A Metabolic Inhibitory Cocktail for Grave Cancers: Metformin, Pioglitazone and Lithium Combination in Treatment of Pancreatic Cancer and Glioblastoma Multiforme

Biochem Genet DOI 10.1007/s10528-016-9754-9

REVIEW

1,2 3 I˙lhan Elmaci • Meric A Altinoz

Received: 1 March 2016 / Accepted: 23 June 2016 Ó Springer Science+Business Media New York 2016

Abstract Pancreatic cancer (PC) and glioblastoma multiforme (GBM) are among the human cancers with worst prognosis which require an urgent need for efficient therapies. Here, we propose to apply to treat both malignancies with a triple combination of drugs, which are already in use for different indications. Recent studies demonstrated a considerable link between risk of PC and diabetes. In experimental models, anti-diabetogenic agents suppress growth of PC, including metformin (M), pioglitazone (P) and lithium (L). L is used in psychiatric practice, yet also bears anti-diabetic potential and selectively inhibits glycogen synthase kinase-3 beta (GSK-3b). M, a biguanide class anti-diabetic agent shows anticancer activity via activating AMP-activated protein kinase (AMPK). Glitazones bind to PPAR-c and inhibit NF-jB, triggering cell proliferation, apoptosis resistance and synthesis of inflammatory cytokines in cancer cells. Inhibition of inflammatory cytokines could simultaneously decrease tumor growth and alleviate cancer cachexia, having a major role in PC mortality. Furthermore, mutual synergistic interactions exist between PPAR-c and GSK-3b, between AMPK and GSK-3b and between AMPK and PPAR-c. In GBM, M blocks angiogenesis and migration in experimental models. Very noteworthy, among GBM patients with type 2 diabetes, usage of M significantly correlates with better survival while reverse is true for sulfonylureas. In experimental models, P synergies with ligands of RAR, RXR and statins in reducing growth of GBM. Further, usage of P was found to be lesser in anaplastic astrocytoma and GBM patients, indicating a protective effect of P against high-grade gliomas. L is accumulated in GBM cells faster and higher than in

& Meric A Altinoz [email protected]

1 Department of Neurosurgery, Memorial Hospital, Istanbul, Turkey 2 Neuroacademy Group, Istanbul, Turkey 3 Department of Immunology, Experimental Medicine Research Center, Istanbul, Turkey 123 Biochem Genet neuroblastoma cells, and its levels further increase with chronic exposure. Recent studies revealed anti-invasive potential of L in GBM cell lines. Here, we propose that a triple-agent regime including drugs already in clinical usage may provide a metabolic adjuvant therapy for PC and GBM.

Keywords Pancreas cancer Á AMPK Á PPAR gamma Á GSK-3b Á Metformin Á Pioglitazone Á Lithium

Introduction: Pancreatic Cancer as a Grave Malignancy with Very Dismal Prognosis

Pancreatic cancer (PC) is a very fatal malignancy due to high rate of advanced stage at disease presentation and lack of an efﬁcient treatment (Ko and Tempero 2009). Its incidence increased over past decades such that over 265,000 people worldwide are diagnosed each year (Jemal et al. 2011). Only 10–15 % of patients are manageable with surgery and more than 95 % of all patients will die within 2 years of diagnosis (Edwards et al. 2005). Due to prominently high mortality rates, PC constitutes the fourth leading cause of cancer deaths in the USA (Jemal et al. 2011). The recent increases in the prevalence of type 2 diabetes mellitus (T2DM) were proposed to contribute to the increase of PC. Roughly half of all PC patients also suffer DM at diagnosis, and roughly half of the DM that is present at the manifestation of PC is of new onset, which developed over 2–3 years preceding PC diagnosis. Thus, this new-onset DM is called as secondary DM (sDM), which will be explained below.

Link Between DM and PC and Epidemiological Studies Showing that Anti-diabetic Drugs Modify Risk of PC

A meta-analysis of three investigations in which 2192 PC patients were compared with 5113 controls revealed a 1.8-fold increase in risk of PC associated with T2DM, although many of the patients classiﬁed as T2DM likely suffered sDM, which manifests close to the PC diagnosis. A meta-analysis encompassing three cohort and six case–control studies found that the RR (relative risk) for PC was two times higher in T1DM patients and ‘‘young-onset’’ diabetics in comparison with nondiabetics (Stevens et al. 2007). Secondary DM emerged as an important group of the total DM population and exerts the highest risk of PC, especially in patients with chronic pancreatitis (CP) (Cui and Andersen 2011). Persons with any form of CP have an elevated risk of PC, which is cumulative over the CP course, such that 4–5 % of patients develop PC over 20 years, a risk which is tenfold to 20-fold greater than the general population. A cohort study revealed that patients with DM and CP coccurence had a very higher risk of PC (HRZ 33.52), compared with healthy subjects (Liao et al. 2012). Although a real estimation of the mortality rates attributable to cancer in DM is complicated due to cardiac disease, basic and clinical data indicate that insulin treatment causes an additional risk for cancer, likely via insulin-like growth factor 1 receptor (IGF-1) signals (Azar and Lyons 2010).

123 Biochem Genet

Physiological insulin concentrations augment PC cell proliferation and glucose utilization via MAP kinase, PI 3-kinase (PI3K) and GLUT-1 (Ding et al. 2000). Many cohort and case–control studies demonstrated that 25–50 % of patients with PC developed DM within 1–3 years before diagnosis (Huxley et al. 2005; Chari et al. 2008). This indicates that recent-onset DM concomitant with PC can be caused by an occult stage of malignancy and denotes that DM is a biomarker of early-stage PC. In addition to the frequent development of DM just before PC diagnosis, the proposal that PC is a cause of new-onset DM is strengthened by several studies. Patients with preneoplastic pancreatic lesions and a family history of PC commonly suffer concomitant DM (Brentnall et al. 1999). Moreover, recent-onset DM accompanying PC could regress following complete tumor removal (Permert et al. 1993; Fogar et al. 1994). In basic studies, PC cell lines induce hyperglycemia in SCID mice and a PC-originated S-100A8 N-terminal peptide is determined as a diabetogenic mediator (Basso et al. 2006). In carcinogen-induced PC, hyperinsulinemia and aberrant insulin secretion patterns are witnessed (Ahren and Andren- Sandberg 1993; Lochhead et al. 2001), which evidence that insulin resistance is a preceeding feature in PC-associated DM. sDM has diverging features from T2DM, yet strategies are necessary for successful discrimination of sDM from the more common T2DM. These high-risk sDM patients could benefit from high-resolution imaging approaches, which would determine suspicious pancreatic parenchymal lesions. A retrospective cohort study of 2122 diabetic patients revealed that PC emerges within 3 years after DM diagnosis in 1 % of the patients older than 50 years (Chari et al. 2005). In another study, PC diagnosis peaked within 3 months after onset of DM (Johnson et al. 2011), while others showed that the interval of DM before diagnosis of PC by primary care providers averaged 6.5 months (Aggarwal et al. 2012). Compared with the control group, PC patients were older, lost more weight, had lower body mass index—BMI and a higher family history of PC. Thus, it is commented that sDM concurrent with PC could be distinguished from the new- onset T2DM based on clinical features, such as lack of a family history for DM, age 65 years or older, recent weight loss of [2 kg or a premorbid or normal BMI \25 kg/m2. sDM triggered by cystic fibrosis, CP, PC or pancreatic resection is generally determined by a lack of the nutrient-triggered secretion of pancreatic polypeptide (PP) and a decreased hepatic insulin sensitivity (Slezak and Andersen 2001; Cui and Andersen 2011). On the other hand, T2DM generally associates with an increase in basal and nutrient-induced release of pancreatic polypeptide (Glaser et al. 1988). The three most frequent subtypes of DM differentiated prominently in metabolic and endocrine features (Cui and Andersen 2011). T1DM associates with a complete deficiency of insulin release and with an absolute requirement for exogenous insulin. Increases in blood sugar and hyperinsulinemia are concomitant in T2DM due to insulin resistance in peripheral tissues mostly linked with obesity. Secondary DM/sDM is linked to benign and malignant lesions of the exocrine pancreas, including acute and chronic pancreatitis, pancreatic trauma and resection, cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy and pancreatic agenesis 123 Biochem Genet and is defined by a serious absence of all pancreatic glucose-regulating hormones (Cui and Andersen 2011). Moreover, PC risk seems to correlate negatively with the interval of DM, with the highest risk of PC detected among patients with DM diagnosed within 1 year. This indicates that many of the diabetic patients suffer from PC-induced DM (sDM), but this subgroup of DM was not noticed in a majority of epidemiological studies. First in 2009, the results of a hospital-based case–control study performed at M. D. Anderson Cancer Center were declared (Li et al. 2009). The study investigated 973 PC patients from 2004 to 2008 (including 259 diabetics) and 863 controls (including 109 diabetics) (Li et al. 2009). The rates of consumption of insulin, insulin-secreting agents, metformin, and other anti-diabetics were determined. Diabetics were compared between cases and controls. The risk of PC was predicted using unconditional logistic regression analysis. Diabetics who consumed metformin had a 62 % lower risk of PC in comparison with those who had not taken metformin, with adjustments for confounders (Li et al. 2009). Furthermore, this difference sustained its significance when the analysis was limited to patients with an interval of diabetes [2 years or those who never used insulin (Li et al. 2009). Very noteworthy, using never users as reference, ever users of insulin or insulin- secreting agents had 4.99-fold and 2.52-fold increased risks for PC (p \ 0.001 and p = 0.005, respectively). When the analysis was limited to patients who were never treated with insulin, the risk association increased for ever users of insulin secretagogues and decreased for ever users of metformin (Li et al. 2009). Another study determined the risk of cancer in T2DM and its modification with anti-hyperglycemic agents (Currie et al. 2009). A retrospective cohort study was performed on people treated in UK general practices, which analyzed patients who developed diabetes [40 years of age, and started treatment with oral anti-diabetics or insulin after 2000. A total of 62,809 patients were divided into four groups according to whether they were treated with metformin only, sulfonylurea, metformin plus sulfonylurea or insulin (Currie et al. 2009). The outcome measures were defined as development of any solid cancer, or cancer of the breast, colon, pancreas or prostate. Metformin monotherapy had the lowest risk of cancer, and moreover, addition of metformin to insulin treatment decreased insulin-associated increase of cancer. Metformin treatment was linked to decreased risk of colon or pancreas cancer, but it did not modify risk of breast or prostate cancer (Currie et al. 2009). Decensi et al. (2010) reported results of a meta-analysis analyzing the effects of metformin on cancer incidence and mortality in diabetics. Eleven studies were pooled for admissibility, reporting 4042 cancers and 529 cancer deaths. A 31 % decrease in overall total relative risk was witnessed in patients who were treated with metformin in comparison with other anti-diabetics. The opposite association reached significance for PC and hepatocarcinoma and was insignificant for cancers of colon, breast and prostate. A trend to a dose–response relationship was also found (Decensi et al. 2010). A retrospective cohort study was reported, which analyzed the survival benefit of metformin in patients with diabetes and PC (Hsu and Saif 2011). The data were collected from the MD Anderson Cancer Center (MDACC) from 2000 to 2009, with additional cases selected from tumor board registry at MDACC. A total of 302 123 Biochem Genet patients were analyzed. It was reported that the median survival was longer in metformin users when compared to nonusers: 16.6 versus 11.5 months (p = 0.0044). A 33 % decrease risk of death also occurred in patients who used metformin compared to those who did not. A meta-analysis was conducted on observational studies which determined the risk of all cancers and specific cancers in association with use of metformin and/or sulfonylureas among T2DM patients (Soranna et al. 2012). Seventeen studies fulfilling inclusion criteria and including 37,632 cancers were accounted after reviewing 401 citations. Metformin usage was associated with significantly decreased risk ratio (RR) of all cancers (summary RR 0.61), especially of PC (0.38, 95 % CI 0.14–0.91). The association between T2DM, glucose-lowering agents (monotherapy with either metformin, sulfonylurea or insulin) and cancer risk in Taiwan was assessed (Hsieh et al. 2012). Using Taiwan’s National Health Research Institutes database of 1,000,000 random subjects from 2000 to 2008, 61,777 patients with T2DM (age C 20 years) were detected and 677,378 enrollees with no record of diabetes. After adjusting for age and sex, it was

Table 1 Metformin and/or AMPK modiﬁcation of cellular proteins in malignant cells Cell cycle and Metabolic Cellular growth, Treatment sensitivity and Cell signaling apoptosis, pathways cancer stem cell related molecules autophagy population

P53: GSK-3b;* EGFR; Radiation sensitivity: pMAPK; Waf1/P21: PGC-1a: Wnt/b-catenin: Gemcitabine sensitivity: mTORC1; P27: Warburg (via decrease of Glutathione; Raptor DVL3) PARP Effect; 5-FHTF: phosphorylation: CD133; cleavage: Acetyl-CoA- Hypoxic response- TCC-2/tuberin Erk activity: carboxylase; CD44; angiogenesis; (via phosphorylation: LC3B1: Insulin, IGF- EpCAM; blockage of HIF-1a) Akt/PKB; 1R; P62: Notch1: Ribosomal protein p70S6K1; GLUT-1; Nanog; synthesis; 4EBP1; MCT4; SIRT; HNF4a; Lipogenic GPCR; genes; STAT3; ChREBP;

PARP poly-ADP ribose polymerase, p62 nucleoprotein p62, LC3B1 component of microtubule-associated proteins, MAP1A and MAP1B, PGC-1a peroxisome proliferator-activated receptor-c coactivator-1a, GLUT-1 glucose transporter 1/SCL2A1, MCT4 monocarboxylate transporter 4, ChREBP carbohydrate- responsive element-binding protein, DVL3 disheveled homolog 3, embryonic segment polarity protein, CD133 prominin-1, stem cell surface glycoprotein, EpCAM epithelial cell adhesion molecule, Notch1 single pass transmembrane protein controlling intercellular interactions between adjacent cells, 5-FHTF (6R,6S)-5-formyl-5,6,7,8-tetrahydrofolate, HIF-1a hypoxia-inducible factor-a, SIRT1 sirtuin-1, a deacetylating enzyme controlling cellular reactions to stressors and longevity, pMAPK phosphorylated mitogen-activated protein kinase, mTORC mammalian target of rapamycin complex, Akt/PKB protein kinase-B, p70S6K1 p70S6 kinase, component of mTORC signaling, 4EBP1 eukaryotic translation initiation factor 4E-binding protein 1, a translation repressor, which interacts with eukaryotic translation initiation factor-4E, HNF4a hepatocyte nuclear factor 4a, transcription factor, GPCR G protein-coupled receptor, serpentine receptor, STAT3 signal transducing activator of transcription-3 * Crosstalk between AMPK activation and GSK-3b inhibition

123 Biochem Genet found that DM patients have signiﬁcantly higher risk of all cancers (OR 1.176; 95 % CI 1.149–1.204, p \ 0.001). Diabetics treated with insulin or sulfonylureas had prominently higher risks of all cancers, compared to those treated with metformin (OR 1.583, p \ 0.001 and OR 1.784; p \ 0.001, respectively). Metformin treatment associated with decreased risk of colon and liver cancer compared to sulfonylureas or insulin, where usage of sulfonylureas was associated with increased risks of breast and lung cancer (Hsieh et al. 2012). The association between use of metformin or other anti-diabetics and risk of PC was analyzed by a case–control study on UK-based general practice research database (GPRD). Cases had a ﬁrst-time diagnosis of PC, and six controls per case were matched on age, sex and general practice. Totally, 2763 patients with PC were determined. Long-term metformin usage was associated with a decreased risk in women (adj. OR 0.4), where both sulfonylurea (C30 prescriptions, adj. OR 1.90) and insulin (C40 prescriptions, adj. OR 2.29) treatments were associated with an increased risk of PC. A meta-analysis was reported in 2013, which analyzed original publications in English released until June 15, 2012 (Zhang et al. 2013). The studies were searched in electronic databases (MEDLINE, ISI Web of Science and EMBASE databases), and relevant reviews were analyzed. According to the eligibility criteria, 37 studies encompassing 1,535,636 participants were selected in terms of data of cancer incidence or mortality. Among metformin consumers compared with nonusers, the SRR for overall cancer incidence was 0.73 and that for mortality was 0.82. The risk reduction for PC was 46 %. Metformin also reduced fatality of liver (SRR, 0.23) and breast cancer (SRR, 0.63) (Zhang et al. 2013).

AMPK as a Target to Control Cancer Growth and Treatment Sensitivity

In Table 1, molecular pathways activated by AMPK and metformin—which activates AMPK—are summarized. AMPK was first defined as a protein kinase activity which phosphorylated and inactivated two major enzymes of fatty acid and sterol biosynthesis: Acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglu- taryl-CoA reductase (HMGR) (Hardie and Alessi 2013). AMPK does not only involve in lipid metabolism, but also in many diverging pathways, both via direct phosphorylation of enzymes and through prolonged effects by phosphorylation of transcription factors and coactivators (Hardie and Alessi 2013). Generally, AMPK mostly blocks anabolic pathways utilizing ATP and NADPH. AMPK is activated in skeletal muscle with exercise and augments muscular glucose uptake, which led to the idea that AMPK-activating drugs might exert therapeutic benefits for T2DM. Indeed, it was found that AMPK is a direct target of metformin, a drug which is used to treat T2DM for over four decades (Hardie and Alessi 2013). AMPK increases fatty acid (FA) uptake via CD36, yet inhibits transcription of lipogenic genes via SREPc. AMPK reduces protein synthesis and translation of ribosome proteins and HIF-1a (hypoxia-inducible factor-1a, an important transcription factor promoting cancer angiogenesis) via blocking mTORC1 (mammalian target of rapamycin complex-1) pathway (Hardie and Alessi 2013). AMPK enhances oxidative 123 Biochem Genet metabolism via PGC-1a and SIRT/sirtuin and substantially attenuates Warburg effect. Warburg effect is a metabolic preference of cancer cells to bypass aerobic metabolism and to augment glycolytic pathways in order to fasten their energy gain (Hardie and Alessi 2013). AMPK activity is dependent on the ratios of ATP/ADP and AMP/ATP, and hence, it acts as a rheostat of the cellular metabolic state. AMPK activation arrests cell cycle via stabilizing p53 (a main tumor suppressor) and the cyclin-dependent kinase inhibitors p21 WAF1 and p27 (Hardie and Alessi 2013). AMPK activation also blocks the synthesis of most macromolecules, including fatty acids, triglycerides, cholesterol, ribosomal RNA and proteins, leading to reduction of cell proliferation. AMPK hinders the anabolic pathways and glycolysis via suppressing mTORC1 by phosphorylating its upstream regulator TSC2 (tuberous sclerosis protein 2/tuberin) and its regulatory subunit Raptor (Hardie and Alessi 2013). Therefore, AMPK inhibition of mTORC1 decreases glycolytic enzymes of cancer cells and GLUT-1 and MCT4 (monocarboxylate transporter 4) transporters, which are essential for glucose uptake and lactate output in tumoral tissues. Glycolytic genes get activated in LKB1-null (LKB1, liver kinase B1 an upstream activator of AMPK and an important tumor-suppressor gene) mice or in AMPK-a1-a2 double null mouse embryo fibroblasts (Hardie and Alessi 2013). AMPK-a2 subunit expression is declined in a percentage of liver cancer, which is linked to increased tumor growth in mouse xenografts, and worse patient prognosis in humans. In cancer- prone mice with heterozygous deletion of PTEN combined with decreased LKB1, tumor (mostly lymphoma) formation can be delayed with metformin (Hardie and Alessi 2013). It was shown that metformin and ionizing radiation activated AMPK in MCF-7 human breast cancer and FSaII fibrosarcoma cell lines (Song et al. 2012). Clinically achievable metformin levels triggered prominent clonogenic cell kill in cancer cells. Furthermore, metformin exerted a specific cytotoxicity toward cancerous but not to non-malignant stem cells. This is of essential importance remembering that the cancer stem cells constitute the most treatment refractory and (posttreatment) repopulating fraction of tumors. Metformin also increased radiosen- sitivity of cancer cells in vitro and significantly increased the radiation-induced growth delay of FSaII tumors (s.c.) in C3H mice. Both metformin and ionizing radiation-activated AMPK inhibited mTOR and its downstream targets S6K1 (ribosomal protein S6 kinase beta-1) and 4EBP1 (eukaryotic translation initiation factor 4E-binding protein 1), essential for proliferation and survival of cancer cells (Song et al. 2012). Another study also showed the importance of AMPK in radiation sensitization of MCF-7 breast cancer cells, which involves sestrin family of stress-responsive genes (SESN2) (Sanli et al. 2012). Sestrins mediate metabolic pathways and aging via modifying the AMPK–mTOR axis. Radiation treatment of MCF-7 cells elevated AMPK, which was repressed via SESN2 siRNA. Furthermore, elevated SESN2 inhibited radiation-induced mTOR signaling and sensitized MCF-7 cells to radiation via AMPK (Sanli et al. 2012). Annonaceous acetogenins (shortened hereafter as A-acets) are polyketides isolated from the plant genus Annonaceae. A-acets exert significant toxicity against a diverse range of cancer cells. The effects of A-acets on 123 Biochem Genet tumor cell lines were analyzed, and it was found that AA005 exhibited the most significant anticancer activity. AA005 depleted ATP, activated AMPK and inhibited mTORC, leading to proliferation inhibition and autophagy in colon cancer cells (Liu et al. 2012). AA005 also blocked cisplatin-induced triggering of mTOR and synergized with cisplatin in anti-proliferative and pro-apoptotic effects in colon cancer cells. Therefore, activation of AMPK and subsequent blockage of mTOR inhibition led to tumor cell autophagy and cisplatin sensitization in colon cancer. Anticancer potential of 3,30-diindolylmethane (DIM) and role of AMPK in antineoplastic activity were studied in vitro and in vivo (Chen et al. 2012a). 30- Diindolylmethane (DIM), an agent gained from the digestion of indole-3-carbinol is abundant in cruciferous plants and exerts anti-tumor activity in vitro and in vivo (Chen et al. 2012a). A formulated DIM (hereafter referred as B-DIM) with higher bioavailability induced apoptosis and inhibited proliferation, angiogenesis and invasion of prostate cancer cells. B-DIM induced AMPK signaling, associated with inhibition of mTOR, decrease of androgen receptor (AR) expression and induction of apoptosis in both androgen-sensitive and androgen-insensitive prostate cancer cells (Chen et al. 2012a). B-DIM also induced AMPK and decreased AR in androgen-insensitive prostate tumor xenografts in SCID mice. AMPK effects on cell proliferation and apoptosis were studied in three breast cancer cell lines (MCF-7, MDA-MB-231 and T47D) with differing p53 and estrogen receptor (ER) status (El-Masry et al. 2012). Induction of AMPK by AICAR and phenformin induced prominent anti-proliferative effects in all breast cancer cell lines, but at varying degrees. Marked cell cycle arrest was observed in T47D cells, while apoptotic actions were prominent in MCF-7 and MDA-MB-231 cells (El- Masry et al. 2012). The role of orotic acid (OA) as a hepato-carcinogen and role of AMPK blockage in this carcinogenic effect were determined (Jung et al. 2012). OA enhanced cell proliferation and attenuated apoptosis in serum-starved SK-Hep1 liver cancer cells, via inhibition of AMPK phosphorylation and subsequent inducing mTORC1 (Jung et al. 2012). Magnolol is a hydroxylated biphenyl compound of Magnolia officinalis with anticancer efficacy and low toxicity. Magnolol exerted several pro-apoptotic effects, such as inducing DNA fragmentation and caspase-3 and PARP (poly-ADP ribose polymerase) cleavage in colon cancer cells. Magnolol triggered the phosphorylation of AMPK in dose- and time-dependent manners (Park et al. 2012). Magnolol also decreased migration and invasion of HCT-116 cells via AMPK (Park et al. 2012). Honokiol is a small molecule polyphenol isolated from magnolia species, which exerts anti-inflammatory, antioxidant and cancer chemopreventive efficacies. When AMPK-null and AMPK-wild-type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown established cell lines were compared, it was found that AMPK was necessary for honokiol-modification of pACC-pS6K axis and also that honokiol increased the expression and cytoplasmic translocation of tumor-suppressor LKB1 in breast cancer cells (Nagalingam et al. 2012). Honokiol also inhibited breast tumorigenesis in vivo (Nagalingam et al. 2012). Metformin significantly attenuated the proliferation of primary B and T lymphoma cells without effecting normal hematopoiesis ex vivo. Metformin induction of AMPK linked to the blockage of mTOR signaling without involvement of AKT (Shi et al. 123 Biochem Genet

2012). Moreover, lymphoma sensitivity to the anti-neoplastic drugs doxorubicin and mTOR inhibitor temsirolimus was significantly increased with metformin. Combi- nation of oral metformin with doxorubicin or temsirolimus induced lymphoma cell autophagy stronger than either agent alone (Shi et al. 2012). Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian tumor cells with diindolylmethane (DIM) for 24 h induced a concentration-dependent autophagy and DIM augmented levels of LC3B, p62 and Atg12 proteins, which increase during autophagic apoptosis (Kandala and Srivastava 2012). Autophagy-blocking agent bafilomycin or chloroquine abolished autophagy triggered by DIM. Moreover, DIM efficiently increased ER stress regulators such as glucose-related protein78 (Grp78), IRE1 and GADD153. A cytosolic calcium chelator BAPT-AM inhibited the phosphorylation of AMPK and also diminished DIM-induced autophagy (Kandala and Srivastava 2012). Inhibitors of AMPK hindered the stimulation of LC3B (light chain-3B) or p62 (nucleoporin 62), indicating that AMPK was necessary in autophagy triggered by DIM. Oral DIM therapy significantly attenuated growth of SKOV3 tumor xenografts (Kandala and Srivastava 2012). Epigallocatechin gallate (EGCG) analogs 4 and 6 were efficient AMPK inducers, and they decreased cell proliferation and increased the cyclin-dependent kinase inhibitor p21 (Chen et al. 2012b). EGCG analogs also suppressed mTOR pathway and decreased the stem cell clones in human breast cancer cells (Chen et al. 2012b). Chemosensitizing potential of silencing of twist1 in non-small cell lung cancer (NSCLC) and role of AMPK in this efficacy were studied (Jin et al. 2012). Twist1 is prominently expressed in primary and metastatic non-small cell lung cancer (NSCLC) and is an important target for lung cancer chemotherapy. Twist1 silencing induced depletion of ATP, activation of AMPK and blockage of mTOR in NSCLC cells. AMPK inhibition of mTOR lowered S6K1 activity (Jin et al. 2012). Inhibition of mTOR/S6K1 attenuated Mcl-1 protein expression and sensitized to cisplatin (Jin et al. 2012). a2 catalytic subunit of AMPK is decreased in hepatocellular carcinoma (HCC) (Lee et al. 2012a). Underexpression of AMPK-a2 was statistically associated with an increased loss of cell differentiation, anaplasia and worse patient prognosis (Lee et al. 2012a). Moreover, ectopic expression of AMPK increased p53 acetylation and stability in HCC cells. The p53 deacetylase SIRT1 was phosphorylated and inactivated by AMPK at Thr344, inducing p53 acetylation and apoptosis of HCC cells (Lee et al. 2012a). Metformin activities were studied on migration of human umbilical vein endothelial cells, on VEGF levels and on angiogenesis in the rat air pouch model (Soraya et al. 2012). Metformin (orally, 50 mg/kg) significantly (p \ 0.01) decreased vascularization in granulomatous tissue by 34 % in rats (Soraya et al. 2012). Metformin at levels between 0.5 and 3 mM significantly (p \ 0.001) reduced VEGF mRNA expression and endothelial cell migration (Soraya et al. 2012). AMPK inducers attenuate cervical cancer cell growth via decreasing DVL3 (segment polarity protein disheveled homolog 3), a procancer positive regulator of Wnt/b-catenin pathway in cervical carcinogenesis (Kwan et al. 2013). Treatment with proteosomal inhibitors AM114 or MG132 partially restored DVL3 levels lowered by metformin. In vivo ubiquitination assay showed that metformin decreased DVL3 via ubiquitin/proteasomal degradation pathway (Kwan et al. 123 Biochem Genet

2013). Anticancer mechanisms of cinnamaldehyde derivative CB-PIC in human SW620 colon cancer cells were investigated (Cho et al. 2013). CB-PIC was significantly cytotoxic on cancer cells, enhanced sub-G1 accumulation and cleaved PARP, hallmarks of apoptosis. CB-PIC increased phosphorylation of AMPK-a and ACC as well as induced the ERK in hypoxic SW620 cells. CB-PIC also lowered HIF-1a, Akt and mTOR in hypoxic SW620 cells. Simultaneous treatment with CB- PIC and metformin increased blockage of HIF-1a and Akt/mTOR and triggered AMPK-a and pACC in hypoxic SW620 cells. Additionally, CB-PIC lowered the proliferation of SW620 cells in athymic mice xenografts, decreased Ki-67, CD34 and CAIX levels and increased pAMPK-a. Noteworthy, CP-PIC showed improved anti-tumor activity in SW620 colon cancer cells under hypoxia than under normoxia. This finding has a special importance when remembering that the hypoxic cells, which accumulate at the G0 phase of the cell cyle, constitute a prominent treatment-resistant tumor population due to their dormant stage. The anticancer efficacy of the ent-kaurane diterpenoid ent-18-acetoxy-7b- hydroxy kaur-15-oxo-16-ene (CrT1) derived from the medicinal plant Croton tonkinensis was studied (Sul et al. 2013). It was questioned whether CrT1 acts via AMPK. CrT1 lowered growth in dose- and time-dependent manners in human SK-HEP1 hepatocarcinoma cells. CrT1 triggered sub-G1 arrest and caspase- mediated apoptosis. CrT1 induced caspase-3, caspase-7, caspase-8, caspase-9 and PARP cleavage, which was hindered by z-VAD-fmk blocking caspase-3 cleavage (Sul et al. 2013). CrT1 enhanced p53 and Bax, but decreased Bcl-2 levels and elevated leakage of Cytochrome-C from mitochondria into the cytoplasm. It was shown that CrT1-induced AMPK blocked the mTOR/(p70)S6K pathway (Sul et al. 2013). The actions of nilotinib on hepatocellular carcinoma (HCC) were investigated (Yu et al. 2013). Nilotinib is an oral tyrosine kinase inhibitor approved for treatment of chronic myelogenous leukemia. Staining with acridine orange and microtubule-associated protein 1 light chain-3 revealed that nilotinib triggered autophagy in dose- and time-dependent manners in HCC cell lines. Nilotinib also elevated AMPK phosphorylation and blocked protein phosphatase PP2A. Inducing PP2A decreased AMPK-mediated nilotinib activation and subsequent autophagy (Yu et al. 2013).

Metformin, an AMPK Activator with General and Pancreas Cancer- Speciﬁc Anti-tumor Actions and Pathways Related to its Anti-neoplastic Potential

The actions of metformin on human PC cell lines ASPC-1, BxPc-3, PANC-1 and SW1990 were determined (Wang et al. 2008a, b). Metformin reduced cells in G1 phase and increased the cells in S phase as well as the apoptosis in all PC cell lines (Wang et al. 2008a, b). Metformin triggered PARP cleavage (an indicator of caspase activation) in all PC cell lines. The pan-caspase inhibitor (VAD-fmk) totally blocked metformin-induced PARP cleavage and apoptosis in ASPC-1, BxPc-3 and PANC-1. The caspase-8-specific inhibitor (IETD-fmk) and the caspase-9-specific inhibitor (LEHD-fmk) partially hindered metformin-induced apoptosis and PARP 123 Biochem Genet cleavage in BxPc-3 and PANC-1 cells (Wang et al. 2008a, b). It was also found that metformin prominently lowered epidermal growth factor receptor (EGFR) and phosphorylated mitogen-activated protein kinase (p-MAPK) in time- and dose- dependent manners in all PC cell lines tested (Wang et al. 2008a, b). A novel crosstalk between insulin and G protein-coupled receptor (GPCR) cascades was defined in human PC cells (Kisfalvi et al. 2009). Insulin increased GPCR signaling via mTOR pathway. Treatment of human PC cells (PANC-1, MIAPaCa-2 and BxPC-3) with insulin (10 ng/mL) for 5 min strongly increased the enhancement of intracellular Ca2? induced by GPCR agonists (e.g., neurotensin, bradykinin and angiotensin-2) (Kisfalvi et al. 2009). Metformin pretreatment completely blocked insulin-induced potentiation of Ca2? signaling but did not effect the actions of GPCR agonists alone. Insulin also increased GPCR agonist-induced growth, measured by DNA synthesis and the cell numbers. Low metformin doses (0.1–0.5 mmol/L) blocked the stimulation of DNA synthesis, and both the anchorage-dependent and anchorage-independent growth induced by insulin and agonists of GPCR (Kisfalvi et al. 2009). Metformin induced prominent and lasting increase in the phosphorylation of AMPK at Thr(172). Moreover, metformin significantly decreased the growth of MIAPaCa-2 and PANC-1 xenografts in nude mice. The effects of varying anti-diabetic drugs on cancer cells (PC: MiaPaCa2, Panc-1; breast cancer: MCF-7, HER18) were determined (Feng et al. 2011). It was found that insulin and glucose induced cancer cell proliferation and induced chemoresistance. On the other hand, metformin and rosiglitazone suppressed cancer cell proliferation and triggered apoptosis. Both drugs modified AKT/mTOR signaling cascade; metformin increased AMPK, whereas rosiglitazone increased chromosome 10 level. Although high insulin and glucose concentrations promoted a chemoresistance, additional treatment with either metformin or rosiglitazone with chemotherapeutics gemcitabine or doxorubicin resulted in an additional inhibition of cell growth and stimulation of apoptosis (Feng et al. 2011). Metabolomic analyses of breast cancer cells exposed to metformin revealed a significant accumulation of 5-formiminotetrahydrofolate (5-FTHF) (Corominas- Faja et al. 2012). Metformin functioned as an anti-folate via altering the carbon flow through the folate-related one-carbon metabolic pathways (Corominas-Faja et al. 2012). Metformin significantly decreased cell survival, clonogenicity, sphere-forming capacity and induced sphere disintegrations in both gemcitabine-sensitive and gemcitabine-resistant PC cells (Bao et al. 2012). Metformin also decreased the cancer stem cell (CSC) markers, CD44, EpCAM, EZH2, Notch1, Nanog and Oct4, and caused reexpression of miRNAs, let-7a, let-7b, miR-26a, miR-101, miR-200b and miR-200c, which are typically lost in PC and especially in pancreatospheres. Reexpression of miR-26a by transfection lowered expression of EZH2 and EpCAM in PC cells. Thus, metformin effects occur at least partially via miRNAs and decreasing cancer stem cell population (Bao et al. 2012). Nine miRNAs were significantly induced in cells treated with metformin. Metformin increased the expression of miR-26a, miR-192 and let-7c in a dose-dependent manner (Li et al. 2012). Forced expression of miR-26a significantly lowered cell 123 Biochem Genet proliferation, invasion, migration and enhanced cell apoptosis, whereas knockdown of miR-26a reversed these effects. Furthermore, it was shown that HMGA1, an oncogene, is a direct target of miR-26a. Xenograft models also evidenced that metformin increased the level of miR-26a and decreased HMGA1 expression in vivo (Li et al. 2012). Gemcitabine combination with anti-IGF-1R antibody R1507 and/or metformin additively inhibited cell proliferation in human PC cell lines, SUIT-2 and MIAPaCa-2 with differential gemcitabine sensitivity (Kawanami et al. 2012). It was defined that drug combinations increased the apoptotic rates in both cell lines. In human PC cells, PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), metformin efficacy to trigger AMPK was prominently higher in comparison with cultures in medium with glucose at 25 mM (Sinnett-Smith et al. 2013). In physiological glucose concentration, metformin blocked mTORC1 activation, DNA synthesis and growth of PDAC cells induced by crosstalk between GPCR and insulin/IGF signaling, at concentrations (0.05–0.1 mM) that were tenfold to 100-fold lower than those used in most previous reports (Sinnett-Smith et al. 2013). Metformin reduced the growth of PANC-1 and MiaPaCa-2 tumors in different xenograft models, including orthotopic implantation (Kisfalvi et al. 2013). Metformin given once daily intraperitoneally at different doses (50–250 mg/kg) to nude mice inhibited the growth of PANC-1 xenografts in a dose-dependent manner. A significant effect of metformin was achieved at 50 mg/kg and maximal effect at 200 mg/kg (Kisfalvi et al. 2013). Metformin reduced phosphorylation of (p70)S6K1 protein and ERK in these xenografts. Metformin also blocked the growth of PC xenografts when administered orally (2.5 mg/mL) either before or after tumor implantation. Metformin, given orally, also blocked the growth of MiaPaCa-2 orthotopical xenografts (Kisfalvi et al. 2013). The effects of low doses of metformin on different subpopulations of PC cells were studied, and it was demonstrated that metformin selectively inhibited the proliferation of CD133?, but not CD24?CD44?ESA? cells (Gou et al. 2013). When cell invasion and in vivo tumor formation was determined, it was revealed that metformin was also efficient to block both. Metformin decreased phospho- Erk and phospho-mTOR-independent of Akt and AMPK phosphorylation. CD133? PC cells are malignant stem cells, which contribute to recurrence, metastasis and treatment resistance in PC (Gou et al. 2013). Human PC cell lines AsPC-1, BxPC-3, PANC-1 and MIAPaCa-2 were cultured in physiological (5 mM) or high (25 mM) glucose conditions in the absence or presence of metformin. The anti-proliferative actions of metformin linked to an activation of AMPK-Thr172 and with a blockage on the insulin/IGF-IR-activation and downstream mediators insulin receptor substrate-1 (IRS-1) and phosphorylated Akt (Karnevi et al. 2013). Exposure to high glucose levels induced higher IGF-1 responses and Akt activation which linked to induced AMPKSer485 phosphorylation and reduced AMPKThr172 phosphorylation, resulting in diminished effects of metformin (Karnevi et al. 2013).

123 Biochem Genet

PPAR-c Ligands: General and Pancreas Cancer-Speciﬁc Anti-tumor Actions

Molecular interactions elicited by thiazolidinediones and PPAR-c activation are summarized in Table 2. PPAR-c is a nuclear receptor serving as a transcription factor to control cell differentiation, apoptosis and glucose metabolism (Kliewer and Wilson 1998). Ligands for PPAR-c such as anti-diabetic drugs of the thiazolidinedione (TDD) class pioglitazone, troglitazone and rosiglitazone (Cho and Momose 2008; Krentz et al. 2008) exert anti-inflammatory effects and reduce PC growth in experimental models (Nakajima et al. 2008; Dong et al. 2009). The anti- inflammatory effects associate with suppression of COX-2 and NF-jB activities, evidencing complex intracellular pathways and their suppression of tumor growth partially occurs via reducing VEGF (Dong et al. 2009). The expression of PPAR-c in human PC and the effects of its ligands on cell growth were studied (Sasaki et al. 2001). Seven human PC cell lines and seven surgically resected human PC tissues were analyzed. PPAR-c was present in all cell lines tested and in 5 out of 7 cancer tissues (71 %), but absent in adjacent normal pancreatic tissues (Sasaki et al. 2001). Proteins in nuclear extracts of the PC cell line PANC-1 specifically bound to the peroxysome proliferator responsive element (PPRE). Cell growth was strongly inhibited with troglitazone and rosiglitazone in dose- and time-dependent manners (p \ 0.01). Oppositely, a non-functional analog of troglitazone did not influence cell growth (Sasaki et al. 2001).

Table 2 Thiazolidindione and/or PPAR-c activation associated modiﬁcations of cellular proteins in malignant cells Cell cycle Metabolic Tumor Treatment sensitivity, Cell pathways angiogenesis apoptosis, autophagy signaling and invasion pathways

WAF1/CIP1:* RXR-PPAR-c VEGF; Gemcitabine sensitivity: STAT3; P27:* complex: COX-2; P53:* Akt/PKB; Cdk2; (via Rb- Adiponectin MMP-2; Caspase-3: E- receptor 2: hypophosphorylation) Actin ﬁlament; Caspase-9: cadherin: Cdk4; Lipoprotein b-Catenin: lipase/LPL: Motility; Bax: Cyclin B1; NF-jB; Hyperlipidemia; uPA; Bcl-2;* Cyclin D1; : ; GSK-3b;* PAI-1 Beclin-2 Cyclin E; AMPK:*

Cdk cyclin-dependent kinase, Rb retinoblastoma protein, RXR retinoid 9 receptor, GSK-3b glycogen synthase kinase-3b, AMPK AMP-activated protein kinase, VEGF vascular endothelial growth factor, COX-2 cyclooxygenase-2, MMP-2 matrix metalloproteinase-2, uPA urokinase-type plasminogen activator, PAI-1 plasminogen activator inhibitor-1, bcl-2 B cell lymphoma oncogen-2, NF-jB nuclear factor of kappa B * Mutual crosstalk of PPAR-c pathway with GSK-3b and AMPK cascades. For other abbreviations, please refer to the explanations of Table 1

123 Biochem Genet

The anticancer activity of two novel analogs of betulinic acid (BA) was determined. BA is a phytochemical triterpenoid from bark extracts and is cytotoxic to malignant cells (Chintharlapalli et al. 2007). A-ring of BA was modified to give a 2-cyano-1-en-3-one moiety, and the effects of the 2-cyano-lup-1-en-3-oxo-20-oic acid (CN-BA), 2-cyano derivative of BA and its methyl ester (CN-BA-Me) were determined in colon and PC cells (Chintharlapalli et al. 2007). CN-BA and CN-BA- Me acted highly cytotoxic to Panc-28 pancreatic and SW480 colon cancer cells. CN-BA and CN-BA-Me also triggered differentiation in 3T3-L1 adipocytes, which exerted a characteristic fat droplet accumulation induced by PPAR-c agonists (Chintharlapalli et al. 2007). Both CN-BA and CN-BA-Me triggered PPAR-c- mediated responses in colon cancer (caveolin1) and PC (p21) cells (Chintharlapalli et al. 2007). The anti-neoplasticity of a new TDD class PPAR-c agonist CS-7017 was studied (Shimazaki et al. 2008). CS-7017 was highly specific for PPAR-c among other PPAR subfamilies. CS-7017 reduced the proliferation of the human anaplastic thyroid tumor cell line DRO and the PC cell line AsPC-1 in vitro at concentrations as low as 10 nM (Shimazaki et al. 2008). The effects of 1,1-bis(30- indoly)-1-(p-substituted phenyl)methanes (C-DIM) on PC were analyzed (Lei et al. 2008). C-DIM exerted structure-dependent induction of PPAR-c and nerve growth factor-induced Ba (Nur77) and triggered receptor-dependent and recepto-independent apoptosis in cancer cells and tumors (Lei et al. 2008). The effects of PPAR-c on angiogenesis of PC were investigated (Dong et al. 2009). PC cell line PANC-1 was exposed to either RXRa ligand 9-cis-RA or a ligand of PPAR-c 15d-PGJ2 or both. 15d-PGJ(2), 9-cis-RA and their combination reduced the proliferation of PANC-1 dose-dependently. 9-cis-RA and 15d-PGJ(2) exerted a synergistic anti-proliferative effect on PC cells. 15d-PGJ2, 9-cis-RA and their combination reduced the expression of VEGF mRNA in PANC-1 cells in dose- and time-dependent manners (Dong et al. 2009). The effects of COX-2 inhibitor NS-398 and/or rosiglitazone on the cell growth and apoptosis were defined in a PC cell line, SW1990 (Sun et al. 2009). NS-398 and rosiglitazone reduced cell proliferation in dose- and time-dependent manners. PCNA index significantly reduced in cells treated with either NS-398 or rosiglitazone. Both NS-398 and rosiglitazone alone and in a synergistical manner triggered apoptosis in SW1990 cells. These agents also suppressed Bcl-2 and increased Bax expression (Sun et al. 2009). PPAR-c ligand, troglitazone reduced the growth of PC cells with special effects on restriction point control of the late G1 phase of the cell cycle (Kawa et al. 2002). Troglitazone reduced the proliferation of six PC cell lines in a dose–response manner, which was inhibited to less than 50 % of control at the concentration of 10 microM. The anti-proliferative action was associated with G1 phase cell cycle arrest through increase of p21 protein. Simultaneously, CDK2 kinase activity was blocked with the hypophosphorylation of Rb protein (Kawa et al. 2002). Troglitazone also induced profound morphologic changes of duct structure with apoptotic cells in the lumen (Kawa et al. 2002). The b-catenin exerts essential roles in intercellular adhesion and signal transduction. Glitazones markedly increased differentiation markers E-cadherin and carcinoembryonic antigen (Ohta et al. 2002). In control cells, b-catenin mainly localized in the cytoplasm and/or nucleus; in glitazone-treated cells, b-catenin 123 Biochem Genet shifted to the plasma membrane, in association with enhanced E-cadherin. Hence, a PPAR-c ligand involves not only in differentiation in PC cells, but also in influencing the E-cadherin/b-catenin system (Ohta et al. 2002). PPAR-c activation by troglitazone inhibited cell invasion and cell migration in PK-1 and PK-9 cells (Motomura et al. 2004). Troglitazone-exposed PK-1 and PK-9 cells became smaller, and their shape shifted from flat to spindle, followed by round; it was shown that modification of actin filament organization by PPAR-c mediated these effects (Motomura et al. 2004). The effects of rosiglitazone and pioglitazone on invasion of human PC cells were determined (Galli et al. 2004). Glitazone inhibited PC cells’ invasiveness, influencing gelatinolytic and fibrinolytic activity independent of PPAR-c and involving reduction of MMP-2 and activating plasminogen activator inhibitor-1 (PAI-1) (Galli et al. 2004). 1,1-Bis(30-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF3) and troglitazone reduced growth of Panc-28 PC cells. DIM-C-pPhCF3 was more potent than troglitazone. Hence, it was used as a model to delineate the mechanism of PPAR-c-mediated reduction of Panc-28 cell growth (Hong et al. 2004). DIM-C-pPhCF3 significantly inhibited G0/G1 ? S phase progression, which was linked to decreased Rb phosphorylation and increased p21 protein, but no effects on p27 or cyclin D1 were witnessed (Hong et al. 2004). p21 was induced via PPAR-c interactions with both specificity protein 1 (Sp1) and Sp4 transcription factors (Hong et al. 2004). The effects of PPAR-c ligands on PC cell invasion and the plasminogen activator system were defined. Expressions of components of the plasminogen activator system [i.e., urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 and uPA receptor] were demonstrated in six human PC cell lines. The PPAR-c ligands 15-deoxy- delta12,14-prostaglandin J2 and ciglitazone reduced PC cell invasion and increased PAI-1 and decreased uPA (urokinase-type plasminogen activator) levels in PC cells (Sawai et al. 2006). N-nitrosobis(2-oxopropyl)amine (BOP) induced pancreatic ductal adenocarcinomas in Syrian golden hamsters, but other rodents were not sensitive to BOP carcinogenesis (Takeuchi et al. 2007). Profound enhancement of serum triglycerides (TGs) and total cholesterol (TC) was witnessed in Syrian golden hamsters, but not C57BL/6 mice, ICR mice, F344 rats and Wistar rats (Takeuchi et al. 2007). In parallel, liver lipoprotein lipase (LPL) activity was lower in hamsters in comparison with mice and rats. Syrian golden hamsters were injected with BOP (10 mg/kg body wt) and subsequently given a diet containing 800 ppm. pioglitazone for 22 weeks (Takeuchi et al. 2007). Pioglitazone induced hepatic LPL mRNA expression and significantly decreased hyperlipidemia (Takeuchi et al. 2007). Concomitantly, the frequency and multiplicity of PCs were significantly lowered by pioglitazone in comparison with controls. The suppression rates were greater in invasive adenocarcinomas than noninvasive ones. The incidence of cholangiocellular carcinoma was also decreased. Thus, inhibition of PC development by pioglitazone is likely due to changes in the serum lipids, and hyperlipidemia could be a contributor in development of PC (Takeuchi et al. 2007). The interactions between the NF-jB and Notch pathways to inhibit PPAR-c expression and to induce PC progression in mice were studied (Maniati et al. 123 Biochem Genet

2011). The majority of human PC have activating mutations in the KRAS proto- oncogene, which lead to induction of the NF-jB pathway and therefore constitutive production of pro-inflammatory cytokines (Maniati et al. 2011). Inhibitor of jB kinase2 (Ikk2), a member of the NF-jB cascade, synergized with basal Notch signaling to augment transcription of Notch target genes (Maniati et al. 2011). The IjB kinase enzyme complex is a member of the upstream NF-jB cascade. The IjBa (inhibitor of jB) protein blocks NF-jB signaling by shading the nuclear localization signals (NLS) of NF-jB proteins and causes its sequestration inactively in the cytoplasm. IKK phosphorylates the inhibitory IjBa protein, which then leads to the dissociation of IjBa from NF-jB. The freeing NF-jB translocates to nucleus and induces the expression of at least 150 genes, some of which bear major anti-apoptotic and pro-inflammatory efficacies. In the Kras(G12D)Pdx1-cre mouse model of PC, genetic deletion of Ikk2 in initiated premalignant epithelia prominently prolonged the interval to pancreatic carcinogenesis and suppressed the classical Notch target genes Hes1 and Hey1 (Maniati et al. 2011). TNF-a triggered NF-jB signaling and, in interaction with basal Notch signals, induced optimal expression of Notch targets (Maniati et al. 2011). TNF-a stimulation caused phosphorylation of histone H3 at the Hes1 promoter, and this signal diminished with Ikk2 deletion. Hes1 reduces expression of PPAR-c, and thus, crosstalk between TNF-a/Ikk2 and Notch sustains the inflammatory profile of transformed cells (Maniati et al. 2011). Cancer cells protect themselves from apoptosis triggered by type I interferons (IFNs) via a ras-MAPK-mediated cascade (Vitale et al. 2012). Since IFN-signaling components STATs are regulated by PPAR-c, the interaction between recombinant IFN-b and the PPAR-c agonist troglitazone was studied. This combination exerted synergistic anti-proliferative effects on BxPC-3 PC cells, via antagonizing the IFN-b-induced activation of STAT3, MAPK and akt and enhancing the binding of both STAT1-associated complexes and PPAR-c to specific DNA responsive elements (Vitale et al. 2012). The synergistic anti-proliferative effects associated with a cell cycle arrest in G0/G1 phase, subsequent to a long-lasting increase of both p21 and p27 expressions. Blockage of MAPK activation and the actions on p21 and p27 expressions, induced by IFN-b and TGZ combination, were due to the reduced activation of STAT3 secondary to troglitazone (Vitale et al. 2012). This combination enhanced autophagy and decreased anti-autophagic bcl-2/beclin-1 complex, which occured via abolishment of the akt-mTOR cascade (Vitale et al. 2012). Ligands for PPAR-c such as pioglitazone and rosiglitazone increased gemcitabine’s cytotoxicity activity on human PC cells in a dose- dependent manner. The synergism was PPAR-c-mediated, since the effect was augmented via PPAR-c overexpression and was blocked by PPAR-c-specific siRNA (Koga et al. 2012). In xenograft models, gemcitabine plus pioglitazone prominently suppressed tumor growth, while single gemcitabine treatment was ineffective (Koga et al. 2012).

123 Biochem Genet

Lithium—LiCl, a GSK-3b Inhibitor with General and Pancreas Cancer- Speciﬁc Anti-tumor Actions

Lithium, the salts of which are efficient for the treatment of several psychiatric diseases (mainly bipolar disorder), is presumed to cause its psychotherapeutic efficacies by acting upon biogenic amine-containing cerebral neurons (Gorkin and Richelson 1979). During lithium maintenance treatment, plasma lithium levels of about 1 mM are achieved. The suitable concentration of lithium in serum for the therapy of bipolar mood disorder is reported to be 0.5–1.0 mM (=mEq/l) (Kurita et al. 2002). When the serum concentration of lithium is higher than 2 mM in patients, toxicity appears, such as nausea, fine tremors, diarrhea, confusion, slurred speech and ataxia (Kurita et al. 2002). Many studies were performed to illuminate the mechanism of action of lithium. Since lithium inhibits inositol 1-monophosphatase and attenuates phosphatidylinositol turnover, it was first proposed that the phosphatidylinositol-mediated signaling pathway is the main target of lithium treatment (Kurita et al. 2002). Nonetheless, lithium concentrations over 4–5 mM are necessary to inhibit phosphatidylinositol turnover and lithium at these concentrations causes toxic side effects in patients. Furthermore, lithium at therapeutic concentrations has no effect on inositol monophosphatase activity in human cerebrospinal fluid (Kurita et al. 2002). GSK-3b is a major regulator of a plethora of signaling cascades, and lithium is a direct inhibitor of GSK-3b (Zhang et al. 2003). Lithium increases the inhibitory N-terminal phosphorylation of GSK-3b. A short peptide derivative of GSK-3b- interaction domain of axin potently inhibited GSK-3b and robustly induced Wnt (Wingless-related integration site)-mediated transcription, similar to lithium (Zhang et al. 2003). Lowering of GSK-3b proteins, either via RNA interference or via suppressing the mouse GSK-3b gene, enhanced N-terminal phosphorylation of GSK-3b, indicating that GSK-3b controls its own phosphorylation. Moreover, N-terminal phosphorylation of GSK-3b can be modified by the GSK-3-dependent protein phosphatase-1 inhibitor-2 complex (Zhang et al. 2003). E-cadherin loss in cancerous tissues associates with dedifferentiation/anaplasia, invasion and metastasis (Ohira et al. 2003). Drosophila DE-cadherin is influenced by Wnt/b-catenin signaling, which was not been shown in mammalian cells until the study of Ohira et al. (2003). The expression of WNT7a, encoded on 3p25, was frequently suppressed in lung cancer, and that loss of E-cadherin or b-catenin was a poor prognostic feature (Ohira et al. 2003). WNT7a stimulates E-cadherin expression via b-catenin in lung cancer cells and contributes to a positive feedback loop. Lithium led to E-cadherin induction via an inositol-independent pathway. Similarly, exposure to mWNT7a specifically triggered free b-catenin and E-cadherin (Ohira et al. 2003). Among transcriptional downregulators of E-cadherin, ZEB1 was associated with E-cadherin loss in lung cancer cells, and its inhibition by RNA interference resulted in stimulation of E-cadherin. Pharmacologic reversal of E-cadherin and WNT7a losses was satisfied with lithium (Ohira et al. 2003). The effect of GSK-3b suppression was investigated on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human prostate cancer

123 Biochem Genet cells (Liao et al. 2003). With exposure to lithium or SB216763, the GSK-3b inhibitors, TRAIL-triggered apoptosis was robustly increased (Liao et al. 2003). TRAIL induction enhanced GSK-3b tyrosine phosphorylation at Y216. Sensitiza- tion to TRAIL was linked to enhanced proteolysis of caspase-8 and its downstream target BID (BH3 interacting-domain death agonist). A caspase-8 inhibitor completely blocked lithium-induced TRAIL sensitization (Liao et al. 2003). The role of GSK-3b in androgen-induced gene expression in human prostate cancer - cells was studied (Liao et al. 2004). Pretreatment of prostate cancer cells expressing wild-type or mutant androgen receptor with the GSK-3b inhibitors, lithium or GF109203X blocked androgen-responsive reporter activity in dose- and time- dependent manners. Also, the expression of two androgen-stimulated gene products, prostate-speciﬁc antigen and matrix metalloproteinase-2, was reduced by the GSK- 3b inhibitors (Liao et al. 2004) (Table 3). Blockage of GSK-3b activity by lithium reduced proliferation of the ovarian cancer cells (Cao et al. 2006). Overexpressing constitutively active form of GSK-3b induced S phase entry, enhanced cyclin D1 expression and increased the proliferation of ovarian cancer cells. GSK-3b inhibition by lithium suppresses the tumors in nude mice inoculated with human ovarian cancer cells (Cao et al. 2006). Inactivation of GSK-3b in human medullary thyroid TT cancer cells with GSK-3b- blocking agents such as lithium and SB216763 reduces proliferation (Kunni- malaiyaan et al. 2007). Anti-proliferative effects of GSK-3b inhibitors linked to a cell cycle arrest via elevation of cyclin-dependent kinase inhibitors such as p21, p27 and p15 and lithium-treated TT xenograft mice had signiﬁcantly less tumor volumes compared with control (Kunnimalaiyaan et al. 2007). It was investigated whether enhancement of Wnt signaling within the bone marrow in myeloma decreases

Table 3 Lithium and/or GSK-3b inhibition associated modiﬁcations of cellular proteins in malignant cells Cell cycle and apoptosis Tumor angiogenesis and Metabolic pathways and cell pathways invasion signaling

WAF1/CIP1:* Motility; RXR-PPAR-c:* P27:* PIK3A; Raf cascade; Bcl-2;* Cadherin-11; Wnt cascade; Caspase-8:* Gli1; BID: E-cadherin: (via Wnt)* b-Catenin:(via Wnt)* NF-jB;

BID BH3 interacting-domain death agonist, pro-apoptotic member of bcl-2 family, PKI3A also known as PI3K/PI-3 kinase, involved in invasion and apoptosis resistance, Cadherin-11 encodes a type II classical cadherin from the cadherin superfamily, integral membrane proteins that mediate calcium-dependent cell–cell adhesion, GLI-1 a protein from the hedgehog cascade, named as GLI due to its ﬁrst isolation from glial brain tumors * Mutual interactions of GSK-3b inhibition with AMPK and PPAR-c pathways. For other abbreviations, please refer to the explanations of Tables 1 and 2

123 Biochem Genet development of osteolytic bone disease. Mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in myeloma bone disease (Edwards et al. 2008). Lithium induced Wnt signaling in osteoblasts and suppressed myeloma bone disease (Edwards et al. 2008). The efficacy of lithium on esophageal cancer (EC) proliferation was investigated (Wang et al. 2008b). Eca-109 cells were exposed to lithium at increasing concentrations (2–30 mMol/L) and time points (0, 2, 4, 6 and 24 h) (Wang et al. 2008b). Lithium decreased the proliferation of Eca-109 cells and a concentration of 20 mMol produced prominent changes in cell cycle with higher number of cells in G2/M phase (Wang et al. 2008b). Lithium blocked GSK- 3b by Ser-9 phosphorylation and stabilized free b-catenin in the cytoplasm. Nonetheless, applied doses were very high and selective targeting of lithium to tumors is necessary, if it could be employed for treatment of esophageal cancer in future. The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancer (Gustin et al. 2009). Gene targeting was performed to knockin PIK3CA mutations into human breast epithelia to reveal novel treatment targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of proliferation in epidermal growth factor and mTOR-independent manner. These features were linked to AKT, ERK and GSK-3b phosphorylation (Gustin et al. 2009). GSK-3b inhibitors lithium and SB216763 selectively reduced proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and reduced the GSK-3b-target gene cyclin-D1 (Gustin et al. 2009). Oral treatment with lithium decreased the growth HCT-116 colon cancer xenografts with mutant PIK3CA compared with isogenic HCT-116 knockout cells carrying only wild-type PIK3CA (Gustin et al. 2009). The cell–cell adhesion molecule cadherin-11 plays essential roles in embryoge- nesis, in cancer cell invasion and in bone metastasis of cancer (Farina et al. 2009. Inhibition of GSK-3b with lithium, GSK-3b-inhibitor BIO and with siRNA suppressed cadherin-11 protein levels (Farina et al. 2009). Medullary thyroid cancer (MTC) is responsible for 14 % of thyroid cancer deaths. MTC has a high recurrence rate and is mostly resistant to current treatments (Adler et al. 2010). The histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) induce the Notch1 pathway. MTC cells were exposed to increasing combinations of up to 20 mM lithium with either 3 mM VPA or 20 muM SBHA for 48 h (Adler et al. 2010). Combined treatment induced active Notch1 and blocked the GSK-3b pathway (Adler et al. 2010). Additive anti-proliferative effects were observed in combination treatment with enhancements of the cleavage of the apoptotic markers caspase-3 and PARP (Adler et al. 2010). The polycomb group gene Bmi1 is essential for neural stem cell self-renewal and increased in several cancers, including medulloblastoma (Korur et al. 2009). Bmi1 is consistently and strongly expressed in glioblastoma multiforme (GBM). Downregulation of Bmi1 by shRNAs triggered differentiation and reduced stem cell markers Sox2 and Nestin. Expression of GSK-3b, which is consistently expressed in primary GBM, also attenuated, which suggest a functional linkage between Bmi1 and GSK-3b (Korur et al. 2009). Blockage of GSK-3b activity by siRNA or lithium also triggered tumor cell differentiation. Tumor apoptosis was also increased, tumor spheroids were 123 Biochem Genet disintegrated, and clonogenicity decreased in a dose-dependent manner (Korur et al. 2009). Lithium was studied if it could enhance the sensitivity of LNCap prostate cancer cells to doxorubicin (Dox), etoposide (Eto) and vinblastine (Vin) (Azimian-Zavareh et al. 2012). Lithium acted cytotoxic in dose- and time-dependent manners. Both Dox (100 or 280 nM) and Vin IC50 (5 nM) doses induced G2/M phase arrest (p \ 0.001) compared with control. Low dose (10 lM) or IC50 (70 lM) Eto doses induced G2/M or S phase arrests, respectively (p \ 0.001). Combination of low dose or IC50 dose of Eto with lithium enhanced apoptosis as demonstrated by high percent of cells in SubG1 (p \ 0.05, p \ 0.01, respectively) (Azimian-Zavareh et al. 2012). Moreover, Eto (10 lM) led to reduced percent of cells in G2/M phase when combined with lithium (Azimian-Zavareh et al. 2012). ZM336372 is small molecule tyrosine kinase modifier, which blocks GSK-3b via phosphorylating it at Ser9 (Deming et al. 2010). Panc-1 and MiaPaCa-2 cells were exposed to ZM336372 or lithium. A dose-dependent enhancement in inactivating phosphorylation of GSK- 3b was witnessed with both ZM336372 and lithium. Growth inhibition with ZM336372 and lithium also occurred dose-dependently. An enhancement of PARP cleavage was shown following treatment with both agents (Deming et al. 2010). Hedgehog signaling cascade exerts a prominent role in the initiation and promotion of pancreatic ductal adenocarcinoma (PDA), and thus, it is a logical target for PDA treatment (Peng et al. 2013). Lithium blocked cell proliferation, G1/S cell cycle progression, triggered apoptosis and reduced tumorigenicity of PDA cells through decreasing the expression and activity of glioma associated oncogene-1 (GLI1; Gli proteins are the mediators of hedgehog signal and regulate cell differentiation and embryonic development). Moreover, lithium increased the anti-tumoral efficacy of gemcitabine (Peng et al. 2013).

Lithium’s Insulinomimetic and Anti-diabetic Effects

In 1980s, weight gains in bipolar disorder patients under lithium treatment prompted investigators to investigate, whether lithium is a diabetogenic agent. Yet, many studies in both experimental animals and humans proved that the reverse is true. Fasting blood sugar was determined in bipolar disorder patients previous to lithium treatment and at intervals during treatment for up to 6 years. The total exposure time to lithium was 495.5 years (Vestergaard and Schou 1987). Even though the patients gained weight during the treatment, their mean blood sugar levels did not increase, and only one patient developed diabetes (Vestergaard and Schou 1987). Lithium could prominently restore insulin sensitivity to normal in diabetic rats, and its insulinomimetic activity seems to be highly speciﬁc for the glycogenic pathway in skeletal muscle (Rossetti 1989). Postmeal plasma glucose increased in diabetic versus control rats and was normalized by addition of lithium and vanadate (Rossetti et al. 1990). In hepatocytes incubated with 20 mM glucose, lithium strongly stimulated glycogen synthesis in cells from both normal and streptozotocin-induced diabetic rats in a concentration- and time-dependent manner; it was related to an

123 Biochem Genet increase in the glycogen synthase activity ratio without an effect on glycogen phosphorylase (Rodriguez-Gil et al. 1993). The increased blood glucose in diabetic rats was about 50 % restored by oral treatment with lithium (0.3 mg/mL) and was completely normalized following vanadate addition (0.05 mg/mL) (Srivastava et al. 1993). Lithium could normalize the decrease of catalase (CAT) and glutathione peroxidase (GSH-PX) but not the lowered superoxide dismutase (SOD) in liver of diabetic rats (Srivastava et al. 1993). Lithium also has an insulin-like effect on glucose transport in skeletal muscle and adipocytes (Tabata et al. 1994; Chen et al. 1998). While lithium had only a minimal effect on basal glucose transport in rat epitrochlearis muscles, it markedly augmented the sensitivity of glucose transport to insulin (Tabata et al. 1994). The increase in glucose transport induced by 300 pM insulin was about 2.5-fold greater with the addition of lithium (Tabata et al. 1994). Lithium also blunted the activation of glycogen phosphorylase by epinephrine (Tabata et al. 1994). These effects were not mediated by adenylate cyclase inhibition, since lithium did not effect neither basal- nor epinephrine-stimulated muscle cAMP concentration (Tabata et al. 1994). Lithium effects of on glucose transport and metabolism in skeletal muscle was very similar to the actions of exercise (Tabata et al. 1994). Lithium also exerts mitogenic and secretagogic actions on insulin-producing pancreatic b cells, which is inhibitable with pertussis toxin, which inactivates GTP-binding proteins by ADP ribosylation (Sjo¨holm 1996). In diabetic rats, hepatic, renal and muscular lithium levels are signiﬁcantly lower in comparison with healthy controls (Hu et al. 1997). Lithium carbonate supplementation reversed this decrease and normalized blood glucose and glycosylated serum protein levels (Hu et al. 1997). After 4 weeks of lithium carbonate supplementation, STZ-mediated destruction of beta cells in the pancreas decreased, fasting blood glucose and 2-h postprandial blood glucose (PBG) decreased, blood lipid peroxides (LPO) decreased and erythrocyte superoxide dismutase (RBC-SOD) and glutathione (GSH) returned to normal, and hepatic LPO decreased and glutathione peroxidase (GSH-Px) increased (Hu et al. 1999). GSK-3b is a negative regulator of insulin signaling pathway (Moh et al. 2008). Two major targets of insulin action, glycogen synthase and insulin receptor substrate-1, are suppressed by GSK-3b, and GSK-3b activity is elevated in diabetic tissues (Kaidanovich and Eldar-Finkelman 2002). Inhibitors of GSK-3 lower hepatic glucose output and augment the synthesis of glycogen from L-glucose (Lochhead et al. 2001). STAT3 sensitizes the insulin signaling through suppression of GSK-3b, and treatment with GSK-3b inhibitor lithium rescued the glucose intolerance and impaired insulin response in STAT3-knockout mice (Moh et al. 2008).

Mutual Interactions with AMPK Activation and GSK-3b Inhibition

A transformation-dependent cell kill was observed via glucose limitation in K-ras- transformed NIH3T3 cells (de Candia et al. 2011). GSK-3b activities in these cells were compared during high versus low glucose availability (de Candia et al.

123 Biochem Genet

2011). Glucose depletion blocked GSK-3b through posttranslational mechanisms, and this inhibition was lesser in normal cells (de Candia et al. 2011). Additional blockage of GSK-3b with lithium, combined with glucose starvation, led to a specific induction of AMPK and significant reduction of proliferation in transformed but not normal cells. These findings evidence potential synergisms between AMPK activation and GSK-3b inhibition specific to malignant cells (de Candia et al. 2011). The BH3 mimetic ABT737 induces autophagy by competitively diminishing the inhibitory interaction between the BH3 domain of Beclin-1 and the anti-apoptotic proteins Bcl-2 and Bcl-X(L), thereby triggering the Beclin 1-dependent allosteric inducing of the pro-autophagic lipid kinase vesicle-mediated vacuolar protein sorting-4 (VPS34) (Malik et al. 2011). ABT737 triggered the activating phosphorylation of AMPK and of the AMPK substrate ACC. ABT737 also diminished the activity of mTOR and caused subsequent dephosphorylation of the mTOR substrate (p70)S6K1 (Malik et al. 2011). Exposure to ABT737 also dephosphorylated and inhibited GSK-3b and Akt, similar to an another structurally unrelated BH3 mimetic, HA14-1. These mechanisms indicate a synergistic autophagy-inducing activity of AMPK activation and GSK-3b inhibition (Malik et al. 2011). GSK-3b has also a major role in Alzheimer’s disease (AD), influencing amyloid-b (Ab) production and neuronal apoptosis (Cai et al. 2012). AMPK signaling controls Ab metabolism, and thus, it was hypothesized that GSK-3b regulated AMPK activation. To verify this idea, the effect of GSK-3b on the expression of AbPP cleavage enzyme (BACE), Ab and AMPK was examined in SH-SY5Y and AbPP695 cells. Three different inhibitors of GSK-3b were employed for this study (Cai et al. 2012). It was shown that lowering of GSK-3b with specific inhibitors strongly lowered Ab generation in human SH-SY5Y and SH-SY5Y-AbPP695 cells via increasing AMPK activity to downregulate Ab (Cai et al. 2012). These findings indicated that GSK-3b inhibition induces AMPK in neuronal cell lines (Cai et al. 2012) and once again suggests potential synergisms between AMPK activation and GSK-3b inhibition. Non-alcoholic fatty liver disease (NAFLD) and pathological adiposity constitute important health problems. Soy isoflavones were tested on NAFLD, pathologic adiposity, de novo lipogenic carbohydrate-responsive element-binding protein (ChREBP) and anti-adipogenic Wnt signaling (Kim and Kang 2012). Isoflavones reduce ChREBP signaling via protein kinase A-PKA and/or AMPK-induced phosphorylation, which hinders ChREBP from binding to the promoter regions of lipogenic enzyme. Isoflavones also directly induced Wnt signaling via estrogen receptor-dependent pathway, which blocked GSK-3b and transactivated T cell factor/lymphoid-enhancer factor (TCF/LEF), the mediator of Wnt signaling (Kim and Kang 2012). Natural isoflavones may be proper agents in treating NAFLD and pathological adiposity via inducing AMPK and inhibiting GSK-3b (Kim and Kang 2012), pathways which may also mutually synergize.

123 Biochem Genet

Mutual Interactions Between GSK-3b and PPAR-c Pathways and Role of GSK-3b in PPAR-c Agonist-Mediated Anti-tumor Activity

The molecular basis of PPAR-c agonist-triggered apoptosis of colon cancer cells was studied (Ban et al. 2010). It was clarified that the inhibitory effect of troglitazone on colon tumor growth was associated with inhibition of NF-jB activity and GSK-3b expression in a dose-dependent manner (Ban et al. 2010). Cells were arrested in G0/G1 phase followed by apoptosis after treatment of troglitazone with simultaneous decreases in the expression of the G0/G1 phase regulatory proteins: Cdk2, Cdk4, cyclin B1, cyclin D1 and cyclin E. Troglitazone also reduced anti-apoptotic protein Bcl-2 and triggered the expression of the pro-apoptotic proteins: caspase-3, caspase-9 and Bax (Ban et al. 2010). Cotreatment of troglitazone with a GSK-3b inhibitor (AR-a014418) or siRNA against GSK-3b significantly increased the suppressive effect of troglitazone on the NF-jB activity, tumor cell proliferation and the expression of G0/G1 phase regulatory proteins and pro-apoptotic proteins (Ban et al. 2010). Since psychiatric and neurodegenerative diseases often associate with perturbed immune status (Liu et al. 2011), it was hypothesized that lithium may act via influencing immune pathways in these patients. Dendritic cells (DCs) play a dominant role in controlling immune responses; hence, the influence of lithium on the development and function of DC was studied. Treatment with lithium during the differentiation of human monocyte-derived immature DCs (iDC) increased CD86 and CD83 expression. On the other hand, the treatment with lithium during LPS- triggered maturation of iDC acted oppositely, indicating that lithium may not induce an unspecific inflammatory response rather it induces a specific acquired immunity (Liu et al. 2011). During iDC differentiation, lithium reduced the activity of GSK- 3b. In addition, lithium activated PPAR-c during iDC differentiation, a pathway not described before (Liu et al. 2011). Elevated CD86 expression by lithium involved the GSK-3b pathway. Lithium modified the expression of CD83 in iDC also through PPAR-c. It was shown that PPAR-c is a downstream target of GSK-3b and was responsible for the lithium modulation of CD86/83 (Liu et al. 2011). Molecular mechanisms of PPAR-c agonist-mediated anti-proliferative effects on prostate cancer cells were analyzed (Ban et al. 2011). The actions of troglitazone on the expression of PPAR-c, GSK-3b and NF-jB activity and prostate cancer cell proliferation were determined. Troglitazone induced the expression of PPAR-c in the nuclei of PC-3 cells, but not in LNCaP cells (Ban et al. 2011). Troglitazone (0–16 lM) decreased cancer cell proliferation at similar rates in both cells via cell cycle arrest in G0/G1 phase and induced apoptosis in a concentration-dependent manner. Troglitazone inhibited the constitutive expression of GSK-3b and activation of NF-jB. Simultaneous treatment with troglitazone and a GSK-3b inhibitor or GSK-3b siRNA significantly increased troglitazone inhibition of the NF-jB activity, the reduction of prostate cancer cell proliferation and induction of apoptosis (Ban et al. 2011). The effects of glitazones on apoptosis and on the serine/ threonine kinase pathway, Akt, and GSK-3b were examined in LNCaP cells. The apoptosis-triggering efficacy of glitazones on prostate cancer cells involved the

123 Biochem Genet inhibition of Akt phosphorylation. Furthermore, glitazones inactivated GSK-3b, which exerts essential roles in prostate cancer growth and in androgen-independent phenotype (Zhu et al. 2012). Very noteworthy, the GSK-3b inhibitor lithium sensitized prostate cancer cells to glitazone cytotoxicity (Zhu et al. 2012). Antioxidant edible anthocyanins reduce obesity and the severity of the metabolic syndrome (Qin and Anderson 2012). Chokeberry extracts were tested whether they could reduce weight gain in rats fed a fructose-rich diet (FRD), and pathways were studied which relate to insulin signaling, adipogenesis and inflammation (Qin and Anderson 2012). Wistar rats were given a FRD to induce insulin resistance, with or without chokeberry extract (CBE) added to the drinking water (100 and 200 mg/kg body weight, daily). Both doses of CBE decreased epididymal fat, blood glucose, cholesterol and LDL cholesterol. CBE also enhanced plasma adiponectin levels and lowered plasma TNF-a and IL6 in comparison with control group. mRNA levels of Gsk-3b decreased. The protein and gene expression of adiponectin and PPAR-c mRNA levels were increased (Qin and Anderson 2012). Gene expressions of inflammatory cytokines such as IL-1b, IL-6 and TNF-a were reduced. These data suggest that the anthocyanidin reduction of insulin resistance occurs via simultaneous triggering of PPAR-c and reduction of GSK-3b, subsequently leading to anti- inflammatory actions (Qin and Anderson 2012).

Mutual Interactions of PPAR-c and AMPK Activation

Pioglitazone is a full PPAR-c agonist and improves insulin sensitivity by increasing blood adiponectin (Kudoh et al. 2011). It was examined whether pioglitazone improves insulin resistance via increase of 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) in 3T3-L1 adipocytes. Pioglitazone significantly enhanced insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Pioglita- zone significantly augmented AdipoR2 expression, but it did not affect AdipoR1 (Kudoh et al. 2011). Forced PPAR-c expression profoundly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3- L1 adipocytes (Kudoh et al. 2011). Pioglitazone significantly increased AMPK phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not trigger phosphorylation of IRS-1, Akt or protein kinase Ck/f (Kudoh et al. 2011). These findings suggest that pioglitazone improved insulin sensitivity, at least partly, via PPAR-c-AdipoR2-mediated AMPK phosphorylation in 3T3-L1 adipocytes. These data also reveal that PPAR-c and AMPK activation can be induced under the same influence, strengthening the proposal that PPAR-c and AMPK activators may synergy in anti-neoplasticity. Obesity causes a chronic inflammation of the adipose tissue, which diminishes its endocrine function and leads to metabolic disorders, such as T2D (Siriwardhana et al. 2013). Edible polyphenols reduce both systemic and adipose tissue inflammation and have the potential to alleviate obesity-associated metabolic disorders (Siriwardhana et al. 2013). Polyphenolic molecules including non- flavonoids, such as curcumin and resveratrol, and flavonoids, such as catechins, quercetin and isoflavones, inhibit NF-jB and MAPK cascades while activating 123 Biochem Genet

AMPK in adipose tissue (Siriwardhana et al. 2013). Dietary polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acid (CLA) and monounsaturated fatty acids (MUFA), such as oleic acid, also have anti-inflammatory effects (Siriwardhana et al. 2013). These include activation of AMPK and PPAR-c, as well as decrease of Toll-like receptors (TLRs) and NF-jB pathway (Siriwardhana et al. 2013). Thus, both AMPK and PPAR-c activation could bear anti-obesity and anti-inflammatory effects, which both are associated with decreased risk of malignancy (Siriwardhana et al. 2013). Besides improving insulin sensitivity, rosiglitazone restores normal vascular function. Adiponectin is a mediator the PPAR-c actions on vessel endothelia in diabetic mice (Wong et al. 2011). In db/db and diet-induced obese mice, PPAR-c activation by rosiglitazone improves endothelium-dependent relaxation of aortae, whereas diabetic mice lacking adiponectin or applied an anti-adiponectin antibody do not respond (Wong et al. 2011). Rosiglitazone triggers adiponectin release from fat explants, and subcutaneous fat transplantation from rosiglitazone-treated mice restores vasodilatation in untreated db/db recipients. Adiponectin induces AMPK/ eNOS and cAMP/PKA pathways in aortae, which enhance NO bioavailability and reduce oxidative stress (Wong et al. 2011). These results reveal that adipocyte- derived adiponectin mediates PPAR-c improvement of endothelial function in diabetes, while also indicating that PPAR-c activation may lead to AMPK activation via adiponectin. Thus, a synergism is suspected to occur between agents activating PPAR-c and AMPK. PPAR-c and AMPK induction have cardioprotective activities against ischemia/reperfusion injury, which intersect and possibly synergize (Morrison and Li 2011). Rosiglitazone (1 lg/g) was injected intravenously 5 min before cardiac reperfusion. Myocardial infarction was significantly alleviated in mice treated with rosiglitazone in comparison with vehicle. Isolated hearts were exposed to 20 min of global, no-flow ischemia in an ex vivo heart perfusion system (Morrison et al. 2011). Postischemic recovery was significantly better with rosiglitazone treatment given at the onset of reperfusion in comparison with vehicle (p \ 0.001). Phospho-AMPK Thr(172) was significantly increased when rosiglitazone was administered 5 min before reperfusion compared with vehicle. Thus, these data once again evidence that AMPK and PPAR-c activation can occur under the same influence and exert anti-inflammation. High-fat diet and low physical activity lead to insulin resistance, non-alcoholic fatty liver disease (NAFLD)/NASH (non-alcoholic steatohepatitis) (Svegliati- Baroni et al. 2011). Recent data revealed an effect of glucagon-like peptide-1 (GLP-1) on liver glucose metabolism (Svegliati-Baroni et al. 2011). The presence of hepatic GLP-1r and the effect of exenatide, a GLP-1 analog, were examined on hepatic signaling. GLP-1r is expressed in human hepatocytes, although reduced in patients with NASH (Svegliati-Baroni et al. 2011). Also, in rats with NASH caused from 3 months of the high-fat diet, GLP-1r and PPAR-c expressions reduce. Treatment of hepatocytes with exenatide increased PPAR-c expression, which also exerted an insulin-sensitizing action via reducing JNK phosphorylation (Svegliati- Baroni et al. 2011). Exenatide also enhanced PKA activity and AMPK phosphorylation. These findings indicate that drugs against NASH can simultaneously activate PPAR-c and AMPK, and thus, PPAR-c and AMPK activation can occur 123 Biochem Genet under same stimuli (Svegliati-Baroni et al. 2011). Anti-diabetic effects of Morinda citrifolia (aka Noni) fermented by fast-fermented soybean paste were studied using a T2DM murine model (Lee et al. 2012b). Six-week-old KK-Ay/TaJcl mice were randomly assigned into four groups: (1) the diabetic control (DC) group, fed with a normal diet; (2) the positive control (PC) group, fed with a healthy food diet; (3) the M. citrifolia (MC) group, fed with an MC-based diet; (4) the fermented M. citrifolia (FMC) group, fed with an FMC-based diet (Lee et al. 2012b). Over an interval of 90 days, food and water intake declined significantly in the FMC and PC groups in comparison with DC group. Blood glucose levels in the FMC group were 211.6–252.2 mg/dL after 90 days, while those in the control group were over 400 mg/dL after 20 days. FMC also reduced glycosylated hemoglobin (HbA1c) levels, restored insulin sensitivity, and strongly decreased serum triglycerides and low-density lipoprotein (LDL) cholesterol (Lee et al. 2012b). A fermented M. citrifolia 70 % ethanolic extract (FMCE) induced PPAR-c and enhanced glucose uptake via stimulation of AMPK. A renal lipid accumulation occurs in diabetes and hypertension, which potentially leads to renal injury (Sakamoto et al. 2012). Angiotensin-2 infusions into rats stimulated lipid deposition in renal tubular epithelial cells. Pioglitazone (2.5 mg/ kg/day) reduced triglycerides in the kidney of the angiotensin-2-induced hyperten- sive rat (Sakamoto et al. 2012). Pioglitazone also increased phosphorylated AMPK. Proteinuria and renal weight in the angiotensin-2-infused rat were significantly decreased by pioglitazone (Sakamoto et al. 2012). Hence, a renoprotective effect of a PPAR-c agonist may involve activation of AMPK and thus supporting the idea of a synergism between PPAR-c ligands and AMPK activators. The effects of PPAR-c and PPAR-a agonist mono- and combination therapy on adipose and skeletal muscle gene expression were determined in relation to insulin sensitivity (Rasouli et al. 2012). Genome-wide transcripts of subcutaneous adipose and skeletal muscle and metabolic phenotypes were determined before and after 10 weeks of pioglitazone and fenofibrate mono- or combination therapy in 26 patients with perturbed glucose tolerance (Rasouli et al. 2012). PPAR-c, alone or in combination with PPAR-a agonists, upregulated genes mediating the TCA cycle, branched-chain amino acid (BCAA) and fatty acid metabolism, PPAR, AMPK, cAMP and insulin signaling pathways. PPAR-c alone or combined with PPAR-a decreased inflammatory responses in adipose tissue (Rasouli et al. 2012). Adiponectin is secreted from adipocytes into blood as high, medium and low molecular weight forms (HMW, MMW and LMW) (Chen et al. 2012c). Each oligomeric form of adiponectin exerts non-overlapping activities, with the HMW oligomer possessing the most efficient insulin-sensitizing efficacy (Chen et al. 2012c). Emodin, a phytochemical agent triggers AMPK in both 3T3-L1 adipocytes and 293T cells. Activation of AMPK by emodin induces the assembly of HMW adiponectin and elevates the ratio of HMW adiponectin to total adiponectin in 3T1- L1 adipocytes (Chen et al. 2012c). It was also shown that emodin activated PPAR-c and induced differentiation and adiponectin expression of 3T3-L1 preadipocytes (Chen et al. 2012c). Hence, the same influence triggered both PPAR-c and AMPK for insulin sensitization. Telmisartan is an angiotensin-2 type-1 receptor blocking drug which restores insulin sensitivity in obesity and insulin resistance (Shiota et al. 123 Biochem Genet

2012). Telmisartan also functions as a partial agonist of the PPAR-c, which is also a target of the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase (SIRT1). Telmisartan corrected insulin sensitivity in obese db/db mice fed a high-fat diet and the size of hypertrophic pancreatic islets. In vitro telmisartan exposure increased expression of Sirt1 mRNA in C2C12 skeletal muscle cells. Induction of Sirt1 mRNA in telmisartan-treated C2C12 myoblasts happened simultaneously with an increase of AMPK phosphorylation and upregulation of NAD?/NADH ratio (Shiota et al. 2012). Thus, PPAR-c and AMPK activation occured simultaneously under the inﬂuence of telmisartan. PPAR-c agonist rosiglitazone alleviates TNF-a- related injury of human neural stem cells (hNSCs) (Chiang et al. 2013). PPAR-c augmented the cell vitality of hNSCs via inhibiting caspase-3 and increasing the levels of two mitochondrial regulators, AMPK and SIRT1 (Chiang et al. 2013). The stimulation of mitochondria by PPAR-c was associated with the activation of the PPAR coactivator1a (PGC-1a) pathway by augmentation of antioxidant defense and mitochondrial systems (Chiang et al. 2013).

Cachexia/Wasting Syndrome with Central Role in Pancreas Cancer Morbidity and Mortality: Association with Inﬂammatory Cascades

About half of all cancer patients suffer a cachexia syndrome, with the frequency higher in patients with solid cancers (Martignoni et al. 2005). A generally accepted definition of cachexia is not available as yet. On the other hand, an unintentional weight loss of [5–10 % of total body weight over a maximum of 6 months is generally assumed as a cachexia indicator. Pancreatic and gastric cancer patients have the highest frequency of cancer cachexia (Martignoni et al. 2005), and PC patients seem to be especially vulnerable, about 80 % of them presenting cachexia at time of diagnosis (Ockenga and Valentini 2005). Peculiarly, PC patients suffer from profound cachexia and decrease in performance status, even when their tumor load is low (Ebrahimi et al. 2004) and the discrepancy of poor prognosis and quite small tumor mass is noteworthy (Ockenga and Valentini 2005). In these patients, death can not be solely explained by the tumor burden or metastases, but rather by metabolic derangements caused by PC (Ockenga and Valentini 2005). Thus, PC cachexia significantly associates with worse prognosis (Cooperman et al. 2000) and contributes to the enworsened quality of life and mortality (Melstrom et al. 2007). PC cachexia occurs due to high resting energy expenditure; an acute-phase response determines a group of patients who are hypermetabolic (Ebrahimi et al. 2004). These patients exert high intrinsic IL-6 production from peripheral blood mononuclear cells (Ebrahimi et al. 2004). Studies also demonstrated that the production of C-reactive protein (CRP, which reflects acute-phase response) by peripheral blood mononuclear cells correlated with IL-6 expression in PC patients and that anti-IL-6 antibodies reduced CRP. A significant correlation between increased levels of several cytokines (IL-8, IL- 10, IL-6 and IL-1RA) and PC phenotypes, including cachexia and asthenia, as well as an association with prognosis (Ebrahimi et al. 2004) was shown. Blood levels of IL-1RA, IL-6, IL-8 and IL-10 all were enhanced significantly (Ebrahimi et al. 123 Biochem Genet

2004). Analyses revealed a correlation between IL-1RA and IL-8, between IL-6 and IL-10 and between IL-8 and IL-10. Patients with higher serum IL-6 levels (5.2 pg/ mL) had lower 1-year survival compared with patients who had low IL-6 levels (Ebrahimi et al. 2004). Higher serum IL-10 levels were also correlated with worse survival compared with lower levels. Serum IL-8 levels were not found to associate with survival. For the multivariate analysis which encompassed only the cytokines as potential confounders, the model selected only high IL-6 levels (Ebrahimi et al. 2004). The correlation between weight loss at the time of sample draw and the IL-8 level was significant. The median weight loss among patients with low IL-8 was 6.5 pounds (range 0–55 pounds) in comparison with a median weight loss of 20 pounds (range 0–80 pounds) among patients with high IL-8 (p \ 0.008). IL-8 is a cytokine/ chemokine, which induces tumor growth both directly and via pro-angiogenic effects. Higher IL-6 levels also associated with poor performance status (Karnofsky or ECOG) and low albumin levels. These data make sense when remembering that IL-6 can induce cachexia and fever and is substantially linked to fever and weight loss in patients with recurrent lymphomas (Ebrahimi et al. 2004). IL-10 is a multifunctional cytokine produced by type-2 helper cells (Th2) as well as monocytes and macrophages, which has immune suppressive effects via the inhibition of Th1-type cytokines, including interferon and IL-2 (Ebrahimi et al. 2004). TNF-a and IL-6 activate proteolysis, insulin resistance, apoptosis and the NF-jB pathway contributing to catabolic processes and subsequent cachexia (Martignoni et al. 2005). Hence, the term ‘‘cytokine driven’’ is often employed in connection with cachexia. In PC cell lines, high expressions of IL-6 and IL-8 occur, likely reflecting high rates of cachexia in PC (Martignoni et al. 2005). IL-6 mRNA expression was significantly higher (p \ 0.01) in tumor specimens of cachectic in comparison with non-cachectic PC patients. Furthermore, IL-6 levels were significantly higher in patients with cachexia compared with patients with chronic pancreatitis and healthy controls (Martignoni et al. 2005). Tissue areas surrounding the tumor did not demonstrate IL-6 immunoreactivity, while IL-6 was strongly present in the cytoplasm of PC cells (Martignoni et al. 2005). IL-6 serum levels were also different in patients with and without cachexia. Combination of TNF-a and IFN-c suppresses myoD and myosin expression via activating NF-jBin myocytes. MyoD is essential for repair and regeneration of muscle fibrils, which indicates that cytokine-mediated sarcopenia may occur via activation of NF-jB through blockage of muscle regeneration. TNF-a and IL-6 are major inflammatory cytokines, which are elevated in chronic inflammation and which also lead to declines of muscle and fat mass. Megestrol is a steroidal and ibuprofen is a nonsteroidal cyclooxygenase inhibitor and both decrease energy expenditure in PC (Uomo et al. 2006). Few studies have been published on this subject, yet the results seem to be encouraging. Multiple studies have demonstrated that the metabolic changes associated with cancer cachexia are unique in comparison with starvation (Melstrom et al. 2007). Cancer patients lose a larger proportion of skeletal muscle mass. There exist three pathways in muscle protein degradation: the lysosomal system, cytosolic proteases and the ubiquitin (Ub)-proteasome pathway (Melstrom et al. 2007). The Ub-proteasome pathway accounts for the main of skeletal muscle 123 Biochem Genet degradation process in cancer cachexia and is induced by several cytokines including TNF-a, interleukin-1b, interleukin-6, IFN-c and proteolysis-inducing factor (Melstrom et al. 2007). Two hundred and twenty-seven PC patients over an 18-month period were studied regarding cachexia and its action on morbidity and mortality. In 40.5 % of the patients, cachexia was present at the time of operation. The cachectic patients exerted a worse nutritional status, represented by lower protein, albumin and hemoglobin (Bachmann et al. 2008). Despite no significant differences in tumor size, lymph node status and CA19-9 levels, the resection rate in patients with cachexia was less (77.8 vs. 48.9 %) due to higher rates of metastases (Bachmann et al. 2008). The survival period in patients with cachexia was significantly shorter in patients both underwent surgery and receiving palliative treatment (Bachmann et al. 2008). Furthermore, cachexia was not necessarily associated with tumor burden. The relationships between the acute-phase response proteins (APRPs), cytokine production and survival in PC patients were examined (Moses et al. 2009). Forty-two patients with PC cachexia and twelve age-matched healthy controls were recruited. The nutritional status, Karnofsky performance score, CRP, serum IL-6 and in vitro monocyte IL-1 and IL-6 production were determined (Moses et al. 2009). The cancer patients had significantly lesser body mass index, Karnofsky performance score and serum albumin, while they concurrently had increased CRP and IL-6 levels (Moses et al. 2009). Both univariate and multivariate analyses demonstrated profound associations between tumor stage, CRP, stimulated IL-6 production and survival. Monocytes in cachectic PC patients are primed to produce high levels of IL-6 upon stimulation and IL-6 overproduction negatively impacted survival. Decreased survival is associated with an elevated APRP (Moses et al. 2009). The multiple effects of cachexia in PC patients were analyzed, in terms of resection rate, effects on pulmonary function, amount of fat and muscle tissue, as well as changes in laboratory parameters (Bachmann et al. 2009). Of 198 patients with PC, 70 % were suffering from weight loss at time of operation, and in 40 % weight loss exceeded 10 % of the stable weight (Bachmann et al. 2009). In patients with cachexia, metastases were significantly more frequent (47 vs. 24 %, p \ 0.001), leading to a significantly reduced resection rate. PC patients with cachexia had more progressed tumor stages and a worse nutritional status. Moreover, patients with cachexia had an impaired lung function and a reduction in fat tissue (Bachmann et al. 2009). Patients with PC and cachexia had lived significantly shorter. If weight loss exceeded 5 %, the resection rate significantly declined, and the changes were more profound if weight loss was 10 % or more (Bachmann et al. 2009). The relationships among body mass index, longitudinal body composition alterations and clinical outcomes in PC patients were explored (Dalal et al. 2012). Records of 41 patients with inoperable PC who participated in a chemoradiation study were reviewed. Sarcopenia was observed in 26 (63 %) patients. At follow-up, weight loss was witnessed in 33 (81 %) patients. The median losses (%) before and after treatment were weight 5 % (p \ 0.001), skeletal muscle (SKM) 4 % (p = 0.003), visceral adipose tissue (VAT) 13 % (p \ 0.001) and subcutaneous adipose tissue 11 % (p = 0.002). In univariate analysis, age, and 123 Biochem Genet losses (%) in weight, SKM and VAT were associated with worse survival. In multivariate analysis, age (hazard ratio = 1.033, p = 0.04) and higher VAT loss (hazard ratio = 2.6 and p = 0.03) remained significant. cDNA-AFLP screening revealed that the genes encoding APRP’s, fibrinogen-a (FGA), fibrinogen-c (FGG), a1-antitrypsin and a2-macroglobulin were induced in the skeletal muscle of PC patients with cachexia (Skorokhod et al. 2012). In parallel, a sixfold upregulation of IL-32 was observed, a cytokine with an important inflammatory role as a direct inducer of TNF-a. Together with a continuing inflammatory response, a significant stimulation of early response genes Egr-1 (early growth response-1) and IER-5 (immediate early response-5) was seen in wasted PC patients (Skorokhod et al. 2012). Egr-1, the master switch of inflammatory responses is directly involved in ‘‘early signal events.’’ Egr-1 also has a major role in the regulation of over 40 target genes, including TNF-a, IL-1b and IL-6, all of which can induce various features of cachexia (Skorokhod et al. 2012). An analysis was made on 2968 pancreatic resections, 408 patients with primary PC who underwent pancreatoduodenectomy and of whom cross-sectional images were available. They were identified and followed up in a prospective database (Pausch et al. 2012). Patients with lower body mass index had a higher 90-day mortality (p = 0.048) and a trend toward greater complication rates and in- hospital mortality, despite a greater comorbidity in obese patients with higher body mass indices (Pausch et al. 2012). Furthermore, patients with large amounts of abdominal wall fat had fewer intra-abdominal abscesses (p = 0.047), lower in- hospital (p = 0.019) and 90-day mortality rates (p = 0.007), and better long-term survival (p = 0.016). It was revealed that in pancreatic cancer, underweight but not obese patients have a poor outcome after pancreatoduodenectomy.

Glioblastoma Multiforme: A Frequent and Very Fatal Malignancy

Gliomas are the most common type of human brain tumors and contain specific histologic subtypes with most common forms as astrocytomas, oligodendrogliomas and ependymomas (Fu et al. 2014). These tumors are classified as grades I–IV on the basis of histopathological and clinical criteria established by the World Health Organization (WHO) (Fu et al. 2014). Generally, WHO grade I gliomas are mostly curable with total surgical resection and rarely evolve into higher-grade lesions (Fu et al. 2014). In contrast, gliomas of WHO grade II or III are invasive, progress to higher-grade lesions and have a poorer outcome (Fu et al. 2014). The grade IV glioma known as glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults and can develop either from de novo (primary GBM) or through progression from low-grade tumors (secondary GBM) (Fu et al. 2014). GBM remains one of the most devastating diseases known to man and affects more than 17,000 patients in the USA alone every year (Soritau et al. 2011). Invasiveness is one of the main hallmarks of primary glial brain tumors, in which malignant cells diffusely infiltrate normal brain tissue and migrate along defined structures of the brain (Nowicki et al. 2008). GBM infiltrates the brain early in its course and makes total neurosurgical resection almost impossible (Soritau 123 Biochem Genet et al. 2011) and is a major reason for the continued poor prognosis (median survival around 12 months) (Nowicki et al. 2008).

Metformin Efﬁcacy in Glioblastoma: Basic Science Evidence

Glycolytic enzymes exist in ample levels, and some were found to be increased in pseudopodia formed by U87 glioma cells (Beckner et al. 2005). The ability of glioma cells to remove the glycolysis inhibitor lactate via gluconeogenesis and incorporation into glycogen led the assumption of supportive mutations (Beckner et al. 2005). Loss of phosphatase and tensin homolog (PTEN) releases glycogenesis from permanent blockage by glycogen synthase kinase-3 (GSK-3) (Beckner et al. 2005). It was assumed that glycolysis in gliomas may propagate migration. Migration of PTEN-mutant U87 cells was investigated for lactate release and support by gluconeogenesis and active PI3K (Beckner et al. 2005). Lactic acid levels plateaued and activation of the PI3K/Akt pathway was witnessed when cells relied only on glycolysis (Beckner et al. 2005). Glycolytic migration was suppressed by inhibiting gluconeogenesis with wortmannin (Beckner et al. 2005). From eight patients with newly diagnosed high-grade gliomas, tumor tissue cultures were established and the sensitivity to metformin and temozolomide (TMZ) was tested (Soritau et al. 2011). Microvascular density (MVD) assay was performed on the tumor samples (Soritau et al. 2011). Seven of the eight cases had a positive correlation between the number of endothelial cells, the phenotype of isolated tumor cells and the response to adjuvant chemoradiotherapy. There was an important difference between TMZ alone and TMZ plus metformin arms in six of the cases in terms of higher tumor inhibition (Soritau et al. 2011). Although the primary path leading to TMZ efficacy is the formation of O-6- methylguanine and apoptotic signaling triggered by O-6-methyl G:T mispairs, that apoptotic signaling goes through a step mediated by AMPK and hence metformin was assumed to augment TMZ efficacy. FOXO3 activation triggers differentiation of glioma-initiating tumor stem cells (GSCs) and metformin was found to activate FOXO3 (Sato et al. 2012). Moreover, systemic administration of metformin reduced the GSCs within established tumors and provided a prominent survival benefit (Sato et al. 2012). The authors have proposed that metformin is the most clinically relevant drug ever reported for targeting of GSCs (Sato et al. 2012). The effects of metformin on the proliferation and invasion of LN18 and LN229 GBM cells were examined under basal conditions or exposed to leptin, a cytokine which involves in glioblastoma development (Ferla et al. 2012). Metformin at concentrations between 2 and 16 mM reduced basal and leptin-stimulated growth of GBM cells in a dose-dependent manner and also significantly blocked their invasion (Ferla et al. 2012). The action of metformin was mediated via upregulation of AMPK and through the downregulation of STAT3 and the Akt/PKB serine/threonine protein kinase (Ferla et al. 2012). Metformin reversed the effects of leptin on the AMPK and STAT3 pathways, but modified Akt activity in a cell-dependent manner (Ferla et al. 2012).

123 Biochem Genet

Metformin reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human GBMs and impaired tumor-initiating cell spherogenesis (Wu¨rth et al. 2013). A higher inhibition of proliferation on CD133- expressing subpopulation was witnessed as compared to CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity (Wu¨rth et al. 2013). Metformin effects in tumor-initiating cell-enriched cultures were linked to a robust inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells (Wu¨rth et al. 2013). The specificity of metformin toward GSCs was confirmed by the lack of significant inhibition of umbilical cord- derived healthy mesenchymal stem cells (Wu¨rth et al. 2013). Metformin blocked LKB1/AMPK/mTOR/S6K1 pathway-dependent cell growth, induced selective cytotoxicity on GSCs by inhibiting the GSC-initiated spherogenesis and blocked the proliferation of CD133? cells, while having a low or null effect on normal human stem cells (Carmignani et al. 2014). Metformin induced both autophagy and apoptosis in glioma cells by inhibiting PI3K/Akt and inducing the MAPK pathways, respectively. Metformin also triggered differentiation of GSCs into non-tumorigenic cells (Carmignani et al. 2014). Considering that metformin easily crosses blood– brain barrier, the authors have commented that a prompt clinical assessment of metformin in glioblastoma patients would represent a valid attempt to improve survival (Carmignani et al. 2014). Liu et al. (2014) surprisingly found that compared with normal tissue, AMPK is constitutively active in human and mouse gliomas. Hence, they questioned whether the anti-proliferative actions of metformin could be AMPK independent in GBMs. Very noteworthy, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation while metformin directly inhibited mTOR by enhancing PRAS40’s association with Raptor (Liu et al. 2014). The authors have speculated that pathophysiological activation of AMPK in glioma cells may be a compensatory mechanism to metabolic stress, yet metformin activation of AMPK would not cause any untoward effects since it activates multiple anti-tumor pathways (Liu et al. 2014). GSCs, responsible for the dismal disease prognosis after conventional treatments, are driven by overactive signaling pathways, such as PI3K/AKT/mTOR and RAS/RAF/MAPK. Hence, Aldea et al. (2014) aimed a target in vitro GSCs by combining metformin as a mTOR inhibitor, with a RAF inhibitor, sorafenib (soraf). GSC cultures were treated with metformin, temozolomide (TMZ), soraf, metformin?TMZ and metformin?soraf, as untreated arm served as control (Aldea et al. 2014). Metformin?soraf exerted the highest anti- proliferative effects in GSCs and non-stem GBM cells (p \ 0.001) (Aldea et al. 2014). Both metformin and soraf alone exhibited a selective cytotoxic effect on GSCs (p \ 0.001), while no effect was detected on non-stem GBM cells (p [ 0.05). These results strongly indicate metformin selectivity for cancer stem cells indicating a great potential to eliminate treatment-resistant tumor cells. Further, metformin displayed synergism with soraf in producing high levels of ROS, decreasing efflux pump activity and generating the highest apoptotic rates when compared to either drug alone (p \ 0.001) (Aldea et al. 2014). Metformin acted cytotoxic and triggered apoptosis in the T98G cells, and caspase-3 levels in the metformin-treated T98G cells were higher than those in the 123 Biochem Genet control cells (Ucbek et al. 2014). Metformin induced apoptosis in the T98G cell line in a concentration-dependent manner (Ucbek et al. 2014). Gritti et al. (2014) demonstrated that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin blocked proliferation in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human GBMs (Gritti et al. 2014). These actions accompanied to metformin-inhibition of a chloride current which is dependent on functional activity of CLIC1 (Gritti et al. 2014). CLIC1 ion channel is selectively active during the G1-S transition via transient membrane insertion (Gritti et al. 2014). Metformin inhibition of CLIC1 induced G1 arrest of GSCs, which was time dependent, and prolonged exposures blocked cell proliferation also for low—albeit clinically more significant— metformin concentrations (Gritti et al. 2014). Further, substitution of Arg29 in the CLIC1 pore region hindered metformin modulation of channel activity (Gritti et al. 2014). CLIC1 was defined not only as a modulator of cell cycle progression in human GSCs but also as one of the main targets of metformin’s anti-proliferative activity (Gritti et al. 2014). Sesen et al. (2015) demonstrated that metformin decreased proliferation and induced cell cycle arrest, autophagy and apoptosis in GBM cells in vitro with activation of AMPK, Redd1 and inhibition of the mTOR. Knockdown of AMPK and Redd1 with siRNA partially, but incompletely, hindered apoptosis induction by metformin indicating both AMPK/Redd1-dependent and AMPK/Redd1-independent effects (Sesen et al. 2015). They further showed that metformin treatment in combination with TMZ and/or irradiation induced a synergistic anti-tumoral response in GBM cell lines (Sesen et al. 2015). Xenografts performed in nude mice also demonstrated that metformin delayed tumor growth (Sesen et al. 2015). Mouhieddine et al. (2015) showed that metformin significantly decreased both the viability and invasion of U251 GBM cells. Metformin effectively blocked the activation induced by TMZ, and a combination of both drugs led to higher reduction of mTOR, 4EBP1 and S6K phosphorylation (Yu et al. 2015). In addition, the combination of the two induced a powerful AMPK activation (Yu et al. 2015). Xenografts performed in nude mice demonstrated that combined treatment significantly reduced tumor growth rates and prolonged median survival of tumor-bearing mice (Yu et al. 2015). The same group also published that TMZ and metformin synergism also encompassed glioma stem cells. Seliger et al. found that metformin at doses as low as 0.01 mM thrice a day was able to inhibit proliferation of susceptible GBM cell lines, with increased lactate secretion, reduced oxygen consumption and activated AMPK signaling (Seliger et al. 2016).

Metformin Efﬁcacy in GBM: Clinical Evidence

Welch and Grommes (2013) retrospectively analyzed 998 GBM patients with DM2 seen from 1998 to 2010. One hundred and twenty-four (12.6 %) patients were affected by DM2 including 34 who developed DM2 after steroid use and 89 who had preexisting DM2 (Welch and Grommes 2013). Median overall survival among diabetic GBMs was 10 months compared with 13 months among non-diabetics; 123 Biochem Genet only 15 % of DM2 patients could achieve permanent steroid taper (Welch and Grommes 2013). Sixty-seven (54 %) were managed with a single anti-diabetic, and, within this monotherapy group, Karnofsky Score, resection status, steroid depen- dency and metformin use were the most important modifiers of survival on multivariate analysis (Welch and Grommes 2013). Although patients treated with sulfonylureas had worse outcomes, patients treated with metformin had higher median survival compared with all other anti-diabetic drugs (Welch and Grommes 2013). Two years later, Adeberg et al. (2015) investigated the influence of diabetes mellitus, corticosteroid therapy and metformin therapy on progression and survival in primary GBM patients. Among 276 patients with primary GBM, forty patients (14.5 %) were identified with a pretherapeutic history of diabetes, and 20 (50 %) of them were treated with metformin (Adeberg et al. 2015). Survival and correlations were assessed via t test and log-rank, univariate and multivariate Cox proportional hazards ratio analyses (Adeberg et al. 2015). Persistent mild and excessive hyperglycemia was correlated with lower survival rates (Adeberg et al. 2015). Corticosteroid usage was associated with lower progression-free and overall survival in the multivariate analysis (Adeberg et al. 2015). No negative influence of diabetes could be detected; however, diabetic patients with metformin therapy demonstrated prolonged progression-free intervals (Adeberg et al. 2015).

Pioglitazone Efﬁcacy in Glioblastoma: Basic Science Evidence

Zang et al. (2003) have shown that 11 GBM cell lines and nine fresh GBM tissue samples from patients expressed abundant amounts of PPAR-c. On the contrary, glia obtained from nine healthy brain tissue samples expressed very low amounts of PPAR-c (Zang et al. 2003). No mutations or polymorphisms of the PPAR-c gene were observed in these cell lines (Zang et al. 2003). The effect of pioglitazone either in the absence or in the presence of a RAR ligand [all-trans retinoic acid (ATRA)] on the proliferation and apoptosis of GBM cells was examined using two GBM cell lines (N39 and DBTRG05MG) (Zang et al. 2003). Pioglitazone and/or ATRA significantly blocked the proliferation and induced apoptosis in both cell lines, which was associated with a decrease of bcl-2 and an increase of bax proteins (Zang et al. 2003). At certain concentrations, glitazones exerted a selective lethality on glioma cells versus astrocytes which was associated with a rapid production of reactive oxygen species (ROS), peroxynitrite and hydrogen peroxide and seemed unrelated to PPAR-c (Pe´rez-Ortiz et al. 2007). ROS production was completely blocked by uncoupling of the electron transport chain and by removal of glucose as an energy substrate (Pe´rez-Ortiz et al. 2007). Additionally, glitazones inhibited state 3 respiration in permeabilized cells, and complex I was the putative target (Pe´rez- Ortiz et al. 2007). Glitazones also depolarized mitochondria and reduced mitochondrial pH (Pe´rez-Ortiz et al. 2007). Further, NO synthase inhibitors revealed that superoxide anion combines with NO to yield peroxynitrite which involved in the cytotoxicity of glitazones in glioma cells (Pe´rez-Ortiz et al. 2007). Cytostatic and anti-invasive mechanisms of pioglitazone and 11-O-hydrox- yfenantrene (IIF), a retinoid-X-receptor (RXR)-ligand in three different GBM cell 123 Biochem Genet lines were studied (Papi et al. 2009). The combination strongly decreased tumor invasion and matrix metalloproteinase-2 and matrix metalloproteinases-9 expression. Combined treatment also profoundly inhibited proliferation and triggered apoptosis in all GBM cell lines tested accompanied by decrease of anti-apoptotic Bcl2 and p53, and increase of pro-apoptotic cytochrome c, cleaved caspase-3, Bax and Bad levels (Papi et al. 2009). These observations were further supported in a murine glioma model in vivo, where oral administration of pioglitazone and IIF significantly reduced tumor volumes (Papi et al. 2009). Very noteworthy, this combination was not only effective when the treatment was started shortly after the intraparenchymal seeding of the glioma cells, but even when started in the last third of the observation period (Papi et al. 2009). Tapia-Pe´rez et al. (2011) compared the effects of several statins and PPAR-c inhibitors rosiglitazone and pioglitazone on human GBM cell lines U87, U138, LN405 and rat GBM, RG II. The cytotoxic effect was determined after 48 and 144 h (Tapia-Pe´rez et al. 2011). A significant cytotoxic effect was shown with a combination of statins plus pioglitazone, which was observed after 48 h and dramatically increased after 144 h (Tapia-Pe´rez et al. 2011). Further, cytotoxicity of the combination of statins and glitazones did not decrease under hypoxia (Tapia-Pe´rez et al. 2011).

Pioglitazone Efﬁcacy in GBM: Clinical Evidence

In a retrospective chart review, the inﬂuence of PPAR-c agonists on the odds of having a high-grade glioma was determined (Grommes et al. 2010). Patients with a diagnosis of anaplastic astrocytoma and GBM between 1999 and 2008 were reviewed and patients with hip fractures served as the control group (Grommes et al. 2010). A total of 1602 hip fracture patients and 302 high-grade glioma patients were deﬁned, and 15 and 16 % were diabetics, respectively (Grommes et al. 2010). PPAR-c agonists were used by 20 % of diabetic hip fracture patients and by 6 % of high-grade glioma patients (Chi-square p value = 0.02) with an odds ratio of 4.08, which indicated a possible anti-neoplastic effect of PPAR-c agonists in gliomas (95 % CI 1.119–14.881) (Grommes et al. 2010).

Lithium Concentrations Achieved During Treatment of Bipolar Disorder and Higher Lithium Uptake of Glioblastoma Cells Over Neuroblastoma Cells

If lithium ions were passively distributed between the intra- and extracellular water, the internal negative membrane potential would cause a cellular accumulation of lithium (Reiser and Duhm 1982). However, the stagnant lithium concentration in nerve and muscle cells and also in non-excitable cells remains in the range of or below the plasma level (Reiser and Duhm 1982). A passive distribution of lithium would yield a ratio for intracellular/extracellular lithium concentration of about 5, as derived from the Nernst equation (Reiser and Duhm 1982). The actual ratios found are below 1 in the glioma-neuroblastoma hybrid cells and in motor neurons (Reiser 123 Biochem Genet and Duhm 1982). It was revealed that not only the Na?–K? pump extrudes lithium ions, but rather a quabain-resistant Na?–Li? countertransport expells this cation, which could act toxic at high levels (Reiser and Duhm 1982). A number of facts about glia indicate that they may also play roles in the therapeutic efficacy of lithium (Gorkin and Richelson 1979). Glia outnumber neurons by a factor of 10 and comprise up to 50 % of the bulk of the brain (Gorkin and Richelson 1979). Glia interact with neurons by several ways, including providing mechanical support and electrical insulation to neighboring neurons, defining anatomical routes for nerve growth during development and regeneration, and acting as biochemical regulators involved in neurotransmitter metabolism and ionic buffering (Gorkin and Richelson 1979). Neuroblastoma cells accumulate lithium in such a manner that veratridine, an alkaloid which specifically opens sodium channels responsible for the action potential, increases both the rate of entry and the steady-state intracellular concentration of lithium (Gorkin and Richelson 1979). At low external concentrations, lithium accumulation by C6 rat glioma cells exceeds that observed in veratridine-stimulated neuroblastoma cells and the level of intracellular lithium in rat glioma cells is inversely proportional to the extracellular potassium concentration (Gorkin and Richelson 1979). However, unlike neuroblastoma cells, in which steady-state levels are attained within 1 h, glioma cells continue to slowly accumulate lithium over a period of several days (Gorkin and Richelson 1979). This time course for lithium accumulation by glioma cells is more consistent with the time course for clinical response (10–14 days). Intracellular lithium levels in glioma cells after 1 h incubation are consistently lower than the levels attained in these cells at the same degree of confluency but chronically ([1 day) exposed to lithium. Furthermore, veratridine has no effect on lithium entry into rat glioma cells. Upon chronic exposure, glioma cells effectively concentrate lithium from low external concentrations (Gorkin and Richelson 1979). Unlike human erythrocytes or neuroblastoma cell line SH-SY5Y, the glioma cell line A1B1 (cloned from the human glioma U251- MG cells) has neither a quabain or phloretin-sensitive component of lithium entry (Saneto and Perez-Polo 1982). Furthermore, glioma cells concentrate significantly more (fourfold) lithium and at a faster rate when compared to the neuroblastoma cell, SH-SY5Y, over the same time period (Saneto and Perez-Polo 1982).

Lithium Against GBM: Experimental Evidence

Glioma invasion is linked to specific molecular changes in extracellular matrix composition, cell adhesion and cytoskeletal dynamics (Nowicki et al. 2008). However, potential anti-invasive therapeutics have not yet successfully translated to the clinic. First in 2008, it was reported that lithium potently and reversibly blocked glioma cell migration and invasion in vitro in different assays and in all cell lines tested (6/6) (Nowicki et al. 2008). Lithiums anti-invasive effect differed from its effects on cell proliferation because: (1) the effects of lithium on migration are of much greater overall magnitude than effects on viability; (2) they occur at lower doses and earlier time points than effects on cell viability; and (3) they are 123 Biochem Genet reversible, even after 96-h treatment, suggesting that the blockage of migration is not due to cytotoxicity (Nowicki et al. 2008). Indeed, lithium caused the retraction of the long protrusions at the front of the migrating cell, which was fully reversible upon withdrawal of treatment (Nowicki et al. 2008). Second, the degree of GSK-3b inhibition (measured by a luciferase reporter assay) showed an inverse correlation with the degree of invasion, suggesting a direct link between GSK-3b inhibition and the blockage of glioma invasion (Nowicki et al. 2008). This was further supported by the finding that siRNA knockdown of either GSK-3a or GSK-3b reduced migration in a wound-healing assay, revealing that both GSK-3 isoforms play a role in glioma invasion (Nowicki et al. 2008). Significant effects of lithium on glioma migration were observed at high concentrations around 5 mM (Nowicki et al. 2008). At the first glance, these levels seem unachievable due to toxic effects described above. Nonetheless, due to the fact that GBM cells continue to accumulate lithium ions at chronic exposure, this efficacy may still be observed in a clinical context. The gene encoding isocitrate dehydrogenase-1 (IDH1) is mutated mainly in secondary GBM (Fu et al. 2014). However, the frequencies of mutations in isocitrate dehydrogenase-2 (IDH2) in these gliomas are rare (Fu et al. 2014). IDH enzymes catalyze the oxidative decarboxylation of isocitrate (ICT) into a-ketoglutarate (a-KG), and their activities depend on either nicotinamide adenine dinucleotide phosphate (NADP?-dependent IDH1 and IDH2) or nicotinamide adenine dinucleotide (NAD?- dependent IDH3) (Fu et al. 2014). Glioma-specific mutations in IDH1 produce a single amino acid substitution at R132 and mutations in IDH2 influence R172 which is the analogous site to R132 in IDH1 (Fu et al. 2014). Mutations in IDH1 and IDH2 lead to simultaneous loss and gain in the production of a-KG and 2-hydroxyglutarate (2-HG), respectively (Fu et al. 2014). Lithium chloride could inhibit cell proliferation of C6 glioma cells both with and without IDH2 mutation, although IDH2 mutation increased the stability of HIF-1a, which enhances tumor resistance and angiogenesis. Moreover, although the b-catenin and HIF-1a increased the secretion of metalloproteinase-2, metalloproteinase-9 in C6 glioma cells carrying IDH2 mutation, the migration potential of lithium-treated C6 glioma cells harboring the IDH2 and its mutant was uniform which indicated lithium could decrease the proliferation and migration potential of C6 glioma cells harboring IDH2 mutation (Fu et al. 2014).

Conclusions

Development of novel and efficient treatment strategies for PC and GBM has vital importance considering their treatment refractoriness and very high mortality. Molecular targeted therapies are underway, yet cancer cells could swiftly bypass the inhibitory effects of these therapies and develop resistance. Moreover, these targeted therapies often have very high costs, which may hinder their usage in less developed countries. Targeting multiple molecular pathways simultaneously and with already in-use cheap drugs would be a logical approach. Until now, classical chemotherapy agents almost always acted via the proliferation differences in malignant versus benign cells, and thus, they were mostly DNA poisons (e.g., cisplatin, cyclophosphamid), DNA replication-blocking agents (e.g., 5-FU, 123 Biochem Genet gemcitabine) or microtubule inhibitors (e.g., vincristine, vinorelbine). In recent years, targeting metabolic differences of cancer cells gained more popularity, although Warburg effect (glycolysis preference of malignant cells to gain energy faster) was described as early as 1950s. AMPK, GSK-3b and PPAR-c are significant modifiers of cancer metabolism, and activation of AMPK and PPAR-c and inhibition of GSK-3b were demonstrated to significantly block both PC and GBM growth. These growth inhibitory effects occur both via decrease in cell proliferation and enhancing intrinsic vulnerability of PC and GBM cells to apoptotic pathways. Thus, these therapies also provide a chance to increase PC and GBM sensivitity to orthodox chemotherapies, such as gemcitabine and TMZ. Moreover, catabolic catastrophe of PC patients with severe cachexia/wasting syndrome can also be targeted simultaneously with drugs modifying AMPK, GSK-3b and PPAR-c, since they could block inflammatory and NF-jB-dependent cascades. If cell culture and animal studies prove our hypothesis and demonstrate a high efficacy of such a triple combination against PC and GBM, further clinical studies would be logical attempts against these grave malignancies.

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