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Rich66 · 11-11-2009, 02:10 AM

http://cancerres.aacrjournals.org/cg...ct/66/21/10269

doi: 10.1158/0008-5472.CAN-06-1500
© 2006 American Association for Cancer Research

Priority Reports

Metformin Is an AMP Kinase–Dependent Growth Inhibitor for Breast Cancer Cells

Mahvash Zakikhani¹, Ryan Dowling², I. George Fantus³, Nahum Sonenberg² and Michael Pollak¹ Departments of ¹ Oncology and ² Biochemistry, McGill University, Montreal, Quebec, Canada and ³ Department of Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
Requests for reprints: Michael Pollak, Cancer Prevention Center, Jewish General Hospital, E-763, 3755 Cote Ste. Catherine Montreal, Quebec, Canada H3T 1E2. Phone: 514-340-8222, ext. 4139; Fax: 514-340-8600; E-mail: Michael.pollak@mcgill.ca.
Recent population studies provide clues that the use of metforminmay be associated with reduced incidence and improved prognosisof certain cancers. This drug is widely used in the treatmentof type 2 diabetes, where it is often referred to as an "insulinsensitizer" because it not only lowers blood glucose but alsoreduces the hyperinsulinemia associated with insulin resistance.As insulin and insulin-like growth factors stimulate proliferationof many normal and transformed cell types, agents that facilitatesignaling through these receptors would be expected to enhanceproliferation. We show here that metformin acts as a growthinhibitor rather than an insulin sensitizer for epithelial cells.Breast cancer cells can be protected against metformin-inducedgrowth inhibition by small interfering RNA against AMP kinase.This shows that AMP kinase pathway activation by metformin,recently shown to be necessary for metformin inhibition of gluconeogenesisin hepatocytes, is also involved in metformin-induced growthinhibition of epithelial cells. The growth inhibition was associatedwith decreased mammalian target of rapamycin and S6 kinase activationand a general decrease in mRNA translation. These results provideevidence for a mechanism that may contribute to the antineoplasticeffects of metformin suggested by recent population studiesand justify further work to explore potential roles for activatorsof AMP kinase in cancer prevention and treatment. (Cancer Res2006; 66(21): 10269-73)

J Cell Mol Med. 2009 Oct 29. [Epub ahead of print]
Metformin attenuates ovarian cancer cell growth in an AMP- kinase dispensable manner.

Rattan R, Giri S, Hartmann L, Shridhar V.
Department of Experimental Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905.
ABSTRACT Metformin, the most widely used drug for type 2 diabetes activates AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -alpha1, -beta1, -gamma1 and -gamma2 isoforms of AMPK subunits. Our studies show that metformin treatment

(1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04),

(2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression,

(3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKalpha and its downstream substrate; ACC (Acetyl Co-carboxylase) and enhanced beta- oxidation of fatty acid,

(4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin mediated effect on AMPK is dependent on LKB1(Liver kinase B1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to downregulate LKB1 in ovarian cancer cells. In contrast, metformin inhibited cell proliferation in both wild type and AMPKalpha1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK dependent as well as independent pathways.

PMID: 19874425 [PubMed - as supplied by publisher]

Oncogene. 2008 Jun 5;27(25):3576-86. Epub 2008 Jan 21.
The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level.

Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger P, Tanti JF, Le Marchand-Brustel Y, Bost F.
INSERM U568, Nice, France.
Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies.

J Mol Signal. 2008 Dec 1;3:18.
Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1.

Zhuang Y, Miskimins WK.
Cancer Biology Research Center, Sanford Research/USD, 1400 West 22nd Street, Sioux Falls, South Dakota, 57105, USA. keith.miskimins@usd.edu.
ABSTRACT: BACKGROUND: The antihyperglycemic drug metformin may have beneficial effects on the prevention and treatment of cancer. Metformin is known to activate AMP-activated protein kinase (AMPK). It has also been shown to inhibit cyclin D1 expression and proliferation of some cultured cancer cells. However, the mechanisms of action by which metformin mediates cell cycle arrest are not completely understood. RESULTS: In this study, metformin was found to inhibit proliferation of most cultured breast cancer cell lines. This was independent of estrogen receptor, HER2, or p53 status. Inhibition of cell proliferation was associated with arrest within G0/G1 phase of the cell cycle. As in previous studies, metformin treatment led to activation of (AMPK) and downregulation of cyclin D1. However, these events were not sufficient for cell cycle arrest because they were also observed in the MDA-MB-231 cell line, which is not sensitive to growth arrest by metformin. In sensitive breast cancer lines, the reduction in cyclin D1 led to release of sequestered CDK inhibitors, p27Kip1 and p21Cip1, and association of these inhibitors with cyclin E/CDK2 complexes. The metformin-resistant cell line MDA-MB-231 expresses significantly lower levels of p27Kip1 and p21Cip1 than the metformin-sensitive cell line, MCF7. When p27Kip1 or p21Cip1 were overexpressed in MDA-MB-231, the cells became sensitive to cell cycle arrest in response to metformin. CONCLUSION: Cell cycle arrest in response to metformin requires CDK inhibitors in addition to AMPK activation and cyclin D1 downregulation. This is of interest because many cancers are associated with loss or downregulation of CDK inhibitors and the results may be relevant to the development of anti-tumor reagents that target the AMPK pathway.

PMID: 19046439 [PubMed - in process]

Cell Cycle. 2009 Mar 15;8(6):909-15. Epub 2009 Mar 26.
Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro.

Alimova IN, Liu B, Fan Z, Edgerton SM, Dillon T, Lind SE, Thor AD.
Department of Pathology, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA.
The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G(1) checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27(kip1) or p21(waf1). It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.

PMID: 19221498 [PubMed - indexed for MEDLINE]

Cancer Res. 2007 Jul 15;67(14):6745-52.
Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth.

Buzzai M, Jones RG, Amaravadi RK, Lum JJ, DeBerardinis RJ, Zhao F, Viollet B, Thompson CB.
Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
The effect of the antidiabetic drug metformin on tumor growth was investigated using the paired isogenic colon cancer cell lines HCT116 p53(+/+) and HCT116 p53(-/-). Treatment with metformin selectively suppressed the tumor growth of HCT116 p53(-/-) xenografts. Following treatment with metformin, we detected increased apoptosis in p53(-/-) tumor sections and an enhanced susceptibility of p53(-/-) cells to undergo apoptosis in vitro when subject to nutrient deprivation. Metformin is proposed to function in diabetes treatment as an indirect activator of AMP-activated protein kinase (AMPK). Treatment with AICAR, another AMPK activator, also showed a selective ability to inhibit p53(-/-) tumor growth in vivo. In the presence of either of the two drugs, HCT116 p53(+/+) cells, but not HCT116 p53(-/-) cells, activated autophagy. A similar p53-dependent induction of autophagy was observed when nontransformed mouse embryo fibroblasts were treated. Treatment with either metformin or AICAR also led to enhanced fatty acid beta-oxidation in p53(+/+) MEFs, but not in p53(-/-) MEFs. However, the magnitude of induction was significantly lower in metformin-treated cells, as metformin treatment also suppressed mitochondrial electron transport. Metformin-treated cells compensated for this suppression of oxidative phosphorylation by increasing their rate of glycolysis in a p53-dependent manner. Together, these data suggest that metformin treatment forces a metabolic conversion that p53(-/-) cells are unable to execute. Thus, metformin is selectively toxic to p53-deficient cells and provides a potential mechanism for the reduced incidence of tumors observed in patients being treated with metformin.

PMID: 17638885 [PubMed - indexed for MEDLINE]

11-11-2009, 02:10 AM	#17
Rich66 Senior Member Join Date: Feb 2008 Location: South East Wisconsin Posts: 3,431	Re: Common Diabetes Drug May 'Revolutionize' Cancer Therapies: Unexpected T-cell Brea http://cancerres.aacrjournals.org/cg...ct/66/21/10269 doi: 10.1158/0008-5472.CAN-06-1500 © 2006 American Association for Cancer Research Priority Reports Metformin Is an AMP Kinase–Dependent Growth Inhibitor for Breast Cancer Cells Mahvash Zakikhani¹, Ryan Dowling², I. George Fantus³, Nahum Sonenberg² and Michael Pollak¹ Departments of ¹ Oncology and ² Biochemistry, McGill University, Montreal, Quebec, Canada and ³ Department of Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada Requests for reprints: Michael Pollak, Cancer Prevention Center, Jewish General Hospital, E-763, 3755 Cote Ste. Catherine Montreal, Quebec, Canada H3T 1E2. Phone: 514-340-8222, ext. 4139; Fax: 514-340-8600; E-mail: Michael.pollak@mcgill.ca. Recent population studies provide clues that the use of metforminmay be associated with reduced incidence and improved prognosisof certain cancers. This drug is widely used in the treatmentof type 2 diabetes, where it is often referred to as an "insulinsensitizer" because it not only lowers blood glucose but alsoreduces the hyperinsulinemia associated with insulin resistance.As insulin and insulin-like growth factors stimulate proliferationof many normal and transformed cell types, agents that facilitatesignaling through these receptors would be expected to enhanceproliferation. We show here that metformin acts as a growthinhibitor rather than an insulin sensitizer for epithelial cells.Breast cancer cells can be protected against metformin-inducedgrowth inhibition by small interfering RNA against AMP kinase.This shows that AMP kinase pathway activation by metformin,recently shown to be necessary for metformin inhibition of gluconeogenesisin hepatocytes, is also involved in metformin-induced growthinhibition of epithelial cells. The growth inhibition was associatedwith decreased mammalian target of rapamycin and S6 kinase activationand a general decrease in mRNA translation. These results provideevidence for a mechanism that may contribute to the antineoplasticeffects of metformin suggested by recent population studiesand justify further work to explore potential roles for activatorsof AMP kinase in cancer prevention and treatment. (Cancer Res2006; 66(21): 10269-73) J Cell Mol Med. 2009 Oct 29. [Epub ahead of print] Metformin attenuates ovarian cancer cell growth in an AMP- kinase dispensable manner. Rattan R, Giri S, Hartmann L, Shridhar V. Department of Experimental Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905. ABSTRACT Metformin, the most widely used drug for type 2 diabetes activates AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -alpha1, -beta1, -gamma1 and -gamma2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKalpha and its downstream substrate; ACC (Acetyl Co-carboxylase) and enhanced beta- oxidation of fatty acid, (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin mediated effect on AMPK is dependent on LKB1(Liver kinase B1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to downregulate LKB1 in ovarian cancer cells. In contrast, metformin inhibited cell proliferation in both wild type and AMPKalpha1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK dependent as well as independent pathways. PMID: 19874425 [PubMed - as supplied by publisher] Oncogene. 2008 Jun 5;27(25):3576-86. Epub 2008 Jan 21. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger P, Tanti JF, Le Marchand-Brustel Y, Bost F. INSERM U568, Nice, France. Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies. J Mol Signal. 2008 Dec 1;3:18. Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1. Zhuang Y, Miskimins WK. Cancer Biology Research Center, Sanford Research/USD, 1400 West 22nd Street, Sioux Falls, South Dakota, 57105, USA. keith.miskimins@usd.edu. ABSTRACT: BACKGROUND: The antihyperglycemic drug metformin may have beneficial effects on the prevention and treatment of cancer. Metformin is known to activate AMP-activated protein kinase (AMPK). It has also been shown to inhibit cyclin D1 expression and proliferation of some cultured cancer cells. However, the mechanisms of action by which metformin mediates cell cycle arrest are not completely understood. RESULTS: In this study, metformin was found to inhibit proliferation of most cultured breast cancer cell lines. This was independent of estrogen receptor, HER2, or p53 status. Inhibition of cell proliferation was associated with arrest within G0/G1 phase of the cell cycle. As in previous studies, metformin treatment led to activation of (AMPK) and downregulation of cyclin D1. However, these events were not sufficient for cell cycle arrest because they were also observed in the MDA-MB-231 cell line, which is not sensitive to growth arrest by metformin. In sensitive breast cancer lines, the reduction in cyclin D1 led to release of sequestered CDK inhibitors, p27Kip1 and p21Cip1, and association of these inhibitors with cyclin E/CDK2 complexes. The metformin-resistant cell line MDA-MB-231 expresses significantly lower levels of p27Kip1 and p21Cip1 than the metformin-sensitive cell line, MCF7. When p27Kip1 or p21Cip1 were overexpressed in MDA-MB-231, the cells became sensitive to cell cycle arrest in response to metformin. CONCLUSION: Cell cycle arrest in response to metformin requires CDK inhibitors in addition to AMPK activation and cyclin D1 downregulation. This is of interest because many cancers are associated with loss or downregulation of CDK inhibitors and the results may be relevant to the development of anti-tumor reagents that target the AMPK pathway. PMID: 19046439 [PubMed - in process] Cell Cycle. 2009 Mar 15;8(6):909-15. Epub 2009 Mar 26. Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Alimova IN, Liu B, Fan Z, Edgerton SM, Dillon T, Lind SE, Thor AD. Department of Pathology, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA. The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G(1) checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27(kip1) or p21(waf1). It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. PMID: 19221498 [PubMed - indexed for MEDLINE] Cancer Res. 2007 Jul 15;67(14):6745-52. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Buzzai M, Jones RG, Amaravadi RK, Lum JJ, DeBerardinis RJ, Zhao F, Viollet B, Thompson CB. Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. The effect of the antidiabetic drug metformin on tumor growth was investigated using the paired isogenic colon cancer cell lines HCT116 p53(+/+) and HCT116 p53(-/-). Treatment with metformin selectively suppressed the tumor growth of HCT116 p53(-/-) xenografts. Following treatment with metformin, we detected increased apoptosis in p53(-/-) tumor sections and an enhanced susceptibility of p53(-/-) cells to undergo apoptosis in vitro when subject to nutrient deprivation. Metformin is proposed to function in diabetes treatment as an indirect activator of AMP-activated protein kinase (AMPK). Treatment with AICAR, another AMPK activator, also showed a selective ability to inhibit p53(-/-) tumor growth in vivo. In the presence of either of the two drugs, HCT116 p53(+/+) cells, but not HCT116 p53(-/-) cells, activated autophagy. A similar p53-dependent induction of autophagy was observed when nontransformed mouse embryo fibroblasts were treated. Treatment with either metformin or AICAR also led to enhanced fatty acid beta-oxidation in p53(+/+) MEFs, but not in p53(-/-) MEFs. However, the magnitude of induction was significantly lower in metformin-treated cells, as metformin treatment also suppressed mitochondrial electron transport. Metformin-treated cells compensated for this suppression of oxidative phosphorylation by increasing their rate of glycolysis in a p53-dependent manner. Together, these data suggest that metformin treatment forces a metabolic conversion that p53(-/-) cells are unable to execute. Thus, metformin is selectively toxic to p53-deficient cells and provides a potential mechanism for the reduced incidence of tumors observed in patients being treated with metformin. PMID: 17638885 [PubMed - indexed for MEDLINE] __________________ Mom's treatment history (link)