Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

Docetaxel radiosensitizes castration-resistant prostate cancer by downregulating CAV-1.

PURPOSE: Docetaxel (DXL), a noted radiosensitizer, is one of the few chemotherapy drugs approved for castration-resistant prostate cancer (CRPC), though only a fraction of CRPCs respond to it. CAV-1, a critical regulator of radioresistance, has been known to modulate DXL and radiation effects. Combining DXL with radiotherapy may create a synergistic anticancer effect through CAV-1 and improve CRPC patients' response to therapy. Here, we investigate the effectiveness and molecular characteristics of DXL and radiation combination therapy in vitro. MATERIALS AND METHODS: We used live/dead assays to determine the IC50 of DXL for PC3, DU-145, and TRAMP-C1 cells. Colony formation assay was used to determine the radioresponse of the same cells treated with radiation with/without IC50 DXL (4, 8, and 12 Gy). We performed gene expression analysis on public transcriptomic data collected from human-derived prostate cancer cell lines (C4-2, PC3, DU-145, and LNCaP) treated with DXL for 8, 16, and 72 hours. Cell cycle arrest and protein expression were assessed using flow cytometry and western blot, respectively. RESULTS: Compared to radiation alone, combination therapy with DXL significantly increased CRPC death in PC3 (1.48-fold, p < .0001), DU-145 (1.64-fold, p < .05), and TRAMP-C1 (1.13-fold, p < .05) at 4 Gy of radiation. Gene expression of CRPC treated with DXL revealed downregulated genes related to cell cycle regulation and upregulated genes related to immune activation and oxidative stress. Confirming the results, G2/M cell cycle arrest was significantly increased after treatment with DXL and radiation. CAV-1 protein expression was decreased after DXL treatment in a dose-dependent manner; furthermore, CAV-1 copy number was strongly associated with poor response to therapy in CRPC patients. CONCLUSIONS: Our results suggest that DXL sensitizes CRPC cells to radiation by downregulating CAV-1. DXL + radiation combination therapy may be effective at treating CRPC, especially subtypes associated with high CAV-1 expression, and should be studied further.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD.

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SUMMARY: Alterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. SUBJECT AREA: Calcium, ROS, Cellular signaling, lung disease.

Inhibition of ribosome assembly factor PNO1 by CRISPR/Cas9 technique suppresses lung adenocarcinoma and Notch pathway: Clinical application.

Growth is crucially controlled by the functional ribosomes available in cells. To meet the enhanced energy demand, cancer cells re-wire and increase their ribosome biogenesis. The RNA-binding protein PNO1, a ribosome assembly factor, plays an essential role in ribosome biogenesis. The purpose of this study was to examine whether PNO1 can be used as a biomarker for lung adenocarcinoma and also examine the molecular mechanisms by which PNO1 knockdown by CRISPR/Cas9 inhibited growth and epithelial-mesenchymal transition (EMT). The expression of PNO1 was significantly higher in lung adenocarcinoma compared to normal lung tissues. PNO1 expression in lung adenocarcinoma patients increased with stage, nodal metastasis, and smoking. Lung adenocarcinoma tissues from males expressed higher PNO1 than those from females. Furthermore, lung adenocarcinoma tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53, suggesting the influence of Tp53 status on PNO1 expression. PNO1 knockdown inhibited cell viability, colony formation, and EMT, and induced apoptosis. Since dysregulated signalling through the Notch receptors promotes lung adenocarcinoma, we measured the effects of PNO1 inhibition on the Notch pathway. PNO1 knockdown inhibited Notch signalling by suppressing the expression of Notch receptors, their ligands, and downstream targets. PNO1 knockdown also suppressed CCND1, p21, PTGS-2, IL-1α, IL-8, and CXCL-8 genes. Overall, our data suggest that PNO1 can be used as a diagnostic biomarker, and also can be an attractive therapeutic target for the treatment of lung adenocarcinoma.

Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-ß-catenin pathway.

Most cancer cells rely on aerobic glycolysis to support uncontrolled proliferation and evade apoptosis. However, pancreatic cancer cells switch to glutamine metabolism to survive under hypoxic conditions. Activation of the Wnt/ß-catenin pathway induces aerobic glycolysis by activating enzymes required for glucose metabolism and regulating the expression of glutamate transporter and glutamine synthetase. The results demonstrate that riluzole inhibits pancreatic cancer cell growth and has no effect on human pancreatic normal ductal epithelial cells. RNA-seq experiments identified the involvement of Wnt and metabolic pathways by riluzole. Inhibition of Wnt-ß-catenin/TCF-LEF pathway by riluzole suppresses the expression of PDK, MCT1, cMyc, AXIN, and CyclinD1. Riluzole inhibits glucose transporter 2 expression, glucose uptake, lactate dehydrogenase A expression, and NAD + level. Furthermore, riluzole inhibits glutamate release and glutathione levels, and elevates reactive oxygen species. Riluzole disrupts mitochondrial homeostasis by inhibiting Bcl-2 and upregulating Bax expression, resulting in a drop of mitochondrial membrane potential. Finally, riluzole inhibits pancreatic cancer growth in KPC (Pdx1-Cre, LSL-Trp53R172H, and LSL-KrasG12D) mice. In conclusion, riluzole can inhibit pancreatic cancer growth by regulating glucose and glutamine metabolisms and can be used to treat pancreatic cancer.

Hypercalciuria switches Ca2+ signaling in proximal tubular cells, induces oxidative damage to promote calcium nephrolithiasis.

Proximal tubule (PT) transports most of the renal Ca2+, which was usually described as paracellular (passive). We found a regulated Ca2+ entry pathway in PT cells via the apical transient receptor potential canonical 3 (TRPC3) channel, which initiates transcellular Ca2+ transport. Although TRPC3 knockout (-/-) mice were mildly hypercalciuric and displayed luminal calcium phosphate (CaP) crystals at Loop of Henle (LOH), no CaP + calcium oxalate (CaOx) mixed urine crystals were spotted, which are mostly found in calcium nephrolithiasis (CaNL). Thus, we used oral calcium gluconate (CaG; 2%) to raise the PT luminal [Ca2+]o further in TRPC3 -/- mice for developing such mixed stones to understand the mechanistic role of PT-Ca2+ signaling in CaNL. Expectedly, CaG-treated mice urine samples presented with numerous mixed crystals with remains of PT cells, which were pronounced in TRPC3 -/- mice, indicating PT cell damage. Notably, PT cells from CaG-treated groups switched their mode of Ca2+ entry from receptor-operated to store-operated pathway with a sustained rise in intracellular [Ca2+] ([Ca2+]i), indicating the stagnation in PT Ca2+ transport. Moreover, those PT cells from CaG-treated groups demonstrated an upregulation of calcification, inflammation, fibrotic, oxidative stress, and apoptotic genes; effects of which were more robust in TRPC3 ablated condition. Furthermore, kidneys from CaG-treated groups exhibited fibrosis, tubular injury and calcifications with significant reactive oxygen species generation in the urine, thus, indicating in vivo CaNL. Taken together, excess PT luminal Ca2+ due to escalation of hypercalciuria in TRPC3 ablated mice induced surplus CaP crystal formation and caused stagnation of PT [Ca2+]i, invoking PT cell injury, hence mixed stone formation.

Ethanol exposure of human pancreatic normal ductal epithelial cells induces EMT phenotype and enhances pancreatic cancer development in KC (Pdx1-Cre and LSL-KrasG12D ) mice.

Alcohol is a risk factor for pancreatic cancer. However, the molecular mechanism by which chronic alcohol consumption influences pancreatic cancer development is not well understood. We have recently demonstrated that chronic ethanol exposure of pancreatic normal ductal epithelial cells (HPNE) induces cellular transformation by generating cancer stem cells (CSCs). Here, we examined whether chronic ethanol treatment induces epithelial-mesenchymal transition in HPNE cells and promotes pancreatic cancer development in KC (Pdx1-Cre, and LSL-KrasG12D ) mice. Our data demonstrate that chronic ethanol exposure of HPNE cells induces SATB2 gene and those cells became highly motile. Ethanol treatment of HPNE cells results in downregulation of E-Cadherin and upregulation of N-Cadherin, Snail, Slug, Zeb1, Nanog and BMI-1. Suppression of SATB2 expression in ethanol-transformed HPNE cells inhibits EMT phenotypes. KC mice fed with an ethanol-containing diet show enhanced pancreatic cancer growth and development than those fed with a control diet. Pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of stem cell markers (CD133, CD44, CD24), pluripotency-maintaining factors (cMyc, KLF4, SOX-2, and Oct-4), N-Cadherin, EMT-transcription factors (Snail, Slug, and Zeb1), and lower expression of E-cadherin than those isolated from mice fed with a control diet. Furthermore, pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of inflammatory cytokines (TNF-α, IL-6, and IL-8) and PTGS-2 (COX-2) gene than those isolated from mice fed with a control diet. These data suggest that chronic alcohol consumption may contribute to pancreatic cancer development by generating inflammatory signals and CSCs.

SATB2 is a novel biomarker and therapeutic target for cancer.

Several studies have confirmed the involvement of cancer stem cells (CSC) in tumour progression, metastasis, drug resistance and cancer relapse. SATB2 (special AT-rich binding protein-2) acts as a transcriptional co-factor and modulates chromatin architecture to regulate gene expression. The purpose of this review was to discuss the pathophysiological roles of SATB2 and assess whether it could be used as a therapeutic target for cancer. SATB2 modulated the expression of those genes which regulated pluripotency and self-renewal. Overexpression of SATB2 gene in normal epithelial cells was shown to induce transformation, as a result transformed cells gained CSC's characteristics by expressing stem cell markers and pluripotency maintaining factors, suggesting its role as an oncogene. In addition, SATB2 induced epithelial-mesenchymal transition (EMT) and metastasis. Interestingly, the expression of SATB2 was positively correlated with the activation of ß-catenin/TCF-LEF pathway. Furthermore, SATB2 silencing inhibited EMT and their positive regulators, and tumour growth, and suppressed the expression of stem cell markers, pluripotency maintaining factors, cell cycle and cell survival genes, and TCF/LEF targets. Based on the cancer genome atlas (TCGA) expression data and published papers, SATB2 alone or in combination with other proteins could be used a diagnostic biomarker for cancer. Although there is no pharmacological inhibitor of SATB2, studies using genetic approaches suggest that SATB2 could be a potential target for cancer treatment and prevention.

Assessment of risk factors, and racial and ethnic differences in hepatocellular carcinoma.

Despite improved screening and surveillance guidelines, significant race/ethnicity-specific disparities in hepatocellular carcinoma (HCC) continue to exist and disproportionately affect minority and disadvantaged populations. This trend indicates that social determinants, genetic, and environmental factors are driving the epidemic at the population level. Race and geography had independent associations with risk of mortality among patients with HCC. The present review discusses the risk factors and issues related to disparities in HCC. The underlying etiologies for these disparities are complex and multifactorial. Some of the risk factors for developing HCC include hepatitis B (HBV) and hepatitis C (HCV) viral infection, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, smoking and alcohol consumption. In addition, population genetics; socioeconomic and health care access; treatment and prevention differences; and genetic, behavioral, and biological influences can contribute to HCC. Acculturation of ethnic minorities, insurance status, and access to health care may further contribute to the observed disparities in HCC. By increasing awareness, better modalities for screening and surveillance, improving access to health care, and adapting targeted preventive and therapeutic interventions, disparities in HCC outcomes can be reduced or eliminated.

Higher expression of SATB2 in hepatocellular carcinoma of African Americans determines more aggressive phenotypes than those of Caucasian Americans.

In the United States, Hepatocellular Carcinoma (HCC) incidence has tripled over the past two decades. The disease has disproportionately affected minority and disadvantaged populations. The purpose of this study was to examine the expression of SATB2 gene in HCC cells derived from African Americans (AA) and Caucasian Americans (CA) and assess its oncogenic potential by measuring cell viability, spheroid formation, epithelial-mesenchymal transition (EMT), stem cell markers and pluripotency maintaining factors in cancer stem cells (CSCs). We compared the expression of SATB2 in human primary hepatocytes, HCC cells derived from AA and CA, and HCC CSCs. Hepatocellular carcinoma cells derived from AA expressed the higher level of SATB2 than those from CA. By comparison, normal human hepatocytes did not express SATB2. Higher expression of SATB2 in HCC cells from AA was associated with greater growth rate, cell viability, colony formation and EMT characteristics than those from CA. Knockout of SATB2 in CSCs by Crispr/Cas9 technique significantly inhibited the expression of SATB2 gene, stem cell markers (CD24, CD44 and CD133), pluripotency maintaining factors (c-Myc, KLF4, SOX2 and OCT4), and EMT compared with non-targeting control group. The expression of SATB2 was negatively correlated with miR34a. SATB2 rescued the miR-34a-mediated inhibition of CSC's viability. These data suggest that SATB2 is an oncogenic factor, and its higher expression may explain the disparity in HCC outcomes among AA.

Melamine induces Ca2+-sensing receptor activation and elicits apoptosis in proximal tubular cells.

Melamine causes renal tubular cell injury through inflammation, fibrosis, and apoptosis. Although melamine affects the rise in intracellular Ca2+ concentration ([Ca2+]i), reactive oxygen species (ROS) production, and proapoptotic pathway activation, the mechanism of upstream Ca2+ signaling is unknown. Because melamine has some structural similarities with l-amino acids, which endogenously activate Ca2+-sensing receptors (CSR), we examined the effect of melamine on CSR-induced Ca2+ signaling and apoptotic cell death. We show here that melamine activates CSR, causing a sustained Ca2+ entry in the renal epithelial cell line, LLC-PK1. Moreover, such CSR stimulation resulted in a rise in [Ca2+]i, leading to enhanced ROS production. Furthermore, melamine-induced elevated [Ca2+]i and ROS production caused a dose-dependent increase in apoptotic (by DAPI staining, DNA laddering, and annexin V assay) and necrotic (propidium iodide staining) cell death. Upon examining the downstream mechanism, we found that transforming growth factor ß1 (TGF-ß1), which increases extracellular matrix genes and proapoptotic signaling, was also upregulated at lower doses of melamine, which could be due to an early event inducing apoptosis. Additionally, cells exposed to melamine displayed a rise in pERK activation and lactate dehydrogenase release resulting in cytotoxicity. These results offer a novel insight into the molecular mechanisms by which melamine exerts its effect on CSR, causing a sustained elevation of [Ca2+]i, leading to ROS generation, fibronectin production, proapoptotic pathway activation, and renal cell damage. Together, these results thus suggest that melamine-induced apoptosis and/or necrosis may subsequently result in acute kidney injury and promote kidney stone formation.

Plasma-aminothiols status and inverse correlation of total homocysteine with B-vitamins in arsenic exposed population of West Bengal, India.

Chronic arsenic toxicity is a serious environmental health problem across the world. Bangladesh and India (particularly the state of West Bengal) are the worst affected countries with such problem. The present study reports plasma-aminothiols (p-aminothiols) like L-cysteine (L-Cys), cysteinyl glycine (Cys-gly), total homocysteine (t-Hcy) and glutathione (GSH) status, and the inverse relationship of t-Hcy with B-vitamins (B1, B6, B9 and B12) in arsenic exposed population of West Bengal, India. Reverse phase HPLC was used to measure p-aminothiols and serum B-vitamins in different arsenic exposed population. Arsenic in drinking water and urine were measured by flow injection analysis system - Atomic Absorption Spectrometry (FIAS-AAS) and Transversely heated graphite atomizer (THGA-AAS) techniques, respectively. Water arsenic exposure was >50 µg/L in 50% population, of which majority (33.58%) belong to the range of >50-500 µg/L and more than 8% were even >1000 µg/L. Urine arsenic (µg/g creatinine) levels increased with arsenic exposure. The variability among p-aminothiols was also observed with higher exposure to arsenic in drinking water. A significant difference between exposed and control population was noticed for plasma L-Cys. The difference of B-vitamins between the population exposed to <50 and >50 µg/L arsenic in drinking water was also found to be significant. B9 and B12 deficiency with increased consumption of arsenic in water corroborates the anemic conditions commonly observed among arsenic exposed population. The aminothiol status indicated oxidative stress in exposed population. This study demonstrated progressive increase in plasma t-Hcy as well as inverse relationships of serum B-vitamins with increased water arsenic concentration.

Work-exposure to PM10 and aromatic volatile organic compounds, excretion of urinary biomarkers and effect on the pulmonary function and heme-metabolism: A study of petrol pump workers and traffic police personnel in Kolkata City, India.

This study focused work-exposure to particulate matter ≤ 10 µm (PM10), volatile organic compounds (VOCs) and biological monitoring of major VOCs (BTEX) to observe the significant effects of traffic related pollutants on respiratory and hematological systems of workers engaged in two occupational settings, petrol pumps and traffic areas of Kolkata metropolitan city, India. PM10 was assessed by personal sampling and particle size distribution by 8-stage Cascade Impactor. VOCs were analysed by gas chromatography-flame ionization detector (GC-FID) and five urinary metabolites, trans trans- mercapturic acid (tt-MA), S-phenyl mercapturic acid (SPMA), hippuric acid (HA), mandelic acid (MA) and methyl hippuric acid (MHA) of VOCs, benzene, toluene, ethyl benzene and xylenes (BTEX) by reverse phase high performance liquid chromatography (HPLC). Pulmonary functions test (PFT) was measured Spirometrically. ∂-aminoleavulinic acid (ALA) and porphobilinogen (PBG) in lymphocytes were measured spectrophometrically following column chromatographic separation. High exposure to PM10, having 50% of particles, ≤ 5.0 µm in both the occupational settings. Exposure to toluene was highest in petrol pumps whereas benzene was highest (104.6 ± 99.0 µg m-3) for traffic police personnel. Workplace Benzene is found many fold higher than the National ambient standard. Air-benzene is correlated significantly with pre- and post-shift tt-MA (p < 0.001) and SPMA (p < 0.001) of exposed workers. Blood cell counts indicated benzene induced hematotoxicity. ALA and PBG accumulation in lymphocytes indicated alteration in heme-metabolism, especially among traffic police. Significant reduction of force exploratory volume in one second (FEV1) and forced vital capacity (FVC) of fuel fillers are observed with increased tt-MA and SPMA. Study revealed PFT impairments 11.11% (6.66% restrictive and 2.22% obstructive and combined restrictive and obstructive type, each) among petrol pumps and 8.3% obstructive type among traffic police.

The natural tumorcide Manumycin-A targets protein phosphatase 1α and reduces hydrogen peroxide to induce lymphoma apoptosis.

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.

GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft.

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.

Resveratrol inhibits growth of orthotopic pancreatic tumors through activation of FOXO transcription factors.

BACKGROUND: The forkhead transcription factors of the O class (FOXO) play a direct role in cellular proliferation, oxidative stress response, and tumorigenesis. The objectives of this study were to examine whether FOXOs regulate antitumor activities of resveratrol in pancreatic cancer cells in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Pancreatic cancer cell lines were treated with resveratrol. Cell viability, colony formation, apoptosis and cell cycle were measured by XTT, soft agar, TUNEL and flow cytometry assays, respectively. FOXO nuclear translocation, DNA binding and transcriptional activities were measured by fluorescence technique, gelshift and luciferase assay, respectively. Mice were orthotopically implanted with PANC1 cells and orally gavaged with resveratrol. The components of PI3K and ERK pathways, FOXOs and their target gene expressions were measured by the Western blot analysis. Resveratrol inhibited cell viability and colony formations, and induced apoptosis through caspase-3 activation in four pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Hs766T, and AsPC-1). Resveratrol induced cell cycle arrest by up-regulating the expression of p21/CIP1, p27/KIP1 and inhibiting the expression of cyclin D1. Resveratrol induced apoptosis by up-regulating Bim and activating caspase-3. Resveratrol inhibited phosphorylation of FOXOs, and enhanced their nuclear translocation, FOXO-DNA binding and transcriptional activities. The inhibition of PI3K/AKT and MEK/ERK pathways induced FOXO transcriptional activity and apoptosis. Furthermore, deletion of FOXO genes abrogated resveratrol-induced cell cycle arrest and apoptosis. Finally, resveratrol-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FOXO1 and FOXO3a, and induction of apoptosis and FOXO target genes. CONCLUSIONS: These data suggest that inhibition of ERK and AKT pathways act together to activate FOXO transcription factors which are involved in resveratrol-mediated pancreatic tumor growth suppression.

Inhibition of PI3K/AKT and MAPK/ERK pathways causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cancer.

BACKGROUND: Mammalian forkhead members of the class O (FOXO) transcription factors, including FOXO1, FOXO3a, and FOXO4, are implicated in the regulation of several biological processes, including the stress resistance, metabolism, cell cycle, apoptosis and DNA repair. The objectives of this study were to examine the molecular mechanisms by which FOXO transcription factors induced cell cycle arrest and apoptosis and enhanced anti-proliferative effects of sulforaphane (SFN, an active compound in cruciferous vegetables) in pancreatic cancer cells. RESULTS: Our data demonstrated that SFN inhibited cell proliferation and colony formation, and induced apoptosis through caspase-3 activation in pancreatic cancer cells. The inhibition of PI3K/AKT and MEK/ERK pathways activated FOXO transcription factors. SFN inhibited phosphorylation of AKT and ERK, and activated FOXO transcription factors, leading to cell cycle arrest and apoptosis. Phosphorylation deficient mutants of FOXO proteins enhanced FOXO transcriptional activity, and further enhanced SFN-induced FOXO activity and apoptosis. SFN induced the expression of p21/CIP1 and p27/KIP1, and inhibited the expression of cyclin D1. CONCLUSION: These data suggest that inhibition of PI3K/AKT and ERK pathways acts together to activate FOXO transcription factor and enhances SFN-induced FOXO transcriptional activity, leading to cell cycle arrest and apoptosis.

Down-regulation of the transcriptional mediator subunit Med1 contributes to the loss of expression of metastasis-associated dapk1 in human cancers and cancer cells.

DAPK1, a ca(+2)/calmodulin regulated serine/threonine kinase, is a major tumor suppressor, whose expression is lost in multiple tumor types. However, the mechanisms contributing to it are unclear. We have recently shown that CCAAT/Enhancer binding protein-beta (C/EBP-beta) is required for the basal and interferon gamma (IFN-gamma)-induced expression of dapk1 in many cell types. C/EBP-beta interacts with the transcriptional Mediator, a multisubunit complex that couples enhancer bound transcription factors to the basal transcriptional machinery in an IFN-gamma dependent manner for regulating dapk1 expression. Specifically, the Med1 (TRAP220/PBP/DRIP220/CRSP220) subunit associates with the enhancer bound C/EBP-beta at the CRE/ATF site of dapk1 in an IFN-gamma dependent manner for stimulating gene expression. Therefore, we investigated if the mechanism responsible for the loss of dapk1 expression in human cancers involves a failure to recruit C/EBP-beta and/or Med1 to the dapk1 promoter. We compared the relative occupancy of these factors at the dapk1 promoter at CRE/ATF sites in normal and cancer cell lines. A significantly lower binding of these factors to the CRE/ATF site of dapk1 promoter occurred in human cancer cell lines than in normal cells. We show that loss of Med1 expression correlates with a corresponding loss of dapk1 expression in a number of primary human lung carcinomas. Med1 levels were significantly lower in cancer cell lines than in normal controls. Importantly, we show that restoration of Med1 induces the expression of dapk1 in these cancer cells and also attenuates their metastatic potential in vivo. Our studies reveal a critical parameter limiting dapk1 expression in cancer cell lines.

Industrial hygiene survey in an aluminium reduction plant in India.

This study reports a work-environmental assessment and workers' exposure in a major prebake type aluminium smelter in India. Levels of known health hazards in and near the main smelting operations viz., the Potroom, the Carbon area, the Butt section, the Rodding shop, the Bath preparing area and the Casthouse were measured. Dustiness in general was high to excessively high. Mean levels of respirable dust (PM10) in air in the three dustiest areas were 24.07 mg/m3 in the Carbon areas, 27.57 mg/m3 in the Bath preparing and 4.44 mg/m3 in the Rodding shop. 40- 60% of the particles were less than 5 microm in size. 0.5- 2.82% particulate fluoride was obtained in the size fraction 0.4- 4.7 microm of the Potroom air. Naturally, exposures to total dusts were very high in these processes. The background levels of NOx and SO2 and fluorides (gaseous and particulate) were found to be within the prescribed Indian Standards. Higher exposures to gaseous and particulate fluoride, 3.85 and 6.53 mg/m3 respectively, were observed among the Rodding shop workers. The levels ofpolycyclic aromatic hydrocarbons (PAHs) were deemed to be excessive in the Carbon area. Measurements of heat stress were made in winter and were found to be lower than the prescribed limit.