Metformin Protects Rat Hepatocytes against Bile Acid-Induced Apoptosis

Background

Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death.

Aim

To evaluate the effects of metformin on hepatocyte cell death.

Methods

Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively.

Results

Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation.

Conclusion

Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.     

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

Introduction

Recently, 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.

Methods

Prostate 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.

Results

Exendin–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.

Conclusion

These 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.     

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

Introduction

The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.

Methods

MDA-MB-468, BT474 and SKBr₃ breast cancer cell lines were treated with metformin and [³H-methyl]choline and [¹⁴C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [³H] Radiolabelled metabolites were determined using thin layer chromatography.

Results

Metformin-treated cells exhibited decreased formation of [³H]phosphocholine but increased accumulation of [³H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [¹⁴C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [¹⁴C(U)]glucose.

Conclusion

This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.     

The anti-proliferative effect of metformin in triple-negative MDA-MB-231 breast cancer cells is highly dependent on glucose concentration: Implications for cancer therapy and prevention

Background

Metformin has been shown to have a strong anti-proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP-activated protein kinase (AMPK). MDA-MB-231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti-cancer agents as well as to the anti-proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA-MB-231 cells to the anti-proliferative effect of metformin.

Methods

MDA-MB-231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by Western blotting.

Results

In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA-MB-231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA-MB-231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin-treatment of MDA-MB-231 cells cultured in normoglycemic conditions and not in hyperglycemic conditions caused a striking activation of AMPK, and an AMPK-dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.

Conclusion

Our data show that normoglycemia sensitizes the triple negative MDA-MB-231 breast cancer cells to the anti-proliferative effect of metformin through an AMPK-dependent mechanism.

General significance

These findings suggest that tight normoglycemic control may enhance the anti-proliferative effect of metformin in diabetic cancer patients.     

Exploring the Altered Dynamics of Mammalian Central Carbon Metabolic Pathway in Cancer Cells: A Classical Control Theoretic Approach

Background

In contrast with normal cells, most of the cancer cells depend on aerobic glycolysis for energy production in the form of adenosine triphosphate (ATP) bypassing mitochondrial oxidative phosphorylation. Moreover, compared to normal cells, cancer cells exhibit higher consumption of glucose with higher production of lactate. Again, higher rate of glycolysis provides the necessary glycolytic intermediary precursors for DNA, protein and lipid synthesis to maintain high active proliferation of the tumor cells. In this scenario, classical control theory based approach may be useful to explore the altered dynamics of the cancer cells. Since the dynamics of the cancer cells is different from that of the normal cells, understanding their dynamics may lead to development of novel therapeutic strategies.

Method

We have developed a model based on the state space equations of classical control theory along with an order reduction technique to mimic the actual dynamic behavior of mammalian central carbon metabolic (CCM) pathway in normal cells. Here, we have modified Michaelis Menten kinetic equation to incorporate feedback mechanism along with perturbations and cross talks associated with a metabolic pathway. Furthermore, we have perturbed the proposed model to reduce the mitochondrial oxidative phosphorylation. Thereafter, we have connected proportional-integral (PI) controller(s) with the model for tuning it to behave like the CCM pathway of a cancer cell. This methodology allows one to track the altered dynamics mediated by different enzymes.

Results and Discussions

The proposed model successfully mimics all the probable dynamics of the CCM pathway in normal cells. Moreover, experimental results demonstrate that in cancer cells, a coordination among enzymes catalyzing pentose phosphate pathway and intermediate glycolytic enzymes along with switching of pyruvate kinase (M2 isoform) plays an important role to maintain their altered dynamics.     

Chromodomain Helicase/ATPase DNA-Binding Protein 1-Like Gene (CHD1L) Expression and Implications for Invasion and Metastasis of Breast Cancer

Background

Chromodomain helicase/ATPase DNA-binding protein 1-like gene (CHD1L), also known as ALC1 (amplified in liver cancer 1 gene), is a new oncogene amplified in many solid tumors. Whether this gene plays a role in invasion and metastasis of breast cancer is unknown.

Methods

Immunohistochemistry was performed to detect the expression of CHD1L in patients with invasive ductal carcinoma and normal mammary glands. Chemotaxis, wound healing, and Transwell invasion assays were also performed to examine cell migration and invasion. Western blot analysis was conducted to detect the expression of CHD1L, MMP-2, MMP-9, pAkt/Akt, pARK5/ARK5, and pmTOR/mTOR. Moreover, ELISA was carried out to detect the expression levels of MMP-2 and MMP-9. Nude mice xenograft model was used to detect the invasion and metastasis of breast cancer cell lines.

Results

CHD1L overexpression was observed in 112 of 268 patients (41.8%). This overexpression was associated with lymph node metastasis (P = 0.008), tumor differentiation (P = 0.020), distant metastasis (P = 0.026), MMP-2 (P = 0.035), and MMP-9 expression (P = 0.022). In the cell experiment, reduction of CHD1L inhibited the invasion and metastasis of breast cancer cells by mediating MMP-2 and MMP-9 expression. CHD1L knockdown via siRNA suppressed EGF-induced pAkt, pARK5, and pmTOR. This knockdown inhibited the metastasis of breast cancer cells into the lungs of SCID mice.

Conclusions

CHD1L promoted the invasion and metastasis of breast cancer cells via the PI3K/Akt/ARK5/mTOR/MMP signaling pathway. This study identified CHD1L as a potential anti-metastasis target for therapeutic intervention in breast cancer.     

Rapamycin Attenuates Splenomegaly in both Intrahepatic and Prehepatic Portal Hypertensive Rats by Blocking mTOR Signaling Pathway

Background

Spleen enlargement is often detected in patients with liver cirrhosis, but the precise pathogenetic mechanisms behind the phenomenon have not been clearly elucidated. We investigated the pathogenetic mechanisms of splenomegaly in both portal hypertensive patients and rats, and tried to identify the possible therapy for this disease.

Methods

Spleen samples were collected from portal hypertensive patients after splenectomy. Rat models of portal hypertension were induced by common bile duct ligation and partial portal vein ligation. Spleen samples from patients and rats were used to study the characteristics of splenomegaly by histological, immunohistochemical, and western blot analyses. Rapamycin or vehicle was administered to rats to determine the contribution of mTOR signaling pathway in the development of splenomegaly.

Results

We found that not only spleen congestion, but also increasing angiogenesis, fibrogenesis, inflammation and proliferation of splenic lymphoid tissue contributed to the development of splenomegaly in portal hypertensive patients and rats. Intriguingly, splenomegaly developed time-dependently in portal hypertensive rat that accompanied with progressive activation of mTOR signaling pathway. mTOR blockade by rapamycin profoundly ameliorated splenomegaly by limiting lymphocytes proliferation, angiogenesis, fibrogenesis and inflammation as well as decreasing portal pressure.

Conclusions

This study provides compelling evidence indicating that mTOR signaling activation pathway plays a key role in the pathogenesis of splenomegaly in both portal hypertensive patients and rats. Therapeutic intervention targeting mTOR could be a promising strategy for patients with portal hypertension and splenomegaly.     

HER Specific TKIs Exert Their Antineoplastic Effects on Breast Cancer Cell Lines through the Involvement of STAT5 and JNK

Background

HER-targeted tyrosine kinase inhibitors (TKIs) have demonstrated pro-apoptotic and antiproliferative effects in vitro and in vivo. The exact pathways through which TKIs exert their antineoplastic effects are, however, still not completely understood.

Methods

Using Milliplex assays, we have investigated the effects of the three panHER-TKIs lapatinib, canertinib and afatinib on signal transduction cascade activation in SKBR3, T47D and Jurkat neoplastic cell lines. The growth-inhibitory effect of blockade of HER and of JNK and STAT5 signaling was measured by proliferation- and apoptosis-assays using formazan dye labeling of viable cells, Western blotting for cleaved PARP-1 and immunolabeling for active caspase 3, respectively.

Results

All three HER-TKIs clearly inhibited proliferation and increased apoptosis in HER2 overexpressing SKBR3 cells, while their effect was less pronounced on HER2 moderately expressing T47D cells where they exerted only a weak antiproliferative and essentially no pro-apoptotic effect. Remarkably, phosphorylation/activation of JNK and STAT5A/B were inhibited by HER-TKIs only in the sensitive, but not in the resistant cells. In contrast, phosphorylation/activation of ERK/MAPK, STAT3, CREB, p70 S6 kinase, IkBa, and p38 were equally affected by HER-TKIs in both cell lines. Moreover, we demonstrated that direct pharmacological blockade of JNK and STAT5 abrogates cell growth in both HER-TKI-sensitive as well as -resistant breast cancer cells, respectively.

Conclusion

We have shown that HER-TKIs exert a HER2 expression-dependent anti-cancer effect in breast cancer cell lines. This involves blockade of JNK and STAT5A/B signaling, which have been found to be required for in vitro growth of these cell lines.     

Correlation between Activation of PI3K/AKT/mTOR Pathway and Prognosis of Breast Cancer in Chinese Women

Background

Abnormal activation of PI3K/AKT/mTOR (PAM) pathway, caused by PIK3CA mutation, KRAS mutation, PTEN loss, or AKT1 mutation, is one of the most frequent signaling abnormalities in breast carcinoma. However, distribution and frequencies of mutations in PAM pathway are unclear in breast cancer patients from the mainland of China and the correlation between these mutations and breast cancer outcome remains to be identified.

Methods

A total of 288 patients with invasive ductal breast cancer were recruited in this study. Mutations in PIK3CA (exons 4, 9 and 20), KRAS (exon 2) and AKT1 (exon 3) were detected using Sanger sequencing. PTEN loss was measured by immunohistochemistry assay. Correlations between these genetic aberrations and clinicopathological features were analyzed.

Results

The frequencies of PIK3CA mutation, KRAS mutation, AKT1 mutation and PTEN loss were 15.6%, 1.8%, 4.4% and 35.3%, respectively. However, except for PTEN loss, which was tied to estrogen receptor (ER) status, these alterations were not associated with other clinicopathological features. Survival analysis demonstrated that PIK3CA mutation, PTEN loss and PAM pathway activation were not associated with disease-free survival (DFS). Subgroup analysis of patients with ER positive tumors revealed that PIK3CA mutation more strongly reduced DFS compared to wild-type PIK3CA (76.2% vs. 54.2%; P = 0.011). PIK3CA mutation was also an independent factor for bad prognosis in ER positive patients.

Conclusions

AKT1, KRAS and PIK3CA mutations and PTEN loss all exist in women with breast cancer in the mainland China. PIK3CA mutation may contribute to the poor outcome of ER positive breast carcinomas, providing evidence for the combination of PI3K/AKT/mTOR inhibitors and endocrine therapy.     

N-3 Polyunsaturated Fatty Acids of Marine Origin and Multifocality in Human Breast Cancer

Objective

The microenvironment of breast epithelial tissue may contribute to the clinical expression of breast cancer. Breast epithelial tissue, whether healthy or tumoral, is directly in contact with fat cells, which in turn could influence tumor multifocality. In this pilot study we investigated whether the fatty acid composition of breast adipose tissue differed according to breast cancer focality.

Methods

Twenty-three consecutive women presenting with non-metastatic breast cancer underwent breast-imaging procedures including Magnetic Resonance Imaging prior to treatment. Breast adipose tissue specimens were collected during breast surgery. We established a biochemical profile of adipose tissue fatty acids by gas chromatography. We assessed whether there were differences according to breast cancer focality.

Results

We found that decreased levels in breast adipose tissue of docosahexaenoic and eicosapentaenoic acids, the two main polyunsaturated n-3 fatty acids of marine origin, were associated with multifocality.

Discussion

These differences in lipid content may contribute to mechanisms through which peritumoral adipose tissue fuels breast cancer multifocality.     

LincRNA‐p21 suppresses development of human prostate cancer through inhibition of PKM2

Objectives

Previously, we found that long intergenic non‐coding RNA‐p21 (lincRNA‐p21) inhibited the development of human prostate cancer. However, the underlying molecular mechanisms are poorly understood. Here, we attempted to investigate the downstream targets of lincRNA‐p21 in prostate cancer.

Materials and methods

Expression of lincRNA‐p21 and PKM2 was determined by qRT‐PCR and Western blot. Lentivirus expressing shPKM2 or shCtrl was used to explore the role of PKM2 on the enhanced cell proliferation and glycolysis of lincRNA‐p21‐silenced prostate cancer cells. A xenograft mouse model was performed to investigate the effect of PKM2 suppression, glycolytic or mammalian target of rapamycin (mTOR) inhibitor on the tumorigenic capacity of lincRNA‐p21‐silenced prostate cancer cells.

Results

We revealed that lincRNA‐p21 silencing in DU145 and LNCaP cells induced up‐regulation of PKM2 and activation of glycolysis, which could be reversed by PKM2 knockdown or rapamycin treatment. We also found that the proliferation and tumorigenesis of lincRNA‐p21‐silenced prostate cancer cells were significantly inhibited after knocking down PKM2. 3‐bromopyruvate (3‐Brpa) or rapamycin treatment largely decreased the tumour burden. Importantly, PKM2 expression was inversely correlated with the lincRNA‐p21 level and the survival of prostate cancer patients.

Conclusions

We demonstrated that lincRNA‐p21 blunted the prostate cancer cell proliferation and tumorigenic capacity through down‐regulation of PKM2. Therefore, targeting PKM2 or glycolysis might be a therapeutic strategy in prostate cancer patients with lowly expressed lincRNA‐p21.

     

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.     

Abnormalities of AMPK Activation and Glucose Uptake in Cultured Skeletal Muscle Cells from Individuals with Chronic Fatigue Syndrome

Background

Post exertional muscle fatigue is a key feature in Chronic Fatigue Syndrome (CFS). Abnormalities of skeletal muscle function have been identified in some but not all patients with CFS. To try to limit potential confounders that might contribute to this clinical heterogeneity, we developed a novel in vitro system that allows comparison of AMP kinase (AMPK) activation and metabolic responses to exercise in cultured skeletal muscle cells from CFS patients and control subjects.

Methods

Skeletal muscle cell cultures were established from 10 subjects with CFS and 7 age-matched controls, subjected to electrical pulse stimulation (EPS) for up to 24h and examined for changes associated with exercise.

Results

In the basal state, CFS cultures showed increased myogenin expression but decreased IL6 secretion during differentiation compared with control cultures. Control cultures subjected to 16h EPS showed a significant increase in both AMPK phosphorylation and glucose uptake compared with unstimulated cells. In contrast, CFS cultures showed no increase in AMPK phosphorylation or glucose uptake after 16h EPS. However, glucose uptake remained responsive to insulin in the CFS cells pointing to an exercise-related defect. IL6 secretion in response to EPS was significantly reduced in CFS compared with control cultures at all time points measured.

Conclusion

EPS is an effective model for eliciting muscle contraction and the metabolic changes associated with exercise in cultured skeletal muscle cells. We found four main differences in cultured skeletal muscle cells from subjects with CFS; increased myogenin expression in the basal state, impaired activation of AMPK, impaired stimulation of glucose uptake and diminished release of IL6. The retention of these differences in cultured muscle cells from CFS subjects points to a genetic/epigenetic mechanism, and provides a system to identify novel therapeutic targets.     

Cancer Stage,Comorbidity, and Socioeconomic Differences in the Effect of Cancer on Labour Market Participation: A Danish Register-Based Follow-Up Study

Purpose

Socioeconomic inequality in return to work after cancer treatment and rehabilitation have been documented, but less is known about its causes. This paper investigates the role played by breast cancer stage at diagnosis and comorbidity.

Methods

We used the comprehensive Danish Cancer Registry to follow 7372 women aged 30-60, who were in the labour force when diagnosed with breast cancer in 2000-06 and survived at least three years. Controls were 213,276 women without breast cancer. Inequalities in employment outlook were estimated as interaction effects in linear regression between educational attainment and disease on employment.

Results

There is significant interaction between education and breast cancer, but it is only marginally affected by including stage and comorbidity in the regression models. Education, breast cancer stage, and comorbidity all have strong effects on later employment, and a considerable amount of the educational effect is mediated by comorbidity and pre-cancer labour market participation and income.

Conclusion

The result of the study is negative in the sense that the stronger effect of breast cancer on employment among low-educated compared to highly educated individuals is not explained by cancer stage or comorbidity. The fact that comorbidity has little impact on inequality may be due to a different social patterning of most comorbidity compared to breast cancer.     

“Double hit” makes the difference

Comment on: Menendez JA, et al. Cell Cycle 2012; 11:2782–92.Metformin (N’, N’-dimethylbiguanide) is an anti-diabetic drug prescribed to more than 100 million patients in the world. In addition to its efficacy for the treatment of diabetes, several recent studies have shown that it has anti-tumoral properties.¹ We and others have shown that metformin targets cancer cell metabolism by inhibiting mitochondrial complex 1 activity.^2,3 This energetic stress leads to a decrease of intracellular ATP concentration, and cancer cells will increase their rate of glycolysis.² This compensatory response is not sufficient to restore ATP levels, but is adequate to maintain viable cells in most of the cancer cells. Indeed, metformin blocks cell growth but can also induce apoptosis in some cancer cell models.⁴ The increase of glycolysis induced by metformin is somehow inconsistent with the observed inhibition of proliferation, since cancer cells use preferentially glycolysis to grow faster. This switch to glycolysis, also known as the “Warburg effect,” is linked to oncogenic transformation⁵ and is accompanied by the hyperactivation of the mTOR pathway. In cancer cells, the increase of glycolysis induced by metformin is associated with a strong inhibition of the mTOR pathway via the AMPK. This new metabolic order established by metformin may explain the paradoxical effect of metformin. In view of the above scenario, Menendez et al. decided to test the synthetic lethality of metformin and combined metformin treatment with glucose starvation. They showed that the treatment of breast cancer cells with metformin alone does not induce apoptosis but arrests cells in G₀/G₁. Glucose starvation by itself induces few apoptosis, but the combination of metformin with the absence of glucose induces massive apoptosis. This is not altogether surprising, since the dual action of metformin and glucose starvation block the two main ways of production of ATP (i.e., mitochondrial respiration and glycolysis) (Fig. 1). This is an interesting observation, which could be valuable for future anticancer therapy; however, glucose starvation is not therapeutically feasible. Thus, the use 2-deoxyglucose (2-DG), an inhibitor of glycolysis, could be useful. We and others found that the combination of 2-DG and metformin inhibits prostate cancer cell proliferation and breast tumor growth in xenograft models.^2,6 Although it induces a slight apoptotic response in vitro, 2-DG alone is not efficient in vivo to alter tumor growth⁶ but improves the curative action of radiotherapy;⁷ similarly, it reinforces metformin action. Another interesting issue raised by Menendez et al. is the use of such dual therapy to target cancer stem cells. Metformin has been shown to selectively kill cancer stem cells and the chemotherapy-resistant subpopulation of cancer stem cells.^8,9 Cancer stem cells greatly depend on aerobic glycolysis to sustain their stemness and immortality. The synthetic lethality induced by metformin and glucose starvation may help to improve chemotherapy action and avoid cancer relapse. In conclusion, targeting cancer cell metabolism with a “dual hit therapy” opens new avenues for the future treatment of cancer.Open in a separate windowFigure 1. The combination of metformin and glucose starvation induces a strong energetic stress. Metformin inhibits the mitochondrial complex 1 and glucose starvation, or 2-DG inhibits ATP production from glycolysis. The combination of the two energetic stresses induces a massive energetic stress and leads to a strong apoptotic response.     

AMPK Activation by A-769662 Controls IL-6 Expression in Inflammatory Arthritis

Objective

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. It is a central regulator of both lipid and glucose metabolism. Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects. In this study, we assessed whether targeted activation of AMPK inhibits inflammatory arthritis in vivo.

Methods

We tested the effect of A-769662, a specific AMPK agonist (60mg/kg/bid) in mouse models of antigen-induced arthritis (AIA) and passive K/BxN serum-induced arthritis. The passive K/BxN serum-induced arthritis model was also applied to AMPKα1-deficient mice. Joints were harvested and subjected to histological analysis. IL-6 expression was measured in both joint tissues and sera by ELISA. The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.

Results

AMPK activation by A-769662 reduced inflammatory infiltration and joint damage in both mouse models. IL-6 expression in serum and arthritic joints was significantly decreased in A-769662-treated mice. AMPKα1 deficient mice mildly elicited an increase of clinical arthritis. IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

Conclusions

AMPK activation by specific AMPK agonist A-769662 suppressed inflammatory arthritis in mice as well as IL-6 expression in serum and arthritic joints. These data suggest that targeted activation of AMPK has a potential to be an effective therapeutic strategy for IL-6 dependent inflammatory arthritis.     

Propofol alleviates hypoxia-induced nerve injury in PC-12 cells by up-regulation of microRNA-153

Background

Although the neuroprotective role of propofol has been identified recently, the regulatory mechanism associated with microRNAs (miRNAs/miRs) in neuronal cells remains to be poorly understood. We aimed to explore the regulatory mechanism of propofol in hypoxia-injured rat pheochromocytoma (PC-12) cells.

Methods

PC-12 cells were exposed to hypoxia, and cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry assay/Western blot analysis, respectively. Effects of propofol on hypoxia-injured cells were measured, and the expression of miR-153 was determined by stem-loop RT-PCR. After that, whether propofol affected PC-12 cells under hypoxia via miR-153 was verified, and the downstream protein of miR-153 as well as the involved signaling cascade was finally explored.

Results

Hypoxia-induced decrease of cell viability and increase of apoptosis were attenuated by propofol. Then, we found hypoxia exposure up-regulated miR-153 expression, and the level of miR-153 was further elevated by propofol in hypoxia-injured PC-12 cells. Following experiments showed miR-153 inhibition reversed the effects of propofol on hypoxia-treated PC-12 cells. Afterwards, we found BTG3 expression was negatively regulated by miR-153 expression, and BTG3 overexpression inhibited the mTOR pathway and AMPK activation. Besides, hypoxia inhibited the mTOR pathway and AMPK, and these inhibitory effects could be attenuated by propofol.

Conclusion

Propofol protected hypoxia-injured PC-12 cells through miR-153-mediataed down-regulation of BTG3. BTG3 could inhibit the mTOR pathway and AMPK activation.

     

Lovastatin Induces Multiple Stress Pathways Including LKB1/AMPK Activation That Regulate Its Cytotoxic Effects in Squamous Cell Carcinoma Cells

Background

Cellular stress responses trigger signaling cascades that inhibit proliferation and protein translation to help alleviate the stress or if the stress cannot be overcome induce apoptosis. In recent studies, we demonstrated the ability of lovastatin, an inhibitor of mevalonate synthesis, to induce the Integrated Stress Response as well as inhibiting epidermal growth factor receptor (EGFR) activation.

Methodology/Principal Findings

In this study, we evaluated the effects of lovastatin on the activity of the LKB1/AMPK pathway that is activated upon cellular energy shortage and can interact with the above pathways. In the squamous cell carcinoma (SCC) cell lines SCC9 and SCC25, lovastatin treatment (1–25 µM, 24 hrs) induced LKB1 and AMPK activation similar to metformin (1–10 mM, 24 hrs), a known inducer of this pathway. Lovastatin treatment impaired mitochondrial function and also decreased cellular ADP/ATP ratios, common triggers of LKB1/AMPK activation. The cytotoxic effects of lovastatin were attenuated in LKB1 null MEFs indicating a role for this pathway in regulating lovastatin-induced cytotoxicity. Of clinical relevance, lovastatin induces synergistic cytotoxicity in combination with the EGFR inhibitor gefitinib. In LKB1 deficient (A549, HeLa) and expressing (SCC9, SCC25) cell lines, metformin enhanced gefitinib cytotoxicity only in LKB1 expressing cell lines while both groups showed synergistic cytotoxic effects with lovastatin treatments. Furthermore, the combination of lovastatin with gefitinib induced a potent apoptotic response without significant induction of autophagy that is often induced during metabolic stress inhibiting cell death.

Conclusion/Significance

Thus, targeting multiple metabolic stress pathways including the LKB1/AMPK pathway enhances lovastatin’s ability to synergize with gefitinib in SCC cells.