Metformin in Lung Cancer: Review of in Vitro and in Vivo Animal Studies

Cancer cells display enhanced growth rates and a resistance to apoptosis. The ability of cancer cells to evade homeostasis and proliferate uncontrollably while avoiding programmed cell death/apoptosis is acquired through mutations to key signaling molecules, which regulate pathways involved in cell proliferation and survival and these mutations allow them to develop resistance to many chemotherapeutic agents, highlighting the need for development of new potent anti-cancer agents. Metformin has long been used as a treatment for type 2 diabetes and has recently attracted attention as a potential agent to be used in the treatment of cancer. The present review summarizes the existing in vitro and in vivo animal studies focusing on the anti-lung cancer effects of metformin and its effects on key proliferative and anti-apoptotic signaling pathways.


Introduction
Cancer cells are characterized by their high rate of proliferation and resistance to apoptotic cell death [1]. There were 1.56 million deaths globally due to lung cancer in 2012, representing approximately 19.4% of all cancer related deaths [2]. It was estimated that, in 2016, new lung cancer cases will account for 14% of all new cancer cases and lung cancer related deaths will account for 26% of all cancer related deaths [3].
Most cancers that originate in the lungs are known as carcinomas, and are subdivided into non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC accounts for approximately 85% of all lung cancer cases [3]. There are three subtypes of NSCLC: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Adenocarcinoma is the most common type, accounting for approximately 40% of all lung cancer cases and is most often characterized by significant production of mucus [4]. Squamous cell carcinoma is more closely related to tobacco smoking than any other type of lung cancer and often occurs centrally in larger bronchi [4,5]. Large cell carcinoma is a heterogeneous group of undifferentiated malignant neoplasms that lack features of other types of lung cancer [4]. SCLC accounts for approximately 15% of all lung cancer cases [3]. Typically, SCLC presents in the central airways and attacks the submucosa layer leading to narrowing of the airways [5]. Compared to NSCLC, SCLC has a shorter doubling time and metastasizes earlier. In fact, over 70% of SCLC patients present with metastatic disease [5].
Cancer cells exhibit various mutations that increase the expression/activation of growth factor (GF) receptor proteins such as epidermal growth factor receptor (EGFR) [6][7][8][9]. Growth factors bind to their receptors, receptor auto-phosphorylation occurs leading to activation of downstream signaling cascades such as the phosphoinositide-3-kinase (PI3K)/Protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway that results in increased survival/proliferation and inhibition of apoptosis [10][11][12][13]. Another pathway activated by GF signaling is the RAS/mitogen activated protein kinases (MAPK) pathway. GF binding to the activated receptor through the involvement of the Grb2/SOS complex renders Ras in the GTP-bound form, activating Raf, MEK and MAPK further downstream [13][14][15][16]. Cancer cells often exhibit mutations that allow them to constitutively enhance proliferation while inhibiting apoptosis/cell death pathways.
Lung cancer is treated with surgery, radiation therapy, chemotherapy, specific and targeted therapy or a combination of these different approaches. Due to a high level of metastasis and post-surgical relapse, chemotherapy has become the most prevalent form of treatment. Unfortunately, cancer cells in general and lung cancer specifically are characterized by mutations and over activation of the PI3K-Akt-mTOR [17,18] and the Ras-MAPK [14,19] cascades that allow them to develop resistance to many first line chemotherapeutic agents highlighting the need to develop new chemotherapeutic agents.
Metformin was first used in medieval Europe, where it was extracted from Galega officinalis also known as the French lilac and it was used to treat polyuria. Metformin is now used as a first-line medication for the treatment of type 2 diabetes mellitus. Research interest on the anticancer effects of metformin was initiated in 2005 in a study by Evans et al., showing reduced risk of cancer in type 2 diabetes patients receiving metformin [20]. This study sparked scientific interest on the anticancer effects of metformin. Further studies examining the anticancer effects of metformin have been performed and many other clinical trials are ongoing [21,22]. For more information on the ongoing clinical trials, the reader can see the following websites https://www.clinicaltrials.gov and https://health-products.canada.ca/ctdb-bdec/index-eng.jsp.
Many studies have been performed using metformin in different cancer cell lines and in in vivo animal models of cancer [23][24][25][26][27][28][29][30][31][32][33]. In the present review, we summarize all in vitro and in vivo animal studies examining the effects of metformin in lung cancer. To establish a systematic literature review, we used the online search engine PubMed. We searched the key phrases: "Metformin" and "Lung Cancer". All published studies pertaining to our topic were included in the current review. In vitro and in vivo studies on the effects of metformin have been summarized in separate sections and sorted by histology and treatment approach (single agent or combination therapy) resulting in a clear, detailed and inclusive summary of the existing literature. This review also focuses on the mechanistic data provided by these studies, which will be beneficial for future research to help focus efforts on identifying the main mechanisms involved in the anticancer action of metformin. The studies presented in the text are also summarized, organized and presented in a table format to allow the reader to extract the information easily. This is a comprehensive systematic review and adds to the existing literature by summarizing all relevant in vitro and in vivo animal studies using metformin in lung cancer.

Effects of Metformin in Lung Cancer: In Vitro Studies
Metformin has been used as a single agent in many different lung cancer cells, and the data obtained indicate numerous anticancer properties ( Table 1). Exposure of A549, RERF-LC-A1, IA-5 and WA-hT cells to metformin (1-20 mM) resulted in a significant decrease in cell proliferation, significant induction of apoptosis as well as decreased colony formation accompanied by a significant induction of G 0 /G 1 cell cycle arrest [34]. Treatment of Calu-1 and Calu-6 cells with metformin (0.3-5 mM) was shown to decrease proliferation in Calu-6 cells which express lower levels of Calveolin-1. This was associated with inhibition of IGF-1 dependent phosphorylation (activation) of the proto-oncogene Akt [35]. FOXO3a, a transcription factor involved in initiation of apoptosis, is phosphorylated by Akt resulting in its nuclear exit and therefore inhibition of apoptosis. IGF-1 dependent FOXO3a phosphorylation and nuclear exit was shown to be inhibited by metformin. Furthermore, it was observed that phosphorylation/activation of the energy sensor AMPK was increased by metformin, an effect that required the involvement of calveolin-1 [35]. Do et al., 2013 treated A549 cells with metformin (1-10 mM) and found that heme oxygenase (HO-1) mRNA and protein levels were suppressed indicating decreased levels of oxidative stress, Nrf2, a transcription factor that regulates production of antioxidant proteins that protect against oxidative damage, was reduced [36]. Additionally, Raf/ERK phosphorylation was also suppressed [36]. Exposure of PC9 cells to metformin (1-32 mM) resulted in an inhibition of proliferation [37]. A study by  found that exposure of A549 and H1975 cells to metformin (5-50 µM) resulted in increased cytotoxicity and decreased thymidine phosphorylase (TP) and excision repair cross-complementation 1 (ERCC1) mRNA and protein levels, both of which are associated with repairing DNA breaks [38]. Interestingly, a significant decrease in MEK/ERK phosphorylation was seen with metformin treatment [38]. Exposure of A549 and A431 cells to metformin (1-10 mM) was found to induce apoptosis and inhibit proliferation as well as reduce Akt and mTOR phosphorylation [39]. Treatment of H1299, GLC82, H1975, CALU-3, CALU-3 GEF-R, H460 and A549 cells with metformin (0.1-20 mM) resulted in inhibition of proliferation and induction of apoptosis [40]. Metformin treatment alone was also found to activate MAPK [40], while in another study in squamous lung cancer cells [41] metformin inhibited MAPK. Interestingly, increased activation of MAPK in breast cancer [42] and adrenal gland cancer [43] was associated with a decrease in cell proliferation and an increase in apoptosis indicating that MAPK activation in specific cells can lead to apoptosis. Exposure of Calu-1 cells to metformin (0.0375-10 mM) resulted in a significant induction of apoptosis which was associated with decreased glucose uptake marked by an inhibition of the key glycolytic enzyme hexokinase-II [44]. These data provide strong evidence that metformin had an inhibitory effect on cancer cell glycolysis, counteracting the Warburg effect [45,46]. A549 cells treated with metformin (0.5-8 mM showed inhibition of proliferation and induction of apoptosis which was associated with increased p38 MAPK phosphorylation [47]. In another study, A549 cells treated with metformin (10 mM) were shown to have decreased expression of B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1). Bmi-1 is an oncogene that alters the cell cycle and apoptosis by promoting tumor cell self-renewal and epithelial to mesenchymal transition (EMT). Metformin's inhibition of EMT has also been reported in several other studies, Kurimoto et al. reported that both PC-9 and HCC-827 adenocarcinoma cells treated with TGF-β and FGF-2 to induce EMT, had elevated levels of PD-L1 (a marker of EMT) and increased resistance to gefitinib and cisplatin, however treatment with metformin (0.1-10 mM) suppressed PD-L1 expression and reversed resistance to gefitinib and cisplatin [48]. Cufi et al. also reported that treatment of MCF-7 breast cancer cells with metformin impeded the TGF-β induced EMT and increased the levels of the epithelial marker E-cadherin [49]. In addition metformin prevented the TGF-β induced cell scattering and accumulation of the mesenchymal marker vimentin in Madin-Darby canine kidney (MDCK) cells) [49]. These metformin effects may be mediated by an increase of miR-15a, miR-128, miR-192 and miR-194 which inhibit Bmi-1 mRNA translation [50]. A study by Li et al., 2015 found that treatment of PC-9 and PC-9GR cells with metformin (5 mM) resulted in decreased markers of pulmonary fibrosis associated with decreased TGF-β activation and reduced levels of the downstream signaling molecules COL1A1, pSMAD2, pSMAD3, pSTAT3, pAKT and pERK [51]. Treatment of H522, H2342, H2405, A549, SPC-A-1, SW900, H1869, SK-MES-1, H661, H1299 and H1281 with metformin (1.25-5 mM) resulted in decreased proliferation and decreased stem cell marker levels [52]. Metformin was also shown to decrease nemo-like kinase (NLK), a member of the MAPK pathway, Nanog, a transcription factor involved in self-renewal of undifferentiated embryonic stem cells as well as the levels of the transcription factors c-Myc and KLF4 [52]. Treatment of A549 cells with metformin (5-50 mM) resulted in increased apoptosis and G 0 /G 1 cell cycle arrest, decreased Bcl-2 and increased Bax protein levels [53]. Exposure of H460 and H1299 cells to metformin (5-20 mM) was shown to inhibit proliferation, induce apoptosis, induce G 0 /G 1 cell cycle arrest, increase AMPK phosphorylation and suppress total and phosphorylated mTOR and p70S6K levels [54].

Effects of Combined Metformin Treatment in Lung Cancer: In Vitro Studies
Apart from all the above-mentioned studies where metformin was used as a monotherapy, more recent studies have examined the effect of metformin treatment in combination with other agents/treatment strategies ( Table 2). Treatment of H520 and H1703 cells with 0.1-1 mM metformin in combination with 1-10 µM gefitinib, an EGFR inhibitor, was found to increase gefitinibs' cytotoxic effect. Metformin potentiated the growth inhibitory effect of gefitinib by decreasing the gefitinib induced MSH2 expression [41]. Furthermore, treatment of PC-9, PC-9GR and H1650-M3 cells with 5 mM metformin and 1-16 µM gefitinib or 2.5-20 µM erlotinib, another EGFR inhibitor, was shown to re-sensitize EGFR-tyrosine kinase inhibitor (TKI) resistant cells and reverse EMT through increased E-cadherin and decreased vimentin and SNAIL expression [55]. In another study H2228 and H3122 cells treated with 5 mM metformin and 400 nM crizotinib, an anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) inhibitor, had increased crizotinib sensitivity, decreased cell proliferation and tumor invasion and increased apoptosis [56]. Additionally, metformin reversed resistance to crizotinib [50]. These effects were accompanied by decreased IGF1-R signaling and a reduction in the IGF-1 induced phosphorylation of mTOR and p70S6K [19]. Treatment with 10 mM metformin and 30 nM trametinib, a MEK inhibitor, was found to be effective in N-RAS mutant lung carcinomas, specifically SW1271 and H2347 cells. This combination was shown to decrease cell viability and reduce activity of the MAPK and PI3K/Akt/mTOR signaling pathways [57]. A549 and H460 cells treated with 0.25-2 mM metformin and 0.25-6 µM sorafenib, a tyrosine kinase inhibitor, resulted in decreased cell proliferation and an increase in AMPK activation [58]. Metformin (2 mM) combined with selumetitnib (0.01-1 µM), a MEK inhibitor, increased apoptosis and decreased proliferation of H358, Calu-3, H1299 and H1975 cells [59]. Furthermore, metformin down regulated GLI1 transcriptional activity as well as decrease the production of MMP-2, MMP-9 and NF-κB [59]. Treatment of AS2 cells with 2.5 µM-2.5 mM metformin and 2.4 µM cisplatin, a platinum based drug that interferes with DNA replication, enhanced cisplatin-induced cytotoxicity, inhibited cisplatin-induced ROS production as well as decreased secretion of VEGF [60]. Treatment of H460 cells with 15.145-60.58 mM metformin and 0.0995-0.199 mM cisplatin or 0.0926-0.1852 mM etoposide, an inducer of DNA strand breaks, decreased proliferation through reduced metabolic viability of these cells [61]. Treatment of H1650 and H1703 cells with 0.1-1 mM metformin and 0.1-1 µM paclitaxel, a drug that targets tubulin, was shown to enhance paclitaxels' cytotoxic effects, decrease DNA excision repair protein (ERCC1) expression and p38 MAPK phosphorylation [62]. In H1299 and H1650 cells treated with 5 mM metformin and 20 µM ciglitazone, a selective PPARγ ligand, an induction of apoptosis, inhibition of growth and reduced PDK1 expression was seen [63]. In HCC4006, NCI-H1975, HCC95, NCI-H2122 and NCI-H3122 cells treatment with 0.01-10 mM metformin in combination with 5 µM salinomycin, a drug with high antimicrobial activity, was shown to promote cell death through inhibition of EGFR, Akt, mTOR and p70S6K signaling [64]. Treatment of A549 and HCC4006 cells with 1-10 mM metformin in combination with 0.1-1 µM salinomycin inhibited the TGF-β induced EMT as well as inhibited cell migration through increased E-cadherin expression [65]. A549, SK-MES-1, H520, SPC-A-1, H1975 and PC-9 cells treated with 3 mM metformin and 100 ng/mL Figitumumab, an antibody against IGF-1R, had reduced PI3K/Akt and MEK/ERK signaling cascade activation [66]. Treatment of A549 cells with 1-4 mM metformin alone or in combination with 200 ng/mL TRAIL protein enhanced TRAIL-mediated tumor cell death and decreased p62 protein levels [67]. A549 and PC9 cells treated with 20 µM metformin in combination with 20 µg/mL β-elemene had reduced growth [68]. It was observed that metformin augments the effects of β-elemene by blockage of Akt signaling and inhibition of DNMT1 expression [68]. In A549, H1299 and SK-MES1 cells treatment with 2.5 µM-5 mM metformin resulted in enhanced response to radiation treatment as was seen by enhanced inhibition of proliferation, induction of cell cycle arrest and induction of apoptosis [69].These effects were associated with activation of the AMPK-p53 pathway, inhibition of Akt and mTOR [69].

Effects of Metformin in Lung Cancer: In Vivo Studies
Metformin has not only been studied as a single agent or as part of a combination treatment in vitro, but has also been studied as both a single agent and as part of a combination treatment in animals (Table 3). In a study where Balb/c mice were injected with A549 or A431 cells then treated with 250 mg/kg/day metformin for 21 days, metformin was found to inhibit growth of K-ras mutant tumors, decreased Akt and mTOR total and phosphorylation levels and induced apoptosis [39]. Mice treated with 5 mg/mL metformin were found to have decreased lung tumorigenesis (tobacco carcinogen, NNK-induced) that was associated with a significant reduction in plasma IGF-1 levels and decreased IGF-1 signaling [70]. Additionally, phosphorylation of multiple RTKs (including EGFR, insulin receptor and VEGF) and GTP-bound Ras levels were decreased and these effects were independent of AMPK [70]. Another study found that nude mice treated with 250 mg/kg/day metformin two days before inoculation with HCC827-pSB388 cells showed decreased tumor growth, reversed IL-6 induced EMT and blocked STAT3 phosphorylation [71].  found that Balb/c mice injected with H1299 or Calu-3 GEF-R cells treated with 200 mg/mL metformin and 150 mg/kg/day gefitinib for 35 days had decreased proliferation and increased apoptosis as well as decreased EGFR phosphorylation and activation of MAPK [40]. Treatment of Balb/c mice injected with PC-9 or PC-9GR cells with 1 mg/mL metformin and 250 mg/L gefitinib for 30 days was found to block tumor growth of TKI-resistant xenografts, decrease IL-6 secretion and expression as well as reverse EMT [55].
Often excellent initial clinical responses are observed with drugs such as gefitinib, however these drugs might promote interstitial lung disease (ILD) which restricts the effectiveness of these agents in treatment. Li et al., 2015 found that treatment of Sprague Dawley (SD) rats with 300 mg/kg metformin attenuated the gefitinib-induced exacerbation of pulmonary fibrosis [51]. Metformin was also found to decrease actin, COL1A1, pSMAD2, pSMAD3, pSTAT3, p-Akt and Erk levels as well as decreased TGF-β activation and protein levels [51]. Pulmonary fibrosis, a type of ILD, has been associated with an increase in cancer risk. Balb/c mice injected with AS2 cells and treated with 500 mg/kg metformin and 4 mg/kg cisplatin showed inhibition of xenograft growth, inhibition of cisplatin-induced ROS production and inhibition of the STAT3 pathway [60]. Balb/c mice injected with A549 cells were treated with 40 mg/kg/day metformin in combination with 5 mg/kg/day cisplatin were found to have decreased tumor size mediated through decreased Bcl-2 protein levels, increased Bax protein levels and increased phosphorylation of p38 MAPK [47]. In another similar study, Balb/c mice injected with A549 cells were treated with 400 mg/kg/day metformin and 30 mg/kg/day sorafenib for 40 days and were shown to have decreased cancer cell proliferation, decreased tumor size, increased AMPK phosphorylation and inhibition of mTOR signaling [58]. Furthermore, treatment of H1299 or H1975 lung cancer cell bearing nude mice with 200 mg/mL metformin and 25 mg/kg selumetinib for 35 days was shown to inhibit EMT through increased SNAIL and decreased vimentin expression, decrease tumor metastatic behavior and decrease production of MMP-2 and MMP-9 through a reduction in NF-κB levels [59]. Balb/c mice injected with A549 or H1299 cells treated with 300 mg/kg/day metformin in combination with 10 Gy ionizing radiation (IR) until euthanasia showed inhibition of xenograft growth, activation of the AMPK-p53 pathway, inhibition of the mTOR-4EBP1 pathway, reduced angiogenesis and enhanced expression of apoptotic markers [69]. Compromised immune system responses play an important role in cancer and in recent years immune related therapies have been developed and shown to have strong clinical benefits [72][73][74]. Some of metformin's anti-cancer effects have been proposed to be immune-mediated [75,76]. Metformin treatment resulted in rejection of solid tumors in normal but not T-cell deficient SCID mice [77], clearly demonstrating an anticancer effect via immune system modulation. Furthermore, treatment of CD8 + lymphocytes with low concentrations of metformin (10 µM) resulted in their protection from apoptosis, ultimately leading to enhanced anti-tumor effects [77].

Effects of Metformin in Lung Cancer: Human Studies
Although the focus of this review is on in vitro and in vivo animal studies of metformin in lung cancer, metformin has already been used in multiple clinical studies. Zhang and colleagues performed a systematic review and meta-analysis of four human studies from 2009 to 2013 and found that, in the four studies, diabetic patients receiving metformin had a significantly lowered risk of lung cancer (RR = 0.71, 95% CI: 0.55-0.95; p = 0.02) [78]. Studies by Hall et al. [79] and Lai et al. [80] investigated the association between diabetes and lung cancer and both reported a reduction in lung cancer risk in patients receiving metformin treatment. Specifically, Lai et al. reported that the risk of lung cancer among diabetic patients receiving metformin was 39%-45% lower [25,80]. Noto et al. performed a meta analyses of articles published before October 2011 and found the risk of lung cancer among type 2 diabetes mellitus metformin users to be 0.67 (95% CI: 0.45-0.99) [81]. Lin et al. identified 750 diabetic patients between the ages of 65-80 who were diagnosed with stage IV NSCLC between 2007 and 2009, the median survival of patients on metformin was five months compared to three months for patient not taking metformin (p < 0.001); the propensity score also showed that metformin use was associated with a statistically significant improvement in survival (HR = 0.80, 95% CI: 0.71-0.89) [27,82]. Tan et al. obtained data on patients with NSCLC who had diabetes from five hospitals in China between January 2004 and March 2009 and found that patients receiving chemotherapy in combination with metformin had better outcomes compared to those receiving chemotherapy in combination with insulin or other diabetic drugs, their overall survival was 20 months, 13.1 months and 13.0 months, respectively [27,83]. A study by Dhillon et al. examined 409 stage I NSCLC and diabetic patients who underwent anatomic resection from 2002 to 2011 and found that diabetes had no effect on overall survival of the patients (p = 0.75), but found that metformin treatment was associated with improved overall survival (p = 0.02) [84]. However, there are also a few studies that report no effect between metformin and risk of lung cancer. Nie et al. reported that in 11 studies found, in a systematic search up to August 2013, there was no significant association between diabetes mellitus patients receiving metformin and lung cancer risk (OR = 0.99, 95% CI: 0.87-1.12) [85]. Similarly, Franciosi et al. reported that in two randomized controlled studies (RCTs) and four observational studies, there was no significant association between metformin and lung cancer risk in RCTs and only a marginal non-significant reduction was found in observational studies in diabetes mellitus patients (OR = 0.83, 95% CI: 0.64-1.06, p = 0.13) [86].
In addition to the studies presented above, there are currently 15 clinical trials as of 11 April 2017 registered on the https://www.clinicaltrials.gov website that are studying the effect of metformin on lung cancer. Of the 15 currently registered studies, there are three studies examining the effects of metformin as a single agent in NSCLC. An additional four studies examining the combination of metformin and gefitinib; the combination of metformin, carboplatin, bevacizumab and paclitaxel; the effects of metformin and Nivolumab; and the combination of metformin, carbohydrate restriction and platinum based chemotherapy, aiming to examine the relationship between metformin and various chemotherapy agents in the treatment of NSCLC. There are three further studies examining the effects of combined metformin and radiotherapy treatment in NSCLC patients. One study is combining metformin with chemotherapy (paclitaxel and carboplatin) and radiotherapy. Another study is examining the effects of metformin with tyrosine kinase inhibitors (TKIs) in NSCLC patients. Additionally, a phase I study of metastatic or unresectable solid tumors is examining the combination of metformin and temsirolimus. The combination of metformin and sirolimus is also being tested in another trial in patients with advanced solid tumors. Finally one study aiming to reduce IGF-1 levels in cancer survivors is examining the effects of metformin treatment alone compared to weight loss in those survivors. Most studies involved advanced stage lung cancer, typically stage III and IV (nine studies), with the remaining studies being in stage I and II, in cancer survivors or in NSCLC patients of all stages. The typical metformin dosage in these trials is between 500 and 2000 mg daily. Sayed et al. [87] conducted a randomized controlled study on stage IV NSCLC non diabetic patients receiving gemcitabine/cisplatin alone or in combination with metformin (500 mg daily) and found that in the combination group compared to the gemcitabine/cisplatin treatment alone, the objective response rate (tumor size reduction) was 46.7% compared to 13.3% (p = 0.109), the overall survival was 12 months compared to 6.5 months (p = 0.119) and the median progression free survival was 5.5 months compared to five months (p = 0.062) respectively. However, there was no significant increase in toxicity in the combination group, and the percentage of patients experiencing nausea was significantly lower in the combination group (26.7%) compared to the gemcitabine/cisplatin group (66.7%) [87]. Overall, this study by Sayed et al. showed that metformin had no statistically significant improvements in objective response rate, overall survival and median progression free survival even though overall survival and median progression free survival improved slightly with metformin treatment and objective response rate improved greatly with metformin treatment, but instead metformin reduced chemotherapy induced-nausea [87].

Conclusions
In summary, all the in vitro and in vivo animal studies presented in this review indicate that metformin alone has potent anti-cancer effects and in combination studies it enhances the effectiveness of other chemotherapy agents or radiation treatment. It should be noted that the concentration of metformin used in in vitro studies are typically 1-10 mM [39,47,50,51] and in some studies as high as 50 mM [53], while the concentration of metformin in blood samples of humans treated with metformin are 2.8-15 µM [88]. We strongly urge researchers to plan future in vitro studies using metformin in the micromolar (µM) range to be physiologically/pharmacologically relevant with the in vivo levels and investigate the mechanism of action.
Although, all the in vitro and in vivo studies summarized in this review indicate potent anticancer effects of metformin, the evidence from clinical studies (meta-analysis and reviews) show both a reduced cancer risk and no response. The closed clinical trial by Sayed et al. [87] did not demonstrate significant results with metformin treatment. Currently, doubt still remains whether the anti-cancer effects of metformin observed in in vitro and in vivo studies will ultimately translate into clinical benefits in the ongoing clinical trials. While whether metformin has a clinically-relevant chemopreventive or anti-cancer effect is not clear at present, we look forward to seeing the evidence from the ongoing human clinical trial studies.