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Numerous compounds for treating human disease have been discovered in nature. Manumycin-A (Man-A) is a natural, well-tolerated microbial metabolite and a potent experimental tumoricide. We recently showed that Man-A stimulated reactive oxygen species (ROS) which were upstream of serine/threonine (Ser/Thr) dephosphorylation and caspase-dependent cleavage of MEK and Akt in lymphoma apoptosis. Conversely, activation-specific, Ser/Thr phosphorylation of MEK and Akt proteins was stable in Man-A-resistant tumors suggesting that stimulation of Ser/Thr PPase activity might be required for Man-A tumoricidal activity. Pre-treatment with Calyculin-A, an equipotent inhibitor of PP1 and PP2A, blocked all downstream effects of Man-A whereas, the PP2A-selective inhibitor, Okadaic acid did not, suggesting that PP1 and not PP2A played a role in Man-A action. Phosphorylation of PP1α on Thr320 inhibits its activity. Hence, we posited that if PP1α was important for Man-A action, then Man-A treatment should promote dephosphorylation of PP1α on Thr320. Indeed, T320 was only dephosphorylated in the tumors that underwent apoptosis. Lastly, stable over-expression of a constitutively active PP1α mimetic (PP1αT320A mutant), elevated basal ROS levels and enhanced Man-A-stimulated apoptosis. Taken together, we conclude that PP1α is an important proximal effector of Man-A mediated lymphoma apoptosis and that the mechanisms of Man-A action warrant further investigation.

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2R gamma(null) mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs.

Metabolic syndrome is common in the general population, but there is little information available on the underlying signaling mechanisms regulating triglyceride (TG) content in the body. In the current study, we have uncovered a role for protein kinase Cbeta (PKCbeta) in TG homeostasis by studying the consequences of a targeted disruption of this kinase. PKCbeta(-/-) mutant mice were considerably leaner and the size of white fat depots was markedly decreased compared with wild-type littermates. TG content in the liver and skeletal muscle of PKCbeta(-/-) mice was also significantly low. Interestingly, mutant animals were hyperphagic and exhibited higher food intake and reduced feed efficiency versus wild type. The protection from obesity involves elevated oxygen consumption/energy expenditure and increased fatty acid oxidation in adipose tissue with concurrent increased mitochondria genesis, up-regulation of PGC-1alpha and UCP-2, and down-regulation of perilipin. The ability of PKCbeta deficiency to promote fat burning in adipocytes may suggest novel therapeutic strategies for obesity and obesity-related disorders.

Transcription factor CCAAT/enhancer-binding protein-beta (C/EBP-beta) regulates a variety of cellular functions in response to exogenous stimuli. We have reported earlier that C/EBP-beta induces gene transcription through a novel interferon (IFN)-response element called gamma-IFN-activated transcriptional element. We show here that IFN-gamma-induced, C/EBP-beta/gamma-IFN-activated transcriptional element-dependent gene expression is regulated by mixed lineage kinases (MLKs), members of the mitogen-activated protein kinase kinase kinase family. MLK3 appears to activate C/EBP-beta in response to IFN-gamma by a mechanism involving decreased phosphorylation of a specific phosphoacceptor residue, Ser(64), within the transactivation domain. Decreased phosphorylation of Ser(64) was independent of IFN-gamma-stimulated ERK1/2 activation and did not require the ERK phosphorylation site Thr(189) located in regulatory domain 2 of C/EBP-beta. Together these studies provide the first evidence that MLK3 is involved in IFN-gamma signaling and identify a novel mechanism of transcriptional activation by IFN-gamma.

This paper studies work exposure of drivers and conductors of special state buses in Kolkata, India to noise, heat, respirable dust and volatile organic compounds (VOCs). Equivalent noise exposures of drivers at work and in-bus noise are evaluated using a precision noise level meter. Thermal conditions like wet bulb globe temperature, relative humidity, and dry-bulb temperature were measured while plying through different traffic routes. Personal sampling techniques are adopted for dust and VOC monitoring and gas chromatography with flame ionisation detector was used to estimate VOCs. Drivers’ exposure to noise depended on the number of trips performed per day and exceeded the recommended American Conference of Governmental Industrial Hygienists’ threshold limit value, mostly after a second trip. The WBGT index revealed strenuous conditions for the working group. Mean dust exposures for drivers and conductors were found to be much higher than the level proposed in the notice of intended changes of ACGIH TLV (1995–96) for diesel particulate matter. Exposure to benzene, toluene, and xylene exhibit higher results than reported elsewhere.

Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-beta (C/EBP-beta) induces gene transcription through a novel IFN response element called the gamma-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626-12632. Here, we describe a new IFN-gamma-stimulated pathway that operates C/EBP-beta-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-gamma to stimulate C/EBP-beta-dependent expression. Sustained ERK activation directly correlated with C/EBP-beta-dependent gene expression in response to IFN-gamma. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-gamma-stimulated gene induction through the gamma-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-beta-induced gene expression required STAT1, but not JAK1. A C/EBP-beta mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-beta to IFN-gamma signaling through ERKs.

s / Pancreatology 13 (2013) e1–e94 e11 mTOR signaling in PaSC. Further, conditioned medium from PanIN cells isolated from KRAS mice enhanced fibrogenic responses in PaSC. Histological analysis of tissue microarrays of patient-matched normal human pancreas parenchyma and PanIN lesions showed profuse active PaSC surrounding both low-grade and advanced PanINs, and acini in adjacent normal parenchymal sections. Conclusions: Our data support a key role for Akt/mTOR signaling in PaSC fibrogenic responses, and indicate that interplay between PanINs and PaSC provide the mechanistic underpinnings of environmental risk factor promotion of tumor progression.