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Sirt1 (Sirtuin 1), AMPK (AMP-activated protein kinase), and eNOS (endothelial nitric oxide synthase) modulate hepatic energy metabolism and inflammation and play a major role in the development of NASH. Cyclic nucleotide phosphodiesterases (PDEs) play an important role in signal transduction by modulating intracellular levels of cyclic nucleotides. We previously found the PDE5 inhibitor sildenafil to synergize with leucine and leucine-metformin combinations in preclinical studies of NASH and obesity. However, efficacy is diminished at higher sildenafil concentrations. Herein, we have successfully modeled the U-shaped sildenafil dose-response in vitro and utilized this model to assess potential mechanisms of this dose-response relationship. Adipocytes and liver cells were treated with leucine (0.5 mM) and different concentrations of sildenafil (1 nM to 100 &micro, M). cAMP, cGMP, and P-AMPK protein expression were used to demonstrate the biphasic response for increasing concentrations of sildenafil. The reversal with higher sildenafil levels was blunted by PDE2 inhibition. These data indicate that sildenafil-mediated increases in cGMP inhibits PDE3 at lower concentrations, which increases cAMP. However, further increases in cGMP from higher sildenafil concentrations activate PDE2 and consequently decrease cAMP, which demonstrates crosstalk between cAMP and cGMP via PDE2, PDE3, and PDE5. These changes in cAMP concentration are further reflected in downstream effects, including AMPK activation.

Background We have previously shown leucine (Leu) to activate Sirt1 by lowering its KM for NAD+, thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving ~ 80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction. Methods Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met–Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies. Results Leu (0.5 mmol/L) + Met (50–100 μmol/L) synergistically activated Sirt1 (p Conclusion Thus, Leu and Met synergize to enable Sirt1 activation at low NAD+ concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met–Leu's full action, which result in improvements in energy metabolism and insulin sensitivity.

// Jheelam Banerjee 1 , Arokya M.S. Papu John 1 , Mohammed H. Al-Wadei 1 , Hildegard M. Schuller 1 1 Experimental Oncology Laboratory, Department of Biomedical & Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA Correspondence to: Hildegard M. Schuller, email: hmsch@utk.edu Keywords: pancreatic cancer, prevention, γ-aminobutyric acid, cyclic adenosine monophosphate, cancer stem cell signaling Received: December 2, 2015 Accepted: May 22, 2016 Published: June 02, 2016 ABSTRACT Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). We have previously shown that these cancers overexpressed stress neurotransmitters and cyclic adenosine monophosphate (cAMP) while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) was suppressed. Using a hamster model, the current study has tested the hypothesis that cAMP decrease by GABA supplementation in the drinking water prevents the development of pancreatitis-associated PDAC. Our data reveal strong preventive effects of GABA supplementation on the development of PDAC and pancreatic intraductal neoplasia (PanIN). ELISA assays and immunohistochemistry revealed significant decreases in the levels of cAMP and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers and phosphorylated signaling proteins, which stimulate cell proliferation, and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP decrease by GABA supplementation inhibits multiple cancer stimulating pathways in cancer stem cells, differentiated cancer cells and the immune system, identifying this approach as promising novel tool for the prevention of PDAC in individuals with a history of smoking and alcoholism.

A small subpopulation of pancreatic cancer cells with characteristics of stem cells drive tumour initiation, progression and metastasis. A better understanding of the regulation of cancer stem cells may lead to more effective cancer prevention and therapy. We have shown that the proliferation and migration of pancreatic cancer cell lines is activated by the nicotinic receptor-mediated release of stress neurotransmitters, responses reversed by γ-aminobutyric acid (GABA). However, the observed cancer inhibiting effects of GABA will only succeed clinically if GABA inhibits pancreatic cancer stem cells (PCSCs) in addition to the more differentiated cancer cells that comprise the majority of cancer tissues and cell lines. Using PCSCs isolated from two pancreatic cancer patients by cell sorting and by spheroid formation assay from pancreatic cancer cell line Panc-1, we tested the hypothesis that nicotine induces the self-renewal of PCSCs. Nicotinic acetylcholine receptors (nAChRs) α3, α4, α5 and α7 were expressed and chronic exposure to nicotine increased the protein expression of these receptors. Immunoassays showed that PCSCs produced the stress neurotransmitters epinephrine and norepinephrine and the inhibitory neurotransmitter GABA. Chronic nicotine significantly increased the production of stress neurotransmitters and sonic hedgehog (SHH) while inducing Gli1 protein and decreasing GABA. GABA treatment inhibited the induction of SHH and Gli1. Spheroid formation and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assays showed significant nicotine-induced increases in self renewal and cell proliferation, responses blocked by GABA. Our data suggest that nicotine increases the SHH-mediated malignant potential of PCSCs and that GABA prevents these effects.

Nonsmall-cell lung cancer (NSCLC) is the leading type of lung cancer and has a poor prognosis. We have shown that chronic stress promoted NSCLC xenografts in mice via stress neurotransmitter-activated cAMP signaling downstream of beta-adrenergic receptors and incidental beta-blocker therapy was reported to improve clinical outcomes in NSCLC patients. These findings suggest that psychological stress promotes NSCLC whereas pharmacologically or psychologically induced decreases in cAMP may inhibit NSCLC. Cancer stem cells are thought to drive the development, progression and resistance to therapy of NSCLC. However, their potential regulation by stress neurotransmitters has not been investigated. In the current study, epinephrine increased the number of cancer stem cell like cells (CSCs) from three NSCLC cell lines in spheroid formation assays while enhancing intracellular cAMP and the stem cell markers sonic hedgehog (SHH), aldehyde dehydrogenase-1 (ALDH-1) and Gli1, effects reversed by GABA or dynorphin B via Gαi -mediated inhibition of cAMP formation. The growth of NSCLC xenografts in a mouse model of stress reduction was significantly reduced as compared with mice maintained under standard conditions. Stress reduction reduced serum levels of corticosterone, norepinephrine and epinephrine while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and opioid peptides increased. Stress reduction significantly reduced cAMP, VEGF, p-ERK, p-AKT, p-CREB, p-SRc, SHH, ALDH-1 and Gli1 in xenograft tissues whereas cleaved caspase-3 and p53 were induced. We conclude that stress neurotransmitters activate CSCs in NSCLC via multiple cAMP-mediated pathways and that pharmacologically or psychologically induced decreases in cAMP signaling may improve clinical outcomes in NSCLC patients.

Understanding the genetic origins and demographic history of Indian populations is important both for questions concerning the early settlement of Eurasia and more recent events, including the appearance of Indo-Aryan languages and settled agriculture in the subcontinent. Although there is general agreement that Indian caste and tribal populations share a common late Pleistocene maternal ancestry in India, some studies of the Y-chromosome markers have suggested a recent, substantial incursion from Central or West Eurasia. To investigate the origin of paternal lineages of Indian populations, 936 Y chromosomes, representing 32 tribal and 45 caste groups from all four major linguistic groups of India, were analyzed for 38 single-nucleotide polymorphic markers. Phylogeography of the major Y-chromosomal haplogroups in India, genetic distance, and admixture analyses all indicate that the recent external contribution to Dravidian- and Hindi-speaking caste groups has been low. The sharing of some Y-chromosomal haplogroups between Indian and Central Asian populations is most parsimoniously explained by a deep, common ancestry between the two regions, with diffusion of some Indian-specific lineages northward. The Y-chromosomal data consistently suggest a largely South Asian origin for Indian caste communities and therefore argue against any major influx, from regions north and west of India, of people associated either with the development of agriculture or the spread of the Indo-Aryan language family. The dyadic Y-chromosome composition of Tibeto-Burman speakers of India, however, can be attributed to a recent demographic process, which appears to have absorbed and overlain populations who previously spoke Austro-Asiatic languages.

STR polymorphisms have proven to be extremely useful in population genetics studies and human identification. The fifteen analysed STR loci were tetra-nucleotide repeats: D5S818, FGA, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51.

The greater Himalayan region demarcates two of the most prominent linguistic phyla in Asia: Tibeto-Burman and Indo-European. Previous genetic surveys, mainly using Y-chromosome polymorphisms and/or mitochondrial DNA polymorphisms suggested a substantially reduced geneflow between populations belonging to these two phyla. These studies, however, have mainly focussed on populations residing far to the north and/or south of this mountain range, and have not been able to study geneflow patterns within the greater Himalayan region itself. We now report a detailed, linguistically informed, genetic survey of Tibeto-Burman and Indo-European speakers from the Himalayan countries Nepal and Bhutan based on autosomal microsatellite markers and compare these populations with surrounding regions. The genetic differentiation between populations within the Himalayas seems to be much higher than between populations in the neighbouring countries. We also observe a remarkable genetic differentiation between the Tibeto-Burman speaking populations on the one hand and Indo-European speaking populations on the other, suggesting that language and geography have played an equally large role in defining the genetic composition of present-day populations within the Himalayas.

Smoking and alcoholism are risk factors for the development of pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). Using a hamster model of pancreatitis-associated PDAC induced by treatment with ethanol in the drinking water and the nicotine-derived nitrosamine 4-methylnitrosamino-(3-pyridyl)-1-butanone (NNK) by subcutaneous injection as well as immunohistochemistry of human PDAC tissue microarrays, we have previously shown that these cancers overexpressed stress neurotransmitters and cAMP while the inhibitory neurotransmitter ã-aminobutyric acid (GABA) was suppressed. Using our hamster model, the current study has tested the hypothesis that cAMP control by GABA supplementation in the drinking water prevents the development of pancreatitis-associated PDAC. Our data reveal strong preventive effects of GABA supplementation on the development of PDAC and pancreatic intraductal neoplasia (PanIN). ELISA assays and immunohistochemistry revealed significant decreases in the levels of cyclic adenosine monophosphate (cAMP) and interleukin 6 accompanied by reductions in the expression of several cancer stem cell markers, phosphorylated signaling proteins associated with cell proliferation and migration in pancreatic exocrine cells of GABA treated animals. We conclude that cAMP control by GABA supplementation inhibits multiple cancer supporting pathways at the level of cancer stem cells, differentiated cancer cells and the immune system, identifying this approach as promising novel tool for the prevention of PDAC in individuals with a history of smoking and alcoholism. Citation Format: Jheelam Banerjee, Arokya M. Papu John, Mohammed H. Al-Wadei, Hildegard M. Schuller. Prevention of pancreatic cancer by cAMP control. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 823.

The Shompen, one of the most isolated and poorly understood contemporary hunter–gatherer populations, inhabit Great Nicobar Island, the southernmost island of the Nicobar archipelago. Morphological imprints in the Shompen were interpreted to favour a mixed Indo-Chinese, Malay, Negrito and Dravidian origin. Analyses of the mitochondrial, Y-chromosomal and autosomal gene pool of contemporary Shompen have revealed low diversity, illustrating a founder effect in the island population. Mitochondrial sequence analyses revealed the presence of two haplogroups of R lineage: B5a, and a newly defined clade, R12. Y-chromosomal analyses demonstrated the occurrence of a single lineage found predominantly in Austro-Asiatic speakers across Asia. With the different types of genetic markers analysed, the Shompen exhibit varying levels of genetic relatedness with the Nicobarese, and Austro-Asiatic speakers of mainland India and Southeast Asia. These genetic analyses provide evidence that the Shompen, an offshoot of the Nicobarese, are descendants of Mesolithic hunter–gatherers of Southeast Asian origin, deriving from at least two source populations.

The analysis of genetic variation, in the nucleotide sequences of mitochondrial DNA, provides unique information in tracing of maternal lineage, determination of population diversity, pharmacogenomics and human identification. This study characterizes the HVR-I and II sequence polymorphism in 80 tribal individuals, belonging to the Austro-Asiatic linguistic family of Chotanagpur plateau, India. A total of 115 polymorphic sites were observed in the sequenced regions and 77 unique haplotypes could be identified.

DNA damage of stromal fibroblastic cells is known to promote tumorigenesis in number of tissues including the breast and pancreas. 69% of prostate cancer patients lose expression of the Tgfbr2 in the stromal compartment. We previously described, transforming growth factor-beta (TGF-ß) responsiveness of the prostate stromal fibroblastic cells can promote tumorigenesis in transgenic mice. It is known TGF-ß promotes DNA stability, however the mechanism is not known. We found that human prostate cancer stroma has significant number of cells with DNA double stranded breaks, as determined by phosphorylated Ser139-histone H2 (γH2AX) immunolocalization. Similarly, prostate stromal cells from mice with a conditional Tgfbr2-knockout were positive for γH2AX staining. Further promoter methylation array analysis of prostate stromal cells from Tgfbr2-knockout and Tgfbr2-flox (control) indicated 17 DNA damage repair genes to be methylated in Tgfbr2-knockout cells versus the control. Thus, we hypothesized that the loss of stromal TGF-ß responsiveness facilitates stromal DNA damage accumulation through epigenetic regulation. We sought to test the role of stromal TGF-ß signaling in epigenetic regulation. Fourteen of 17 genes were RT-PCR verified to be silenced in Tgfbr2-KO cells and re-expressed following 5-azaDC (de-methylating agent) treatment. Homologous genes were found to be epigenetically silenced in human prostate CAF (carcinoma associated fibroblasts) cultured from primary tumors compared to NAF (normal prostate fibroblasts) from 10 prostate cancer patients. A downstream mediator of TGF-ß signaling includes Smad3 activation. Thus, a similar methylation array analysis of Smad3-knockout prostate stromal cells was performed. We found none of the DNA damage repair genes to be silenced in Smad3-KO cells. Next, we measured DNA methyltransferase-1 (DNMT1) mRNA, protein and activity in Tgfbr2-KO and Tgfbr2-flox cultured prostate stromal cells. Interestingly, protein expression and activity of DNMT1 was 3-fold greater in Tgfbr2-KO stroma over Tgfbr2-flox cells, in the absence of DNMT1 mRNA expression differences. DNMT3b differed little between the two cell types. The use of proteasome inhibitor, MG-132, suggested DNMT1 to be down regulated by TGF-ß in a post-translational manner. Finally, tissue recombinant experiments of Tgfbr2-KO prostate stromal cells with LNCaP, human prostate cancer epithelia, developed large tumors in orthotopic grafts. Similar grafts with wild type and Smad3-KO stromal cells with LNCaP epithelia developed small tumors. Strikingly Tgfbr2-KO stromal cells treated with 5-azaDC and subsequently grafted with LNCaP cells developed smaller tumors. These results for the first time support the role of TGF-ß signaling on stromal DNA stability in tumorigenesis and support a mechanism for TGF-ß responsiveness results in epigenetic silencing of DNA damage repair genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1429.

Introduction: A previously described mouse model, Tgfbr2-fspKO, with a conditional stromal knockout of the TGF-beta type II receptor (Tgfbr2), developed prostatic intra-epithelial neoplasia (PIN) lesions and subsequently progressed to prostate adenocarcinoma. Tissue recombination of wild-type (WT) prostatic organoids with 100% Tgfbr2-floxE2/floxE2 prostatic stroma resulted in grafts with normal prostatic glandular architecture, whereas recombination with 100% Tgfbr2-fspKO stroma resulted in grafts with PIN lesions. Interestingly, only grafts made with a mixture of WT and KO stroma resulted in prostatic adenocarcinoma. Furthermore, loss of stromal Tgfbr2 expression occurs in a proportion of human prostate cancer tissues. The objective of the study was to elucidate the mechanism(s) contributing to the cancer progression in the context of a mixed heterogeneous stroma. Method: Prostate stromal-epithelial interactions were described by a hybrid computational model involving a hypothesized 2-step mechanism (initial transformation followed by invasion), where these steps are controlled by stromal-derived paracrine morphogenic factors. Biological parameters for the candidate morphogens (Wnt3a and SDF-1/CXCL12) were assessed by qPCR and ELISA from stromal mixing co-culture experiments. Results: Modeling indicated that the expression of the initiating morphogen increases as a function of the % of Tgfbr2-KO stromal cells. The incidence of invasion promoted by the pro-invasive morphogen was biphasic with a 50/50 mixture of WT and Tgfbr2-KO stromal cells resulting in maximal cancer progression. Wnt3a is produced at 4-fold higher levels in Tgfbr2-KO vs WT stroma and antibody mediated depletion of Wnt3a from stromal conditioned media reduced the growth rate of LNCaP cells. SDF-1 is produced at nearly 4-fold higher levels in WT vs. Tgfbr2-KO stroma and SDF-1 mRNA expression is synergistically increased when WT and Tgfbr2-KO stroma are co-cultured. Additionally, CD90 expression is heterogeneously expressed by stromal tissues associated with primary human prostate cancer as compared to benign tissue as demonstrated by immunofluorescence staining. Conclusions: The model and data support the importance of stromal heterogeneity in the development of prostatic adenocarcinoma in two independent tumorigenic steps. Each step is facilitated by a different stromal component, so that stromal heterogeneity results in the maximal tumorigenesis. Tgfbr2-KO stromal cells produce an altered cytokine profile that drives the proliferation of the epithelial compartment and likely primes the WT stromal cells to produce additional cytokines, like SDF-1, that promote epithelial invasion and cancer progression. While most studies tend to focus on the altered stroma, this study suggests that the WT stroma cells play a necessary and important role required as a rate-limiting step for prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1428.

Stromal cells promote prostate cancer initiation and progression. Metastasized prostate cancers form osteoblastic bone lesions. The prostate cancer associated stroma expresses alpha-smooth muscle actin (alpha-SMA). Additionally, 69% of human prostate cancer tissues lost stromal TGFbeta type II receptor (Tgfbr2) expression. Conditional stromal knockout of the Tgfbr2 mouse model developed prostate adenocarcinoma. In parallel to the primary site, we observed human prostate cancer associated stroma in bone metastasis gained the expression of androgen receptor and alpha-SMA, which were negative in the normal bone marrow. Tgfbr2 expression was also lost in prostate cancer associated stroma in bone marrow, compared to its high expression in the normal bone marrow. This led us to question whether the stromal cells from the primary site or the bone marrow contribute the prostate cancer osteoblastic bone lesion development. In vitro, the conditioned media from Tgfbr2-flox or Tgfbr2-KO mouse prostate stromal cells (PSC) or bone marrow stromal cells (BMSC) were incubated on the C42B prostate cancer epithelia. Expressions of PTHrP, RANKL osteolytic factors and ET1, RUNX2, BMP2 osteoblastic factors by C42B cells were analyzed by qRT-PCR. The Tgfbr2-flox PSC conditioned media significantly increased the C42B cell expression of all the osteolytic and osteoblastic factors examined compared to C42B cells without conditioned media. However, the Tgfbr2-KO PSC conditioned media decreased the expression of PTHrP and RANKL, while further increased the expression of osteoblastic factors, ET1, RUNX2 and BMP2 by C42B cells by at least 2-fold. Conditioned media of Tgfbr2-flox and Tgfbr2-KO BMSC increased the expression of C42B cells on osteolytic factors (10-fold PTHrP and 2-fold RANKL), but neither has significant effect on expression of osteoblastic factors. Importantly, Tgfbr2-KO PSC and BMSC conditioned media increased C42B proliferation similarly by 2-fold over that of respective Tgfbr2-Tgfbr2-flox conditioned media. We found that the reduced PTHrP and RANKL expression was associated with respective promoter methylation, when C42B cells were incubated with Tgfbr2-KO PSC, but not BMSC. Epigenetic silencing of such osteolytic genes, would suggest for the first time, that the prostate stroma could influence osteoblastic tumor at a distant site. So, GFP labeled C42B cells, following pre-incubation with PSC or BMSC conditioned media, were injected into the tibia of SCID mice. X-rays of the bone lesions and fluorescence imaging of GFP-expressing tumor growth were measured. The data suggested, that the stromal cells from primary prostates, but not from the second metastasis site of bone marrow, promotes prostate cancer growth in the bone and osteoblastic bone lesion development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1418.

Phylogenetic analysis of human complete mitochondrial DNA sequences has largely contributed to resolving phylogenies and antiquity of different lineages belonging to the majorhaplogroups L, N and M (East-Asian lineages). In the absence of whole mtDNA sequence information of M lineages reported in India that exhibits highest diversity within the sub-continent, the present study was undertaken to provide a detailed analysis of this haplogroup to precisely characterize the lineages and unravel their intricate phylogeny. The phylogenetic tree constructed from sequencing information of twenty four whole mtDNA genome revealed novel substitutions in the previously defined M2a and M6 lineages. The most striking feature of this phylogenetic tree is the formulation of a new lineage M30, distinguished by the presence of 12007 transition, and comprises of the recently defined M18 and a potential new sub-lineage possessing substitution at 16223 and 16300. M30 further branches into M30a sub-lineage, defined by 15431 and 195A substitution. The age of M30 lineage was estimated at 33,042 YBP, indicating a more recent expansion time than M2 (49,686 YBP). Contradictory to earlier reports, the M5 lineage does not always include a 12477 substitution, and is more appropriately defined by a transversion at 10986A. The phylogenetic tree also identifies a potential new lineage M* with HVSI sequence 16223,16325. No new substitutions were found in M25 and the M3 mt DNA genome could only be tentatively rooted by 16126 mutation. M4 and M*(16251, 16267) lineages could not be resolved distinctly. This study describes seven new basal mutations and fourteen lineages that substantially contribute to the present understanding of superhaplogroup M. The phylogenetic tree supported by median-joining network helps in distinctly identifying the genetic relation between different M lineages that could not be achieved solely by control region sequence information. Although high control region diversity has been reported in the different M lineages distributed in India, complete sequencing of M* and defined lineages suggests that these mt DNA genomes emerged from a limited number of branches arising from the M trunk.