Antiproliferative Effects Of Metformin Research Articles

Metformin Induces a Caspase 3-Unrelated Apoptosis in Human Colorectal Cancer Cell Lines HCT116 and SW620.

To study the effects of metformin on the proliferation and growth of human colorectal cancer cell lines HCT116 and SW620. The antiproliferative effect of metformin was assayed using an MTS reagent and its ability to inhibit colony formation was demonstrated using a clonogenic assay. Flow cytometry using YO-PRO-1/PI was performed to examine the effects of metformin on apoptosis and cell death of HCT116 and SW620. Caspase 3 activities were measured in caspase-3 activity tests using a caspase-3 activity kit. Furthermore, Western blots were performed with anti-PARP1, anti-caspase 3, and anti-cleaved caspase 3 to confirm whether caspase activation was present or not. Both MTS proliferation assays and clonogenic assays showed that metformin inhibited the proliferation and growth of HCT116 and SW620 cells in a concentration-dependent manner. Flow cytometric analysis identified early apoptosis and metformin-induced cell death in both cell lines. However, caspase 3 activity could not be detected. Cleavage of both PARP1 and pro-caspase 3 was not observed in the Western blot, confirming the absence of caspase 3 activations. This present study suggests a caspase 3-unrelated apoptosis mechanism of metformin-induced cell death in human colorectal cancer cell lines HCT116 and SW620.

NDUFS3 knockout cancer cells and molecular docking reveal specificity and mode of action of anti-cancer respiratory complex I inhibitors.

Inhibition of respiratory complex I (CI) is becoming a promising anti-cancer strategy, encouraging the design and the use of inhibitors, whose mechanism of action, efficacy and specificity remain elusive. As CI is a central player of cellular bioenergetics, a finely tuned dosing of targeting drugs is required to avoid side effects. We compared the specificity and mode of action of CI inhibitors metformin, BAY 87-2243 and EVP 4593 using cancer cell models devoid of CI. Here we show that both BAY 87-2243 and EVP 4593 were selective, while the antiproliferative effects of metformin were considerably independent from CI inhibition. Molecular docking predictions indicated that the high efficiency of BAY 87-2243 and EVP 4593 may derive from the tight network of bonds in the quinone binding pocket, although in different sites. Most of the amino acids involved in such interactions are conserved across species and only rarely found mutated in human. Our data make a case for caution when referring to metformin as a CI-targeting compound, and highlight the need for dosage optimization and careful evaluation of molecular interactions between inhibitors and the holoenzyme.

Abstract 903: Metformin antitumoral activity is exclusively mediated by the membrane functional expression of the Chloride Intracellular Channel 1 in glioblastoma stem cells

Abstract Glioblastoma (GB) is the deadliest brain tumor, representing a major clinical challenge in the field. Despite standard of care together with cutting-edge strategies, life expectancy stands invariantly poor. Tumor heterogeneity and its high-grade of invasiveness make it difficult to be surgically resected, and recurrence is inevitable. There is scientific consensus that tumor relapse originates from progenitor/stem-like cells known as glioblastoma stem cells (GSCs) displaying properties of self-renewal and multi-lineage differentiation that contribute to tumor mass heterogeneity. Thus, GSCs represent the target of newly designed therapies. In recent years, it has been highlighted the anti-cancer properties of metformin, the first-line drug in Type-2 diabetes (T2D) treatment. Metformin assumption in T2D patients has been positively linked with a decrease in the risk of several cancer types and cancer-related mortality, including glioblastoma. Additionally, numerous studies demonstrated metformin's anticancer properties against GSCs. Despite metformin's mechanism of action on cancer cells has not been clarified yet, its effect has been proposed to target the oxidative phosphorylation (OXPHOS) pathway. Considering that GSCs have been reported to shuttle between glycolysis and OXPHOS energetic pathways, their metabolic plasticity should prevent the antiproliferative effect of metformin. However, metformin-treated GSCs show a phenotype superimposable to that caused by the blockage of the transmembrane form of the chloride intracellular channel 1 (tmCLIC1). tmCLIC1 involvement in the progression of GB has been assessed deeply in vitro as well as in vivo. In addition, its peculiar localization and enrichment on GSCs' membrane renders it a valuable pharmacological target for GB. Here, we show that in glioblastoma, the transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) works as a privileged and unique metformin membrane receptor. Nuclear magnetic resonance experiments on live cells confirm the binding to tmCLIC1. Metformin impairs tmCLIC1 activity by a specific binding on a single amino acid, the arginine29. Its mutation demonstrates that metformin-depending tmCLIC1 block is prevented, failing to reduce glioblastoma cells proliferation in 2D and 3D models as well. Moreover, in vivo experiments on zebrafish embryos closely mirror the behavior observed in vitro. Our results demonstrate that tmCLIC1 is essential for metformin antineoplastic effect in glioblastoma and prove a direct binding between the drug and its target. Considering tmCLIC1 contribution to glioblastoma progression, the present work paves the way for future investigations intended to develop a strategy aimed to enhance metformin-tmCLIC1 interaction. Ivan Verduci was supported by a AIRC fellowship for Italy. Citation Format: Ivan Verduci, Francesca Cianci, Riccardo Cazzoli, Gaetano Cannavale, Stefania Castiglione, Matteo Ranucci, Luca Palloni, Beatrice Balboni, Stefania Faletti, Stefania Girotto, Giuliana Pelicci, Saverio Minucci, Alessandro Fantin, Tullio Florio, Michele Mazzanti. Metformin antitumoral activity is exclusively mediated by the membrane functional expression of the Chloride Intracellular Channel 1 in glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 903.

A functional screen with metformin identifies microRNAs that regulate metabolism in colorectal cancer cells

Metformin inhibits oxidative phosphorylation and can be used to dissect metabolic pathways in colorectal cancer (CRC) cells. CRC cell proliferation is inhibited by metformin in a dose dependent manner. MicroRNAs that regulate metabolism could be identified by their ability to alter the effect of metformin on CRC cell proliferation. An unbiased high throughput functional screen of a synthetic micoRNA (miRNA) library was used to identify miRNAs that impact the metformin response in CRC cells. Experimental validation of selected hits identified miRNAs that sensitize CRC cells to metformin through modulation of proliferation, apoptosis, cell-cycle and direct metabolic disruption. Among eight metformin sensitizing miRNAs identified by functional screening, miR-676-3p had both pro-apoptotic and cell cycle arrest activity in combination with metformin, whereas other miRNAs (miR-18b-5p, miR-145-3p miR-376b-5p, and miR-718) resulted primarily in cell cycle arrest when combined with metformin. Investigation of the combined effect of miRNAs and metformin on CRC cell metabolism showed that miR-18b-5p, miR-145-3p, miR-376b-5p, miR-676-3p and miR-718 affected glycolysis only, while miR-1181 only regulated CRC respiration. MicroRNAs can sensitize CRC cells to the anti-proliferative effects of metformin. Identifying relevant miRNA targets may enable the design of innovative therapeutic strategies.

Antiproliferative effects of metformin in cellular models of pheochromocytoma

Pheochromocytomas (PCCs) are rare neuroendocrine tumors derived from adrenal medulla chromaffin cells. Malignancy and recurrence are rare but demand effective treatment. Metformin exerts antiproliferative effects in several cancer cell lines. We thus evaluated the effects of metformin on cell viability and proliferation, cellular respiration and AMPK-AKT-mTOR-HIFA proliferation pathway on a rat PCC cell line (PC12-Adh). We then addressed metformin's effects on the AMPK-AKT-mTOR-HIFA pathway on two human primary cultures: one from a VHL-mutant PCC and other from a sporadic PCC. Metformin (20 mM) inhibited PC12-Adh cell proliferation, and decreased oxygen consumption, ATP production and proton leak, in addition to loss of mitochondrial membrane potential. Further, metformin induced AMPK phosphorylation and impaired AMPK-PI3k-AKT-mTOR pathway activation. The mTOR pathway was also inhibited in human VHL-related PCC cells, however, in an AMPK-independent manner. Metformin-induced decrease of HIF1A levels was likely mediated by proteasomal degradation. Altogether our results suggest that metformin impairs PCC cellular proliferation.

Metformin inhibits gastric cancer cell proliferation by regulation of a novel Loc100506691-CHAC1 axis

Long noncoding RNAs (lncRNAs) are a group of nonprotein coding transcripts that play a critical role in cancer progression. However, the role of lncRNA in metformin-induced inhibition of cell growth and its biological function in gastric cancer remain largely unknown. In this study, we identified an oncogenic lncRNA, Loc100506691, the expression of which was decreased in gastric cancer cells with metformin treatment. Moreover, Loc100506691 was significantly overexpressed in gastric cancer compared with adjacent normal tissues (p < 0.001), and high Loc100506691 expression was significantly correlated with poor survival of patients with gastric cancer. Additionally, Loc100506691 knockdown could significantly suppress gastric cancer cell growth in vitro, and ectopic Loc100506691 expression accelerated tumor growth in an in vivo mouse model. Analysis of the cell cycle revealed that Loc100506691 knockdown induced cell cycle arrest at the G2/M phase by impairing cell entry from the G2/M to G1 phase. Loc100506691 negatively regulated CHAC1 expression by modulating miR-26a-5p/miR-330-5p expression, and CHAC1 knockdown markedly attenuated Loc100506691 knockdown-induced gastric cancer cell growth and motility suppression. We concluded that anti-proliferative effects of metformin in gastric cancer may be partially caused by suppression of the Loc100506691-miR-26a-5p/miR-330-5p-CHAC1 axis.

Metal Complex Formation and Anticancer Activity of Cu(I) and Cu(II) Complexes with Metformin.

Metformin has been used for decades in millions of type 2 diabetes mellitus patients. In this time, correlations between metformin use and the occurrence of other disorders have been noted, as well as unpredictable metformin side effects. Diabetes is a significant cancer risk factor, but unexpectedly, metformin-treated diabetic patients have lower cancer incidence. Here, we show that metformin forms stable complexes with copper (II) ions. Both copper(I)/metformin and copper(II)/metformin complexes form adducts with glutathione, the main intracellular antioxidative peptide, found at high levels in cancer cells. Metformin reduces cell number and viability in SW1222 and K562 cells, as well as in K562-200 multidrug-resistant cells. Notably, the antiproliferative effect of metformin is enhanced in the presence of copper ions.

Metformin impairs cisplatin resistance effects in A549 lung cancer cells through mTOR signaling and other metabolic pathways.

Lung cancer is the leading cause of cancer-associated death worldwide and exhibits intrinsic and acquired therapeutic resistance to cisplatin (CIS). The present study investigated the role of mTOR signaling and other signaling pathways after metformin (MET) treatment in control and cisplatin-resistant A549 cells, mapping pathways and possible targets involved in CIS sensitivity. MTT, flow cytometry, clonogenic assay, western blotting, proteomic analysis using the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) approach and reverse transcription-quantitative PCR were performed. The results revealed that CIS treatment induced mTOR signaling pathway overactivation, and the mTOR status was restored by MET. MET and the mTOR inhibitor rapamycin (RAPA) decreased the viability in control and resistant cells, and decreased the cell size increase induced by CIS. In control cells, MET and RAPA decreased colony formation after 72 h and decreased IC50 values, potentiating the effects of CIS. Proteomics analysis revealed important pathways regulated by MET, including transcription, RNA processing and IL-12-mediated signaling. In CIS-resistant cells, MET regulated the apoptotic process, oxidative stress and G2/M transition. Annexin 4 (ANXA4) and superoxide dismutase 2 (SOD2), involved in apoptosis and oxidative stress, respectively, were chosen to validate the SILAC analysis and may represent potential therapeutic targets for lung cancer treatment. In conclusion, the chemosensitizing and antiproliferative effects of MET were associated with mTOR signaling and with potential novel targets, such as ANXA4 and SOD2, in human lung cancer cells.

Metformin Induces Apoptosis and Inhibits Notch1 in Malignant Pleural Mesothelioma Cells.

Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related cancer arising from the mesothelial cells lining the pleural cavity. MPM is characterized by a silent clinical progression and a highly resistance to conventional chemo/radio-therapies. MPM patients die in a few months/years from diagnosis. Notch signaling is a well-conserved cell communication system, which regulates many biological processes. In humans, the dysregulation of Notch pathway potentially contributes to cancer onset/progression, including MPM. Metformin is the first-line drug used to treat type 2 diabetes mellitus. Metformin is proven to be an effective antitumor drug in preclinical models of different types of cancer. To date, clinical efficacy is being studied in many clinical trials. In this study, the anti-proliferative effect of metformin on MPM cells and the putative involvement of Notch1 as a mediator of metformin activities, were investigated. MPM cells showed high levels of Notch1 activation compared to normal pleural mesothelial cells. Furthermore, metformin treatment hampered MPM cell proliferation and enhanced the apoptotic process, accompanied by decreased Notch1 activation.

Metformin suppresses the proliferation and invasion through NF-kB and MMPs in MCF-7 cell line

Abstract Objective MCF-7 cells, a breast cancer cell line, are used for experiments of estrogen receptor (ER)-positive breast cancer and many sub-clones representing different classes of ER-positive tumors. We aimed to determine the efficacy of metformin, a potential anti-cancer agent, on the cell proliferation, and the expressions of NF-kB (p65), MMP-2 and MMP-9 in MCF-7 cell line. Materials and methods MCF-7 cells (human breast adenocarcinoma) were treated with elevating doses of metformin (0–50 mM) for 24 h. The anti-proliferative effect of metformin was studied by BrdU proliferation assay, and the expression levels of NF-kB (p65), MMP-2 and MMP-9 were analyzed by immunocytochemical staining. Results The percentage of cell proliferation was reduced significantly by 10 and 50 mM doses of metformin (p &lt; 0.001). The expression levels of nuclear NF-kB (p65), MMP-9 and MMP-2 were considerably reduced in 50 mM metformin treated cells while the expression of cytoplasmic NF-kB (p65) elevated compared to control group (p &lt; 0.05). Ten millimolar metformin also reduced expression of MMP-9 significantly (p &lt; 0.05). Conclusion Metformin may act on the proliferation, and the processes of invasion and metastasis of MCF-7 cells through blocking NF-kB, which is intensely expressed in breast cancer cells, and through diminishing the expression of MMP-2 and MMP-9 significantly.

Heme Oxygenase-1 Inhibition Sensitizes Human Prostate Cancer Cells towards Glucose Deprivation and Metformin-Mediated Cell Death.

High levels of heme oxygenase (HO)-1 have been frequently reported in different human cancers, playing a major role in drug resistance and regulation of cancer cell redox homeostasis. Metformin (MET), a drug widely used for type 2 diabetes, has recently gained interest for treating several cancers. Recent studies indicated that the anti-proliferative effects of metformin in cancer cells are highly dependent on glucose concentration. The present work was directed to determine whether use of a specific inhibitor of HO-1 activity, alone or in combination with metformin, affected metastatic prostate cancer cell viability under different concentrations of glucose. MTT assay and the xCELLigence system were used to evaluate cell viability and cell proliferation in DU145 human prostate cancer cells. Cell apoptosis and reactive oxygen species were analyzed by flow cytometry. The activity of HO-1 was inhibited using a selective imidazole-based inhibitor; genes associated with antioxidant systems and cell death were evaluated by qRT-PCR. Our study demonstrates that metformin suppressed prostate cancer growth in vitro and increased oxidative stress. Disrupting the antioxidant HO-1 activity, especially under low glucose concentration, could be an attractive approach to potentiate metformin antineoplastic effects and could provide a biochemical basis for developing HO-1-targeting drugs against solid tumors.

Antiproliferative Effects of Metformin in Triple Negative MDA‐MB 231 Breast Cancer Cells Exposed to Glucose‐Starved And 2‐Deoxyglucose

IntroductionWe have previously reported that 50μM of the anti‐diabetic drug metformin protects endothelial cells against hyperglycemia‐induced endothelial dysfunction and senescence thus providing support for the vascular protective benefits of metformin when used in diabetes 1,2. However, when 2mM (but not 50μM) metformin was used in a cell culture protocol with tumor murine microvascular endothelial in a glucose‐starved (GS) or 2‐deoxyglucose (2DG; 5mM) media pro‐survival autophagy and the mTOR pathway were inhibited and cell viability was reduced 3. These data supported other published evidence from both clinical and ex vivo cell‐based studies suggesting the utility of metformin for the treatment of various cancers. We therefore postulated that triple‐negative breast cancer (TNBC) that does not benefit from hormonal or HER2 targeted therapies may also respond to a metformin and GS/2DG protocol and investigated the effects of metformin in MDA‐MB 231 TNBC cells that were cultured in the presence of 2DG.MethodsMDA‐MB 231 cells were treated with 2DG (10mM) in a glucose free media for 48h in the absence/presence of metformin (2mM). Western blot analysis (n=3–4); cell proliferation (MTT assay; n=5); migration (Radius Cell Migration Assay; n=4) and viability (propidium iodide assay; n=5) were performed. Significance was determined by ANOVA.ResultsCell proliferation rate significantly decreased (95%) in 1) GS + metformin when compared to non‐treated GS (88%) and 2) 2DG exposed cells treated with metformin (46%) versus cells that were treated with either metformin (20%) or 2DG (28%) alone. Cell viability significantly (P&lt;0.05) decreased in GS treated with metformin (34%) when compared to non‐treated GS (8%). In addition cell migration in cells exposed to 2DG and treated with metformin was reduced. Western blot analysis revealed that treatment with metformin significantly decreased (fold change) the levels of LC3A‐II (2.4&amp;1.8) and LC3B‐II (2.3&amp;1.6), pmTOR (S2448; 1.6&amp;1.4) and downstream p4E‐BP1(T36/47; 1.5&amp;1.3), pS6(S235/236; 4.7&amp;2.8) and pS6(S240/244; 3.2&amp;1.8) in GS and 2DG‐exposed cells + 2mM metformin.ConclusionResults show that using mM metformin in TNBC cells in a GS media containing 2DG offers a potential adjunct anti‐cancer protocol for the treatment of TNBC.Support or Funding InformationSupported by a Qatar Foundation National Priorities Research Program grant (NPRP: 04‐910‐3‐244) and a Junior Scientist Research Experience Program grant (JSREP: 03‐016‐3‐009)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of apoptosis by metformin and progesterone in estrogen-induced endometrial hyperplasia in rats: involvement of the bcl-2 family proteins

This study compared the antiproliferative effects of metformin and progesterone, via examination of the Bcl-2/Bax-caspase apoptotic pathway in estrogen-induced endometrial hyperplasia (EH) in 40 rats. Two rats died after bilateral oophorectomy, and 1 week after surgery, the remaining 38 were randomly divided into three groups: the first (control, n = 12) received 4 mg/kg 17β estradiol hemihydrate (E); the second (n = 13) received 4 mg/kg 17β estradiol hemihydrate and 50 mg/kg metformin (E + M); and the third (n = 13) received 4 mg/kg 17β estradiol hemihydrate and 1 mg/day medroxiprogesterone acetate (E + MPA). Histological markers and Bcl-2, Bax and caspase 9 expression were analyzed. Luminal epithelial thickness, density of gland and epithelial height was significantly higher in group E than in groups E + M and E + MPA. Histopathologic parameters were similar between the E + M and E + MPA groups. Bcl-2/Bax ratio was significantly decreased in the E + M and E + MPA groups and caspase 9 expression levels were significantly increased in the E + M and E + MPA groups, compared with the control group. In addition, Bcl-2/Bax ratio and caspase 9 expression were similar between the E + M and E + MPA groups. The data indicate that metformin reduces estrogen-induced EH in rats, via activation of the caspase-dependent mitochondrial apoptotic pathway, to the same degree as progesterone.

Modulation of the anti-tumor activity of VEGF blockade by metformin.

e23009 Background: The payoff from first-generation angiogenesis inhibitors was commonly temporary, due to the development of intrinsic as well as acquired resistance. Apart from the vascular resistance due to circumventing VEGF blockade by proangiogenic factors, resistance has also been associated with assortment of resistant clones. Accordingly, a combination strategy was proposed to overcome such resistance. Metformin, as a potential candidate, has received considerable concern as an anticancer agent. Methods: IN-VITRO WORK;Cells were divided into normoxic and hypoxic groups. For both, four subgroups were developed; control, metformin, glucose deprivation and a subgroup in which cells were subjected to both glucose deprivation and metformin treatment. Cells were then subjected to analysis for Apoptosis, Cell cycle progression and western blot. IN-VIVO WORK; Tumor growth was assessed IGROV-1 subcutaneous xenografts. Animal were divided into four groups; control, Metformin, Bevacizumab and a fourth group receiving both drugs. Tumor growth was assessed along treatments and at time of sacrifice.After sacrificing animals, tumors were further analyzed for; necrosis, apoptosis, proliferative capacity, activation of AMPK and its downstream effectors. Results: Our in-vitro results suggested an anti-proliferative effect of metformin in terms of apoptosis, cell cycle arrest, AMPK activation and mTOR inhibition at various time points. In-vivo experiments revealed a significant decrease of tumor weight and proliferative capacity in tumors treated with both drugs and a trend toward a decrease in tumor growth along the treatment. Yet, there was no difference between the bevacizumab and the combination groups regarding AMPK activation suggesting another mechanism to exert the combination effect. The effect of Metformin on CSC percentage and viability was tested. Glucose deprivation plus hypoxia lead to increase in CSC population, while adding metformin to bevacizumab reverse this augmentation in CSC numbers. Conclusions: Study represents a proof of concept study demonstrating a positive modulatory effect of metformin addition to bevacizumab. Additional experimental work on a wider scale is running to ascertain the current results.

Anti-proliferative effect of metformin on a feline injection site sarcoma cell line independent of Mtor inhibition

Metformin is an oral hypoglycemic drug that has been shown to inhibit cancer cell proliferation via up-regulation of AMPK (AMP-activated protein kinase), and possibly inhibition of mTOR (mammalian target of rapamycin). The purpose of this study was to evaluate the effects of metformin on a feline injection site sarcoma cell line. Cells from a feline injection site sarcoma cell line were treated with metformin at varied concentrations. A dose-dependent decrease in cell viability following metformin treatment was observed, with an IC50 of 8.0mM. Using flow cytometry, the mechanism of cell death was determined to be apoptosis or necrosis. To evaluate the role of mTOR inhibition in metformin-induced cell death, Western blot was performed. No inhibition of mTOR or phosphorylated mTOR was found. Although metformin treatment leads to apoptotic or necrotic cell death in feline injection site sarcoma cells, the mechanism does not appear to be mediated by mTOR inhibition.

MiR-27a-mediated antiproliferative effects of metformin on the breast cancer cell line MCF-7.

Metformin was demonstrated to have effects on breast cancer, and microRNA-27a (miR-27a) is a prognostic marker for breast cancer progression and patient survival. AMPKα2 was found to be a suppressor in breast cancer MCF-7 cells. Therefore, the present study aimed to explain this phenomenon in regards to the relationship between microRNAs (miRNAs) and their target genes and to predict how AMPKα2 may be a downstream target gene of miR-27a, thus exploring the new mechanism of metformin in the treatment of breast cancer regarding miRNAs. The MTT assay was used to assess whether metformin can inhibit the growth of breast cancer MCF-7 cells. The levels of miR-27a and AMPKα2 mRNA were examined using RT-PCR, and the expression levels of AMPKα2 and caspase-3 were determined by western blot analyses after MCF-7 cells were treated with metformin. The association of miR-27a and AMPKα2 was confirmed by transfecting cells with miR-27a mimics, miR-27a inhibitors and its negative control (NC), respectively. A luciferase assay was conducted to detect the miR-27a binding to the AMPKα2 3'-untranslated region (3'-UTR). The results of the MTT assay showed that metformin suppressed the growth of MCF-7 cells in a dose-and time‑dependent manner. miR-27a was downregulated, and AMPKa2 was upregulated after intervention with metformin, and caspase-3 was activated. Transfection tests showed that the expression of AMPKα2 was downregulated in the MCF-7 cells after transfection of the miR-27a mimics. The luciferase assay verified the binding of miR-27a to the AMPKα2 3'-UTR. In conclusion, metformin inhibited MCF-7 cell growth, and miR-27a plays a vital role in this process by targeting AMPKα2.

MiR-27a-mediated antiproliferative effects of metformin on the breast cancer cell line MCF-7

miR-27a-mediated antiproliferative effects of metformin on the breast cancer cell line MCF-7

The anti-proliferative effect of metformin in a model of adrenocortical carcinoma

The anti-proliferative effect of metformin in a model of adrenocortical carcinoma

Combined Treatment with Exendin-4 and Metformin Attenuates Prostate Cancer Growth.

IntroductionRecently, the pleiotropic benefits of incretin-based therapy have been reported. We have previously reported that Exendin–4, a glucagon-like peptide–1 (GLP–1) receptor agonist, attenuates prostate cancer growth. Metformin is known for its anti-cancer effect. Here, we examined the anti-cancer effect of Exendin–4 and metformin using a prostate cancer model.MethodsProstate cancer cells were treated with Exendin–4 and/or metformin. Cell proliferation was quantified by growth curves and 5-bromo–2′-deoxyuridine (BrdU) assay. TUNEL assay and AMP-activated protein kinase (AMPK) phosphorylation were examined in LNCaP cells. For in vivo experiments, LNCaP cells were transplanted subcutaneously into the flank region of athymic mice, which were then treated with Exendin–4 and/or metformin. TUNEL assay and immunohistochemistry were performed on tumors.ResultsExendin–4 and metformin additively decreased the growth curve, but not the migration, of prostate cancer cells. The BrdU assay revealed that both Exendin–4 and metformin significantly decreased prostate cancer cell proliferation. Furthermore, metformin, but not Exendin–4, activated AMPK and induced apoptosis in LNCaP cells. The anti-proliferative effect of metformin was abolished by inhibition or knock down of AMPK. In vivo, Exendin–4 and metformin significantly decreased tumor size, and further significant tumor size reduction was observed after combined treatment. Immunohistochemistry on tumors revealed that the P504S and Ki67 expression decreased by Exendin–4 and/or metformin, and that metformin increased phospho-AMPK expression and the apoptotic cell number.ConclusionThese data suggest that Exendin–4 and metformin attenuated prostate cancer growth by inhibiting proliferation, and that metformin inhibited proliferation by inducing apoptosis. Combined treatment with Exendin–4 and metformin attenuated prostate cancer growth more than separate treatments.

Antitumoral effects of metformin on cancer stem cells

Epidemiological and preclinical studies suggested that metformin, the first-line drug for type-2 diabetes, exerts direct antitumoral activity. Clinical trials are currently ongoing for several human tumors, although the molecular mechanisms of metformin antiproliferative activity are still not completely identified. Several studies reported the involvement of AMP-activated kinase-dependent pathway, and the downstream effectors (e.g., mTOR) in metformin metabolic activity. Nevertheless, contrasting evidence was obtained on the involvement of this pathway in metformin antitumor effect, and several reports showed AMPK-independent antiproliferative activity. According to the hierarchical theory of tumorigenesis, cancer stem cells (CSC) are considered the real pharmacological target to obtain cancer remission. Here, we show that metformin inhibits proliferation of human glioblastoma cells, selectively acting on CSC viability, through the direct inhibition of chloride intracellular channel-1 (CLIC1). CLIC1 chloride current is specifically induced upon translocation and transient membrane insertion of this channel during cell cycle progression. By means of patch clamp experiments, we show that the antiproliferative effect of metformin correlates with the inhibition of CLIC1 current, causing the arrest of the G1-S transition. Furthermore, point mutation of the putative CLIC1 pore region impairs metformin interaction with the channel, highlighting an inhibitory activity from the extracellular side. This effect is rather specific for CSC, since no cytotoxic effects, using metformin at the same concentration, were observed in human normal stem cells. These findings highlight the role of CLIC1 as a principal target of metformin's antiproliferative activity in human CSCs, paving the way for novel and needed pharmacological approach for cancer treatment.