Molecular Target Of Metformin Research Articles

Current findings on the antitumor effects of metformin on esophageal squamous cell carcinoma (Review).

Esophageal squamous cell carcinoma (ESCC) is an intractable type of cancer that requires novel therapeutic modalities, since the therapeutic outcomes are often inadequate, even in response to multidisciplinary treatment. The antitumor effect of metformin, an antidiabetic drug, has been reported in esophageal cancer; however, its effects are diverse. Since various multidisciplinary therapies are used in ESCC, the antitumor effect of metformin is expected to be synergistic in some treatment strategies. The present review summarizes the antitumor effects of metformin and discusses its use in combination with existing therapies. The present study reviewed relevant studies where the molecular targets of metformin (AMPK and inflammatory system signals) were described, followed by the classification and organization of its antitumor effects, and subsequently summarized the current research on its antitumor effects, especially in ESCC. A number of studies have reported that metformin prevents the development of ESCC and exerts its antitumor effects through various pathways. In addition, metformin has been shown to inhibit tumor growth, induce apoptosis, inhibit cancer cell invasion, migration and angiogenesis into the tumor, and decrease tumor malignancy, such as metastasis. Furthermore, it may modulate host tumor immunity in a tumor-suppressive manner and is expected to improve prognosis following treatment for ESCC. Notably, metformin may be beneficial in combination with chemotherapy, such as cisplatin, and radiation. By contrast, it has been shown to potentially induce resistance to 5-fluorouracil. Finally, the effects of metformin in combination with other therapies are discussed in the present study, and perspectives on the potential benefits of metformin for future ESCC treatment are presented. In conclusion, the present review may be useful in improving the understanding of the wide range of antitumor effects of metformin. Although some concerning points remain, using metformin in ESCC treatment could be promising. Notably, more knowledge needs to be accumulated regarding the effects of metformin on ESCC.

Effect of metformin in hypothalamic astrocytes from an immunocompromised mice model

Astrocytes are glial cells that play key roles in neuroinflammation, which is a common feature in diabetic encephalopathy and aging process. Metformin is an antidiabetic compound that shows neuroprotective properties, including in inflammatory models, but astroglial signaling pathways involved are still poorly known. Interferons α/β are cytokines that participate in antiviral responses and the lack of their signaling increases susceptible to viral infections. Here, we investigated the effects of metformin on astrocytes from hypothalamus, a crucial brain region related to inflammatory processes. Astrocyte cultures were derived from interferon α/β receptor knockout (IFNα/βR-/-) and wild-type (WT) mice. Metformin did not change the expression of glial fibrillary acidic protein but caused an anti-inflammatory effect by decreasing pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β), as well as increasing gene expression of anti-inflammatory proteins interleukin-10 and Nrf2 (nuclear factor erythroid derived 2 like 2). However, nuclear factor κB p65 and cyclooxygenase 2 were downregulated in WT astrocytes and upregulated in IFNα/βR-/- astrocytes. AMP-activated protein kinase (AMPK), a molecular target of metformin, was upregulated only in WT astrocytes, while sirtuin 1 increased in both mice models. The expression of inducible nitric oxide synthase was decreased in WT astrocytes and heme oxygenase 1 was increased in IFNα/βR-/- astrocytes. Although loss of IFNα/βR-mediated signaling affects some effects of metformin, our results support beneficial roles of this drug in hypothalamic astrocytes. Moreover, paradoxical response of metformin may involve AMPK. Thus, metformin can mediate glioprotection due its effects on age-related disorders in non-diabetic and diabetic encephalopathy individuals.

Molecular mechanisms of action of metformin: latest advances and therapeutic implications.

Metformin is among the most widely used antidiabetic drugs. Studies over the past few years have identified multiple novel molecular targets and pathways that metformin acts on to exert its beneficial effects in treating type 2 diabetes as well as other disorders involving dysregulated inflammation and redox homeostasis. In this mini-review, we discuss the latest cutting-edge research discoveries on novel molecular targets of metformin in glycemic control, cardiovascular protection, cancer intervention, anti-inflammation, antiaging, and weight control. Identification of these novel targets and pathways not only deepens our understanding of the molecular mechanisms by which metformin exerts diverse beneficial biological effects, but also provides opportunities for developing new mechanistically based drugs for human diseases.

Molecular targets of metformin against ovarian cancer based on network pharmacology.

The objective of this study was to analyze potential targets of metformin against ovarian cancer (OC) through network pharmacology. Pharmacodynamic targets of metformin were predicted using the Bioinformatics Analysis Tool for the molecular mechanism of traditional Chinese medicine (BATMAN), Drugbank, PharmMapper, SwissTargetPrediction, and TargetNet databases. R was utilized to analyze the gene expression of OC tissues, normal/adjacent noncancerous tissues, and screen differentially expressed genes (DEGs) in the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) + Genotype-Tissue Expression (GTEx) datasets. STRING 11.0 was utilized to explore the protein-protein interaction (PPI) of metformin target genes differentially expressed in OC. Cytoscape 3.8.0 was used to construct the network and screen the core targets. Additionally, gene ontology (GO) annotation and enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for the common targets of metformin and OC through the DAVID 6.8 database. A total of 95 potential common targets of metformin and OC were identified from the intersection of 255 potential pharmacodynamic targets of metformin and 10,463 genes associated with OC. Furthermore, 10 core targets were screened from the PPI network [e.g., interleukin (IL) 1B, KCNC1, ESR1, HTR2C, MAOB, GRIN2A, F2, GRIA2, APOE, PTPRC]. In addition, it was shown in GO enrichment analysis that the common targets were mainly associated with biological processes (i.e., response to stimuli or chemical, cellular processes, and transmembrane transport), cellular components (i.e., plasma membrane, cell junction, and cell projection), and molecular functions (i.e., binding, channel activities, transmembrane transporter activity, and signaling receptor activities). Furthermore, it was indicated by KEGG pathway analysis that the common targets were enriched in metabolic pathways. The critical molecular targets and molecular pathways of metformin against OC were preliminarily determined by bioinformatics-based network pharmacology analysis, providing a basis, and reference for further experimental studies.

Metformin Attenuates Hyperglycaemia-Stimulated Pro-Fibrotic Gene Expression in Adventitial Fibroblasts via Inhibition of Discoidin Domain Receptor 2.

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a Collagen receptor tyrosine kinase, has an obligate regulatory role in Collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and overexpression approaches, the present study examined whether DDR2 is a target of metformin and whether, by targeting DDR2, it inhibits Fibronectin and Collagen type I expression in rat aortic adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-β1/SMAD2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of Fibronectin and Collagen type I, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis and possibly cardiac fibrosis associated with diabetic cardiomyopathy.

P–300 Metformin: new therapeutic approach for endometriosis-associated infertility

Abstract Study question Could endometriosis-associated infertility be mitigated by metformin? Summary answer Metformin treatment restored fertility to control rates in endometriosis-induced mice that present lower fertility. No effect was observed on sham-operated mice. What is known already Endometriosis is a gynaecological disorder characterized by ectopic vascularized endometrial tissue growth, mainly in pelvic cavity, which provokes pain and infertility. Corroborating observations in women, endometriosis decreases oocyte quality and pregnancy success in rodents, without affecting the number of ovulations, resorption rate and fetal weight. Thus, animal models of endometriosis constitute a valuable tool to elucidate the pathophysiology of the disease and putative pharmacological therapies. Metformin is widely used for diabetes type–2 treatment, reducing glucose, oxidative stress and inflammation. Due to its antioxidant properties, metformin has shown to induce regression of endometrial implants in a rat model of endometriosis. Study design, size, duration B6CBA/F1 female mice were randomly divided in groups and subjected to treatment: 1-Endometriosis (n = 20); 2-Sham-operated (n = 12); 3-Endometriosis with metformin (n = 20); 4-Sham-operated with metformin (n = 20). Endometriosis was surgically induced by heterologous transplantation of endometrium from one donor in receptors from the same strain mice. Implants were confirmed and monitored by ultrasound. 50mg/kg/day of metformin was orally administrated during 3 months to Groups 3 and 4. Half of mice in each group were mated to fertility study. Participants/materials, setting, methods Endometriomas were monitored at 3 timepoints during the experiments. Biometric parameters of mice and number of implantation sites, fetuses and fetal weight were recorded. Fertility rates were assessed by the average number of fetuses in each group. Histological characterization of ovary, uterus, endometriomas, and peritoneal tissue at the implant site was performed by Hemathoxylin & Eosin (H&E) staining. Statistical study among groups was carried out and significant differences were considered for student t-test < 0.05. Main results and the role of chance A decrease of 30% of fertility rate was verified in mice with endometriosis (p = 0,01); treatment with metformin was able to revert this decrease (p = 0,04). Interestingly, no differences in fertility were found in sham-operated mice under metformin treatment relatively with those of group 2 (p = 0,16). Although the number of absorptions observed in mice of the endometriosis group was higher, no statistical difference was reached comparatively with other groups. No biometrical differences were found between mice with endometriosis receiving metformin and those that do not receive the drug. Regarding H&E staining we verified that endometriomas showed histologically resemblances to uterus. Moreover, endometriomas from mice without treatment with metformin were dark brown, recalling for human endometriomas called “chocolate” cysts, while the endometriomas from mice who were treated with metformin were visually clearly. We postulate that these findings owes to a metformin-mediated decrease of oxidative imbalance and inflammatory response, induction of regression of endometriomas and regulation of oestrogen secretion. Limitations, reasons for caution Extrapolation of data from animal models to human needs caution, considering that endometriosis pattern differs between species. Also, further investigation, focused in identification of molecular targets of metformin and molecular pathways activated in endometriosis, is needed and in course. Wider implications of the findings: With these results, we indicate metformin as a novel and safe strategy to mitigate endometriosis-related oxidative stress and indeed could be used as a valid pharmacological approach to ameliorate endometriosis-associated infertility. Trial registration number Not applicable

Obesity Connected Metabolic Changes in Type 2 Diabetic Patients Treated With Metformin.

Metformin is widely used in the treatment of Type 2 Diabetes Mellitus (T2DM). However, it is known to have beneficial effects in many other conditions, including obesity and cancer. In this study, we aimed to investigate the metabolic effect of metformin in T2DM and its impact on obesity. A mass spectrometry (MS)-based metabolomics approach was used to analyze samples from two cohorts, including healthy lean and obese control, and lean as well as obese T2DM patients on metformin regimen in the last 6 months. The results show a clear group separation and sample clustering between the study groups due to both T2DM and metformin administration. Seventy-one metabolites were dysregulated in diabetic obese patients (30 up-regulated and 41 down-regulated), and their levels were unchanged with metformin administration. However, 30 metabolites were dysregulated (21 were up-regulated and 9 were down-regulated) and then restored to obese control levels by metformin administration in obese diabetic patients. Furthermore, in obese diabetic patients, the level of 10 metabolites was dysregulated only after metformin administration. Most of these dysregulated metabolites were dipeptides, aliphatic amino acids, nucleic acid derivatives, and urea cycle components. The metabolic pattern of 62 metabolites was persistent, and their levels were affected by neither T2DM nor metformin in obesity. Interestingly, 9 metabolites were significantly dysregulated between lean and obese cohorts due to T2DM and metformin regardless of the obesity status. These include arginine, citrulline, guanidoacetic acid, proline, alanine, taurine, 5-hydroxyindoleacetic acid, and 5-hydroxymethyluracil. Understanding the metabolic alterations taking place upon metformin treatment would shed light on possible molecular targets of metformin, especially in conditions like T2DM and obesity.

Screening Novel Molecular Targets of Metformin in Breast Cancer by Proteomic Approach.

Metformin is a commonly prescribed antihyperglycemic drug, and has been investigated in vivo and in vitro for its effect to improve the comorbidity of diabetes and various types of cancers. Several studies investigated the therapeutic mechanisms of metformin on cancer cells, but the exact mechanism of metformin’s effect on the proteomic pathways of cancer cells is yet to be further investigated. The main objective of our research line is to discover safe and alternative therapeutic options for breast cancer, we aimed in this study to design a novel “bottom up proteomics workflow” in which proteins were first broken into peptides to reveal their identity, then the proteomes were precisely evaluated using spectrometry analysis. In our study, metformin suppressed cell proliferation and induced apoptosis in human breast carcinoma cell line MCF-7 with minimal toxicity to normal breast epithelial cells MCF-10. Metformin induced apoptosis by arresting cells in G1 phase as evaluated by flow cytometric analysis. Moreover, The G1 phase arrest for the MCF-7 has been confirmed by increased expression levels of p21 and reduction in cyclin D1 level. Additionally, metformin increased the expression levels of p53, Bax, Bad while it reduced expression levels of Akt, Bcl-2, and Mdm2. The study employed a serviceable strategy that investigates metformin-dependent changes in the proteome using a literature-derived network. The protein extracts of the treated and untreated cell lines were analyzed employing proteomic approaches; the findings conveyed a proposed mechanism of the effectual tactics of metformin on breast cancer cells. Metformin proposed an antibreast cancer effect through the examination of the proteomic pathways upon the MCF-7 and MCF-10A exposure to the drug. Our findings proposed prolific proteomic changes that revealed the therapeutic mechanisms of metformin on breast cancer cells upon their exposure. In conclusion, the reported proteomic pathways lead to increase the understanding of breast cancer prognosis and permit future studies to examine the effect of metformin on the proteomic pathways against other types of cancers. Finally, it suggests the possibility to develop further therapeutic generations of metformin with increased anticancer effect through targeting specific proteomes.

Abstract 5919: Identification of a novel molecular interaction, targeted by Metformin, between breast cancer and white adipose tissue progenitors

Abstract The white adipose tissue (WAT) contains a resident population of progenitors with mesenchymal- or endothelial-like phenotype, able to promote breast cancer (BC) progression (Orecchioni et al., 2013). We recently characterized a novel molecular axis underneath the interaction between BC and WAT progenitors, possibly explaining the acquisition of a cancer-associated phenotype by WAT progenitor cells and the establishment of a permissive tumor microenvironment. GM-CSF balance was identified to be the principal regulator of the transcriptional alteration occurring in WAT progenitors exposed to BC: GM-CSF, produced by BC cells, dramatically changed the secretion profile of progenitors, enhancing its own production through a positive regulatory loop and inducing the expression of several factors involved in inflammation (IL-1β), ECM remodeling and angiogenesis (MMP9). IL-1β was strongly down-regulated by GM-CSF neutralization in WAT progenitors in vitro and in preclinical models. Induction of IL-1β by GM-CSF was able, in turn, to up-regulate MMP9 in the same cells. The markedly increased expression of MMP9 was already observed in BC-associated WAT progenitors, being addressed to increased local and metastatic tumor growth in obese mice. GM-CSF production was significalntly enhanced in the presence of concomitant obesity in preclinical models of BC, both in orthotopic models (MMTV-neu/ErbB2; 4T1) and in spontaneous tumor-bearing mice (MMTV-PyMT). GM-CSF neutralization in several obese syngeneic models of BC significantly reduced immunosuppressive cells in tumors and in the surrounding WAT, including T-regs (CD4+CD25brightCD127low), Granulocytic myeloid-derived suppressor cells (G-MDSCs, CD11b+Ly6C-Ly6G+), tumor-associated macrophages (TAMs, F4/80+CD206+MCHIIlow) and tumor infiltrating lymphocytes (TILs, CD8+PD1+). Different BC types produce distinct amounts of GM-CSF: triple negative BC (TNBC) expressed higher levels compared to hormone receptors positive cells, correlating GM-CSF expression to higher tumor grade. In the context of targeting WAT-BC interaction, Metformin was able to impair tumor growth, metastasis and angiogenesis in preclinical models (Orecchioni et al., 2015) and affecting immune cells, including T-regs, TAMs and MDSCs. From our data, GM-CSF was identified to be a new target of Metformin in both BC and WAT progenitors, suggesting a novel inhibitory mechanism mediated by the drug. Metformin displayed the same efficacy in reducing BC growth, metastatic spread and immune system regulation, when compared to GM-CSF specific neutralization in obese syngeneic models. Taken together, these results indicate GM-CSF as a new molecular target of Metformin and support the role of this novel mechanism in BC progression. Ongoing studies also suggest that this pathway might be induced in WAT progenitors by other solid tumors, including non-Hodgkin’s B-lymphoma. Citation Format: Francesca Reggiani, Valentina Labanca, Patrizia Mancuso, Andrea Manconi, Francesco Bertolini. Identification of a novel molecular interaction, targeted by Metformin, between breast cancer and white adipose tissue progenitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5919. doi:10.1158/1538-7445.AM2017-5919

Effect of Metformin on the Medullary Thyroid Cancer Cells

Background. Medullary thyroid cancer (MTC) is associated with activation of mTOR signalingpathways. Recent studies showed that the anti-diabetic agent metformin decreases proliferation of cancer cellsthrough AMPK-dependent inhibition of mTOR.The objective of current study — assessment of the effect of metformin on MTC cells.Materials and Methods. Performed growth, viability, migration and resistance to anoikis assays using twoMTC-derived cell lines (TT and MZ-CRC-1). Expressions of molecular targets of metformin were examined inMTC cell lines and in 14 human MTCs tissue samples.Results. We found that metformin inhibited growth and decreased expression of Cyclin D1 in MTC cells. Treatmentwith metformin was associated with inhibition of mTOR/p70S6K/pS6 signaling and with down-regulation ofpERK in both TT and MZ-CRC-1 cells. Metformin had no significant effects on pAKT in the cell lines examined.Metformin inducible AMPK activation was noted only in TT cells. Treatment with AMPK inhibitor (Compound C)or AMPK silencing did not prevent growth-inhibitory effects of metformin in TT cells. Metformin had no effect on MTC cell migration, but reduced the ability of cells to form multi-cellular spheroids in non adherent conditions.Immunostaining of human MTC showed over-expression of Cyclin D1 in all tumors compared to corresponding normal tissue. Activation of mTOR/p70S6K was detected in 8/14 (57.1 %) of examined tumors.Conclusions. Together these findings indicate that growth inhibitory effects in MTC cells are associated withdown-regulation of both mTOR/6SK and pERK signaling pathways. Expression of metformin’s molecular targetsin human MTC cells suggests its potential utility for the treatment of MTC in patients.

Treatment With Metformin Is Associated With Higher Remission Rate in Diabetic Patients With Thyroid Cancer

Clinical trials demonstrated that metformin increases the efficiency of systemic therapy in cancer patients. We examined whether the efficacy of conventional treatment of differentiated thyroid cancer (DTC) is affected by therapy with metformin in diabetic patients. We compared the rate of complete response (CR) between diabetics who were treated with metformin (group MF+; n = 34) or not treated (group MF-; n = 21) and control nondiabetic patients (group C; n = 185). We also examined the effects of metformin on DTC cells in vitro. The groups were comparable in terms of age, sex, body mass index, diabetes management, frequencies of multifocal tumor growth, extrathyroidal extension, and locoregional and distant metastases. Tumor size was significantly smaller in the MF+ group compared with the MF- and C groups (1.37 ± 0.97 vs 2.44 ± 1.49 vs 2.39 ± 1.73 cm, respectively; P = .026). A multivariate model revealed that extrathyroidal extension (P = .018), distant metastases (P < .0001), and lack of treatment with metformin of diabetics (P < .0001) decreased the likelihood of CR. A Cox hazards model revealed that age (P = .025), locoregional metastases (P = .022), distant metastases (P = .003), and lack of treatment with metformin of patients with diabetes (P = .014) are associated with increased risk for shortened progression-free survival. In vitro data revealed that metformin inhibited cancer cell growth, activated cAMP-inducible protein kinase (5'-AMP-activated protein kinase [AMPK]), and down-regulated p70S6K/pS6. Metformin potentiated H₂O₂-inducible activation of AMPK but attenuated pERK and p70S6K. Tumors from MF+ patients demonstrated a lower level of phospho-p70S6K compared with the MF- group. Tumor size is smaller in patients treated with metformin, suggesting inhibition of tumor growth by the drug. Among diabetics, the absence of metformin therapy is an independent factor for decreased likelihood of CR and increased risk of shorter progression-free survival. In vitro data suggest that p70S6K/pS6 is likely a molecular target of metformin in DTC cells.

Metformin, Independent of AMPK, Induces mTOR Inhibition and Cell-Cycle Arrest through REDD1

Metformin is a widely prescribed antidiabetic drug associated with a reduced risk of cancer. Many studies show that metformin inhibits cancer cell viability through the inhibition of mTOR. We recently showed that antiproliferative action of metformin in prostate cancer cell lines is not mediated by AMP-activated protein kinase (AMPK). We identified REDD1 (also known as DDIT4 and RTP801), a negative regulator of mTOR, as a new molecular target of metformin. We show that metformin increases REDD1 expression in a p53-dependent manner. REDD1 invalidation, using siRNA or REDD1(-/-) cells, abrogates metformin inhibition of mTOR. Importantly, inhibition of REDD1 reverses metformin-induced cell-cycle arrest and significantly protects from the deleterious effects of metformin on cell transformation. Finally, we show the contribution of p53 in mediating metformin action in prostate cancer cells. These results highlight the p53/REDD1 axis as a new molecular target in anticancer therapy in response to metformin treatment.

Role of organic cation transporter 3 (SLC22A3) and its missense variants in the pharmacologic action of metformin.

The goals of this study were to determine the role of organic cation transporter 3 (OCT3) in the pharmacological action of metformin and to identify and functionally characterize genetic variants of OCT3 with respect to the uptake of metformin and monoamines. For pharmacological studies, we evaluated metformin-induced activation of AMP-activated protein kinase, a molecular target of metformin. We used quantitative PCR and immunostaining to localize the transporter and isotopic uptake studies in cells transfected with OCT3 and its nonsynonymous genetic variants for functional analyses. Quantitative PCR and immunostaining showed that OCT3 was expressed high on the plasma membrane of skeletal muscle and liver, target tissues for metformin action. Both the OCT inhibitor, cimetidine, and OCT3-specific short hairpin RNA significantly reduced the activating effect of metformin on AMP-activated protein kinase. To identify genetic variants in OCT3, we used recent data from the 1000 Genomes and the Pharmacogenomics of Membrane Transporters projects. Six novel missense variants were identified. In functional assays, using various monoamines and metformin, three variants, T44M (c.131C>T), T400I (c.1199C>T) and V423F (c.1267G>T) showed altered substrate specificity. Notably, in cells expressing T400I and V423F, the uptakes of metformin and catecholamines were significantly reduced, but the uptakes of metformin, 1-methyl-4-phenylpyridinium and histamine by T44M were significantly increased more than 50%. Structural modeling suggested that these two variants may be located in the pore lining (T400) or proximal (V423) membrane-spanning helixes. Our study suggests that OCT3 plays a role in the therapeutic action of metformin and that genetic variants of OCT3 may modulate metformin and catecholamine action.