mM Of Metformin Research Articles

Metformin Improves Burn Wound Healing by Modulating Microenvironmental Fibroblasts and Macrophages.

Metformin, a biguanide, exerts different functions through various signaling pathways. In order to investigate the function and mechanism of metformin in burn wounds, we established burn rat models, subcutaneously injected metformin to treat the wounds, and observed the morphologies and the expression of collagen I, collagen III, fibronectin, and pro-inflammatory markers. In vitro experiments were performed to investigate the effects of metformin on the proliferation, migration, and collagen I synthesis of the mouse embryonic fibroblast (NIH 3T3) cell line and on the proliferation, apoptosis, and immune response of the mouse mononuclear macrophage (RAW 264.7) cell line. Finally, we studied the regulatory effects of metformin on a co-culture of RAW 264.7/NIH 3T3 cells. We found that 100 mM of metformin reduced dermal thickness, collagen I deposition, and mRNA expression of IL1β and CCL2 in rat burn wounds. In vitro experiments revealed that metformin inhibited the proliferation of NIH 3T3 and RAW 264.7 cells. Metformin attenuated NIH 3T3 cell migration via the AMPK/mTOR pathway and attenuated collagen I synthesis through the TGFβ1/Smad3 pathway. Metformin inhibited the apoptosis of RAW 264.7 cells induced by 10 μg/mL LPS. Metformin downregulated the mRNA expression of IL1β and CCL2 in RAW 264.7 cells under 1 μg/mL LPS induction by inhibiting NF-κB p65 phosphorylation. In a RAW 264.7/NIH 3T3 co-culture, metformin attenuated collagen I synthesis in NIH 3T3 cells by inhibiting RAW 264.7 paracrine secretion of TGF-β1. This provides new evidence related to the development of metformin for potentially improving burn wound healing.

Metformin Inhibits STAT3 and MAPK Signaling through AMPK Activation in CSF3RT618I Cells

Introduction: In the light of the discovery of the CSF3R mutation in chronic neutrophilic leukemia (CNL) patients, the T618I mutation demonstrated to be a molecular marker and a possible target for new therapies due to the JAK/STAT signaling pathway activation [N Engl J Med (2013) 368(19):1781-90]. Since no curative treatment options are available for patients diagnosed with CNL, it is of interest the study new therapeutical approaches for these patients. In this regard, the treatment of JAK2V617F murine models with metformin, an FDA-approved drug for type 2 diabetes, reduced splenomegaly, extramedullary hematopoiesis, and colony formation of HSPC [Cell Death Dis. 2018;9(3):311]. Hence, we characterized the preclinical cellular and molecular effects of metformin in CSF3RT618I models. Methods: The in vitro experiments were performed in Ba/F3 cells transduced retrovirally with CSF3RT618I overexpression (OE) model. Cells were treated with metformin (1-20 mM) to access cell viability and survival. Protein expression was evaluated by western blot. For in vivo evaluation, eight-week-old female NSG mice were transplanted with 2x106 Ba/F3 CSF3RT618I cells, resuspended in Matrigel: PBS (1:1 ratio). After the tumor formation (2 days), mice were treated for 10 days by intraperitoneal injection of vehicle (PBS) or metformin (125 mg/kg, n=10/group). Tumor volume was measured using the formula (V = W2 × L × 0.52), where W and L are the smallest and largest diameters, respectively. Ex vivo primary cells from a patient diagnosed with CNL harboring the CSF3RT618I mutation were used to assess the effects of metformin treatment. The primary bone marrow mononuclear cells were evaluated regarding cell survival and clonogenic capacity upon metformin treatment. All experiments were approved by the Animal and Human Ethics Committee of the Institution. Statistical analyses were performed by Mann-Whitney or ANOVA test and Bonferroni post-test. A p-value < 0.05 was considered. Results: After 24 and 48 hours, Ba/F3 CSF3RT618I treated with 1, 5, 10 and 20 mM of metformin presented a reduction in cell viability in a dose- and time-dependent manner (p < 0.01). The IC50 for metformin treatment for 24, 48, and 72 hours was 17.44, 1.3, and 3.2 mM, respectively. Increased frequency of apoptotic cells was observed at 5, 10, and 20 mM of metformin at times of 24, 48, and 72 hours (p < 0.01). Molecularly, CSF3RT618I cells displayed reduced STAT3 and ERK phosphorylation and increased AKT/mTOR activation, upon metformin 5 mM treatment for 24 hours. AMPK activation was observed in cells treated with 1-20 mM of metformin compared to vehicle. In vivo, metformin treatment decreased the tumor burden of CSF3RT618I transplanted mice after 9 days of treatment (vehicle versus metformin mean ± SEM: 731.9±90.14 vs. 481.6±38.14; p = 0.035). At the protein level, we observed inhibition in Cyclin D1 and the presence of caspase 3 cleavage in the isolated tumor from the metformin-treated mice. Ex vivo metformin treatment of the primary cells isolated from the CNL patient harboring CSF3RT618I resulted in increased apoptosis, with 51.94% (p < 0.05), 52.40% (p < 0.05), and 63.1% (p < 0.001), of apoptosis induction upon treatment with 5, 10 and 20 mM. Metformin ex vivo treatment strongly reduced hematopoietic progenitor cells colony formation on primary CNL CSF3RT618I peripheral blood (83.5% at 10 mM and 100% at 20 mM) and bone marrow mononuclear cells (100% at 10 mM and 98% at 20 mM). Conclusions: We provide evidence that metformin exerts anticancer activity in CSF3RT816I cells, by downregulation of STAT3 and MAPK signaling through AMPK activation. These results support the idea that metformin can be repurposed in the CNL clinics, as a therapeutical option.

Synergistic Effects of Metformin-Lapatinib Combination on the Expression of Bax in SK-BR3 Cells

Background: HER2-positive breast cancers tend to be more aggressive and are associated with poorer outcomes than other types of breast cancer. Metformin, the first-line medication for the treatment of type 2 diabetes, has anticancer activity against various cancerous cells. Objectives: We assessed the cytotoxic effect of metformin in combination with Lapatinib on the SK-BR3 cells. Methods: Following culturing cells, IC50 of metformin and lapatinib were calculated using MTT assay after 48h of treatment with different concentrations of metformin and lapatinib alone and in combination. The level of pro-apoptotic protein Bax expression was measured by western blot analysis. Results: Metformin and lapatinib could significantly inhibit the cell viability of SKBR-3 in a dose-dependent manner, and the minimum cytotoxic effect of these drugs was observed after 48 h at 5 mM and 50 nM, respectively. Several combinations of metformin with lapatinib showed a synergistic efficacy on the cell viability inhibition, which could decrease the IC50 of lapatinib from 500 nM to at least 200 nM. The strongest synergistic cytotoxic effect on the cell viability was observed at 40 mM of metformin plus 400 nM of lapatinib. Furthermore, after 48h of co-treatment with metformin-lapatinib combination, the level of pro-apoptotic Bax expression was significantly increased at 10 mM of metformin plus 200 nM of lapatinib. Conclusions: This study demonstrated that the metformin-lapatinib combination may be a valuable candidate for breast cancer patients with HER2 overexpression. However, further studies are needed to make a definitive conclusion.

Metformin attenuates steroidogenesis in ovarian follicles of the broiler breeder hen.

The follicular hierarchy in broiler breeder chicken ovary is often deranged due to excessive ovarian follicular recruitment, resulting in a condition that resembles polycystic ovary syndrome (PCOS) in women. Metformin is widely prescribed to correct PCOS and has been shown to affect granulosa cell functions in humans and rodent models. The objectives of this study are to determine the effects of metformin on signal transduction pathways, gene expression related to steroidogenesis, and progesterone secretion from granulosa cells isolated from the most recently recruited preovulatory and prehierarchical follicles of broiler breeder chickens. Granulosa cells were treated with 0, 1, 10, or 20 mM of metformin in the presence of FSH. The abundance of pAMPK, pACC, pERK, and pAkt was determined by Western blotting. The expression of genes related to progesterone biosynthesis was quantified by qPCR. Progesterone concentrations in culture media were quantified by ELISA. Metformin treatment did not have an effect on the abundance of pAMPK and pACC in prehierarchical follicles but significantly decreased the abundance of pERK and pAkt in a dose-dependent manner in preovulatory and prehierarchical follicles. The expression of genes related to steroidogenesis such as FSHR, STAR, CYP11A1, HSD3B, and progesterone secretion was significantly decreased in response to metformin treatment in a dose-dependent manner. Our data suggest that metformin treatment attenuates progesterone secretion via AMPK-independent pathways in granulosa cells of prehierarchical and preovulatory follicles of broiler breeder hens. Further studies are required to determine if metformin administration could ameliorate ovarian dysfunction in obese broiler breeder hens.

A Novel Mechanism of High Dose Radiation Sensitization by Metformin.

Introduction: Metformin, the most widely used treatment for diabetes, is lethal to cancer cells and increases in toxicity when used in combination with radiation. In addition to various molecular and metabolic mechanisms that have been previously proposed, the studies presented provide evidence of an additional, novel mechanism of sensitization following high dose radiotherapy; the magnitude of sensitization depends on the microenvironmental levels of glucose and oxygen which are in turn affected by high dose radiation.Methods: Cancer cells (A549 and MCF7) were studied in vitro under various controlled conditions. Endpoints included clonogenic cell survival and ROS expression measured by DHE and DCFDA. CD1 nu/nu athymic mice implanted with A549 cells received metformin alone (200 mg/kg, i.p.), radiation alone (15 Gy) or a combination of metformin and radiation; the effect of treatment sequence on efficacy was assessed by tumor growth delay and histology. In a separate set of experiments, tumor blood flow was measured using a tracer clearance technique using SPECT after the administration of metformin alone, radiation alone and the combined treatment.Results: In vivo, metformin provided equally effective tumor growth delay when given 24 h after radiation as when given 1 h or 4 h before radiation, an observation not previously reported and, in fact, unexpected based on published scientific literature. When drug followed radiation, the tumors were histologically characterized by massive cellular necrosis. In vitro, cancer cells when glucose depleted and/or hypoxic were preferentially killed by metformin, in a drug dose dependent manner. A549 cells exposed to 5.0 mM of metformin was reduced seven fold in survival when in a glucose deprived as compared to a low-glucose medium (0 vs. 1.0 g/L). Finally, using a SPECT detector to follow the washout of a radioactive tracer, it was shown that a high single dose of radiosurgery (15 Gy) could dramatically inhibit blood flow and presumably diminish glucose and oxygen.Discussion: Insight into the best timing of drug and radiation administration is gained through an understanding of the mechanisms of interaction. A new mechanism of metformin sensitization by high dose radiation is proposed based on the blood flow, glucose and oxygen.

Inhibition of Chloride Intracellular Channel 1 (CLIC1) as Biguanide Class-Effect to Impair Human Glioblastoma Stem Cell Viability.

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC50 values significantly lower than metformin, (range 0.054–0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanide-structured molecules, characterized by high antitumor efficacy and safe toxicological profile.

Metabolic changes associated with metformin potentiates Bcl-2 inhibitor, Venetoclax, and CDK9 inhibitor, BAY1143572 and reduces viability of lymphoma cells.

Metformin exerts direct anti-tumor effects by activating AMP-activated protein kinase (AMPK), a major sensor of cellular metabolism in cancer cells. This, in turn, inhibits pro-survival mTOR signaling. Metformin has also been shown to disrupt complex 1 of the mitochondrial electron transport chain. Here, we explored the lymphoma specific anti-tumor effects of metformin using Daudi (Burkitt), SUDHL-4 (germinal center diffuse large B-cell lymphoma; GC DLBCL), Jeko-1 (Mantle-cell lymphoma; MCL) and KPUM-UH1 (double hit DLBCL) cell lines. We demonstrated that metformin as a single agent, especially at high concentrations produced significant reductions in viability and proliferation only in Daudi and SUDHL-4 cell lines with associated alterations in mitochondrial oxidative and glycolytic metabolism. As bcl-2 proteins, cyclin dependent kinases (CDK) and phosphoinositol-3- kinase (PI3K) also influence mitochondrial physiology and metabolism with clear relevance to the pathogenesis of lymphoma, we investigated the potentiating effects of metformin when combined with novel agents Venetoclax (bcl-2 inhibitor), BAY-1143572 (CDK9 inhibitor) and Idelalisib (p110δ- PI3K inhibitor). Co-treating KPUM-UH1 and SUDHL-4 cells with 10 mM of metformin resulted in 1.4 fold and 8.8 fold decreases, respectively, in IC-50 values of Venetoclax. By contrast, 3-fold and 10 fold reduction in IC-50 values of BAY-1143572 in Daudi and Jeko-1 cells respectively was seen in the presence of 10 mM of metformin. No change in IC-50 value for Idelalisib was observed across cell lines. These data suggest that although metformin is not a potent single agent, targeting cancer metabolism with similar but more effective drugs in novel combination with either bcl-2 or CDK9 inhibitors warrants further exploration.

Metformin Reshapes the Methylation Profile in Breast and Colorectal Cancer Cells

With no sharp cure, breast cancer still be the major and the most serious life-threatening disease worldwide. Colorectal is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women. In the present investigation, colon cancer cells (CaCo-2) and breast cancer cells (MCF-7) were treated with elevated doses of metformin (MET) for 48h. Cell count was assessed using trypan blue test, and the cytotoxicity was evaluated using MTT assay. Methylation-specific PCR was performed on the bisulfite-treated DNA against two tumor suppressor genes; RASSF1A and RB. Results indicated that: in breast cancer, the cell count was decreased significantly (P>0.005) after being treated with 5, 10, 20, 50, and 100 mM of MET. The elevated concentration had increased reduction percentages on the MCF-7 cells, as 5 mM and 100 mM have yielded 35% and 93.3% reduction in cell viability, respectively. Colon cancer cells have responded to the doses of MET differently, as for the 5 mM and the 100 mM, it gave 88% and 60% reduction in cells viability, respectively. Cytotoxicity assay revealed that 5 mM and 100 mM of MET caused breast cancer cells to loss 61.53% and 85.16% of its viability, respectively, whereas colon cancer cells have responded to the 5 mM and 100 mM of MET by reducing the cells viability with 96.91% and 96.24%, respectively. No RB promoter methylation was detected in colon cells, while RASSF1A was partially methylated. In the MCF-7 breast cancer cells, both RASSF1A and RB were partially methylated.

Enhanced cytocompatibility and osteoinductive properties of sol-gel-derived silica/zirconium dioxide coatings by metformin functionalization.

The aim of this study was to evaluate the pro-osteogenic properties of sol-gel-derived silica/zirconium dioxide coatings functionalized with 1 mM of metformin. The matrices were applied on 316L stainless steel using dip-coating technique. First of all, physicochemical properties of biomaterials were evaluated. Surface morphology and topography was determined using energy-dispersive X-ray spectroscopy and atomic force microscopy. The chemical composition was evaluated using Fourier transform infrared spectroscopy. Further, wettability and surface free energy were characterized. Cytocompatibility of biomaterials was tested invitro using model of human multipotent mesenchymal stromal cells isolated from adipose tissue. The influence of biomaterials on cells morphology and proliferation was determined. Osteogenic effect of obtained biomaterials was evaluated in terms of their influence on secretory activity of human multipotent mesenchymal stromal cells isolated from adipose tissue and matrix mineralization. Analysis was performed in relation to the control cultures i.e. maintained on pure SS316L substrate and SS316L covered with silica/zirconium dioxide. Obtained results indicate that silica/zirconium dioxide_metformin coatings ameliorated metabolic and proliferative activity of human multipotent mesenchymal stromal cells isolated from adipose tissue, as well as promoted their proper growth and adhesion. The human multipotent mesenchymal stromal cells isolated from adipose tissue cultured on biomaterials were characterized by typical fibroblast-like morphology. The addition of metformin to the silica/zirconium dioxide coatings improved functional differentiation of human multipotent mesenchymal stromal cells isolated from adipose tissue. Osteogenic cultures on silica/zirconium dioxide_metformin were characterized by formation of well-developed osteonodules rich in calcium and phosphorous. Moreover, human multipotent mesenchymal stromal cells isolated from adipose tissue cultured on silica/zirconium dioxide_metformin synthesized increased amount of alkaline phosphatase, bone morphogenetic protein 2 and osteopontin, both on messenger RNA and protein level. Obtained biomaterials modulate cellular plasticity of human multipotent mesenchymal stromal cells isolated from adipose tissue promoting their osteogenic differentiation, thus may find application in broadly defined tissue engineering.

Abstract 4742: Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists

Abstract We have already found that a nuclear receptor PXR can modulate the cytotoxicity of CDDP for cancer cells, and ketoconazole, a PXT antagonist, can be an agent enhancing the antitumor activity of CDDP due to increase of the intracellular platinum content as the mechanism. However, nuclear receptor ligands have a variety of functions, and there is a possibility that PXR antagonist may enhance the cytotoxicity of CDDP through the mechanism other than the inhibition of transporter-mediated efflux of CDDP. The goal of this study was to compare the enhancing ability of three PXR antagonists, ketoconazole (KTZ), phenethyl isothiocyanate (PEITC) and metformin (MFM), for the antitumor effect of CDDP and clarify the contribution of inhibition of the efflux transporter as the mechanism of PXR antagonist. Cell lines used in this study were human hepatoma cell line HepG2. Changes in mRNA expression were assessed by real-time RT-PCR, and the ability of apoptosis induction was assessed as caspase-3 activity. Total intracellular platinum contents were determined by ICP-AES. When HepG2 cells were treated for 24 hr with 10 μM of CDDP, no change of caspase-3 activity was observed when compared to control cells. Also, 30 μM of KTZ, 30 μM of PEITC or 5 mM of MFM had no affect on the caspase-3 activity in treated cells compared with control cells. When KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 24-hr treatment with 10 μM of CDDP, the caspase-3 activity was significantly increased to 461%, 355% and 326% of the control, respectively, in comparison with that in CDDP alone. When HepG2 cells were treated for 8 hr with 25 μM of CDDP, the intracellular platinum content was 110 ng/mg protein. On the other hand when KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 8-hr treatment with 25 μM of CDDP, the intracellular platinum content was apparently increased to 227%, 491% and 161% of CDDP alone, respectively. Three PXR antagonists resulted in an increase in the intracellular platinum content that seems to be due to inhibition of the platinum efflux transporter, but there is no correlation between increase in intracellular platinum content and enhancement of antitumor activity by PXR antagonists. These results concluded that PXR antagonist can be an agent enhancing the antitumor activity of CDDP, but the ability of PXR antagonist for enhancing antitumor activity of CDDP can not be determined from only the increase in the intracellular platinum content. Citation Format: Shuichi Kishimoto, Megumi Yasuda, Megumi Tomita, Yuki Yamane, Ryosuke Suzuki, Shoji Fukushima. Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists. [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 4742.

Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line.

Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure.

Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells.

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.

Abstract P6-04-22: Metformin is an effective drug to target the bevacizumab driven cancer stem cell stimulation in murine 4T1 breast cancer cell line

Abstract Hypoxia-driven cancer stem cell (CSC) stimulation limits the effectiveness of antiangiogenic agents in breast cancer. The use of antiangiogenic drugs in combination with CSC-targeting drugs is a reasonable strategy to optimize cancer treatment. Metformin has been demonstrated to be efficient against multiple forms of cancer. Recent reports have demonstrated metformin can inhibit CSCs proliferation. We analyzed the influence of metformin and its combination with bevacizumab on CSC population in breast cancer. The MDA-MB-231, MCF7, 67nr and 4T1 cell lines were exposed to different concentration of metformin (0 mM, 1 mM, 5mM and 10 mM) in vitro during 72 hours. Using the trypan blue exclusion assay, we estimated the percentage of dead cells in culture. We observed a dose-time dependent citotoxicity of metformin among all cell lines. Using a dose of 10 mM of metformin, the 4T1 cells were cultured for 48 hours and the flow cytometry was performed to analyze the PI / Anexin expression. The mean percentage of PI+ cells in the control group was 12.8 ± 1.2%. The mean percentage of PI+ in metformin treated group was 22.7 ± 2.2% (p = 0.005). We did not observe any difference in anexin expression. To determine the role of ALDH expression as a marker for breast CSCs in 4T1 cell line, we sorted Aldefluorhigh 4T1 cells and performed a limiting dilution assay. We observed the prevalence of CSCs in Aldefluorhigh 4T1 cells was 1/455 cells (CI 95% from 1,948 to 107). The prevalence in Aldefluor- and the Aldefluorwt 4T1 cells was 1/5,361 cells (CI 95% from 21,557 to 1,333) and 1/2,086 cells (CI 95% from 7,466 to 583), respectively (p = 0.06). The pairwise test for differences in stem cell frequency between Aldefluorhigh and Aldefluor- cells was significant (p = 0.01). In order to determine whether metformin interferes with breast CSC population in vitro and in vivo, we treated 4T1 cells in culture (10 nM) and 4T1 tumor-bearing mice with intraperitoneal (n = 4) metformin (2,100 mg/kg/day) alone or in combination with bevacizumab (5mg/kg s.c. twice a week). In the in vitro assay, the mean percentage of Aldefluorhigh 4T1 cells was 28.8 ± 4.06% in the control group. After 48 hours of metformin exposure, the mean percentage of Aldefluorhigh 4T1 cells was 15.6 ± 3.5% (p = 0.01). In the in vivo assay, tumors were smaller in mice treated with metformin or bevacizumab alone or in combination. However, there is a significant increase in Aldefluorhigh cell population (p = 0.04) and ALDH1A1 gene expression (real time RT-PCR; p&amp;lt; 0.0001) in bevacizumab treated tumors. We did not observed an increase in Aldefluorhigh cell population and ALDH1A1 gene expression in tumors from mice treated with metformin/bevacizumab combination. Our results demonstrate metformin is an effective drug to target the bevacizumab driven cancer stem cell stimulation in murine 4T1 breast cancer cell line. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-22.

Metformin Induces G1 Cell Cycle Arrest and Inhibits Cell Proliferation in Nasopharyngeal Carcinoma Cells

It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP-activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1-50 mM of metformin in a dose- and time-dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666-1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time-dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1) (T389), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (T37/46) and S6 (S235/236) in C666-1 cells. In a summary, metformin prevents proliferation of C666-1 cells by down-regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK-mediated inhibition of mTORC1 signaling may be involved in this process.

Abstract 1748: Metformin overcomes cisplatin-resistance in triple negative breast cancer

Abstract Background: Triple negative breast cancers (TNBC) account for approximately 15% of breast cancer but 30% of deaths. The majority of TNBC are basal breast cancers that have higher rate of relapses as compared with other breast cancer subtypes, indicating there exists a sub-population of tumor cells that are highly resistant to chemotherapies. Platinum-based agents and PARP inhibitors have shown efficacy in basal breast cancers with BRCA1/2 mutation background. In this study, we expand the analysis to basal breast cancer lines without BRCA1/2 mutations. Results and Methods: We treated a panel of basal breast cancer lines (HCC70, MDA-MB-468, BT549, HCC1143) with cisplatin and observed that 6 µM of cisplatin were able to kill &amp;gt;99% of a confluent plate of these cell lines. The cisplatin-resistant population, however, behaved very differently in these lines. Cisplatin-resistant MDA-MB-468 cells had similar phenotype as cisplatin-sensitive cells and they repopulated the whole plate in a short time. Cisplatin-resistant cells in the other 3 TNBC lines had a different phenotype than the cisplatin-sensitive cells and remained quiescent for 2-3 weeks before proliferating in a vertical fashion. The vertical string of cells eventually detached from the plate and kept expanding as clusters of cells in suspension. Screening through various drugs, we observed that 2 mM of metformin as a combined treatment with cisplatin was able to irradicate these cisplatin-resistant cells in both MDA-MB-468 and BT549 cell lines. Additionally, we demonstrated that using aminoimidazole carboxamide ribonucleotide (AICAR), an AMP kinase (AMPK) activator, and Rapamycin, an mTORC1 inhibitor had the same effect as metformin indicating the AMPK-mTOR axis might be important for the survival of cisplatin-resistant cells. Conclusions: TNBC without BRCA1/2 mutation are sensitive to cisplatin treatment but possess a sub-population of resistant cells. We demonstrated that cisplatin-resistant cells in TNBC lines behaved differently after cisplatin treatment and that combined treatment of metformin and cisplatin may be able to overcome this resistance. Future experiments include comparing this combination with the combined treatment of cisplatin and PARP inhibitor, which might have effect in cisplatin-sensitive cells but not in cisplatin-resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2011-1748

Phosphorylation and Activation of AMP-Activated Protein Kinase (AMPK) by Metformin in the Human Ovary Requires Insulin

Metformin is commonly used to treat women with polycystic ovary syndrome, but its precise mechanism of action is unclear, and it even appears to have direct ovarian effects. At the cellular level, it may act either via an insulin-dependent pathway or an independent pathway by activating AMP-activated protein kinase (AMPK). In the ovary, metformin directly decreased estradiol and progesterone production by human granulosa cells, and inhibition of progesterone production by metformin in rat granulosa cells caused an increase in phosphorylated AMPK (pAMPK). We investigated whether metformin activates AMPK in the human ovary by looking for changes in phosphorylation of AMPK and its downstream target acetyl CoA carboxylase (ACC). mRNA and protein for α1 and α2 AMPK subunits were present in all human ovarian tissue. Neither 100 nm nor 2 mm of metformin affected subunit expression. After 1 or 4 h, neither dose of metformin increased pAMPK or pACC, although after 1 h, the addition of insulin significantly enhanced pAMPK, whereas insulin alone had no effect on phosphorylation of either AMPK or ACC. The addition of compound C, an inhibitor of AMPK, negated the effect of metformin in the presence of insulin on pAMPK. This effect on AMPK was not due to a change in the ADP/ATP ratio measured by HPLC. In summary, the presence of insulin was required to cause a metformin-induced increase in pAMPK in these human ovarian cells. Although previous data suggest that metformin may act via an insulin-independent pathway, our results therefore imply that insulin may be required to initiate an effect.