Toran Sanli

Metformin inhibits growth and enhances radiation response of non-small cell lung cancer (NSCLC) through ATM and AMPK.

Background:We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR).Methods:Human NSCLC cells, mouse embryonic fibroblasts from wild-type and AMP-activated kinase (AMPK) α1/2-subunit−/− embryos (AMPKα1/2−/−-MEFs) and NSCLC tumours grafted into Balb/c-nude mice were treated with IR and MET and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays and immunohistochemistry (IHC).Results:Metformin (2.5 μM–5 mM) inhibited proliferation and radio-sensitised NSCLC cells. Metformin (i) activated the ataxia telengiectasia-mutated (ATM)–AMPK–p53/p21cip1 and inhibited the Akt–mammalian target of rapamycin (mTOR)–eIF4E-binding protein 1 (4EBP1) pathways, (ii) induced G1 cycle arrest and (iii) enhanced apoptosis. ATM inhibition blocked MET and IR activation of AMPK. Non-small cell lung cancer cells with inhibited AMPK and AMPKα1/2−/−-MEFs were resistant to the antiproliferative effects of MET and IR. Metformin or IR inhibited xenograft growth and combined treatment enhanced it further than each treatment alone. Ionising radiation and MET induced (i) sustained activation of ATM–AMPK–p53/p21cip1 and inhibition of Akt–mTOR–4EBP1 pathways in tumours, (ii) reduced expression of angiogenesis and (iii) enhanced expression of apoptosis markers.Conclusion:Clinically achievable MET doses inhibit NSCLC cell and tumour growth and sensitise them to IR. Metformin and IR mediate their action through an ATM–AMPK-dependent pathway. Our results suggest that MET can be a clinically useful adjunct to radiotherapy in NSCLC.

Ionizing radiation activates AMP-activated kinase (AMPK): a target for radiosensitization of human cancer cells.

PURPOSE Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. METHODS AND MATERIALS Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. RESULTS IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK alpha subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21(waf/cip) as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. CONCLUSIONS We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21(waf/cip) and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21(waf/cip) pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

AMP-activated protein kinase (AMPK) beyond metabolism: A novel genomic stress sensor participating in the DNA damage response pathway

AMP-activated protein kinase (AMPK), an established metabolic stress sensor, has gained popularity in cancer biology due to its ability to control cellular growth and mediate cell cycle checkpoints in cancer cells in response to low energy levels. AMPK is a key effector of the tumor suppressor liver kinase B 1 (LKB1) which inhibits the cellular growth mediator mammalian target of rapamycin (mTOR) and activates checkpoint mediators such as p53 and the cyclin dependent kinase inhibitors p21cip1 and p27kip1. However, recent work describes a novel function for AMPK as a sensor of genomic stress and a participant of the DNA damage response (DDR) pathway. Ionizing radiation and chemotherapy activate AMPK in cancer cells to mediate signal transduction downstream of ataxia telangiectasia mutated (ATM) to activate p53- p21cip1/p27kip1 and inhibit mTOR. We discuss evidence on the transcriptional and post-translational regulation of AMPK by ionizing radiation and the role of the enzyme as a mediator of chemo- and radiation sensitivity in epithelial cancer cells. Furthermore, we review data on the participation of AMPK in cytokinesis and observations suggesting a physical association of this enzyme with the mitotic apparatus. The evidence available to date suggests that AMPK is a point of convergence of metabolic and genomic stress signals, which (1) control the activity of growth mediators, (2) propagate DDR, and (3) mediate the anti-proliferative effects of common cytotoxic cancer therapy such as radiation and chemotherapy. This highlights the importance of targeting AMPK with novel cancer therapeutics.

Sestrin2 Modulates AMPK Subunit Expression and Its Response to Ionizing Radiation in Breast Cancer Cells

Background The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth. Methods and Findings Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKα1β1γ1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKα1 and AMPKβ1 subunit phosphorylation, and co-localized with phosphorylated AMPKα-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKα1β1γ1, as well as mRNA levels of LKB1, AMPKα1, and AMPKβ1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism. Conclusions Our results suggest that in breast cancer cells SESN2 is associated with AMPK, it is involved in regulation of basal and IR-induced expression and activation of this enzyme, and it mediates sensitization of cancer cells to IR.

Resveratrol enhances prostate cancer cell response to ionizing radiation. Modulation of the AMPK, Akt and mTOR pathways

BackgroundProstate cancer (PrCa) displays resistance to radiotherapy (RT) and requires radiotherapy dose escalation which is associated with greater toxicity. This highlights a need to develop radiation sensitizers to improve the efficacy of RT in PrCa. Ionizing radiation (IR) stimulates pathways of IR-resistance and survival mediated by the protein kinase Akt but it also activates the metabolic energy sensor and tumor suppressor AMP-Activated Protein Kinase (AMPK). Here, we examined the effects of the polyphenol resveratrol (RSV) on the IR-induced inhibition of cell survival, modulation of cell cycle and molecular responses in PrCa cells.MethodsAndrogen-insensitive (PC3), sensitive (22RV1) PrCa and PNT1A normal prostate epithelial cells were treated with RSV alone (2.5-10 μM) or in combination with IR (2-8 Gy). Clonogenic assays, cell cycle analysis, microscopy and immunoblotting were performed to assess survival, cell cycle progression and molecular responses.ResultsRSV (2.5-5 μM) inhibited clonogenic survival of PC3 and 22RV1 cells but not of normal prostate PNT1A cells. RSV specifically sensitized PrCa cells to IR, induced cell cycle arrest at G1-S phase and enhanced IR-induced nuclear aberrations and apoptosis. RSV enhanced IR-induced expression of DNA damage (γH2Ax) and apoptosis (cleaved-caspase 3) markers as well as of the cell cycle regulators p53, p21cip1 and p27kip1. RSV enhanced IR-activation of ATM and AMPK but inhibited basal and IR-induced phosphorylation of Akt.ConclusionsOur results suggest that RSV arrests cell cycle, promotes apoptosis and sensitizes PrCa cells to IR likely through a desirable dual action to activate the ATM-AMPK-p53-p21cip1/p27kip1 and inhibit the Akt signalling pathways.

Lovastatin Sensitizes Lung Cancer Cells to Ionizing Radiation: Modulation of Molecular Pathways of Radioresistance and Tumor Suppression

Introduction: In this study, we investigated the effect of the 3-hydroxy-3-methylgutaryl-CoA reductase inhibitor lovastatin, as a sensitizer of lung cancer cells to ionizing radiation (IR). Methods: A549 lung adenocarcinoma cells were treated with 0 to 50 &mgr;M lovastatin alone or in combination with 0 to 8 Gy IR and subjected to clonogenic survival and proliferation assays. To assess the mechanism of drug action, we examined the effects of lovastatin and IR on the epidermal growth factor (EGF) receptor and AMP-activated kinase (AMPK) pathways and on apoptotic markers and the cell cycle. Results: Lovastatin inhibited basal clonogenic survival and proliferation of A549 cells and sensitized them to IR. This was reversed by mevalonate, the product of 3-hydroxy-3-methylgutaryl-CoA reductase. Lovastatin attenuated selectively EGF-induced phosphorylation of EGF receptor and Akt, and IR-induced Akt phosphorylation, in a mevalonate-sensitive fashion, without inhibition on extracellular signal-regulated kinase 1/2 phosphorylation by either stimulus. IR phosphorylated and activated the metabolic sensor and tumor suppressor AMPK, but lovastatin enhanced basal and IR-induced AMPK phosphorylation. The drug inhibited IR-induced expression of p53 and the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, but caused a redistribution of cells from G1-S phase (control and radiated cells) and G2-M phase (radiated cells) of cell cycle into apoptosis. The latter was also evident by induction of nuclear fragmentation and cleavage of caspase 3 by lovastatin in both control and radiated cells. Conclusions: We suggest that lovastatin inhibits survival and induces radiosensitization of lung cancer cells through induction of apoptosis, which may be mediated by a simultaneous inhibition of the Akt and activation of the AMPK signaling pathways.

Ionizing radiation regulates the expression of AMP-activated protein kinase (AMPK) in epithelial cancer cells: Modulation of cellular signals regulating cell cycle and survival

PURPOSE To analyze the (i) expression of AMPK in a variety of epithelial cancer cells, (ii) regulation of AMPK subunit expression by ionizing radiation (IR) and (iii) impact of AMPK on signaling pathways regulating cell cycle and survival. METHODS AND MATERIALS Human lung, prostate, and breast normal and cancer cells were treated with 0 or 8 Gy IR and mRNA and protein levels of AMPK were evaluated by RT-PCR and immunoblotting 24 or 48 h later. Untreated and radiated wild type (WT) and AMPKα(-/-) mouse embryonic fibroblasts (MEFs) were analyzed by immunoblotting using total- and phosphorylation-specific antibodies. Histone H2Ax was examined by fluorescence microscopy. The cell cycle and survival of WT and AMPKα(-/-) MEFs was also evaluated following 8 Gy by IR. RESULTS AMPK subunits were found widely expressed in normal and cancer epithelial cells. IR increased subunit protein levels and stimulated gene transcription in cancer cells. AMPKα(-/-)-MEFs showed enhanced basal total levels of ATM and phosphorylation of its substrates histone H2Ax, but inhibited response of these markers and of checkpoint kinase Chk2 phosphorylation to IR. AMPKα(-/-)-MEFs showed increased basal levels of p53 and cyclin-dependent kinase inhibitors p21(cip1), but lack of response of both genes to IR. These cells had increased basal levels and activation of the Akt-mTOR-p70(S6K)/4-EBP1 signalling pathway. IR increased Akt, p70(S6K) and 4-EBP1 phosphorylation in WT-MEFs, but this was reduced in AMPKα(-/-)-MEFs. AMPKα(-/-)-MEFs failed to arrest at the G2-M checkpoint after IR and showed a trend for radio-resistance in proliferation assays. CONCLUSIONS AMPK is widely expressed in human normal and cancer epithelial cells and its gene transcription, protein levels, and enzymatic activity is stimulated by IR. Work with AMPKα knockout cells suggests that AMPK (i) may mediate a suppressive regulation on basal expression and activity of ATM and its downstream effector pathways Chk2/p53-p21(cip1) and Akt-mTOR, (ii) facilitates the normal response of these pathways to IR and, (iii) mediates the IR-induced G2-M checkpoint.

Metformin: On Ongoing Journey across Diabetes, Cancer Therapy and Prevention

Cancer metabolism is the focus of intense research, which witnesses its key role in human tumors. Diabetic patients treated with metformin exhibit a reduced incidence of cancer and cancer-related mortality. This highlights the possibility that the tackling of metabolic alterations might also hold promising value for treating cancer patients. Here, we review the emerging role of metformin as a paradigmatic example of an old drug used worldwide to treat patients with type II diabetes which to date is gaining strong in vitro and in vivo anticancer activities to be included in clinical trials. Metformin is also becoming the focus of intense basic and clinical research on chemoprevention, thus suggesting that metabolic alteration is an early lesion along cancer transformation. Metabolic reprogramming might be a very efficient prevention strategy with a profound impact on public health worldwide.

Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts

IntroductionEarlier, we showed that in cancer cells, AMP-activated kinase (AMPK) participates in a signal transduction pathway involving ATM-AMPK-p53/p21cip1 which is activated by ionizing radiation (IR) to mediate G2-M arrest and enhanced cytotoxicity. We also observed that AMPK modulates ATM expression and activity and the IR response of the Akt-mTOR pathway. Since the ATM, AMPK and Akt pathways are key targets of novel radio-sensitizing therapeutics, we examined the chronic modultion of expression and activity of those pathways by IR alone in xenograft models of lung cancer.MethodsImmuno-compromised mice were grafted with human lung A549 and H1299 cells, were treated with a single fraction of 0 or 10 Gy, and left to grow for 8 weeks. Extracted tumors were subjected to lysis and immunoblotting or fixation and immunohistochemical analysis.ResultsIR inhibited significantly xenograft growth and was associated with increased expression of Ataxia Telengiectasia Mutated (ATM) and enhanced phosphorylation of two ATM targets, H2Ax and checkpoint kinase Chk2. Irradiated tumours showed increased total AMPK levels and phosphorylation of AMPK and its substrate Acetyl-CoA Carboxylase (ACC). IR led to enhanced expression and phosphorylation of p53 and cyclin dependent kinase inhibitors p21cip1 and p27kip1. However, irradiated tumours had reduced phosphorylation of Akt, mTOR and it‘s target translation initiation inhibitor 4EBP1. Irradiated xenografts showed reduced microvessel density, reduced expression of CD31 but increased expression of hypoxia-induced factor 1A (HIF1a) compared to controls.ConclusionIR inhibits epithelial cancer tumour growth and results in sustained expression and activation of ATM-Chk2, and AMPK-p53/p21cip1/p27kip1 but partial inhibition of the Akt-mTOR signaling pathways. Future studies should examine causality between those events and explore whether further modulation of the AMPK and Akt-mTOR pathways by novel therapeutics can sensitize lung tumours to radiation.

Lifestyle Factors and MicroRNAs: A New Paradigm in Cancer Chemoprevention.

MicroRNAs (miRNAs) are characterized as small RNA molecules that modulate gene transcription in a posttranslational manner. Functionally, miRNAs play important roles in a diverse number of biological processes, including cell development, differentiation, proliferation, and apoptosis. Consequently, changes in the expression pattern of miRNAs have been associated with multiple human pathologies, including cancer. Based on these alterations, distinct miRNAs can be utilized as markers for cancer risk evaluation or used in tumour detection. Recent evidence has indicated that lifestyle factors, such as nutrition, physical activity, and glycemic control provide health benefits through regulation of miRNA expression. In this review, we provide a concise overview of miRNA regulation, biosynthesis, and their expression patterns in normal and malignant tissue. We then summarize the emerging knowledge of how lifestyle factors, including nutrients, exercise, and hypoglycemic agents modify miRNAs and are involved in cancer prevention. Finally, we conclude by providing recommendation for future investigations into novel agents that can modulate miRNAs and act as chemotherapeutic agents against cancer.