Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions

Introduction

Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines.

Material and Methods

MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells.

Results

Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation.

Conclusion

We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.     

Hormesis in Cholestatic Liver Disease; Preconditioning with Low Bile Acid Concentrations Protects against Bile Acid-Induced Toxicity

Introduction

Cholestasis is characterized by accumulation of bile acids and inflammation, causing hepatocellular damage. Still, liver damage markers are highest in acute cholestasis and drop when this condition becomes chronic, indicating that hepatocytes adapt towards the hostile environment. This may be explained by a hormetic response in hepatocytes that limits cell death during cholestasis.

Aim

To investigate the mechanisms that underlie the hormetic response that protect hepatocytes against experimental cholestatic conditions.

Methods

HepG2.rNtcp cells were preconditioned (24 h) with sub-apoptotic concentrations (0.1–50 μM) of various bile acids, the superoxide donor menadione, TNF-α or the Farsenoid X Receptor agonist GW4064, followed by a challenge with the apoptosis-inducing bile acid glycochenodeoxycholic acid (GCDCA; 200 μM for 4 h), menadione (50 μM, 6 h) or cytokine mixture (CM; 6 h). Levels of apoptotic and necrotic cell death, mRNA expression of the bile salt export pump (ABCB11) and bile acid sensors, as well as intracellular GCDCA levels were analyzed.

Results

Preconditioning with the pro-apoptotic bile acids GCDCA, taurocholic acid, or the protective bile acids (tauro)ursodeoxycholic acid reduced GCDCA-induced caspase-3/7 activity in HepG2.rNtcp cells. Bile acid preconditioning did not induce significant levels of necrosis in GCDCA-challenged HepG2.rNtcp cells. In contrast, preconditioning with cholic acid, menadione or TNF-α potentiated GCDCA-induced apoptosis. GCDCA preconditioning specifically reduced GCDCA-induced cell death and not CM- or menadione-induced apoptosis. The hormetic effect of GCDCA preconditioning was concentration- and time-dependent. GCDCA-, CDCA- and GW4064- preconditioning enhanced ABCB11 mRNA levels, but in contrast to the bile acids, GW4064 did not significantly reduce GCDCA-induced caspase-3/7 activity. The GCDCA challenge strongly increased intracellular levels of this bile acid, which was not lowered by GCDCA-preconditioning.

Conclusions

Sub-toxic concentrations of bile acids in the range that occur under normal physiological conditions protect HepG2.rNtcp cells against GCDCA-induced apoptosis, which is independent of FXR-controlled changes in bile acid transport.     

Hyperreactivity of Blood Leukocytes in Patients with NAFLD to Ex Vivo Lipopolysaccharide Treatment Is Modulated by Metformin and Phosphatidylcholine but Not by Alpha Ketoglutarate

Introduction and Aims

Toll-like receptor 4 and proinflammatory cytokines play a central role in the progression of nonalcoholic fatty liver disease. We investigated IL-1, IL-6 and TNFα production and toll-like receptor 4 in both—obese and lean patients with non-alcoholic fatty liver disease who met different sets of metabolic syndrome criteria and linked the results with the disease burden.

Materials and Methods

95 subjects were divided into four groups depending on the following criteria: presence or absence of metabolic syndrome and/or non-alcoholic fatty liver disease, glucose tolerance (prediabetes or normoglycemia) and BMI value (obese or lean). We determined the levels of IL-1β, IL-6, TNFα, and monocyte toll-like receptor 4 expression in fresh blood as well as in blood cultures treated with lipopolysaccharide with or without metformin, alphaketoglutarate or phosphatidylcholine supplementation.

Results

The blood leukocytes of patients with non-alcoholic fatty liver disease are hypersensitive to lipopolysaccharide treatment and produce elevated levels of pro-inflammatory cytokines in response to ex vivo treatment with lipopolysaccharide. Moreover, they overexpress toll-like receptor-4. Hyperreactivity was typical mainly for obese patients with non-alcoholic fatty liver disease together with metabolic syndrome and decreased with the severity of disease. Metformin was the most effective in attenuation of hyperreactivity in all groups of patients with non-alcoholic fatty liver disease, but in obese patients the effectiveness of metformin was weaker than in lean. The reduction of cytokine level by metformin was accompanied by the decrease in toll-like receptor-4 expression. phosphatidylcholine also attenuated hyperreactivity to lipopolysaccharide but mainly in obese patients. Alpha ketoglutarate did not modulate cytokines’ level and toll-like receptor 4 expression in non-alcoholic fatty liver disease patients.

Conclusions

Metformin and phosphatidylcholine attenuated lipopolysaccharide induced toll-like receptor 4 overexpression and overproduction of pro-inflammatory cytokines; however, their efficacy depended on combined presence of non-alcoholic fatty liver disease, metabolic syndrome and obesity.     

Zerumbone suppresses IKKα, Akt,and FOXO1 activation,resulting in apoptosis of GBM 8401 cells

Background

Zerumbone, a sesquiterpene compound isolated from subtropical ginger, Zingiber zerumbet Smith, has been documented to exert antitumoral and anti- inflammatory activities. In this study, we demonstrate that zerumbone induces apoptosis in human glioblastoma multiforme (GBM8401) cells and investigate the apoptotic mechanism.

Methods

We added a caspase inhibitor and transfected wild-type (WT) IKK and Akt into GBM 8401 cells, and measured cell viability and apoptosis by MTT assay and flow cytometry. By western blotting, we evaluated activation of caspase-3, dephosphorylation of IKK, Akt, FOXO1 with time, and change of IKK, Akt, and FOXO1 phosphorylation after transfection of WT IKK and Akt.

Results

Zerumbone (10∽50 μM) induced death of GBM8401 cells in a dose-dependent manner. Flow cytometry studies showed that zerumbone increased the percentage of apoptotic GBM cells. Zerumbone also caused caspase-3 activation and poly (ADP-ribose) polymerase (PARP) production. N-benzyloxycarbonyl -Val-Ala-Asp- fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, hindered zerumbone-induced cell death. Transfection of GBM 8401 cells with WT IKKα inhibited zerumbone-induced apoptosis, and zerumbone significantly decreased IKKα phosphorylation levels in a time-dependent manner. Similarly, transfection of GBM8401 cells with Akt suppressed zerumbone-induced apoptosis, and zerumbone also diminished Akt phosphorylation levels remarkably and time-dependently. Moreover, transfection of GBM8401 cells with WT IKKα reduced the zerumbone-induced decrease in Akt and FOXO1 phosphorylation. However, transfection with WT Akt decreased FOXO1, but not IKKα, phosphorylation.

Conclusion

The results suggest that inactivation of IKKα, followed by Akt and FOXO1 phosphorylation and caspase-3 activation, contributes to zerumbone-induced GBM cell apoptosis.     

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 Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background

Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model.

Methodology/Principal Findings

Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH.

Conclusions/Significance

These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.     

Prenatal Metformin Exposure in Mice Programs the Metabolic Phenotype of the Offspring during a High Fat Diet at Adulthood

Aims

The antidiabetic drug metformin is currently used prior and during pregnancy for polycystic ovary syndrome, as well as during gestational diabetes mellitus. We investigated the effects of prenatal metformin exposure on the metabolic phenotype of the offspring during adulthood in mice.

Methods

Metformin (300 mg/kg) or vehicle was administered orally to dams on regular diet from the embryonic day E0.5 to E17.5. Gene expression profiles in liver and brain were analysed from 4-day old offspring by microarray. Body weight development and several metabolic parameters of offspring were monitored both during regular diet (RD-phase) and high fat diet (HFD-phase). At the end of the study, two doses of metformin or vehicle were given acutely to mice at the age of 20 weeks, and Insig-1 and GLUT4 mRNA expressions in liver and fat tissue were analysed using qRT-PCR.

Results

Metformin exposed fetuses were lighter at E18.5. There was no effect of metformin on the maternal body weight development or food intake. Metformin exposed offspring gained more body weight and mesenteric fat during the HFD-phase. The male offspring also had impaired glucose tolerance and elevated fasting glucose during the HFD-phase. Moreover, the expression of GLUT4 mRNA was down-regulated in epididymal fat in male offspring prenatally exposed to metformin. Based on the microarray and subsequent qRT-PCR analyses, the expression of Insig-1 was changed in the liver of neonatal mice exposed to metformin prenatally. Furthermore, metformin up-regulated the expression of Insig-1 later in development. Gene set enrichment analysis based on preliminary microarray data identified several differentially enriched pathways both in control and metformin exposed mice.

Conclusions

The present study shows that prenatal metformin exposure causes long-term programming effects on the metabolic phenotype during high fat diet in mice. This should be taken into consideration when using metformin as a therapeutic agent during pregnancy.     

Persistent Inflammation and Endothelial Activation in HIV-1 Infected Patients after 12 Years of Antiretroviral Therapy

Objective

The study investigated markers of inflammation and endothelial activation in HIV infected patients after 12 years of successful combination antiretroviral treatment (cART).

Methods

Inflammation and endothelial activation were assessed by measuring levels of immunoglobulins, β2-microglobulin, interleukin (IL) 8, tumor necrosis factor α (TNFα), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), sE-Selectin, and sP-Selectin.

Results

HIV infected patients had higher levels of β2-microglobulin, IL-8, TNFα, and sICAM-1 than uninfected controls, and HIV infected patients lacked correlation between platelet counts and sP-Selectin levels found in uninfected controls.

Conclusion

Discrete signs of systemic and vascular inflammation persist even after very long term cART.     

Metformin Changes the Relationship between Blood Monocyte Toll-Like Receptor 4 Levels and Nonalcoholic Fatty Liver Disease—Ex Vivo Studies

Background

Toll-like receptor 4 (TLR4) contributes to the development of NAFLD (nonalcoholic fatty liver disease) and MetS (metabolic syndrome). It is unclear whether anti-diabetic metformin affects TLR4 expression on blood monocytes, thereby protecting or improving inflammatory parameters. Therefore, we investigated TLR4 in patients with NAFLD meeting different sets of MetS criteria and linked the results with the disease burden.

Methods

70 subjects were characterized and divided into three groups: (I) healthy individuals, (II) nonobese with NAFLD and without MetS, and (III) prediabetic, obese with NAFLD and MetS. We determined the concentrations of IL-1β, IL-6, TNFα, and monocyte TLR4 levels in fresh blood as well as in blood cultures with or without metformin supplementation.

Results

The characteristics of the study groups revealed a significant association between NAFLD and BMI, MetS and inflammatory parameters, and TLR4. In ex vivo studies, 100 μM of metformin decreased the TLR4 level by 19.9% (II group) or by 35% (III group) as well as IL-1β and TNFα production. A stepwise multiple regression analysis highlighted a strong effect of metformin on attenuation of the link between TLR4 and NAFLD, and TNFα.

Conclusion

We concluded that, by attenuation of the blood monocyte TLR4 level, metformin reduced their inflammatory potential—critical after recruitment these cells into liver. However, this finding should be confirmed after in vivo metformin administration.     

Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes

Introduction

AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.

Methods

We assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.

Results

Knockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.

Conclusion

LKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.     

Pyrrolidinyl caffeamide against ischemia/reperfusion injury in cardiomyocytes through AMPK/AKT pathways

Background

Coronary heart disease is a leading cause of death in the world and therapy to reduce injury is still needed. The uncoupling of glycolysis and glucose oxidation induces lactate accumulation during myocardial ischemia/reperfusion (I/R) injury. Cell death occurs and finally leads to myocardial infarction. Caffeic acid, one of the major phenolic constituents in nature, acts as an antioxidant. Pyrrolidinyl caffeamide (PLCA), a new derivative of caffeic acid, was synthesized by our team. We aimed to investigate the effect of PLCA on hypoxia/reoxygenation (H/R) in neonatal rat ventricular myocytes (NRVM) and on myocardial I/R in rats.

Results

Cardiomyocytes were isolated and subjected to 6 h hypoxia followed by 18 h reperfusion. PLCA (0.1 to 3 μM) and metformin (30 μM) were added before hypoxia was initiated. PLCA at 1 μM and metformin at 30 μM exerted similar effects on the improvement of cell viability and the alleviation of cell apoptosis in NRVM after H/R. PLCA promoted p-AMPK, p-AKT, and GLUT4 upregulation to induce a cardioprotective effect in both cell and animal model. The accumulation of cardiac lactate was attenuated by PLCA during myocardial I/R, and infarct size was smaller in rats treated with PLCA (1 mg/kg) than in those treated with caffeic acid (1 mg/kg).

Conclusions

AMPK and AKT are synergistically activated by PLCA, which lead facilities glucose utilization, thereby attenuating lactate accumulation and cell death. The cardioprotective dose of PLCA was lower than those of metformin and caffeic acid. We provide a new insight into this potential drug for the treatment of myocardial I/R injury.     

Akt regulates IL-10 mediated suppression of TNFα-induced cardiomyocyte apoptosis by upregulating Stat3 phosphorylation

Background

We have already reported that TNF-α increases cardiomyocyte apoptosis and IL-10 treatment prevented these effects of TNF-α. Present study investigates the role of Akt and Jak/Stat pathway in the IL-10 modulation of TNF-α induced cardiomyocyte apoptosis.

Methodology/Principal findings

Cardiomyocytes isolated from adult Sprague Dawley rats were exposed to TNF-α (10 ng/ml), IL-10 (10 ng/ml) and TNF-α+IL-10 (ratio 1) for 4 h. Exposure to TNF-α resulted in an increase in cardiomyocyte apoptosis as measured by flow cytometry and TUNEL assay. IL-10 by itself had no effect, but it prevented TNF-α induced apoptosis. IL-10 treatment increased Akt levels within cardiomyocytes and this change was associated with an increase in Jak1 and Stat3 phosphorylation. Pre-exposure of cells to Akt inhibitor prevented IL-10 induced Stat3 phosphorylation. Furthermore, in the presence of Akt or Stat3 inhibitor, IL-10 treatment was unable to block TNF-α induced cardiomyocyte apoptosis.

Conclusion

It is suggested that IL-10 modulation of TNF-α induced cardiomyocyte apoptosis is mediated by Akt via Stat3 activation.     

The Vitamin D Receptor Agonist BXL-01-0029 as a Potential New Pharmacological Tool for the Treatment of Inflammatory Myopathies

Objective

This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects.

Methods

Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC₅₀ determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis.

Results

BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls.

Conclusions

Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.     

Opposing Functions of Akt Isoforms in Lung Tumor Initiation and Progression

Background

The phosphatidylinositol 3-kinase–regulated protein kinase, Akt, plays an important role in the initiation and progression of human cancer. Mammalian cells express three Akt isoforms (Akt1–3), which are encoded by distinct genes. Despite sharing a high degree of amino acid identity, phenotypes observed in knockout mice suggest that Akt isoforms are not functionally redundant. The relative contributions of the different Akt isoforms to oncogenesis, and the effect of their deficiencies on tumor development, are not well understood.

Methods

Here we demonstrate that Akt isoforms have non-overlapping and sometimes opposing functions in tumor initiation and progression using a viral oncogene-induced mouse model of lung cancer and Akt isoform-specific knockout mice.

Results

Akt1 ablation significantly delays initiation of lung tumor growth, whereas Akt2 deficiency dramatically accelerates tumorigenesis in this mouse model. Ablation of Akt3 had a small, not statistically significant, stimulatory effect on tumor induction and growth by the viral oncogene. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and Ki67 immunostaining of lung tissue sections revealed that the delayed tumor induction in Akt1^−/− mice was due to the inhibitory effects of Akt1 ablation on cell growth and survival. Conversely, the accelerated growth rate of lung tumors in Akt2^−/− and Akt3^−/− mice was due to increased cell proliferation and reduced tumor cell apoptosis. Investigation of Akt signaling in tumors from Akt knockout mice revealed that the lack of Akt1 interrupted the propagation of signaling in tumors to the critical downstream targets, GSK-3α/β and mTOR.

Conclusions

These results demonstrate that the degree of functional redundancy between Akt isoforms in the context of lung tumor initiation is minimal. Given that this mouse model exhibits considerable similarities to human lung cancer, these findings have important implications for the design and use of Akt inhibitors for the treatment of lung cancer.     

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.