition of mitochondrial respiration, and provide a rationale to explore metformin and phenformin as hypoxic radiosensitizers.

Treatment with antidiabetic biguanide drugs directly impacts the function of multiple pituitary cell types from two non-human primate models

Treatment with antidiabetic biguanide drugs directly impacts the function of multiple pituitary cell types from two non-human primate models

Salting-out assisted liquid-liquid extraction coupled with hydrophilic interaction chromatography for the determination of biguanides in biological and environmental samples

A new salting-out assisted liquid-liquid extraction (SALLE) sample preparation method for the determination of the polar anti-diabetic biguanide drugs (metformin, buformin and phenformin) in blood plasma, urine and lake water samples were developed. The SALLE was performed by mixing samples (plasma (0.2mL), urine or lake water (1.0mL)) with acetonitrile (0.4mL for plasma, 0.5mL for urine or lake water), sodium hydroxide powder was then added for the phase separation. The effects of type of salting-out reagent, type of extraction solvent, volumes of acetonitrile and sample, amount of sodium hydroxide, vortexing and centrifugation times on the extraction efficiency were investigated. The upper layer, containing the biguanides, was directly injected into a HPLC unit using ZIC-HILIC column (150mm×2.1mm×3.5μm) and was detected at 236nm. The method was validated and calibration curves were linear with r2>0.99 over the range of 20–2000μgL−1 for plasma and 5–2000μgL−1 for urine and lake water samples. The limits of detection were in the range (3.8–5.6)μgL−1, (0.8–1.5)μgL−1 and (0.3–0.8)μgL−1 for plasma, urine and lake water, respectively. The accuracies in the three matrices were within 87.3–103%, 87.4–109%, 82.2–109% of the nominal concentration for metformin, buformin and phenformin, respectively. The relative standard deviation for inter- and intra −day precision were in the range of 1.0–17% for all analytes in the three matrices.

Results from 11C-metformin-PET scans, tissue analysis and cellular drug-sensitivity assays questions the view that biguanides affects tumor respiration directly

The anti-diabetic biguanide drugs metformin (METF) and phenformin (PHEN) may have anti-cancer effects. Biguanides suppress plasma growth factors, but nonetheless, the view that these mitochondrial inhibitors accumulate in tumor tissue to an extent that leads to severe energetic stress or alleviation of hypoxia-induced radioresistance is gaining ground. Our cell studies confirm that biguanides inhibits cell proliferation by targeting respiration, but only at highly suprapharmacological concentrations due to low drug retention. Biodistribution/PET studies of 11C-labeled metformin (11C-METF) revealed that plasma bioavailability remained well below concentrations with metabolic/anti-proliferative in vitro effects, following a high oral dose. Intraperitoneal administration resulted in higher drug concentrations, which affected metabolism in normal organs with high METF uptake (e.g., kidneys), but tumor drug retention peaked at low levels comparable to plasma levels and hypoxia was unaffected. Prolonged intraperitoneal treatment reduced tumor growth in two tumor models, however, the response did not reflect in vitro drug sensitivity, and tumor metabolism and hypoxia was unaffected. Our results do not support that direct inhibition of tumor cell respiration is responsible for reduced tumor growth, but future studies using 11C-METF-PET are warranted, preferably in neoplasia’s originating from tissue with high drug transport capacity, to investigate the controversial idea of direct targeting.

Voltammetric and capillary electrophoretic study of scavenger kinetics of methylglyoxal by antidiabetic biguanide drugs

A novel electroanalytical approach is used to get insight into scavenger kinetics and mechanism of methylglyoxal (MG) by biguanides metformin, phenformin and 1-phenylbiguanide under the physiological conditions (pH7.41 and 37°C). The approach is based on monitoring the time profiles of concentrations of the protonated biguanides by ion transfer voltammetry (ITV) at the polarizable room-temperature ionic liquid membrane. Effects of the reactant's concentrations and pH lead us to propose a mechanism including an acid-base reaction of biguanide, which separates the parallel reactions of the protonated and deprotonated biguanide with methylglyoxal. Reaction rate increases considerably in the sequence metformin<phenformin<1-phenylbiguanide. Kinetic data confirm the previous finding that scavenging of methylglyoxal by metformin is a relatively slow process. Optimization of the background electrolyte enables to use capillary electrophoresis (CE) to detect simultaneously the protonated biguanide and the MG anion in the reaction mixture. CE analysis then yields the mole-to-mole ratios of the reacted biguanides to MG in the range 0.6–0.7 pointing to the simultaneous formation of a 1:1 and a 1:2 reaction products. Usefulness of ion voltammetry or amperometry for further kinetic studies of biguanides, as well as for their assays, can be envisaged.

Abstract 3084: The effects of NT-1044, a novel AMPK activator, on endometrial cancer cell proliferation, apoptosis, cell stress, and tumor growth

Abstract Objectives: Obesity and diabetes are associated with increased risk and worse outcomes in endometrial cancer (EC). Anti-diabetic biguanide drugs such as metformin may have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Metformin requires organic cation transporters (OCTs) for entry into cells, and NT-1044 (NovaTarg Therapeutics) is an AMPK activator designed to have greater affinity for two of these transporters, OCT1 and 3. We sought to compare the effects of NT-1044 on cell proliferation in human EC cell lines and on tumor growth in an endometrioid EC mouse model. Methods: Cell proliferation was assessed in two EC cell lines, ECC-1 and Ishikawa, by MTT assay after exposure to NT-1044. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by flow cytometry. Reactive oxygen species (ROS) were measured using a DCFH-DA assay. Western immunoblotting was performed to evaluate the effects of NT-1044 on the downstream targets of the AMPK/mTOR pathway and proteins related to cell cycle and cellular stress. For in vivo studies, we utilized the LKB1f/f/p53f/f mouse model of endometrioid endometrial cancer. Mice were treated with placebo or NT-1044 (200 mg/kg/day, oral gavage) following tumor onset for 4 weeks. Results: NT-1044 significantly inhibited proliferation in a dose-dependent manner in both EC cell lines after 72 hours of exposure (IC50 218 uM for ECC-1; 87 uM for Ishikawa). Treatment with NT-1044 resulted in G1 cell cycle arrest, induced apoptosis and increased ROS production in both cell lines. NT-1044 increased phosphorylation of AMPK and decreased phosphorylation of S6, a key downstream target of the mTOR pathway. Expression of the cell cycle proteins CDK4, CDK6 and cyclin D1 decreased in a dose dependent-fashion while cellular stress protein expression was induced in both cell lines. As compared to placebo, NT-1044 inhibited endometrial tumor weight in the LKB1f/f/p53f/f mice by 56% (p = 0.031). Conclusions: NT-1044 suppressed EC cell growth through G1 cell cycle arrest, induction of apoptosis, cellular stress and inhibition of the AMPK/mTOR pathway. In addition, NT-1044 inhibited EC tumor growth in vivo. More work is needed to determine if this novel biguanide will be beneficial in the treatment of women with EC, a disease strongly impacted by obesity and diabetes. Citation Format: Dario R. Roque, Weiya Z. Wysham, Chunxiao Zhou, Ken W. Batchelor, Wendy R. Brewster, Victoria L. Bae-Jump. The effects of NT-1044, a novel AMPK activator, on endometrial cancer cell proliferation, apoptosis, cell stress, and tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3084.

The enhancement of oxidative DNA damage by anti-diabetic metformin, buformin, and phenformin, via nitrogen-centered radicals

Metformin (N,N-dimethylbiguanide), buformin (1-butylbiguanide), and phenformin (1-phenethylbiguanide) are anti-diabetic biguanide drugs, expected to having anti-cancer effect. The mechanism of anti-cancer effect by these drugs is not completely understood. In this study, we demonstrated that these drugs dramatically enhanced oxidative DNA damage under oxidative condition. Metformin, buformin, and phenformin enhanced generation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) in isolated DNA reacted with hydrogen peroxide (H2O2) and Cu(II), although these drugs did not form 8-oxodG in the absence of H2O2 or Cu(II). An electron paramagnetic resonance (EPR) study, utilizing alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide as spin trapping agents, showed that nitrogen-centered radicals were generated from biguanides in the presence of Cu(II) and H2O2, and that these radicals were decreased by the addition of DNA. These results suggest that biguanides enhance Cu(II)/H2O2-mediated 8-oxodG generation via nitrogen-centered radical formation. The enhancing effect on oxidative DNA damage may play a role on anti-cancer activity.

Metformin for Prevention and Treatment of Colon Cancer: A Reappraisal of Experimental and Clinical Data.

In the 70-80th of last century, it has been shown that the antidiabetic biguanide drugs phenformin (PF) and buformin (BF) can exert an inhibitory action on carcinogenesis in animal models and increase from 5 to 10-years survival of cancer patients. Since 2005, after first evidence publication of the capacity of metformin (MF), another biguanide, to prevent development of malignant tumors in individuals with type 2 diabetes (T2D) and suppress tumorigenesis in mice, the burst of studies started in this field focused on breast, pancreas, prostatic, endometrial and some other cancers. Colorectal cancer (CRC) is the most prevalent among digestive system cancers. In this mini-review are presented the available data on the capacity of antidiabetic biguanide drugs to prevent colorectal carcinogenesis and growth in vitro and in vivo in experimental models and clinical observations.

Abstract 4462: The effects of NT-1044, a novel AMPK activator, on endometrial cancer cell proliferation and apoptosis

Abstract Objectives: Obesity and diabetes are associated with increased risk and worse outcomes in endometrial cancer (EC). Anti-diabetic biguanide drugs such as metformin may have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Metformin requires organic cation transporters (OCTs) for entry into cells, and NT-1044 (NovaTarg Therapeutics) is an AMPK activator designed to have greater affinity for two of these transporters, OCT1 and 3. We sought to compare the effects of NT-1044 on cell proliferation and apoptosis in human EC cell lines. Methods: Cell proliferation was assessed in two EC cell lines, ECC-1 and Ishikawa, by MTT assay after exposure to NT-1044. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by flow cytometry. Phosphorylated (phos)-S6, phos-AMPK, Cyclin D-1 and CDK-6 were evaluated by Western immunoblotting. Results: NT-1044 significantly inhibited proliferation in a dose-dependent manner in both EC cell lines after 72 hours of exposure (IC50 218 uM for ECC-1; 87 uM for Ishikawa). Treatment with NT-1044 resulted in G1 cell cycle arrest and apoptosis in both cell lines. NT-1044 increased phosphorylation of AMPK and decreased phosphorylation of S6, a key downstream target of the mTOR pathway. Expression of Cyclin-D1 and CDK-6 also decreased in a dose dependent-fashion in both cell lines. Conclusions: NT-1044 suppressed EC cell growth through G1 cell cycle arrest, inducing apoptosis, and inhibition of the mTOR pathway. More work is needed to determine if this novel biguanide will be beneficial in the treatment of women with EC, a disease strongly impacted by obesity and diabetes. Citation Format: Dario R. Roque, Weiya W. Wysham, Chunxiao Zhou, Ken Batchelor, Wendy R. Brewster, Victoria L. Bae-Jump. The effects of NT-1044, a novel AMPK activator, on endometrial cancer cell proliferation and apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4462. doi:10.1158/1538-7445.AM2015-4462

Abstract 3113: Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in a genetically engineered mouse model of serous ovarian cancer

Abstract Objectives: Anti-diabetic biguanide drugs have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Phenformin is a biguanide that is associated with a higher risk of lactic acidosis than is seen in metformin. In vitro and in vivo studies suggest that phenformin may be more potent for inhibiting tumor growth than metformin. We sought to examine the efficacy of phenformin in primary cultures of human ovarian cancers (OCs) as well as in a genetically engineered mouse model of serous OC. Methods: Serous OC tumors were collected from consenting patients, at the time of their surgical debulking for primary culture. Cell proliferation was assessed by MTT assay. Apoptosis was evaluated by Annexin V-FITC assay using Cellometer. Effects of phenformin on phosphorylated-AMPK and -S6 expression was documented by Western immunoblotting. For the in vivo studies, we utilized the KpB++ serous OC mouse model. This is a more aggressive derivative of the K18-gT121+/-; p53fl/fl;Brca1fl/fl (KpB) genetically engineered OC mouse model. The KpB++ mice were treated with placebo or phenformin (2 mg/kg body weight in drinking water) following tumor onset for one month. Immunohistochemical analysis was performed on the ovarian tumors after treatment with placebo or phenformin, in regards to Ki-67 expression, cleaved caspase 3 (a marker of apoptosis), phosphorylated AMPK and phosphorylated S6. Individual slides were digitized using the Aperio ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed using Aperio ImageScope software. Results: Phenformin significantly inhibited cell proliferation in a dose-dependent manner in 100% (7/7) of the primary cultures of ovarian tumors, within 48 to 72 hours of exposure (p=0.00001-0.015, IC50 range of 0.1-5 mM). Treatment with phenformin resulted in the induction of apoptosis in 71.4% (5/7) of the ovarian tumors. Western immunoblot analysis demonstrated that phenformin increased phosphorylation of AMPK and decreased phosphorylation of S6. Phenformin significantly inhibited tumor growth in the KpB++ mice (n=8-13 animals per group) by 68% as compared to placebo treated controls, after one month of treatment (p=0.016). As compared to the placebo treated control animals, phenformin was found to statistically decrease Ki67 staining, inhibit phosphorylated S6 expression and increase both cleaved caspase 3 and phosphorylated AMPK expression (p= 0.01-0.04). Conclusions: In primary culture of human serous OC tumors, phenformin potently inhibited cell growth and increased apoptotic cell death through AMPK activation and mTOR pathway inhibition. In vivo studies using the KpB++ mouse model found that phenformin was highly efficacious in inhibiting tumor growth. Although the risk/benefit ratio clearly favors metformin over phenformin for the treatment of diabetes, this may not hold true for the treatment of cancer. Citation Format: Amanda L. Jackson, Xiaoyun Han, Joshua E. Kilgore, Chunxiao Zhou, Liza Makowski, Victoria Bae-Jump. Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in a genetically engineered mouse model of serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3113. doi:10.1158/1538-7445.AM2014-3113

Abstract 1312: Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines

Abstract Objectives: Epidemiologic and pre-clinical studies suggest that anti-diabetic biguanide drugs, such as metformin and buformin, may have anti-tumorigenic effects and behave as chemosensitizers. Thus, we evaluated the effects of buformin alone and in combination with paclitaxel on proliferation in human endometrial cancer (EC) cell lines as well as the impact of buformin on apoptosis and adhesion/invasion. Methods: The ECC-1 and Ishikawa EC cell lines were treated with varying doses of buformin alone and in combination with paclitaxel. Cell growth was determined by MTT assay. Cell cycle progression was assessed by Cellometer. Apoptosis was evaluated by Annexin V-FITC assay. Invasion was demonstrated by transwell invasion assay. Adhesion was assessed by ELISA. Effects on AMPK and S6 expression were documented by Western blotting. Results: Buformin inhibited proliferation in a dose-dependent manner in the EC cell lines (IC50 1000 μM for ECC-1, 10μM for Ishikawa, p=0.00001-0.0010). Treatment with buformin resulted in G1 arrest in the ECC-1 cell line and G2 arrest in the Ishikawa cell line. Buformin induced apoptosis in the Ishikawa cell line, but not in the ECC-1 cell line. Treatment with buformin resulted in inhibition of adhesion (p=0.42-0.0009) and invasion (p=0.00001-0.227) in both EC cell lines. Western blotting analysis demonstrated that buformin increased phosphorylation of AMPK and decreased phosphorylation of S6. Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC50 values of 0.1-0.5 nM and 1-5 nM for Ishikawa and ECC-1 cells, respectively. To assess synergy of paclitaxel and buformin, the combination index (CI) was calculated by the method of Chou and Talalay. Simultaneous exposure of cells to various doses of paclitaxel in combination with buformin (0.1-1 μM) resulted in a significant synergistic anti-proliferative effect (CI &amp;lt;1, range 0.009-0.85). Conclusions: Buformin inhibited proliferation in EC cell lines, via AMPK activation and mTOR pathway inhibition. Inhibition of adhesion/invasion was also seen with buformin. Buformin acted synergistically with paclitaxel to inhibit cell proliferation. More work is needed to determine if buformin and other biguanides alone or in combination with cytotoxic agents will be beneficial in the treatment of women with EC. Citation Format: Joshua Kilgore, Amanda Jackson, Haifeng Qiu, Chunxiao Zhou, Paola Gehrig, Victoria Bae-Jump. Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1312. doi:10.1158/1538-7445.AM2014-1312

Abstract 1031: The effects of NT1014, a novel AMPK activator, on ovarian cancer cell proliferation and apoptosis.

Abstract Introduction: Anti-diabetic biguanide drugs, such as metformin, have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. NT1014 (NovaTarg Therapeutics) is a novel AMPK activator that enters cells specifically through the organic cation transporter, OCT1. Thus, we sought to assess the effect of NT1014 on cell proliferation and apoptosis in human ovarian cancer cell lines. Methods: Ovarian cancer cell proliferation was assessed by MTT assay after exposure to NT1014. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by Cellometer. Phosphorylated-S6, pan-S6, and OCT1 were evaluated by Western immunoblotting analysis. Results: NT1014 significantly inhibited proliferation in a dose-dependent manner in both ovarian cancer cell lines (IC50 1000 μM for SKOV3, 500 μM for IGROV1), within 48 hours of exposure. Treatment with NT1014 resulted in G1 cell cycle arrest in both cell lines. Apoptosis occurred in IGROV1 cells exposed to NT1014, but not in SKOV3 cells. Western immunoblot analysis demonstrated that NT1014 decreased phosphorylation of the S6 protein, a key downstream target of the mTOR pathway. Expression of OCT1 was also decreased in a dose-dependent fashion. Conclusion: NT1014 suppressed ovarian cancer cell growth, predominantly through G1 cell cycle arrest, and resulted in inhibition of the mTOR pathway. More work is needed to determine if metformin and related novel agents, such as NT1014, will be beneficial in the treatment of women with ovarian cancer. Citation Format: Joshua Kilgore, Amanda L. Jackson, Haifeng Qiu, Chunxiao Zhou, Kenneth Batchelor, Paola Gehrig, Victoria Bae-Jump. The effects of NT1014, a novel AMPK activator, on ovarian cancer cell proliferation and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1031. doi:10.1158/1538-7445.AM2013-1031

Abstract 2442: Antitumorigenic effects of phenformin in human ovarian cancer cell lines.

Abstract Introduction: Anti-diabetic biguanide drugs, such as metformin, have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Phenformin is another biguanide with anti-diabetic activity that was withdrawn from the market due to a risk of lactic acidosis that was higher than that seen with metformin. In vitro and in vivo studies suggest that phenformin may be more potent for inhibiting tumor growth than metformin. Thus, our objective was to assess the effect of phenformin on proliferation and apoptosis in ovarian cancer cell lines. Methods: Three ovarian cell lines, SKOV3, IGROV1, and HEY, were used. Cell proliferation was assessed by MTT assay after exposure to phenformin and after exposure to phenformin under varying glucose levels. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by Cellometer. Western immunoblotting was performed to determine expression of the downstream targets of phenformin, including AMPK, ribosomal protein S6, cyclin D and E2, cyclin dependent kinase (CDK) 4 and 6, p21 and p27. Inhibition of adhesion by phenformin was assessed by in vitro adhesion assay. Results: Phenformin potently inhibited proliferation in a dose-dependent manner in all three ovarian cancer cell lines (IC50 = 2.5mM for SKOV3 and HEY, 1mM for IGROV1 at 48 hours) (p = 0.0035 – 0.00001). Inhibition of proliferation was found at normal, low and high glucose concentrations, but greater potency was observed under low glucose concentrations. Treatment with phenformin resulted in G1 cell cycle arrest and induction of apoptosis. Cellular adhesion was decreased by 14-32% in the ovarian cancer cell lines at a phenformin dose of 2.5mM (p = 0.009 – 0.0001). Western immunoblot analysis demonstrated that phenformin induced phosphorylation of AMPK, its immediate downstream mediator, within 18 hours of exposure. In parallel, treatment with phenformin decreased phosphorylation of the S6 protein, a key target of the mTOR pathway. Phenformin was also found to increase the cell cycle inhibitors, p21 and p27, and decrease cyclin D, cyclin E2, CDK4 and CDK 6, corresponding with G1 arrest in these cell lines. Conclusion: Phenformin potently inhibited ovarian cancer cell growth via G1 arrest and increased apoptosis. Although the risk/benefit ratio clearly favors metformin over phenformin for the treatment of diabetes, this may not hold true for the treatment of cancer if phenformin were found to have superior anti-tumorigenic activity. Continued work is needed to explore the benefits of both metformin and phenformin for the treatment of ovarian cancer and the optimal therapeutic index. Citation Format: Amanda L. Jackson, Joshua E. Kilgore, Haifeng Qiu, Chunxiao Zhou, Paola A. Gehrig, Victoria L. Bae-Jump. Antitumorigenic effects of phenformin in human ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2013-2442