The combination of metformin and 2 deoxyglucose inhibits autophagy ...

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May 14, 2010 - ence initially termed the “Warburg effect” remains one of the main features that dis- tinguish normal cells from tumor cells. Many studies have ...
Autophagic Punctum

Autophagy 6:5, 670-671; July 1, 2010; © 2010 Landes Bioscience

The combination of metformin and 2-deoxyglucose inhibits autophagy and induces AMPK-dependent apoptosis in prostate cancer cells Issam Ben Sahra, Jean-François Tanti and Frédéric Bost* INSERM; U895; Centre Méditerranéen de Médecine Moléculaire (C3M); Team 7; Cellular and Molecular Physiopathology of Obesity and Diabetes; Nice, France; and Université de Nice Sophia-Antipolis; Faculté de Médecine; Institut Signalisation et pathologies; Nice, France

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argeting cancer cell metabolism is a promising new 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.

Key words: metformin, 2 deoxyglucose, prostate cancer, metabolism, AMPK, apoptosis, autophagy, p53 Submitted: 05/14/10 Revised: 05/20/10 Accepted: 05/21/10 Previously published online: www.landesbioscience.com/journals/ autophagy/article/12434 *Correspondence to: Frédéric Bost; Email: [email protected] Punctum to: Ben Sahra I, Laurent K, Giuliano S, Ponzio G, Gounon P, Le Marchand-Brustel Y, Giorgetti-Peraldi S, Cormont M, Bertolotto C, Deckert M, Auberger P, Tanti J-F, Bost F. Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53 dependent apoptosis in prostate cancer cells. Cancer Res 2010; 70:2465–75; PMID:20215500 ; DOI: 10.1158/0008-5472.CAN-09-2782.

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The metabolic fragility of cancer cells, which preferentially utilize glycolysis to metabolize glucose, rather than undergo oxidative phosphorylation, has recently received a lot of attention. This difference initially termed the “Warburg effect” remains one of the main features that distinguish normal cells from tumor cells. Many studies have tried to take advantage of this difference to specifically target cancer cells using metabolic perturbators such as 2-deoxyglucose. 2DG is an inhibitor of glucose metabolism, since it inhibits hexokinase, the first rate-limiting enzyme of glycolysis. Treatment with 2DG leads to intracellular ATP depletion and induces autophagy in prostate cancer cells (Fig. 1). Because of the tumor dependence on glycolysis, 2DG has been considered as a potential anti-cancer agent and association/combination of chemotherapeutic agents and 2DG has been used successfully in mice. Metformin

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is a widely used anti-diabetic drug prescribed to almost 120 million people for the treatment of type 2 diabetes. It lowers hyperglycemia by inhibiting hepatic glucose production. Numerous recent studies show that metformin decreases cancer cell viability and tumor growth in xenograft models. Furthermore, retrospective epidemiological studies reveal a decrease in the incidence of cancer in patients treated with metformin. Metformin hampers the respiratory chain complex I in hepatocytes and inhibits oxygen consumption in colon cancer cells. Unlike 2DG, metformin does not induce autophagy in prostate cancer cells; instead, it blocks cell cycle progression in G0 /G1 (Fig. 1). However, similar to 2DG, metformin activates the AMPactivated protein kinase (AMPK), a major sensor of energy metabolism, which then inhibits mTOR and downregulates anabolic pathways. We decided to study the combination of 2DG and metformin, two drugs which target the two primary sources of cell energy and may represent a major advantage over traditional chemotherapies. Cancer cells become resistant to chemotherapy by inactivating normal pathways of cell death and activating cell survival pathways. We show that metformin and 2DG act synergistically to induce a massive depletion of intracellular ATP, lead to a stronger inhibitory effect of cell viability than either drug alone, and affect more significantly prostate cancer cells than normal epithelial prostate cells. Indeed, this combination blocks cell cycle in G2 /M and induces p53-dependent apoptosis after 48 hours of treatment. We

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Figure 1. (A) Metformin alone inhibits the complex I of mitochondria and induces cell cycle arrest. 2-deoxyglucose (2DG) alone inhibits glycolysis and induces autophagy. (B) The addition of metformin to 2DG drastically decreases the intracellular concentration of ATP and induces a switch from 2DGinduced autophagy to apoptosis.

also show that the induction of apoptosis is dependent on the AMPK/mTOR pathway (Fig. 1). Such implication of AMPK in apoptosis is not surprising since AICAR, which mimics energy depletion, has already been reported to induce apoptosis through AMPK in B-cell chronic lymphocytic leukemia and pancreatic beta cells. Importantly, we demonstrate in this paper that the addition of metformin to 2DG induces a shift from autophagy to apoptosis. Indeed, increasing concentrations of 2DG leads to the augmentation of LC3-II and the formation of autophagosomes in prostate cancer cells. The addition of metformin to 2DG-treated cells leads to the total disappearance of LC3-I and LC3-II, and a decrease in Beclin 1 expression, a protein required for autophagy. However, beclin 1 is also a tumor suppressor gene, which reduces malignant phenotype and the ability for anchorage-independent growth in MCF7, a breast cancer cell line. This role is in

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contradiction with the antiproliferative effect of metformin, which triggers apoptosis in combination with 2DG although Beclin 1 was reduced. Thus, we suggest that Beclin 1 has a survival function by blocking the onset of the apoptotic cascade, and metformin is the trigger of apoptosis in cells engaged in the survival response induced by 2DG. A similar conclusion has been made in breast cancer cells in response to DNA damage after downregulation of beclin 1 expression with shRNA. Metformin, as an activator of AMPK and an inhibitor of mTOR, should potentially induce autophagy. However, only one study demonstrates that metformin induces autophagy in cancer cells suggesting that, depending on cell type, activation of AMPK is not automatically associated with the induction of autophagy. Other activators of AMPK, such as AICAR, do not systematically induce autophagy and are even reported to inhibit autophagy.

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The process of autophagy has been identified as an important mechanism of resistance to cellular stress (energy deprivation, genotoxic stress, etc.). We demonstrate in this study that metformin triggers a switch from a survival process induced by nutrient deprivation to cell death. Our study reinforces the growing interest in metabolic perturbators in cancer therapy and highlights the potential use of the combination of metformin and 2DG as an anti-cancer treatment. Acknowledgements

We thank Y. Le Marchand Brustel for carefully reading the manuscript. This research was supported by INSERM, University of Nice Sophia-Antipolis, Association pour la Recherche sur le Cancer (grant# 1018), and the Ministère de la Recherche (IBS). F. Bost and J.F. Tanti are CNRS investigators.

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