Metformin_ Prevention of genomic instability and ...

Mutat Res Gen Tox En 827 (2018) 1–8

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Metformin: Prevention of genomic instability and cancer: A review a

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Masoud Najafi , Mohsen Cheki , Saeed Rezapoor , Ghazale Geraily , Elahe Motevaseli , Carla Carnovalef, Emilio Clementig,h, Alireza Shirazid a

Radiology and Nuclear Medicine Department, School of Paramedical Sciences, Kermanshah University of Medical Science, Kermanshah, Iran Department of Radiologic Technology, Faculty of Paramedicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Radiology, Faculty of Paramedical, Tehran University of Medical Sciences, Tehran, Iran d Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran e Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran f Department of Biomedical and Clinical Sciences L. Sacco, Unit of Clinical Pharmacology, ASST Fatebenefratelli-Sacco University Hospital, Università di Milano, Milan, Italy g Scientific Institute, IRCCS E. Medea, Bosisio Parini, Lecco, Italy h Unit of Clinical Pharmacology, Department of Biomedical and Clinical Sciences, Consiglio Nazionale delle Ricerche Institute of Neuroscience, L. Sacco University Hospital, Università di Milano, Milan, Italy b c

A R T I C L E I N F O

A B S T R A C T

Keywords: DNA damage Reactive oxygen species Oxidative stress

The diabetes drug metformin can mitigate the genotoxic effects of cytotoxic agents and has been proposed to prevent or even cure certain cancers. Metformin reduces DNA damage by mechanisms that are only incompletely understood. Metformin scavenges free radicals, including reactive oxygen species and nitric oxide, which are produced by genotoxicants such as ionizing or non-ionizing radiation, heavy metals, and chemotherapeutic agents. The drug may also increase the activities of antioxidant enzymes and inhibit NADPH oxidase, cyclooxygenase-2, and inducible nitric oxide synthase, thereby limiting macrophage recruitment and inflammatory responses. Metformin stimulates the DNA damage response (DDR) in the homologous end-joining, homologous recombination, and nucleotide excision repair pathways. This review focuses on the protective properties of metformin against genomic instability.

1. Introduction Genomic instability is an abnormal increase in mutations to the genome, which can be transferred to offspring cells. Cytotoxic agents, such as non-ionizing and ionizing radiation, free radicals, and metals, attack the genome thorough mechanisms that include generation of reactive oxygen species (ROS) and damage to nuclear or mitochondrial DNA, cell membranes, and enzymes [1]. These processes contribute to genomic instability, increasing mutation rates and resulting in the development of aggressive phenotypes and cancer. DNA damage may also lead to changes in the activities of genes involved in DNA repair, cell division, oncogenes, and tumor suppressor genes. Genomic instability is associated with increased mutation rate in genomic DNA; this may result from chronic ROS production, inhibition of antioxidant system enzymes, inflammation, and/or epigenetic changes [123–127]. Genomic instability has been examined by several endpoints, including chromosome rearrangements and aberrations, gene amplification, aneuploidy, micronuclei (MN) formation, microsatellite instability, and gene mutations [2]. Many compounds may mitigate

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genomic instability direct scavenging of ROS, hydrogen donation to reactive free radicals, inducing and/or altering the levels of endogenous enzymes for detoxifying ROS, and enhancing the DNA damage repair pathway [3,4]. Metformin is prescribed to over 120 million patients worldwide as treatment of choice for type 2 diabetes. The drug is considered safe, as its glucose-lowering actions are not accompanied by hypoglycaemia [5]. Metformin has many biological effects, including anti-inflammatory, anti-apoptotic, anticancer, hepatoprotective, cardioprotective, otoprotective, renoprotective, radioprotective and radiosensitizing, and antioxidant activities [6–15]. Several processes are implicated in the preventive action of metformin against genomic instability. One mechanism is protection against oxidative stress [16]. Metformin scavenges free radicals, prevents damage to nuclear and mitochondrial DNA, and enhances DNA repair [17]. In this article, we discuss the evidence for protective effects of metformin against DNA damage and cancer caused by genotoxic agents, and consider possible mechanisms for these effects.

Corresponding author. E-mail addresses: [email protected], [email protected] (M. Cheki).

https://doi.org/10.1016/j.mrgentox.2018.01.007 Received 22 September 2017; Received in revised form 28 December 2017; Accepted 15 January 2018 1383-5718/ © 2018 Elsevier B.V. All rights reserved.

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4. Mechanisms of the protective effect of metformin 4.1. Metformin and cellular metabolism 4.1.1. Metformin and ROS Metformin’s protective effects have been have associated with oxidative stress, DNA damage and DNA damage repair; however, there is no consensus concerning the role of the drug. Several studies have shown that metformin reduces ROS generation induced by stressors, thereby protecting cells and ameliorating genomic instability and possibly cancer risk [16]. Metformin can detoxify ROS as a direct or indirect free radical scavenger, through donation of a H atom from its CH3 or NH groups, and by up-regulation of thioredoxin activity [25–28]. The ability of metformin to counteract oxidative damage was confirmed in several experiments [29–31]. Metformin can reduce the level of intracellular ROS and γH2AX or Ataxia Telangiectasia Mutated (ATM) protein kinase activation in normal mitogenically stimulated lymphocytes [32]. It decreases insulin-induced intracellular ROS production and DNA damage in normal rat kidney epithelial cells [33]. In diabetic animals, by decreasing oxidative stress, metformin reduces the number of micronucleated erythrocytes [34]; micronuclei (MN) are an indicator of genome instability [35]. We have shown that metformin does not cause DNA damage in human blood lymphocytes [36] in vitro. Our results are in agreement with those of Sant'Anna et al. [37]. Other studies showed non-genotoxicity of metformin in rat and mouse bone marrow cells [12,38]. In contrast, other studies have reported that metformin causes increased DNA damage signalling, i.e, increased γH2AX expression in hepatoma cells [130] and increased γH2AX focus formation in pancreatic cancer cells [131]. These studies hypothesized that DNA-damaging effects of metformin are due to diminished DNA repair resulting from ATP depletion [130] or metformin-induced AMPK-dependent activation and consequent downregulation of the mTOR signaling pathway [131]. Onarn et al. found that metformin, at pharmacological concentrations, has no modifying effect on chemically induced DNA damage in cultured human lymphocytes, despite its partial protective effect against lipid peroxidation. However, higher metformin concentrations increased cumene hydroperoxide (CumOOH)-induced DNA damage [46]. Amador et al. reported that chronic treatment of Chinese hamster ovary (CHO-K1) cells with metformin may be genotoxic [39]. The concentrations of metformin used in these two studies (114.4 and 572 μg/ml, respectively) are 40–170-fold greater than the recommended therapeutic plasma level [40]. Harishankar et al. compared type 2 diabetes patients receiving metformin to healthy controls, and reported increased MN frequencies in the patients; however, type 2 diabetes can cause oxidative stress that could elevate MN frequencies [41]. Disagreements in the literature regarding metformin and DNA damage demonstrate the need for further investigation. Both genetic and environmental factors are associated with genotoxicity [41]. Table 1 summarizes the protective effects of metformin against genotoxicity induced by exogenous and endogenous agents. Fig. 2 illustrates mechanisms of action of metformin in response to toxicant-induced genomic instability. Fig. 3 illustrates possible molecular mechanisms for the protective effects of metformin.

Fig. 1. Chemical structure of metformin (CAS 1115-70-4).

2. Metformin Metformin (Fig. 1) is a biguanide derived from the perennial plant, Galega officinalis (French Lilac, also known as Goat’s Rue, Italian Fitch, or Professor weed). Its medicinal use dates back to ancient Egypt and medieval Europe; tea infusions were used to treat polyuria and halitosis, both of which are now recognised as symptoms of diabetes [18]. In the 1920s, guanidine was identified as the active component of galega, and was used to synthesize several anti-diabetic compounds; in the late 1950s, French scientists linked two guanidine rings together, producing better tolerated anti-diabetic agents, metformin and phenformin. Metformin was approved for the treatment of hyperglycemia in Britain (1958), Canada (1972), and the USA (1995) [19–21]. In the 1970s, phenformin and buformin, more potent, lipophilic biguanides, were withdrawn from the market, due to an adverse reaction, lactic acidosis; this led to increased use of metformin [18–21]. Metformin has remained the first-line therapeutic option for type two diabetes, with approximately 120 million patients taking the drug, world-wide [22]. The therapeutic plasma level of metformin is 0.5–2.5 mg/l (3–15 μM) [23]. Oral bioavailability is 50–60%; absorption takes place from the small intestine; 90% of the dose is eliminated unchanged in the urine in 12 h [20]. Metformin does not bind to plasma proteins. Its plasma half-life in humans is 6.2 h, with maximum plasma concentration, 1–2 mg/l, reached 1–2 h after an oral dose of 500–1000 mg [20]. Currently, the approved dose of metformin is 1–2.55 g daily, administered twice daily, for a 60 kg patient. The most frequent side effects associated with metformin are gastrointestinal, with over half of patients able to tolerate the maximum daily dose; however, it has been reported that 5% of patients are unable to tolerate any dose. The rare event of lactic acidosis occurs in approximately 3 per 100,000 patients and appears to be linked to renal insufficiency, impairing clearance and resulting in extremely high plasma levels of the drug. Thus, metformin is contraindicated in patients with substantial renal dysfunction. Anaemia due to vitamin B12 malabsorption and deficiency is also noted as a rare event [20,24]. 3. Search strategy We carried out a comprehensive search of Thomson ISI’s Web of Science, MEDLINE (PubMed), Google Scholar, and Scopus databases for articles published from Jan. 1990 to May 2017, using the search term “metformin” combined with the terms “DNA damage”, “DNA repair”, “genotoxicity”, " reactive oxygen species”, “antioxidant”, “cellular respiration”, and “inflammation”. We reviewed published articles and the bibliographies of selected manuscripts. The abstracts/titles of all articles identified by electronic searches were screened to determine whether they met the following inclusion criteria: (a) full abstract available online; (b) manuscript written in English; (c) focussed on or including the protective effect of metformin against DNA damage; (d) focussed on DNA repair by metformin; (e) focussed on antioxidant effects of metformin; (f) focussed on immunomodulatory activities of metformin; (g) focussed on anti-inflammatory effects of metformin; (h) focussed on epigenetic modifications by metformin; (i) focussed on anticancer effects of metformin.

4.1.2. Metformin and the antioxidant system Metformin activate endogenous repair systems, preventing ROS toxicity. The drug enhances the activity of the AMPK pathway and increases the expression of thioredoxin through the forkhead transcription factor 3 and TRX functions [30,42]. The consequent decrease in ROS levels may reduce genomic instability and possibly cancer risk [16,43,44]. Administration of metformin to rats decreased frequencies of MN 2

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Table 1 Protective effects of metformin against genotoxicity induced by exogenous and endogenous agents. Test System

Routea

Agent

Endpointsb

Results

Ref.

animals animals animals animals in vitro in vitro

Oral Oral Oral Oral – –

adriamycin streptozotocin nicotinamide-streptozotocin ionising radiation ionising radiation cumene hydroperoxide

↓ ↓ ↓ ↓ ↓ ↓

[12] [38] [34] [15] [36] [117]

in in in in

vitro vitro vitro vitro

– – – –

↓ ↓ ↓ ↓

[33] [118] [13] [29]

in vitro

–

insulin paraquat paraquat endogenous DNA damage in Hutchinson-Gilford Progeria Syndrome fibroblasts spontaneous DNA damage in Fanconi anemia – patient–derived fibroblast cells ionising radiation ionising radiation metformin metformin

MN MN, Total CAs MN γH2AX MN, NPB, Total CAs DNA fragmentation, comet assay tail factor % MN, % DNA in tail γH2AX foci, 8-oxo-dG γH2AX foci γH2AX foci RCF, CB

↓

[119]

γH2AX foci, comet assay tail factor% γH2AX foci CAs, % DNA damage (comet), MN MN

↑ ↑ ↑ ↑

[130] [131] [39] [41]

in vitro in vitro in vitro, animals human a b

intraperitoneal injection

Route of drug delivery. MN; Micronuclei, NPB; Nucleoplasmic bridges, CAs; Chromosomal aberrations, RCF; Radial chromosome formation, CB; chromosomal breaks.

glutathione reductase, and decreased MDA and H2O2 levels in murine livers damaged by CCl4 [45]. These studies show that metformin can exert protective effects on DNA through antioxidant activity. Stimulation of the antioxidant system against other toxicants has also been demonstrated [46]. Alhaider et al. showed that pretreatment of rats with metformin restored the depletion of GSH and the induction of ROS levels induced by streptozotocin, which might contribute to the nephroprotective effect of metformin in diabetes [47]. In contrast, some authors found that metformin increases ROS in cancer cells, when given alone or in combination with ionising radiation [14].

and chromosome berrations induced by hyperglycaemia, via increased glutathione (GSH) and decreased malondialdehyde (MDA) [38]. Doxorubicin-induced reductions of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities in adult mouse cardiomyocytes were reversed by metformin [8]. Administration of metformin to rats before cisplatin injection reduced the levels of MDA and total ROS, and restored changes of the activities of GSH, CAT, glutathione transferase, and SOD in kidney tissues [11]. Similar results were obtained after pretreatment of mice with metformin prior to adriamycin [12]. A study by Dai et al. showed that metformin elevated GSH levels, and the actions of SOD, CAT, glutathione peroxidase and

Fig. 2. Proposed mechanism of action of metformin in response to toxicant-induced genomic instability.

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Fig. 3. Molecular mechanisms for the protective effects of metformin.

inflammation. NOXs activation is persistent leading to sustained ROS production [55]. NOX1 and NOX2 can suppress AMPK and induce malignancy [56]. Stimulation of AMPK by metformin can thus bypass at least in part the tumourigenic activity of NOXs. It also inhibits NOXs directly through AMPK activation [57]. Metformin suppresses NOX4 upregulation. Upregulation of NOX4 by some stimulators, including ionizing radiations and chemotherapeutic agents, is associated with increased ROS production and DNA damage [58–60]. Evidence indicates that the TGF-β-NOX4 pathway is the most important pathway for ROS production, DNA damage and genomic instability in bone marrow [61]. Metformin through suppression of this pathway may protect cells against genomic instability induced by ionizing radiation or other cytotoxic agents [62], an effect to which the effects described above on mitochondria may also contribute [63]. Indeed, administration of metformin resulted in decreased DNA damage and stem cell senescence in bone marrow [15].

4.1.3. Metformin and cellular respiration Mitochondria are the most important source of free radical production in cells and this flux of oxidative stress can cause DNA damage and genomic instability [48]. Mitochondria may produce excessive ROS in a process termed “ROS-induced ROS generation” [49]. Complex 1 (NADH-ubiquinone oxidoreductase) of the electron transfer chain (ETC) is the main source of ROS in this organelle [50]. When defense mechanisms, including CAT and mnSOD [51], are overwhelmed, mitochondrial genome instability (mtGI) ensues, with mutations in mitochondrial DNA (mtDNA) and overall mitochondrial malfunction. mtGI contributes to development of several types of human malignancy, including colorectal, gastric, breast, and kidney cancers [52]. Metformin inhibits complex I; this may be one of the mechanisms whereby it protects cells against the genotoxicity of mitochondrial ROS. Inhibition of complex I results in decreased ATP/increased AMP levels [51]. AMP stimulates the phosphorylation and activation of the AMPactivated protein kinase (AMPK), an enzyme that promotes the activity of p53, playing a key role in DNA repair [52].

4.1.6. Metformin and insulin-induced ROS A high level of insulin growth factor-1 (IGF1) has been reported to be associated with tumorigenesis. IGF-1 can induce DNA damage and mutagenesis through stimulation of genes involved in reduction/oxidation systems, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and NOXs [64–67]. It also activates the PI3K/ Akt/mTOR pathway, which results in inhibition of apoptosis, cell proliferation, and tumorigenesis [68,69]. Metformin, by reduction in IGF-1 binding to its receptor, suppresses the PI3K/Akt/mTOR pathway [70]. Metformin also suppresses mTOR and proliferation of prostate cancer cells through increased REDD1 gene expression which is itself regulated by p53 [71].

4.1.4. Metformin and AMPK AMPK protects cells against physiological and pathological stresses, hypoxia, and heat shock, but it also mediates cell cycle checkpoints, inhibits pro-survival growth pathways, and modulates mitotic progression [53]. Although the mechanisms of action of metformin on DNA damage responses (DDR) remain to be elucidated, control of AMPK activity has indeed a central role. It has been reported that metformin has an antiproliferative effect associated with cell cycle arrest in the G0G1 phase, cell apoptosis, and cell death, which are mediated by oxidative stress and AMPK activation [56]. Metformin protects skin against UVB-induced injury by enhancing DNA repair capacity. The researchers suggested that metformin promotes UVB-induced DNA damage repair and growth control. Moreover, they showed that metformin reduces UVB-induced skin tumourigenesis. They found that metformin promotes UVB-induced DNA damage repair through AMPK activation, whereas it decreases cell proliferation through AMPK-independent ERK inhibition. Furthermore, metformin increased XPC levels, suggesting that AMPK positively regulates the NER pathway through upregulation of XPC [54].

5. Metformin, inflammation and the immune system Inflammation contributes to carcinogenesis via stimulation of ROS and NO production. Suppression of mismatch repair (MMR) enzymes, such as 8-oxoguanine glycosylase (ogg1), and BER enzymes, such as O6alklyguanine-DNA alkyltransferase (AGT) are examples of suppression of DNA repair by reactive species generated during inflammation [72,73,122]. As a result, inflammation causes accumulation of DNA damage and mutations of tumour suppressor genes, which may result in genetic instability and carcinogenesis [74,121]. In in vitro, in vivo, and human studies, metformin was found to reduce inflammation by decreasing the levels of the inflammatory

4.1.5. Metformin and NADPH oxidase NADPH oxidases (NOXs) are a family of potent ROS-producing enzymes activated in response to stimuli such as pathogens, diabetes, and 4

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metformin, can inhibit acetyl-CoA production and fatty acid synthesis. Galdieri et al. showed that this effect of metformin on prostate and ovarian cancer cells is responsible for histone acetylation [93].

cytokines Il-6, TNF-α, the activity of COX-2, and c-Jun N-terminal kinase (JNK) and of the proinflammatory transcription factors NF-kB and STAT3 and of Saa1 and Saa2 [52,75,76]. In a study by Kelly et al., metformin treatment resulted in decreased IL-1 as well as increased production of anti-inflammatory IL-10 in macrophages. These changes were associated with decreased ROS production [77]. Another mechanism for the anti-inflammatory effect of metformin is stimulation of the SIRT1 signalling pathway. Upregulation of SIRT1 by AMPK results in the amelioration of oxidative stress and decreased DNA damage. As a result, activation of SIRT1 signalling can decrease release of DAMPs and the subsequent inflammatory responses [78]. Moreover, SIRT1 has some tumour suppressor activity, as it can downregulate βcatenin and survivin [79]. CD8+ cytotoxic T lymphocytes (CTLs) recognize and kill tumour cells via the tumour-specific class I major histocompatibility complex molecule (MHC-I) [80]. Under some conditions, upregulation of immunosuppressive factors, such as transforming growth factor (TGF)-β1, intercellular adhesion molecule 1 (ICAM-1), and vascular endothelial growth factor (VEGF), may attenuate the tumour-suppression activity of immune cells [81]. Since the activity of CTLs is strictly controlled by AMPK and mitochondrial fatty acid oxidation, treatment of mice with metformin leads to increased CD8T cell survival and activity [82]. Pearce et al. proposed that the changes in AMPK and mitochondrial fatty acid oxidation induced by metformin are a strategy for modulation of T cell responses [83].

7.1. Anti-cancer effects of metformin Interest in repurposing metformin for treatment of cancer was prompted by epidemiological and clinical evidence [128,129]. Epidemiological studies suggest that diabetic patients treated with metformin have reduced incidence and mortality from a range of neoplasms [94–98]. In particular, metformin reduced the incidence of breast cancer in a study of 22,621 women [99]. Further studies confirmed the protective effect of metformin against breast cancer induction [100,101]. In 2005, Evans et al. found that metformin therapy was associated with reduced risk for cancer in a group of 11, 876 patients with type 2 diabetes. The protective effect increased with dose and duration of metformin use [102]. In 2006, Bowker et al. found that metformin use decreased cancer mortality; in a study of 10,309 patients diagnosed with type 2 diabetes, sulfonylurea or exogenous insulin users were more likely to die of cancer-related causes than were metformin users [103]. More recently (2009), Libby et al. found a negative correlation between metformin use and risk of cancer in 8000 diabetic patients. Metformin users were 4% less likely to be diagnosed with cancer, 3% less likely to die of cancer, and had a 1-year prolongation of cancer diagnosis from the start of metformin usage [104]. Jiralerspong et al. found that diabetic breast cancer patients treated with metformin had a 24% pathological complete response (pCR) to standard neoadjuvant chemotherapy, while non-users had only 8% pCR. Despite this, metformin did not improve the estimated 3-year relapse-free survival rate [105]. Meta-analysis confirmed a 31% reduction in overall cancer incidence or mortality among diabetic metformin users [106]. The reduction was statistically significant for pancreatic and hepatocellular cancer, but not for colon, breast, or prostate cancer. A dose-response relationship was noted for each cancer type [107]. A recent retrospective study found that the outcomes of chemotherapy in non-small cell lung carcinoma patients who were also diabetic were better (increased progression-free survival and overall survival) if they were taking metformin vs. other anti-diabetic agents. A large prospective study in Taiwan found that although diabetes increases cancer incidence 2-fold, diabetic patients using metformin (at doses as low as 500 mg/day) have a cancer incidence which is the same, or less, than the incidence for non-diabetics [107]. Retrospective epidemiological studies are subject to confounding factors; indeed, obesity and prolonged hyperinsulinaemia also influence cancer risk, burden, and prognosis [108]. Patients are not randomised to metformin vs other therapies, so there are likely to be underlying metabolic differences between treatment groups. Nevertheless, these studies were designed independently and carried out in different settings; they demonstrate clinically important benefits that justify further clinical evaluation of metformin in cancer.

6. Metformin, ATM, γH2AX and DNA repair ATM, a 370 kDa protein of the PIKK family, is a mediator of the DDR. Via DNA damage repair enzymes, including RAD50, Nijmegen breakage syndrome 1 (NBS1), and meiotic recombination 11 (MRE11), the DDR recruits ATM to DNA double-strand breaks (DSBs [84,85]. ATM then initiates a network of signalling events leading to DNA repair, cell cycle arrest, or survival. When ATM is activated, histone H2AX is phosphorylated on serine 139 (γH2Ax), and orchestrates the recruitment of repair complexes at sites of DNA damage [86]. AMPK participates in ATM signalling events which protect cells against genomic stress and modulate cell survival [84]. Vazquez-Martin et al. found that metformin activates ATM and checkpoint kinases-2 (Chk2) in human epithelial pre-cancerous lesions. Chk2 is a protein kinase that, along with other proteins, is involved in the ATM pathway following DNA damage. This study showed that metformin induces phosphorylation of H2AX to γH2AX. This effect of metformin stimulates the ATM/Chk2 pathway even in the absence of DNA damage [87]. However, phosphorylation of H2AX was not detected in ovarian cancer in response to metformin [88] and metformin does not induce DDR in an ovarian cancer cell line [89]. Halicka et al. showed that metformin leads to decreased constitutive Ser139-phosphorylation of H2AX and constitutive activation of ATM [32]. 7. Metformin and epigenetic modifications

7.2. Clinical evidence Histone acetylation is an epigenetic modification regulated by the enzymatic activity of histone acetyltransferases and deacetylases [90]. Metformin-activated AMPK enhances the activity of histone deacetylase SIRT1, reducing acetylation of p53 and expression of p21 in human umbilical vascular endothelial cells (HUVECs) [91]. Also, in human hepatocellular carcinoma (HepG2) cells, a low concentration of metformin suppresses the deacetylase activity of SIRT1 on p53, leading to increased p53 acetylation and cell senescence [120]. Histone acetylation is a novel strategy for cancer treatment based on increasing expression of tumor suppressor genes. Metformin causes changes in histone acetylation via mitochondrial biosynthetic limitation. Metformin can alter histone H3 acetylation in cancer-prone cells, inhibiting carcinogenesis. This effect may be mediated through reduction of acetyl-CoA substrate [92]. In addition, AMPK, activated by

Prospective clinical data in non-diabetic patients also motivate the evaluation of metformin in cancer. A study of non-diabetic breast cancer survivors without clinical recurrence, and with normal baseline insulin levels, demonstrated 22% decreased insulin levels after only six months of metformin [109]. Treatment with metformin for 1 month, 250 mg/d, reduced aberrant crypt foci in the rectal epithelium (an endoscopic surrogate marker of colorectal cancer risk), vs. placebo, supporting metformin’s chemopreventive potential [110]. Recently, a pre-operative trial examined the effect of metformin on non-diabetic women with operable invasive breast cancer, who received 1000 mg metformin twice daily for two weeks. A significant reduction in levels of KI-67, a nuclear protein that may be necessary for cellular proliferation, and changes in expression of genes involved in metabolism and cancer 5

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signaling, were seen [111]. Interim analyses of on-going clinical trials have demonstrated that metformin is safe and tolerable in cancer patients and the drug may have anti-proliferative potential when used as sole anti-cancer treatment [112]. Currently, according to National Institute of Health online databases (www.clinicaltrials.gov), there are more than 50 clinical trials underway investigating, prospectively, metformin’s prophylactic and antineoplastic activities.

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