The impact of type 2 diabetes and antidiabetic drugs on cancer cell growth

Despite investigations into mechanisms linking type 2 diabetes and cancer, there is a gap in knowledge about pharmacotherapy for diabetes in cancer patients. Epidemiological studies have shown that diabetic cancer patients on different antidiabetic treatments have different survival. The clinically relevant question is whether some antidiabetic pharmacotherapeutic agents promote cancer whereas others inhibit cancer progression. We investigated the hypothesis that various antidiabetic drugs had differential direct impact on cancer cells using four human cell lines (pancreatic cancer: MiaPaCa2, Panc‐1; breast cancer: MCF7, HER18). We found that insulin and glucose promoted cancer cell proliferation and contributed to chemoresistance. Metformin and rosiglitazone suppressed cancer cell growth and induced apoptosis. Both drugs affected signalling in the protein kinases B (AKT)/mammalian target of rapamycin pathway; metformin activated adenosine monophosphate (AMP)‐activated protein kinase whereas rosiglitazone increased chromosome ten level. Although high insulin and glucose concentrations promoted chemoresistance, the combination of metformin or rosiglitazone with gemcitabine or doxorubicin, resulted in an additional decrease in live cancer cells and increase in apoptosis. In contrast, exenatide did not have direct effect on cancer cells. In conclusion, different types of antidiabetic pharmacotherapy had a differential direct impact on cancer cells. This study provides experimental evidence to support further investigation of metformin and rosiglitazone as first‐line therapies for type 2 diabetes in cancer patients.     

Metformin rescues cell surface major histocompatibility complex class I (MHC-I) deficiency caused by oncogenic transformation

Active avoidance by tumor cells from attack and elimination by immune cells is an emerging cancer hallmark that is achieved primarily through decreasing the levels of major histocompatibility complex class I (MHC-I) at the cancer cells’ surface. Deficiencies in MHC-I antigen-restricted immunosurveillance may be intertwined with an altered, Warburg-like cancer cell-intrinsic metabolism, another emerging hallmark of cancer that involves a switch from mitochondrial respiration to glycolysis to efficiently support large-scale biosynthetic programs that are required for active cell proliferation. We recently envisioned that intervention strategies aimed at reversing the bioenergetic signature of cancer cells (e.g., the antidiabetic biguanide metformin) should correct oncogene (e.g., HER2)-driven MHC-I defects, thus preventing immune escape of oncogene transformants. First, we explored how metformin treatment impacted mitochondrial biogenesis in cultured breast cancer cells overexpressing the membrane tyrosine kinase receptor HER2, the best-characterized downregulator of MHC-I. Metformin exposure was found to dose-dependently increase the expression levels of cytochrome c oxidase I and mitochondrial succinate dehydrogenase, which are encoded by mitochondrial and nuclear DNA, respectively. Second, we explored whether metformin-enhanced mitochondrial biogenesis might significantly alter the MHC-I status in breast carcinoma cells. MHC-I expression, as assessed by flow cytometry using an anti-HLA-ABC monoclonal antibody, was fully restored (up to ~25-fold upregulation) in MHC-I-negative HER2 gene-amplified carcinoma cells. These findings may help delineate a previously unrecognized mechanism through which metformin (and metformin-like drugs) may enable a cancer patient’s own immune system to mount an efficient anti-metastasis response that can prevent or delay disease recurrence. Restored antigenicity and immunogenicity of tumor cells may represent a previously unrecognized primary mode of action underlying the cancer-preventive effects of metformin.     

Metformin suppresses growth of human head and neck squamous cell carcinoma via global inhibition of protein translation

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer in the world; the main risk factors are alcohol and tobacco use. Advancements in therapies have yet to improve the prognosis of HNSCC. the connection between diabetes and cancer is being recognized, and metformin has been shown to decrease cancer incidence in diabetic patients. Accordingly, here, for the first time, we investigated metformin''s efficacy on the growth and viability of human HNSCC FaDU and Detroit 562 cells. our results show that metformin treatment (5–20 mM) dose-dependently inhibits the growth of both cell lines. In FaDU cells, metformin caused 18–57% and 35–81% growth inhibition after 48 and 72 h treatments, respectively. Similarly, in Detroit 562 cells, 48 and 72 h metformin treatment resulted in 20–57% and 33–82% inhibition, respectively. Mechanistically, metformin caused G₁ arrest, which coincided with a decrease in the protein levels of Cdks (2, 4 and 6), cyclins (D1 and e) and Cdk inhibitors (p15, p16, p18 and p27) but no change in p19 and p21. Metformin also decreased the levels of oncogenic proteins Skp2 and β-Trcp. In other studies, metformin decreased the phosphorylation of 4E-BP1 at Ser65, Thr37/46 and Thr70 sites but drastically increased the phosphorylation of EF2 at Thr56 and AMPK at Thr172, which results in global translational inhibition. In summary, the observed wide spectrum of mechanistic effects of metformin on HNSCC cells provides support for the anticancer capability of the drug and its potential use in future therapies.Key words: metformin, head and neck cancer, cell viability, growth inhibition, cell cycle arrest, chemoprevention     

Tangeretin boosts the anticancer activity of metformin in breast cancer cells via curbing the energy production

BackgroundBreast cancer is the first leading cause of women cancer-related deaths worldwide. While there are many proposed treatments for breast cancer, low efficacy, toxicity, and resistance are still major therapeutic obstacles. Thus, there is a need for safer and more effective therapeutic approaches. Because of the direct link between obesity and carcinogenesis, energy restriction mimetic agents (ERMAs) such as the antidiabetic agent, metformin was proposed as a novel antiproliferative agent. However, the anticancer dose of metformin alone is relatively high and impractical to be implemented safely in patients. The current work aimed to sensitize resistant breast cancer cells to metformin's antiproliferative effect using the natural potential anticancer agent, tangeretin.MethodsThe possible synergistic combination between metformin and tangeretin was initially evaluated using MTT cell viability assay in different breast cancer cell lines (MCF-7, MDA-MB-231, and their resistant phenotype). The possible mechanisms of synergy were investigated via Western blotting analysis, reactive oxygen species (ROS) measurement, annexin/PI assay, cell cycle analysis, and wound healing assay.ResultsThe results indicated the ability of tangeretin to improve the anticancer activity of metformin. Interestingly, the improved activity was almost equally observed in both parental and resistant cancer cells, which underlines the importance of this combination in cases of the emergence of resistance. The synergy was mediated through the enhanced activation of AMPK and ROS generation in addition to the improved inhibition of cell migration, induction of cell cycle arrest, and apoptosis in cancer cells.ConclusionThe current work underscores the importance of metformin as an ERMA in tackling breast cancer and as a novel approach to boost its anticancer activity via a synergistic combination with tangeretin.     

Metformin extends life span of HER-2/neu transgenic mice and in combination with melatonin inhibits growth of transplantable tumors in vivo

Population studies have shown that treatment with the antidiabetic biguanide metformin significantly reduced cancer risk. In our animal studies, metformin delayed the onset of mammary adenocarcinoma (MAC) in transgenic HER-2/neu mice but not the onset of spontaneous mammary tumors in female SHR mice. Pineal hormone also inhibits mammary carcinoma development in HER2/neu transgenic mice as well as in female SHR mice. Here we demonstrated that a combination of metformin and melatonin significantly inhibits growth of transplanted tumors in mice. Metformin (1.2 mg/ml in drinking water) increased mean life span by 8% and MAC latency by 13.2% (p     

Fibroblasts rescue oral squamous cancer cell from metformin-induced apoptosis via alleviating metabolic disbalance and inhibiting AMPK pathway

Metformin is an antidiabetic drug widely used for the treatment of type 2 diabetes. Growing evidence suggests that it may exert antitumor effects in vivo and in vitro. However, even with the promising potency on defeating cancer cells, the pre-clinical and epidemiological studies of metformin on various kinds of cancers are not satisfactory, and the reasons and underlying mechanisms remain unknown. Since cancer is a complex system, dependent on a promoting microenvironment, we hypothesize that the interactions between cancer cells and their neighborhood fibroblasts are essential for metformin resistance. To test this, we used a cell co-culture model closely mimicking the in vivo interactions and metabolic exchanges between normal stromal cells (NOFs) and oral squamous cancer cells (OSCC). Here we show that while metformin can significantly inhibit cell growth and induce apoptosis of OSCC cultured alone in a dose-dependent manner through activating p-AMPK^T172 and modulating Bcl-2, Bax, and cleaved PARP. However, when OSCC are co-cultured with NOFs the metformin effects on OSCC cells are annihilated. NOFs are rescuing OSCC from metformin – induced apoptosis, at least partially, through inhibiting the activity of AMPK and PARP, maintaining mitochondrial membrane potential and increasing the oxidative stress. Our results indicate that metformin effects on oral cancer cells are modulated by the microenvironment and that this has to be taken into consideration in the context of developing a new combination of drugs for oral cancer treatment.     

Metformin lowers Ser-129 phosphorylated α-synuclein levels via mTOR-dependent protein phosphatase 2A activation

Phospho-Ser129 α-synuclein is the modified form of α-synuclein that occurs most frequently within Parkinson''s disease pathological inclusions. Here we demonstrate that the antidiabetic drug metformin significantly reduces levels of phospho-Ser129 α-synuclein and the ratio of phospho-Ser129 α-synuclein to total α-synuclein. This effect was documented in vitro in SH-SY5Y and HeLa cells as well as in primary cultures of hippocampal neurons. In vitro work also elucidated the mechanisms underlying metformin''s action. Following metformin exposure, decreased phospho-Ser129 α-synuclein was not strictly dependent on induction of AMP-activated protein kinase, a primary target of the drug. On the other hand, metformin-induced phospho-Ser129 α-synuclein reduction was consistently associated with inhibition of mammalian target of rapamycin (mTOR) and activation of protein phosphatase 2A (PP2A). Evidence supporting a key role of mTOR/PP2A signaling included the finding that, similar to metformin, the canonical mTOR inhibitor rapamycin was capable of lowering the ratio of phospho-Ser129 α-synuclein to total α-synuclein. Furthermore, no decrease in phosphorylated α-synuclein occurred with either metformin or rapamycin when phosphatase activity was inhibited, supporting a direct relationship between mTOR inhibition, PP2A activation and protein dephosphorylation. A final set of experiments confirmed the effectiveness of metformin in vivo in wild-type C57BL/6 mice. Addition of the drug to food or drinking water lowered levels of phospho-Ser129 α-synuclein in the brain of treated animals. These data reveal a new mechanism leading to α-synuclein dephosphorylation that could be targeted for therapeutic intervention by drugs like metformin and rapamycin.     

二甲双胍抗肿瘤作用及其分子机制的研究进展

二甲双胍是全球范围内治疗2型糖尿病最常用的药物之一,具有使用方便、疗效好、价格低廉且毒副作用小等优点。近年来大量的流行病学研究及体内外实验研究发现二甲双胍能够用于多种肿瘤的治疗及预防,然而其分子机制尚不十分明确;主要包括调节体内胰岛素/IGF-1轴、激活AMPK信号通路、调控micro RNAs的表达、活化Caspase分子、阻断AGEs-RAGE系统等,这些机制为将来二甲双胍应用于肿瘤的预防及临床治疗提供了重要的理论依据。本文针对糖尿病治疗药物二甲双胍在抗肿瘤中的作用及其分子机制进行全面综述。     

Diabetes,metformin use,and colorectal cancer survival in postmenopausal women

Background: Observational studies have associated metformin use with lower colorectal cancer (CRC) incidence but few studies have examined metformin's influence on CRC survival. We examined the relationships among metformin use, diabetes, and survival in postmenopausal women with CRC in the Women's Health Initiative (WHI) clinical trials and observational study. Methods: 2066 postmenopausal women with CRC were followed for a median of 4.1 years, with 589 deaths after CRC diagnosis from all causes and 414 deaths directly attributed to CRC. CRC-specific survival was compared among women with diabetes with metformin use (n = 84); women with diabetes with no metformin use (n = 128); and women without diabetes (n = 1854). Cox proportional hazard models were used to estimate associations among metformin use, diabetes and survival after CRC. Strategies to adjust for potential confounders included: multivariate adjustment with known predictors of colorectal cancer survival and construction of a propensity score for the likelihood of receiving metformin, with model stratification by propensity score quintile. Results: After adjusting for age and stage, CRC specific survival in women with diabetes with metformin use was not significantly different compared to that in women with diabetes with no metformin use (HR 0.75; 95% CI 0.40–1.38, p = 0.67) and to women without diabetes (HR 1.00; 95% CI 0.61–1.66, p = 0.99). Following propensity score adjustment, the HR for CRC-specific survival in women with diabetes with metformin use compared to non-users was 0.78 (95% CI 0.38–1.55, p = 0.47) and for overall survival was 0.86 (95% CI 0.49–1.52; p = 0.60). Conclusions: In postmenopausal women with CRC and DM, no statistically significant difference was seen in CRC specific survival in those who used metformin compared to non-users. Analyses in larger populations of colorectal cancer patients are warranted.     

The combination of metformin and 2 deoxyglucose inhibits autophagy and induces AMPK-dependent apoptosis in prostate cancer cells

Targeting cancer cell metabolism is a new promising strategy to fight cancer. Metformin, a widely used antidiabetic agent, and 2-deoxyglucose (2DG) drastically affect cancer cell metabolism. Recently, we showed that the combination of the two drugs was much more harmful for cancer cells than the treatment with metformin or 2DG alone. At the cellular level, this combination leads to p53- and AMPK-dependent apoptosis. Furthermore, we showed that metformin inhibits 2DG-induced autophagy, decreases beclin 1 expression and triggers a switch from a survival process to cell death.     

Metformin reverses multidrug resistance and epithelial–mesenchymal transition (EMT) via activating AMP-activated protein kinase (AMPK) in human breast cancer cells

Breast cancer is the most frequently diagnosed tumor type and the primary leading cause of cancer deaths in women worldwide and multidrug resistance is the major obstacle for breast cancer treatment improvement. Emerging evidence suggests that metformin, the most widely used antidiabetic drug, resensitizes and cooperates with some anticancer drugs to exert anticancer effect. However, there are no data regarding the reversal effect of metformin on chemoresistance in breast cancer. In the present study, we investigated the resistance reversal effect of metformin on acquired multidrug-resistant breast cancer cells MCF-7/5-Fu derived from MCF-7 breast cancer cells and innate multidrug-resistant MDA-MB-231 breast cancer cells, and we found that metformin resensitized MCF7/5-FU and MDA-MB-231 to 5-fluorouracil (5-FU), adriamycin, and paclitaxel. We also observed that metformin reversed epithelial–mesenchymal transition (EMT) phenotype and decreased the invasive capacity of MCF7/5-FU and MDA-MB-231 cells. However, there were no significant changes upon metformin-treated MCF7 cells. Moreover, we found metformin treatment activated AMPK signal pathway in MCF7/5-FU and MDA-MB-231 cells and compound C, the AMPK inhibitor, could partly abolish the resensitization and EMT reversal effect of metformin. To the best of our knowledge, we are the first to report that metformin can resensitize multidrug-resistant breast cancer cells due to activating AMPK signal pathway. Our study will help elucidate the mechanism of chemoresistance and establish new strategies of chemotherapy for human breast cancer.     

Cancer and aging: More puzzles,more promises?

In this issue of Cell Cycle, a new paper shows that metformin, an oral antidiabetic drug that activates AMP-activated protein kinase, prolongs both mean and maximal life span and prevents reproductive aging of female mice. Unexpectedly, metformin did not decrease the incidence of cancer in this mice strain. Here, we discuss the relationship between aging and cancer, the mechanism of metformin action, and the prospects of using this compound for life span extension in humans.     

Regulation of dual specificity phosphatases in breast cancer during initial treatment with Herceptin: a Boolean model analysis

Background

25% of breast cancer patients suffer from aggressive HER2-positive tumours that are characterised by overexpression of the HER2 protein or by its increased tyrosine kinase activity. Herceptin is a major drug used to treat HER2 positive breast cancer. Understanding the molecular events that occur when breast cancer cells are exposed to Herceptin is therefore of significant importance. Dual specificity phosphatases (DUSPs) are central regulators of cell signalling that function downstream of HER2, but their role in the cellular response to Herceptin is mostly unknown. This study aims to model the initial effects of Herceptin exposure on DUSPs in HER2-positive breast cancer cells using Boolean modelling.

Results

We experimentally measured expression time courses of 21 different DUSPs between 0 and 24 h following Herceptin treatment of human MDA-MB-453 HER2-positive breast cancer cells. We clustered these time courses into patterns of similar dynamics over time. In parallel, we built a series of Boolean models representing the known regulatory mechanisms of DUSPs and then demonstrated that the dynamics predicted by the models is in agreement with the experimental data. Furthermore, we used the models to predict regulatory mechanisms of DUSPs, where these mechanisms were partially known.

Conclusions

Boolean modelling is a powerful technique to investigate and understand signalling pathways. We obtained an understanding of different regulatory pathways in breast cancer and new insights on how these signalling pathways are activated. This method can be generalized to other drugs and longer time courses to better understand how resistance to drugs develops in cancer cells over time.

     

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.     

Cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis

Background

A growing body of evidence has suggested that metformin potentially reduces the risk of cancer. Our objective was to enhance the precision of estimates of the effect of metformin on the risk of any-site and site-specific cancers in patients with diabetes.

Methods/Principal Findings

We performed a search of MEDLINE, EMBASE, ISI Web of Science, Cochrane Library, and ClinicalTrials.gov for pertinent articles published as of October 12, 2011, and included them in a systematic review and meta-analysis. We calculated pooled risk ratios (RRs) for overall cancer mortality and cancer incidence. Of the 21,195 diabetic patients reported in 6 studies (4 cohort studies, 2 RCTs), 991 (4.5%) cases of death from cancer were reported. A total of 11,117 (5.3%) cases of incident cancer at any site were reported among 210,892 patients in 10 studies (2 RCTs, 6 cohort studies, 2 case-control studies). The risks of cancer among metformin users were significantly lower than those among non-metformin users: the pooled RRs (95% confidence interval) were 0.66 (0.49–0.88) for cancer mortality, 0.67 (0.53–0.85) for all-cancer incidence, 0.68 (0.53–0.88) for colorectal cancer (n = 6), 0.20 (0.07–0.59) for hepatocellular cancer (n = 4), 0.67 (0.45–0.99) for lung cancer (n = 3).

Conclusion/Significance

The use of metformin in diabetic patients was associated with significantly lower risks of cancer mortality and incidence. However, this analysis is mainly based on observational studies and our findings underscore the more need for long-term RCTs to confirm this potential benefit for individuals with diabetes.     

Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions

Introduction

Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines.

Material and Methods

MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells.

Results

Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation.

Conclusion

We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.     

Therapeutic effects of metformin in breast cancer: involvement of the immune system?

Breast cancer and associated diabetes mellitus have gained raising interest as an elevated risk of breast cancer prognosis resulting in increased mortality in diabetic patients. In this context, the long-acting insulin analog glargine and other antidiabetics have been discussed to promote tumorigenesis. In contrast, the biguanide class oral antidiabetic metformin has been shown capable of enhancing cell cycle arrest and inducing apoptosis as well as reducing growth factor signaling. Consequently, several studies are underway to evaluate a possible role of metformin in breast cancer treatment. Although mechanisms involved are not definitely clear yet, here, we discuss metformin’s anticancer effects including the potential impact of the immune system.