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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
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.
Although recent studies in vitro and in vivo indicate that the polyphenol resveratrol (RSV) has anti-diabetic properties, the exact mechanisms involved are not known. In the present study, we ...examined the effects of RSV and the mechanism of regulation of glucose uptake in skeletal muscle cells.
In L6 myotubes RSV (100μM) induced maximum stimulation of glucose (2DG) uptake (201±8.90% of control, p<0.001), an effect that was similar to insulin action. RSV-stimulated glucose uptake was abolished by AMPK inhibition. In the presence of the sirtuin inhibitor nicotinamide, RSV-stimulated 2DG uptake and AMPK phosphorylation were abolished. RSV did not stimulate significant translocation of GLUT4 or GLUT1 transporters. However, treatment with indinavir, a GLUT4 specific inhibitor, blocked RSV-stimulated glucose uptake. We propose that RSV elevates glucose uptake in muscle cells through a mechanism that involves sirtuins and AMPK and possibly stimulation of GLUT4 transporter intrinsic activity.
Progressive decline in skeletal muscle mass and function are growing concerns in an aging population. Diet and physical activity are important for muscle maintenance but these requirements are not ...always met. This highlights the potential for nutritional supplementation. As a primary objective, we sought to assess the effect of a novel combination of L-Carnitine, creatine and leucine on muscle mass and performance in older subjects.
Forty-two healthy older adults aged 55-70 years were randomized to receive either a novel L-Carnitine (1500 mg), L-leucine (2000 mg), creatine (3000 mg), Vitamin D3 (10 μg) (L-Carnitine-combination) product (
= 14), L-Carnitine (1500 mg) (
= 14), or a placebo (
= 14) for eight weeks. We evaluated body mass by DXA, upper and lower strength by dynamometry, and walking distance by a 6-min walk test at baseline and after eight weeks of intervention. These measures, reflecting muscle mass, functional strength and mobility have been combined to generate a primary composite score. Quality of life, blood safety markers, and muscle biopsies for protein biomarker analysis were also conducted at baseline and the end of the study.
The primary composite outcome improved by 63.5 percentage points in the L-Carnitine-combination group vs. placebo (
= 0.013). However, this composite score did not change significantly in the L-Carnitine group (
0.232), and decreased slightly in the placebo group (
0.534). Participants supplemented with the L-Carnitine-combination showed a 1.0 kg increase in total lean muscle mass (
= 0.013), leg lean muscle mass (0.35 kg,
0.005), and a 1.0 kg increase in lower leg strength (
= 0.029) at week 8. In addition, these increases were significant when compared to the placebo group
0.034,
0.026, and
0.002, respectively). Total mTOR protein expression was increased in participants in the L-Carnitine-combination group at the end of the study compared to the baseline (
= 0.017). This increase was also significant when compared to the placebo (
0.039), suggesting that the increase in muscle mass and strength was due to new protein synthesis and mTOR pathway activation.
The trial did reach its primary objective. L-Carnitine combined with creatine and L-leucine significantly improved the composite score which reflects muscle mass and strength, at the end of the study compared to placebo. The combination showed an increase in mTOR protein level, a driver for increased muscle mass which translated to an improvement in muscle strength. This new combination may provide a potential nutritional intervention to promote muscle growth and improved physical functioning in older adults.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Mammary carcinomas have been associated with a high-fat diet, and the rate of breast cancer in overweight post-menopausal women is up to 50% higher than in their normal-weight counterparts. ...Epidemiological studies suggest that prolactin (PRL) plays a role in the progression of breast cancer. The current study examined breast cancer as a metabolic disease in the context of altered fatty acid catabolism by examining the effect of PRL on carnitine palmitoyl transferase 1 (CPT1), an enzyme that shuttles long-chain fatty acids into the mitochondrial matrix for β-oxidation. The effect of PRL on the adenosine 5'-monophosphate-activated protein kinase (AMPK) energy sensing pathway was also investigated.
MCF-7 and MDA-MB-231 breast cancer cells and 184B5 normal breast epithelial cells treated with 100 ng/ml of PRL for 24 hr were used as in vitro models. Real-time PCR was employed to quantify changes in mRNA levels and Western blotting was carried out to evaluate changes at the protein level. A non-radioactive CPT1 enzyme activity assay was established and siRNA transfections were performed to transiently knock down specific targets in the AMPK pathway.
PRL stimulation increased the expression of CPT1A (liver isoform) at the mRNA and protein levels in both breast cancer cell lines, but not in 184B5 cells. In response to PRL, a 20% increase in CPT1 enzyme activity was observed in MDA-MB-231 cells. PRL treatment resulted in increased phosphorylation of the α catalytic subunit of AMPK at Thr172, as well as phosphorylation of acetyl-CoA carboxylase (ACC) at Ser79. A siRNA against liver kinase B1 (LKB1) reversed these effects in breast cancer cells. PRL partially restored CPT1 activity in breast cancer cells in which CPT1A, LKB1, or AMPKα-1 were knocked down.
PRL enhances fatty acid β-oxidation by stimulating CPT1 expression and/or activity in MCF-7 and MDA-MB-231 breast cancer cells. These PRL-mediated effects are partially dependent on the LKB1-AMPK pathway, although the regulation of CPT1 is also likely to be influenced by other mechanisms. Ultimately, increased CPT1 enzyme activity may contribute to fueling the high energy demands of cancer cells. Targeting metabolic pathways that are governed by PRL, which has already been implicated in the progression of breast cancer, may be of therapeutic benefit.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prostate 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.
Androgen-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.
RSV (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, p21(cip1) and p27(kip1). RSV enhanced IR-activation of ATM and AMPK but inhibited basal and IR-induced phosphorylation of Akt.
Our 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-p21(cip1)/p27(kip1) and inhibit the Akt signalling pathways.
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.
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 p21
cip1
and p27
kip1
. 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- p21
cip1
/p27
kip1
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.
Earlier, 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.
Immuno-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.
IR 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.
IR 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.
Abstract 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 p21cip1 , but lack of response of both genes to IR. These cells had increased basal levels and activation of the Akt-mTOR-p70S6K /4-EBP1 signalling pathway. IR increased Akt, p70S6K 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-p21cip1 and Akt-mTOR, (ii) facilitates the normal response of these pathways to IR and, (iii) mediates the IR-induced G2-M checkpoint.
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