Reversal of obesity-driven aggressiveness of endometrial cancer by metformin.

BACKGROUND: Obesity and diabetes are associated with increased risk and worse outcomes for endometrial cancer. Metformin is a widely prescribed generic drug for the treatment of type II diabetes and metabolic syndrome and may also have anti-tumorigenic effects. Thus, we assessed the metabolic anti-tumorigenic effects of metformin in (1) human endometrial cancer cell lines under varying glucose concentrations, and (2) a novel genetically engineered mouse model of endometrioid endometrial cancer under obese and lean conditions. METHODS: The effects of metformin on cytotoxicity, apoptosis, cell cycle progression, and the AMPK/mTOR/S6 and MAPK pathways were assessed in ECC-1 and Ishikawa cells under low, normal and high glucose conditions. The impact of metformin treatment on tumor growth under obese and lean conditions was evaluated using a novel LKB1fl/fl p53fl/fl mouse model of endometrial cancer. Global, untargeted metabolomics was used to identify (1) obesity-associated differences between endometrial tumors and (2) the obesity-dependent effects of metformin in the endometrial tumors. RESULTS: Hypoglycemic conditions significantly enhanced the sensitivity of the cells to metformin in regards to its anti-proliferative and apoptotic effects, as compared to hyperglycemic and normal glucose conditions. Metformin inhibited tumor growth in both the obese and lean mice, which metformin-induced inhibition of tumor progression in obese mice was significantly greater than in lean mice. Metabolomic profiling in endometrial cancer tissues revealed significant differences between obese- and lean-mice. Enhanced energy metabolism was seen in obese- versus lean-mice as evidenced by increases in glycolytic and oxidative phosphorylation intermediates. In addition, dramatic increases in lipid biosynthesis and lipid peroxidation were found in the obese- versus lean-mice, whereas metformin obviously reversed the obesity-driven upregulation of lipid and protein biosynthesis in the obese mice. CONCLUSIONS: The obese state promoted tumor aggressiveness in the LKB1fl/fl p53fl/fl mouse model, accompanied by increases in energy metabolism, lipid biosynthesis, and markers of lipid peroxidation. Metformin had increased efficacy against endometrial cancer in obese versus lean mice and reversed the detrimental metabolic effects of obesity in the endometrial tumors. Taken together, it is likely that the unique metabolic milieu underlies metformin's improved efficacy in treating endometrial cancer which develop in an obese host environment.

Erratum: Atorvastatin exhibits anti-tumorigenic and anti-metastatic effects in ovarian cancer in vitro.

[This corrects the article on p. 2478 in vol. 7, PMID: 29312801.].

Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in an orthotopic mouse model of serous ovarian cancer.

Obesity and diabetes have been associated with increased risk and worse outcomes in ovarian cancer (OC). The biguanide metformin is used in the treatment of type 2 diabetes and is also believed to have anti-tumorigenic benefits. Metformin is highly hydrophilic and requires organic cation transporters (OCTs) for entry into human cells. Phenformin, another biguanide, was taken off the market due to an increased risk of lactic acidosis over metformin. However, phenformin is not reliant on transporters for cell entry; and thus, may have increased potency as both an anti-diabetic and anti-tumorigenic agent than metformin. Thus, our goal was to evaluate the effect of phenformin on established OC cell lines, primary cultures of human OC cells and in an orthotopic mouse model of high grade serous OC. In three OC cell lines, phenformin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, caused cellular stress, inhibited adhesion and invasion, and activation of AMPK and inhibition of the mTOR pathway. Phenformin also exerted anti-proliferative effects in seven primary cell cultures of human OC. Lastly, phenformin inhibited tumor growth in an orthotopic mouse model of serous OC, coincident with decreased Ki-67 staining and phosphorylated-S6 expression and increased expression of caspase 3 and phosphorylated-AMPK. Our findings demonstrate that phenformin has anti-tumorigenic effects in OC as previously demonstrated by metformin but it is yet to be determined if it is superior to metformin for the potential treatment of this disease.

Dual inhibition of glycolysis and glutaminolysis as a therapeutic strategy in the treatment of ovarian cancer.

Cancer cell metabolism is required to support the biosynthetic demands of cell growth and cell division, and to maintain reduction oxidaton (redox) homeostasis. This study was designed to test the effects of glucose and glutamine on ovarian cancer cell growth and explore the inter-relationship between glycolysis and glutaminolysis. The SKOV3, IGROV-1 and Hey ovarian cancer cell lines were assayed for glucose, pyruvate and glutamine dependence by analyzing cytotoxicity, cell cycle progression, apoptosis and ATP production. As determined by MTT assay, glucose stimulated cell growth while the combination of glucose, glutamine and pyruvate resulted in the greatest stimulation of cell proliferation. Furthermore, 2-deoxy-glucose (2-DG) and 3-bromopyruvate (3-BP) induced apoptosis, caused G1 phase cell cycle arrest and reduced glycolytic activity. Moreover, 2-DG in combination with a low dose of aminooxyacetate (AOA) synergistically increased the sensitivity to 2-DG in the inhibition of cell growth in the ovarian cancer cell lines. These studies suggest that dual inhibition of glycolysis and glutaminolysis may be a promising therapeutic strategy for the treatment of ovarian cancer.

Atorvastatin exhibits anti-tumorigenic and anti-metastatic effects in ovarian cancer in vitro.

Ovarian cancer is the 8th most common cancer in women, and the 5th leading cause of cancer-related deaths among women in the United States. Statins have been shown to have promising anti-tumorigenic activity in many types of cancers. We sought to determine the effects of atorvastatin (ATO) on cell proliferation in ovarian cancer and identify the mechanisms by which ATO inhibits cell growth in this disease. ATO inhibited cell proliferation of both the Hey and SKOV3 ovarian cancer cells in a dose-dependent manner. The anti-proliferative activity of ATO in the ovarian cancer cell lines was associated with induction of apoptosis, autophagy, cellular stress and cell cycle G1 arrest via inhibition of AKT/mTOR and activation of the MAPK pathways. Moreover, ATO inhibited cell adhesion and invasion as well as decreased expression of VEGF and MMP9. c-Myc was downregulated in ovarian cancer cells exposed to ATO. Inhibition of c-Myc by JQ1 synergistically increased the sensitivity of ovarian cancer cells to ATO. This data suggests that ATO may have a therapeutic role in the treatment of ovarian cancer and warrant further exploration in clinical trials.

NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo.

BACKGROUND: NT1014 is a novel biguanide and AMPK activator with a high affinity for the organic cation-specific transporters, OCT1 and OCT3. We sought to determine the anti-tumorigenic effects of NT1014 in human ovarian cancer cell lines as well as in a genetically engineered mouse model of high-grade serous ovarian cancer. METHODS: The effects of NT1014 and metformin on cell proliferation were assessed by MTT assay using the human ovarian cancer cell lines, SKOV3 and IGROV1, as well as in primary cultures. In addition, the impact of NT1014 on cell cycle progression, apoptosis, cellular stress, adhesion, invasion, glycolysis, and AMPK activation/mTOR pathway inhibition was also explored. The effects of NT1014 treatment in vivo was evaluated using the K18 - gT121+/-; p53fl/fl; Brca1fl/fl (KpB) mouse model of high-grade serous ovarian cancer. RESULTS: NT1014 significantly inhibited cell proliferation in both ovarian cancer cell lines as well as in primary cultures. In addition, NT1014 activated AMPK, inhibited downstream targets of the mTOR pathway, induced G1 cell cycle arrest/apoptosis/cellular stress, altered glycolysis, and reduced invasion/adhesion. Similar to its anti-tumorigenic effects in vitro, NT1014 decreased ovarian cancer growth in the KpB mouse model of ovarian cancer. NT1014 appeared to be more potent than metformin in both our in vitro and in vivo studies. CONCLUSIONS: NT1014 inhibited ovarian cancer cell growth in vitro and in vivo, with greater efficacy than the traditional biguanide, metformin. These results support further development of NT1014 as a useful therapeutic approach for the treatment of ovarian cancer.

Buformin exhibits anti-proliferative and anti-invasive effects in endometrial cancer cells.

OBJECTIVE: Biguanides are anti-diabetic drugs that are thought to have anti-tumorigenic effects. Most pre-clinical studies have focused on metformin for cancer treatment and prevention; however, buformin may be potentially more potent than metformin. Given this, our goal was to evaluate the effects of buformin on cell growth, adhesion and invasion in endometrial cancer cell lines. METHODS: The ECC-1 and Ishikawa endometrial cancer cell lines were used. Cell proliferation was assessed by MTT assay. Apoptosis and cell cycle analysis was performed by FITC Annexin V assay and propidium iodide staining, respectively. Adhesion was analyzed using the laminin adhesion assay. Invasion was assessed using the transwell invasion assay. The effects of buformin on the AMPK/mTOR pathway were determined by Western immunoblotting. RESULTS: Buformin and metformin inhibited cell proliferation in a dose-dependent manner in both endometrial cancer cell lines. IC50s were 1.4-1.6 mM for metformin and 8-150 µM for buformin. Buformin induced cell cycle G1 phase arrest in the ECC-1 cells and G2 phase arrest in the Ishikawa cells. For both ECC-1 and Ishikawa cells, treatment with buformin resulted in induction of apoptosis, reduction in adhesion and invasion, activation of AMPK and inhibition of phosphorylated-S6. Buformin potentiated the anti-proliferative effects of paclitaxel in both cell lines. CONCLUSION: Buformin has significant anti-proliferative and anti-metastatic effects in endometrial cancer cells through modulation of the AMPK/mTOR pathway. IC50 values were lower for buformin than metformin, suggesting that buformin may be more potent for endometrial cancer treatment and worthy of further investigation.

Everolimus exhibits anti-tumorigenic activity in obesity-induced ovarian cancer.

Everolimus inhibits mTOR kinase activity and its downstream targets by acting on mTORC1 and has anti-tumorigenic activity in ovarian cancer. Clinical and epidemiologic data find that obesity is associated with worse outcomes in ovarian cancer. In addition, obesity leads to hyperactivation of the mTOR pathway in epithelial tissues, suggesting that mTOR inhibitors may be a logical choice for treatment in obesity-driven cancers. However, it remains unclear if obesity impacts the effect of everolimus on tumor growth in ovarian cancer. The present study was aimed at evaluating the effects of everolimus on cytotoxicity, cell metabolism, apoptosis, cell cycle, cell stress and invasion in human ovarian cancer cells. A genetically engineered mouse model of serous ovarian cancer fed a high fat diet or low fat diet allowed further investigation into the inter-relationship between everolimus and obesity in vivo. Everolimus significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, reduced invasion and caused cellular stress via inhibition of mTOR pathways in vitro. Hypoglycemic conditions enhanced the sensitivity of cells to everolimus through the disruption of glycolysis. Moreover, everolimus was found to inhibit ovarian tumor growth in both obese and lean mice. This reduction coincided with a decrease in expression of Ki-67 and phosphorylated-S6, as well as an increase in cleaved caspase 3 and phosphorylated-AKT. Metabolite profiling revealed that everolimus was able to alter tumor metabolism through different metabolic pathways in the obese and lean mice. Our findings support that everolimus may be a promising therapeutic agent for obesity-driven ovarian cancers.

The HMG-CoA reductase inhibitor, simvastatin, exhibits anti-metastatic and anti-tumorigenic effects in ovarian cancer.

Ovarian cancer is the 5th leading cause of cancer death among women in the United States. The mevalonate pathway is thought to be a potential oncogenic pathway in the pathogenesis of ovarian cancer. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) inhibitor, is a widely used drug for inhibiting the synthesis of cholesterol and may also have anti-tumorigenic activity. Our goal was to evaluate the effects of simvastatin on ovarian cancer cell lines, primary cultures of ovarian cancer cells and in an orthotopic ovarian cancer mouse model. Simvastatin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, and caused cellular stress via reduction in the enzymatic activity of HMGCR and inhibition of the MAPK and mTOR pathways in ovarian cancer cells. Furthermore, simvastatin induced DNA damage and reduced cell adhesion and invasion. Simvastatin also exerted anti-proliferative effects on primary cell cultures of ovarian cancer. Treatment with simvastatin in an orthotopic mouse model reduced ovarian tumor growth, coincident with decreased Ki-67, HMGCR, phosphorylated-Akt and phosphorylated-p42/44 protein expression. Our findings demonstrate that simvastatin may have therapeutic benefit for ovarian cancer treatment and be worthy of further exploration in clinical trials.

Simvastatin, an HMG-CoA reductase inhibitor, exhibits anti-metastatic and anti-tumorigenic effects in endometrial cancer.

OBJECTIVE: Our goal was to evaluate the effects of simvastatin on endometrial cancer cell lines and primary cultures of endometrial cancer cells. METHODS: Cell proliferation in the ECC-1 and Ishikawa endometrial cancer cell lines and primary cultures of endometrial cancer cells was assessed by MTT assay. Apoptosis and cell cycle were detected by Annexin V assay and propidium iodide staining, respectively. Reactive oxygen species and cell adhesion were assessed using ELISA assays. Invasion was analyzed using a transwell invasion assay. Mitochondrial DNA damage was confirmed using qPCR. The effects of simvastatin on the AKT/mTOR and MAPK pathways were determined by Western blotting. RESULTS: Simvastatin inhibited cell proliferation in a dose-dependent manner in both endometrial cancer cell lines and 5/8 primary cultures of endometrial cancer cells. Simvastatin treatment resulted in G1 cell cycle arrest, a reduction in the enzymatic activity of HMG-CoA, induction of apoptosis as well as DNA damage and cellular stress. Treatment with simvastatin resulted in inhibition of the MAPK pathway and exhibited differential effects on the AKT/mTOR pathway in the ECC-1 and Ishikawa cells. Minimal change in AKT phosphorylation was seen in both cell lines. An increase in phosphorylated S6 was seen in ECC-1 and a decrease was seen in Ishikawa. Treatment with simvastatin reduced cell adhesion and invasion (p<0.01) in both cell lines. CONCLUSION: Simvastatin had significant anti-proliferative and anti-metastatic effects in endometrial cancer cells, possibly through modulation of the MAPK and AKT/mTOR pathways, suggesting that statins may be a promising treatment strategy for endometrial cancer.