Metformin Inhibited Cell Proliferation Research Articles

Metformin regulates autophagy via LGMN to inhibit choriocarcinoma

Choriocarcinoma has the problem of chemotherapy insensitivity and recurrence. Metformin may be a promising candidate to restrict choriocarcinoma progress because of its indirect and direct beneficial role on inhabitations of cancer cells without severe adverse side effects. In this study, metformin pressed the proliferation and invasion of choriocarcinoma JAR cells in vitro and the growth of the JAR subcutaneous xenografts in vivo. The high throughput sequencing and bioinformatics technology identified the low expression of legumain (LGMN) in lysosomal pathway caused by metformin, which was upregulated in human choriocarcinoma tissues compared with the early pregnancy tissues. As elevating metformin concentration and treatment time, the mRNA and protein expression of LGMN both depressed in two choriocarcinoma cell lines (JAR and JEG-3). LGMN was involved in metformin-mediated inhibition of cell proliferation and invasion. Furthermore, metformin induced autophagy via inhibiting LGMN through AKT/mTOR/LC3II signaling pathway of choriocarcinoma. Autophagy inhibitor could depress metformin-induced autophagy and improve cell proliferation and invasion ability dropped by metformin, while autophagy inducer could partially reverse the change of cell proliferation and invasion modulated by combination of metformin and LGMN overexpression. These results indicated that metformin inhibited cell proliferation and invasion ability by inducing autophagy in a LGMN-dependent manner so as to play a role in the treatment of choriocarcinoma.

Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.

Metformin inhibits proliferation of oral squamous cell carcinoma cells by suppressing proteolysis of nerve growth factor receptor

ObjectiveWe aimed to explore the effects of metformin on oral squamous cell carcinoma (OSCC) cell proliferation and the associated molecular mechanisms. MethodsWe established an OSCC model in SCC15 cells overexpressing nerve growth factor receptor (NGFR) or the N-terminal region (aa 1–250; NGFR-N), and assessed cell proliferation by CCK-8 assay, colony formation assay, and cell cycle analysis. Levels of NGFR and related genes and proteins were detected by qPCR and western blotting, and NGFR and NGFR-N affinity for p53 was assessed by immunoprecipitation assay. Additionally, the effects of NGFR and NGFR-N on p53 binding with its downstream gene promoters were analyzed by chromatin immunoprecipitation. ResultsMetformin inhibited OSCC cell proliferation and blocked NGFR proteolysis, thereby reducing the generation of its intracellular domain and NGFR-N. Moreover, compared with NGFR, NGFR-N showed higher affinity for p53 and more strongly inactivated p53 to promote cell proliferation. Furthermore, upregulation of NGFR-N downregulated levels of p53-specific downstream transcripts and proteins, whereas these levels were significantly upregulated in metformin-treated cells overexpressing NGFR. ConclusionsThese results showed that metformin inhibited cell proliferation by suppressing NGFR proteolysis, thereby promoting its antitumor effect in OSCC and offering novel insight into a role for metformin in OSCC treatment.

Metformin Treatment Sensitizes Human Laryngeal Cancer Cell Line Hep- 2 to 5-Fluorouracil

Background: Larynx cancer (LCa) is the most common head and neck cancer and accounts for 1-2.5% of all human cancers worldwide. Metformin, an oral anti-diabetic drug, has been recently shown to have anti-cancer activity in various cancer types, and there are several studies in the literature pointing to its potential to sensitize cancer cells to chemotherapeutic drugs. Objective: This study was aimed at exploring the anti-cancer effects of metformin alone or in combination with 5-fluorouracil (5-FU) on Hep-2 cells. Methods: The effects of metformin and/or 5-FU on the proliferative, clonogenic, and apoptotic potential of Hep-2 cells were evaluated with Cell Viability Detection Kit-8, soft agar assay and Annexin VFITC Apoptosis assay, respectively. Migratory and invasive potential of cells was tested using scratch, transwell migration and Matrigel invasion assays. Gene expression of cells exposed to metformin and/or 5-FU was profiled using RT2 mRNA PCR Array plates. Results: Treatment of Hep-2 cells with metformin inhibited cell proliferation by inducing apoptosis, and suppressed cell migration. Besides, treatment of metformin along with 5-FU improved the antiproliferative and anti-migratory effects of 5-FU. However, unexpectedly, metformin was found to enhance cellular invasion and reverse the inhibitory effect of 5-FU on the invasive potential of Hep-2 cells. Conclusion: Our findings suggest that metformin might be used as an adjuvant agent in the treatment of LCa. However, the potential of metformin to promote the invasion of cancer cells should not be neglected.

Metformin inhibits cell proliferation in SKM-1 cells via AMPK-mediated cell cycle arrest

Metformin, a widely used antidiabetic drug, has previously been demonstrated to exert anti-cancer effects in certain hematological malignancies, but its effects on the transformation of myelodysplastic syndromes to acute myeloid leukemia (AML-MDS) remain unclear. The present study aimed to investigate the effects of metformin on SKM-1 cells (an AML-MDS cell line) and its underlying mechanisms. SKM-1 cells were treated with different concentrations of metformin. Cell proliferation was assayed by CCK-8. Apoptosis and cell cycle phases were detected by flow cytometry, while cell cycle related proteins and AMPK were tested by Western blot. SKM-1 cells were transfected with LV-AMPKα1-RNAi to reduce the expression of AMPK. Metformin inhibited cell proliferation in a dose and time dependent manner by inducing G0/G1 phase arrest rather than apoptosis induction. Metformin promoted the expression of p-AMPK, P53, P21CIP1 and P27KIP1, while inhibited the expression of CDK4 and CyclinD1. AMPK knockdown attenuated the effects of metformin on SKM-1 cells. These findings suggested that metformin inhibited proliferation of SKM-1 cells, potentially through an AMPK-mediated cell cycle arrest.

Metformin reverses mesenchymal phenotype of primary breast cancer cells through STAT3/NF-\u03baB pathways

BackgroundBreast cancer currently is the most frequently diagnosed neoplasm and the leading cause of death from cancer in women worldwide, which is mainly due to metastatic disease. Increasing our understanding of the molecular mechanisms leading to metastasis might thus improve the pharmacological management of the disease. Epithelial-mesenchymal transition (EMT) is a key factor that plays a major role in tumor metastasis. Some pro-inflammatory cytokines, like IL-6, have been shown to stimulate phenotypes consistent with EMT in transformed epithelial cells as well as in carcinoma cell lines. Since the EMT is one of the crucial steps for metastasis, we studied the effects of metformin (MTF) on EMT.MethodsCytotoxic effect of MTF was evaluated in eight primary breast cancer cell cultures by crystal violet assay. EMT markers and downstream signaling molecules were measured by Western blot. The effect of MTF on cell proliferation and cell migration were analyzed by MTT and Boyden chamber assays respectively.ResultsWe observed that the response of cultured breast cancer primary cells to MTF varied; mesenchymal cells were resistant to 10 mM MTF and expressed Vimentin and SNAIL, which are associated with a mesenchymal phenotype, whereas epithelial cells were sensitive to this MTF dose, and expressed E-cadherin but not mesenchymal markers. Further, exposure of mesenchymal cells to MTF down-regulated both Vimentin and SNAIL as well as cell proliferation, but not cell migration. In an in vitro IL-6-induced EMT assay, primary breast cancer cells showing an epithelial phenotype underwent EMT upon exposure to IL-6, with concomitant activation of STAT3 and NF-κB; addition of MTF to IL-6-induced EMT reversed the expression of the mesenchymal markers Vimentin and SNAIL, decreased pSTAT3 Y705 and pNF-κB S536 and increased E-cadherin. In addition, downregulation of STAT3·activation was dependent on AMPK, but not NF-κB phosphorylation. Further, MTF inhibited cell proliferation and migration stimulated by IL-6.ConclusionThese results suggest that MTF inhibits IL-6-induced EMT, cell proliferation, and migration of primary breast cancer cells by preventing the activation of STAT3 and NF-κB. STAT3 inactivation occurs through AMPK, but not NF-κB.

The Antitumor Effects of Metformin on Gastric Cancer In Vitro and on Peritoneal Metastasis.

Gastric cancer (GC) with peritoneal metastasis remains difficult to treat. The anti-diabetic drug metformin exerts various antitumor effects via the 5'-adenosine monophosphate-activated protein kinase (AMPK) pathway and nuclear factor-kappa B (NF-ĸB). Therefore, we evaluated the antitumor effects of metformin for GC in vitro and on peritoneal metastasis. The human GC cell lines MKN1, MKN45, KATO-III and SNU-1 were used. The antiproliferative effect was evaluated in vitro with 0.5 mM or 25 mM glucose and in vivo using tumor xenograft peritoneal models of metastasis. The protein expression of AMPK, liver kinase B1 (LKB1) and NF-ĸB in tumors was examined by western blotting. Metformin inhibited cell proliferation in all GC lines and sensitivity was increased under low-glucose conditions in vitro. Metformin also suppressed peritoneal metastasis. In tumors, metformin reduced the numbers of proliferating cells and NF-ĸB expression, but a similar trend was not noted for AMPK. Metformin may be a useful drug for the treatment of GC with peritoneal metastasis.

Metformin suppresses growth and adrenocorticotrophic hormone secretion in mouse pituitary corticotroph tumor AtT20 cells.

Pituitary corticotroph tumors lead to excess adrenocorticotrophic hormone (ACTH) secretion, resulting in Cushing's disease (CD), which is associated with significant mortality. Standard treatments include neurosurgery, radiotherapy and medical therapy. Both surgery and radiotherapy have undesirable complications and high recurrence rates. At present, there is only one medical option available that targets pituitary adenoma and ACTH secretion, the drug pasireotide. However, hyperglycemia is common during pasireotide treatment. In addition, some patients have discontinued pasireotide treatment because of hyperglycemia-related adverse events or uncontrolled diabetes. New medical treatments directly targeting the corticotroph cells and suppressing ACTH secretion are urgently required. Metformin is a commonly used antidiabetic drug that has been widely used to control the hyperglycemia that occurs in patients with CD, which is secondary to both cortisol excess and pasireotide treatment. Recent studies suggest that metformin has direct anticancer activities against many tumor cell lines. In the present study, we investigated whether metformin exerts an anti-tumor effect by directly targeting pituitary corticotroph tumors and exploring the underlying mechanisms. Using the mouse corticotroph tumor cells, AtT20 cells, we report that metformin inhibited cell proliferation, promoted cell apoptosis and decreased ACTH secretion but did not block the cell cycle in cells. The apoptosis induced by metformin was accompanied by increased caspase-3 activity. Meanwhile, metformin down-regulated the anti-apoptotic protein B cell lymphoma 2 (Bcl-2) but up-regulated the pro-apoptotic protein Bcl2-associated X (BAX), which suggests the involvement of the mitochondrial-mediated apoptosis pathway. Furthermore, metformin promoted AMP-activated protein kinase (AMPK) phosphorylation but inhibited insulin-like growth factor-1 receptor (IGF-1R) expression, protein kinase B (PKB/AKT) phosphorylation and mammalian target of rapamycin (mTOR) phosphorylation. Finally, the IGF-1R inhibitor picropodophyllin (PPP) significantly inhibited the cell proliferation of AtT20 cells. We conclude that metformin inhibits cell proliferation and induces apoptosis in AtT20 cells by activating AMPK/mTOR and inhibiting IGF-1R/AKT/mTOR signaling pathways. Metformin may have direct antitumor activity against pituitary corticotroph tumors.

Stimulating effect of palmitate and insulin on cell migration and proliferation in PNT1A and PC3 prostate cells: Counteracting role of metformin.

A potential association between obesity and prostate cancer has been proposed. Metformin, an antidiabetes drug, has antiproliferative effects being proposed for cancer treatment. However, under intense proliferative stimulation conditions such as those found in obesity, its efficacy is still uncertain. Thus, we analyzed the effects of saturated fatty acid and/or insulin under high concentrations, with or without metformin, on the proliferation and migration of prostate cells. Human prostate epithelial cell lines non-tumor (PNT1A) and tumor (PC3) were treated with control media (DMEM, C), palmitate (100 µM, HF), and/or insulin (50 µU, HI) with or without metformin (100 µM) for 24 or 48 h. Both PNT1A and PC3 cells had greater proliferation when treated with HF, while HI treatment stimulated only PNT1A. Metformin inhibited cell proliferation caused by HF in both cell lines, but it did not block the proliferative action of HI in PNT1A cells. PNT1A increased cell migration after all treatments, while only HF influenced PC3; metformin inhibited the migration stimulated by all obese microenvironments. Both HF and HI treatments in PNT1A and HF treatment in PC3 augmented vimentin expression, resulting in a higher epithelial-mesenchymal transition (which, in turn, could influence cell migration). Metformin inhibited vimentin expression in both normal and tumor cells. Although HF treatment had increased AMPK activation, it also increased the levels of activated ERK1/2, which could be responsible for high cell proliferation in both cell lines. In contrast, HI decreased AMPK activation in both cell lines, whereas it increased ERK1/2 levels in PNT1A and decreased them in PC3 (reflecting greater cell proliferation only in non-tumor cells). Metformin maintained high activation of AMPK and decreased ERK1/2 levels after HF in both cell lines and only after HI in PNT1A, which was able to decrease the cell proliferation triggered by these treatments. Higher concentrations of palmitate on PC3 cells and palmitate and insulin on PNT1A cells stimulate cellular activities that could favor cancer progression. Metformin inhibited most of these stimuli, showing the efficacy of this drug for cancer adjuvant therapy in obese patients (a group at increased risk for the development of prostrate cancer).

Metformin inhibits ovarian cancer via decreasing H3K27 trimethylation.

Metformin has been used for the treatment of type II diabetes mellitus for decades. Recently, used of metformin in the therapy of diverse human cancer types has received widespread attention, while the underlying mechanisms have been not fully elucidated. In the current study, 5-ethynyl-20-de-oxyuridine assay to detect cell proliferation, flow cytometry to detect apoptosis, scratch wound healing and Transwell migration assay to detect cell migration capacity. The current study reported that metformin inhibited cell proliferation and migration, and promoted apoptosis in ovarian cancer cells, particularly under normoglycemic conditions in vitro. Metformin treatment significantly promoted the phosphorylation of AMP-activated protein kinase (AMPK), and reduced histone H3 lysine 27 trimethylation (H3K27me3) and polycomb repressor complex 2 (PRC2) levels. Additionally, overexpression of EZH2 to increase H3K27me3 abrogated the effect of metformin on the cell proliferation, migration and apoptosis in SKOV3 and ES2 cells. Similar to metformin, another AMPK agonist, 2-deoxy-D-glucose, reduced the H3K27me3 level and PRC2 expression. In cells pretreated with Compound C, an AMPK inhibitor, metformin was not able to induce AMPK phosphorylation or reduce H3K27me3. Metformin-mediated AMPK activation and H3K27me3 inhibition were more robust in cells exposed to low glucose (5.5 mM) compared with those exposed to high glucose (25 mM). These findings implicate H3K27me3 repression mediated by AMPK phosphorylation in the antitumor effect of metformin in ovarian cancer, indicating that metformin alters epigenetic modifications by targeting PRC2 and supports the use of metformin in treatment of patients with epithelial ovarian cancer without diabetes.

High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer.

ObjectivesSTAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin.MethodsIn Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the in vivo efficacy of metformin.ResultsExpression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. In vitro, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. In vivo, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins.ConclusionsThese results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.

Effects of prolonged exposure to low dose metformin in thyroid cancer cell lines.

Background: Thyroid cancer is generally associated with an excellent prognosis, but there is significant long-term morbidity with standard treatment. Some sub-types however have a poor prognosis. Metformin, an oral anti-diabetic drug is shown to have anti-cancer effects in several types of cancer (breast, lung and ovarian cancer). The proposed mechanisms include activation of the Adenosine Mono-phosphate-activated Protein Kinase (AMPK) pathway and inhibition of the mTOR pathway (which promotes growth and proliferation). By inhibiting hepatic gluconeogenesis and increasing glucose uptake by muscles, metformin decreases blood glucose and circulating Insulin levels.Aims: Explore the effect of metformin on the growth and proliferation of thyroid cancer cell lines.Methods: The effects of metformin on thyroid cancer cell lines (FTC-133, K1E7, RO82-W-1, 8305C and TT) and normal thyroid follicular cells (Nthy-ori 3-1) were investigated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay for cell proliferation; clonogenic assays; FACS analysis for apoptosis and cell cycle, H2A.X phosphorylation (γH2AX) assay for DNA repair and scratch assay for cell migration.Results: Metformin inhibited cell proliferation and colony formation at 0.03 mM and above and inhibited cell migration at 0.3 mM. At concentrations of 0.1 mM and above metformin increased the percentage of apoptotic cells and induced cell cycle arrest in G0/G1 phase at minimum concentration of 0.3 mM. Unlike previous reports, no effect on DNA repair response was demonstrated.Conclusion: Metformin suppressed growth of all thyroid cancer cell lines, at concentrations considered to be within in the therapeutic range for diabetic patients on metformin (<0.3 mM).

Metformin Induces p53-Dependent Mitochondrial Stress in Therapy-Sensitive and -Resistant Lymphoma Pre-Clinical Model and Primary Patients Sample with B-Cell Non-Hodgkin Lymphoma (NHL)

Introduction: Metformin, a guanidine originally derived for Galega officinalis (French lilac), has been widely prescribed to type II diabetics since 1950 and well known for its safety toxicity profile. Recently, metformin use was associated with a decrease in risk cancer development and lower cancer-related mortality among breast, colorectal, prostrate, lung, hepatic and ovarian cancer patients. Our group previously reported that the use of metformin during front-line chemo-immunotherapy (i.e. R+CHOP) improved the clinical outcome of diffuse large B-cell lymphoma (DLBCL). The mechanism(s) of action underlying the antitumor effect of metformin remains not to be fully elucidated. Previous research done in our laboratory revealed that metformin inhibited cell proliferation through repressing PCNA and p21 proteins. Here, we report that metformin activates tumor suppressor p53 by suppressing MDMX in pre-clinical lymphoma models.Methods: A panel of rituximab-sensitive (RSCL), rituximab-resistant (RRCL) cell lines and primary tumor cells isolated from B-cell lymphoma patients were exposed to escalating doses of metformin (0-64mM). Changes in cell viability were determined by Presto Blue (Sigma) assay in cell lines and Titer Glo in patient samples. Changes in MDMX, MDM2 and p53 expression levels were determined by western blotting after exposure cells to metformin. MDMX-MDM2-p53 interactions and p53 ubiquitination following in vitro exposure to metformin were determined by immunoprecipitation of p53 and probing for MDMX, MDM2, ubiquitin and p53 in RSCL and RRCLs. Loss of Dψm or induction of apoptosis following metformin exposure were assessed by DiOC6 or Annexin V/PI staining and flow cytometry. Oxidative stress induced by metformin was measured by flow cytometry using dihydrorhodamine-123 (DHR-123) dye.Result: Metformin induced a dose-and time- dependent cell death in cell lines and primary patient tumor cells. In vitro exposure of lymphoma cell to metformin resulted in a decrease in MDMX levels. Immunoprecipitation studies demonstrated that following exposure to metformin, MDMX bound less to p53 leading to less p53 ubiquitination. In vitro exposure of RSCL or RRCL to metformin resulted in the expression of p53 regulated BH3 single domain proteins (Noxa and Puma). Moreover, metformin repressed mitochondrial potential, induced reactive oxidative species (ROS) generation and triggered apoptosis.Conclusion: Our data suggests that metformin had anti-tumor activity against RSCL, RRCL and primary tumor cells isolated from lymphoma patients. The down-regulation of MDMX and re-activation p53 function following metformin exposure may contribute to the disruption in the mitochondria potential, generation of ROS and induction of apoptosis observed in our models RSCL and RRCL. Our finding highlights a potential role for metformin in the treatment of B-cell malignancies. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Funds) DisclosuresCzuczman:MorphoSys: Consultancy; Boehringer-Ingelheim: Other: Advisory Board; Celgene: Employment; Immunogen: Other: Advisory board.

Abstract 1161: Metformin has an inhibitory effect on cell proliferation but does not induce death in colorectal cancer

Abstract Metformin, a widely used and well-tolerated antidiabetic drug, may reduce cancer risk and improve prognosis of certain malignancies. In vitro and in vivo studies on several cancer models have evaluated the potential antitumorigenic effects of metformin and many clinical trials are looking to its use for suppressing tumor growth. However, the mechanism for its anti-cancer effect remains uncertain and needs to be clarified. Here we investigated the anti-cancer activity of metformin on colorectal cancer growth by treating three colorectal cancer cell lines, HT29, HCT116, HCT116p53-/-, with the drug. We found that, at a dose of 5mM, metformin reduced cell proliferation in all the colorectal cancer cells analyzed, as shown by BrdU incorporation assays. Metformin induced cell cycle arrest in G0/G1 phase that was accompanied by a strong decrease of cyclin D1, c-Myc expression, and inhibition of pRB phosphorylation. The antiproliferative activity of the drug resulted mediated by suppression of mTOR and IGF-1/AKT pathways. In particular, we observed inactivation of mTOR and of its downstream targets S6 and 4EBP1, both in an AMPK dependent and independent way, and also downregulation of the activity of IGF1R. We analysed the biological mechanisms by which metformin inhibited cell proliferation by evaluating its ability to induce apoptosis, autophagy or senescence, as observed in other cancer cell lines. Unexpectedly, we found that metformin did not stimulate any of these mechanisms in colorectal cancer cells. In support of these results, a colony formation assay showed that metformin did not arrest growth of cells, but only slowed down cell ability of forming colonies. Our findings highlight that metformin inhibits the proliferation of colorectal cancer cell lines as a consequence of promoting cell cycle arrest in the G0/G1 phase, but does not induce cell death. Further investigations are needed to better elucidate the mechanisms altered by the drug in colorectal cancer and caution should be used when treating cancer patients with metformin. Citation Format: Angela Mogavero, Maria Valeria Maiorana, Claudia Bertan, Fabio Bozzi, Marco A. Pierotti, Manuela Gariboldi. Metformin has an inhibitory effect on cell proliferation but does not induce death in colorectal cancer. [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 1161. doi:10.1158/1538-7445.AM2015-1161

Metformin Affects the Proliferation Cell Nuclear Antigen (PCNA) and p21 Protein Interaction Resulting in Direct Anti-Tumor Activity and Enhances the Cytotoxic/Biological Effects of Chemotherapy Agents or Rituximab in Lymphoma in Vitro and in Vivo Pre-Clinical Models

Abstract Most cancer cells predominantly produce adenosine triphosphate (ATP) by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria. This shift in cellular metabolism is know as the Warburg effect and is primarily observed in rapidly growing tumors including aggressive B-cell lymphoma and is thought to be a consequence of the progression to cancer rather than the cause of it. Altering the glucose metabolism in cancer cells appears to be an attractive strategy in cancer medicine. Previously, we demonstrated that the acquirement of rituximab resistance was associated with an increase in the Warburg effect leading to concomitant chemotherapy resistance in lymphoma pre-clinical models. The use of metformin, an oral biguanide widely used to treat insulin resistance conditions, during chemotherapy has been associated with improved clinical outcomes in solid tumor patients receiving chemotherapy. In a retrospective analysis, we demonstrated that the use of metformin during front-line chemo-immunotherapy (i.e. R+CHOP) improved the clinical outcome of diffuse large B-cell lymphoma (DLBCL). In an attempt to characterize the mechanism by which metformin affects the biology of DLBCL, we studied the metabolic and signaling changes in rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) exposed to metformin. A panel of RSCL and RRCL were exposed to metformin. Changes in Ki67, proliferation cell nuclear antigen (PCNA), and its regulator (p21) were determined by flow cytometry or Western blotting respectively. Ki67/PCNA/p21 changes were correlated to cell cycle distribution, cell viability and chemotherapy sensitivity. For in vivo studies, SCID mice were inoculated via tail vein injection (iv) with Raji cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose on Day 3,7,10,14), metformin (at 2ug/ml in drinking water from day 3 till the end of experiment) or metformin with rituximab. Differences in survival (measured as the time for limb paralysis development) were evaluated by log-rank test between treatment arms. Metformin in combination with rituximab (mean survival not reached at 69+/- 5.3 days) lead to an improved survival than rituximab (mean survival 57.1+/-4.2 days) (P=0.05). In vitro exposure of RSCL/RRCL to metformin inhibited cell proliferation (measure by, MTT assay, alamar blue reduction, cell titer glow assay, and ki67 staining) in a dose-dependent manner and enhanced the anti-tumor activity of chemotherapy drugs. Perhaps related to this effect, metformin exposure induced G1 phase cell cycle arrest in both RSCL and RRCL. Using the flow cytometry technology (FITC-labeled anti-KI67 antibody/PI-DNA staining), we detected a decrease in Ki67 of RSCL/RRCL distributed in the G1 and S phase following metformin exposure. In vitro exposure of RSCL and RRCL to metformin increased p21 and reduced PCNA levels. Immuno-precipitation of p21 following metformin drug exposure increased in the interaction between p21 and PCNA. Our data suggests that metformin inhibits the proliferation of RSCL or RRCL and enhances the anti-tumor activity of chemotherapy agents or rituximab in lymphoma pre-clinical models (in vitro and in vivo). Perhaps related to the biological effects observed, p21 and PCNA, play a pivotal role on the anti-tumor activity of metformin in lymphoma pre-clinical models. Our finding highlights a potential role for metformin in the treatment of B-cell malignancies. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.

A-activated protein kinase is required for cell survival and growth in hela-s3 cellsin vivo

Activation of the AMP-dependent protein kinase (AMPK) is linked to cancer cell survival in a variety of cancer cell lines, particularly under conditions of stress. As a potent activator of AMPK, metformin has become a hot topic of discussion for its effect on cancer cell. Here, we report that AMPK activated by metformin promotes HeLa-S3 cell survival and growth in vivo. Our results show that metformin inhibited cell proliferation in MCF-7 cells, but not in LKB1-deficient HeLa-S3 cells. Re-expression of LKB-1 in HeLa-S3 cells restored the growth inhibitory effect of metformin, indicating a requirement for LKB-1 in metformin-induced growth inhibition. Moreover, AMPK activation exerted a protective effect in HeLa-S3 cells by relieving ER stress, modulating ER Ca(2+) storage, and finally contributing to cellular adaptation and resistance to apoptosis. Our findings identify a link between AMPK activation and cell survival in HeLa-S3 cells, which demonstrates a beneficial effect of AMPK activated by metformin in cancer cell, and suggests a discrete re-evaluation on the role of metformin/AMPK activation on tumor cell growth, proliferation, and on clinical application in cancer therapy.

Another Surprise from Metformin: Novel Mechanism of Action via K-Ras Influences Endometrial Cancer Response to Therapy

Metformin is an oral biguanide commonly used for the treatment of type II diabetes and has recently been demonstrated to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Using both in vitro and in vivo models, we examined the effects of metformin on endometrial tumors with defined aberrations in the PI3K/PTEN/mTOR and MAPK signaling pathways to understand metformin mechanism of action and identify clinically useful predictors of response to this agent. In vitro assays of proliferation, cytotoxicity, and apoptosis were used to quantify the effects of metformin on endometrial cancer cell lines with mutations in the PI3K/PTEN/mTOR and MAPK signaling pathways. The in vivo effects of oral metformin on tumor progression were further examined using xenograft mouse models of endometrial cancer. K-Ras localization was analyzed by confocal microscopy using GFP-labeled oncogenic K-Ras and by immunoblot following subcellular fractionation. Metformin inhibited cell proliferation, induced apoptosis, and decreased tumor growth in preclinical endometrial cancer models, with the greatest response observed in cells harboring activating mutations in K-Ras. Furthermore, metformin displaces constitutively active K-Ras from the cell membrane, causing uncoupling of the MAPK signaling pathway. These studies provide a rationale for clinical trials using metformin in combination with PI3K-targeted agents for tumors harboring activating K-Ras mutations, and reveal a novel mechanism of action for metformin.

K-ras gene mutation as a predictor of cancer cell responsiveness to metformin

An increasing number of studies support the use of metformin, a common antidiabetic drug, as a novel anticancer therapeutic. However, its mechanism of action has yet to be identified. In the current study, metformin was observed to effectively inhibit the growth of the K-ras mutant but not wild-type tumors in vivo. The antitumor effects of metformin were mediated by the induction of apoptosis and inhibition of proliferation in vivo. In addition, metformin induced apoptosis in the K-ras mutant tumors, A549 and PANC-1, but not in the K-ras wild-type tumor, A431, in vitro. Similarly, at lower concentrations, metformin inhibited cell proliferation in the K-ras mutant, but not in the K-ras wild-type tumor cells in vitro. These observations indicate that tumors with K-ras mutations are sensitive to metformin therapy. In addition, metformin significantly arrested K-ras mutant and wild-type tumor cells in G1 phase in vitro and metformin downregulated two important downstream effectors of the Ras signaling pathway in K-ras mutant tumors. Metformin was concluded to function as a potential K-ras-targeting agent that has potential for cancer therapy.

Activation of AMP-Activated Protein Kinase Inhibits the Proliferation of Human Endothelial Cells

AMP-activated protein kinase (AMPK) is an evolutionary conserved energy-sensing enzyme that regulates cell metabolism. Emerging evidence indicates that AMPK also plays an important role in modulating endothelial cell function. In the present study, we investigated whether AMPK modulates endothelial cell growth. Treatment of cultured human umbilical vein endothelial cells with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 6,7-dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), or metformin inhibited cell proliferation and DNA synthesis. The antiproliferative action of AICAR was largely prevented by the adenosine kinase inhibitor 5'-iodotubercidin and mimicked by infecting endothelial cells with an adenovirus expressing constitutively active AMPK. In contrast, pharmacological blockade of endothelial nitric oxide synthase or heme oxygenase-1 activity failed to reverse the inhibition of endothelial cell growth by AICAR. Flow cytometry experiments revealed that pharmacological activation of AMPK arrested endothelial cells in the G₀/G₁ phase of the cell cycle, and this was associated with increases in p53 phosphorylation and p53, p21, and p27 protein expression and decreases in cyclin A protein expression and retinoblastoma protein phosphorylation. In addition, silencing p21 and p27 expression partially restored the mitogenic response of AMPK-activated cells. Finally, activation of AMPK by AICAR blocked the migration of endothelial cells after scrape injury and stimulated tube formation by endothelial cells plated onto Matrigel-coated plates. In conclusion, these studies demonstrate that AMPK activation inhibits endothelial cell proliferation by elevating p21 and p27 expression. In addition, they show that AMPK regulates endothelial cell migration and differentiation and identify AMPK as an attractive therapeutic target in treating diseases associated with aberrant endothelial cell growth.

Metformin attenuates ovarian cancer cell growth in an AMP‐kinase dispensable manner

Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -α1, -β1, -γ1 and -γ2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKα and its downstream substrate; acetyl co-carboxylase (ACC) and enhanced β-oxidation of fatty acid and (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin-mediated effect on AMPK is dependent on liver kinase B1 (LKB1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to down-regulate LKB1 in ovarian cancer cells. In contrast, met formin inhibited cell proliferation in both wild-type and AMPKα1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK-dependent as well as AMPK-independent pathways.