Hwyda A. Arafat

Human Epicardial Adipose Tissue Is a Source of Inflammatory Mediators

Background—Inflammatory mediators that originate in vascular and extravascular tissues promote coronary lesion formation. Adipose tissue may function as an endocrine organ that contributes to an inflammatory burden in patients at risk of cardiovascular complications. In this study, we sought to compare expression of inflammatory mediators in epicardial and subcutaneous adipose stores in patients with critical CAD. Methods and Results—Paired samples of epicardial and subcutaneous adipose tissues were harvested at the outset of elective CABG surgery (n=42; age 65±10 years). Local expression of chemokine (monocyte chemotactic protein [MCP]-1) and inflammatory cytokines (interleukin [IL]-1&bgr;, IL-6, and tumor necrosis factor [TNF]-&agr;) was analyzed by TaqMan real-time reverse transcription–polymerase chain reaction (mRNA) and by ELISA (protein release over 3 hours). Significantly higher levels of IL-1&bgr;, IL-6, MCP-1, and TNF-&agr; mRNA and protein were observed in epicardial adipose stores. Proinflammatory properties of epicardial adipose tissue were noted irrespective of clinical variables (diabetes, body mass index, and chronic use of statins or ACE inhibitors/angiotensin II receptor blockers) or plasma concentrations of circulating biomarkers. In a subset of samples (n=11), global gene expression was explored by DNA microarray hybridization and confirmed the presence of a broad inflammatory reaction in epicardial adipose tissue in patients with coronary artery disease. The above findings were paralleled by the presence of inflammatory cell infiltrates in epicardial adipose stores. Conclusions—Epicardial adipose tissue is a source of several inflammatory mediators in high-risk cardiac patients. Plasma inflammatory biomarkers may not adequately reflect local tissue inflammation. Current therapies do not appear to eliminate local inflammatory signals in epicardial adipose tissue.

Anti-inflammatory effects of the Nigella sativa seed extract, thymoquinone, in pancreatic cancer cells

BACKGROUND Both hereditary and sporadic forms of chronic pancreatitis are associated with an increased risk of developing pancreatic ductal adenocarcinoma (PDA). Inflammation has been identified as a significant factor in the development of solid tumour malignancies. We have recently shown that thymoquinone (Tq), the major constituent of Nigella sativa oil extract, induced apoptosis and inhibited proliferation in PDA cells. Tq also increased p21 WAF1 expression, inhibited histone deacetylase (HDAC) activity, and induced histone hyperacetylation. HDAC inhibitors have been shown to ameliorate inflammation-associated cancer. In this study, we evaluated the anti-inflammatory potential of Tq in PDA cells in comparison with that of a specific HDAC inhibitor, trichostatin A (TSA). METHODS PDA cells were treated with or without Tq (25-75 microM), with or without pre-treatment of tumour necrosis factor (TNF)-alpha (25 ng/ml). The effect of Tq on the expression of different proinflammatory cytokines and chemokines was analysed by real-time polymerase chain reaction (PCR). Luciferase-labelled promoter studies evaluated the effect of Tq on the transcription of monocyte chemoattractant protein-1 (MCP-1) and nuclear factor-kappaB (NF-kappaB). The effect of Tq on the constitutive and TNF-alpha-induced activation and nuclear translocation of NF-kappaB was examined by ELISA and immunohistochemistry. RESULTS Tq dose- and time-dependently significantly reduced PDA cell synthesis of MCP-1, TNF-alpha, interleukin (IL)-1beta and Cox-2. At 24 h, Tq almost completely abolished the expression of these cytokines, whereas TSA had a less dramatic effect. Tq, but not TSA, significantly and dose-dependently reduced the intrinsic activity of the MCP-1 promoter. Tq also inhibited the constitutive and TNF-alpha-mediated activation of NF-kappaB in PDA cells and reduced the transport of NF-kappaB from the cytosol to the nucleus. CONCLUSIONS Our data demonstrate previously undescribed anti-inflammatory activities of Tq in PDA cells, which are paralleled by inhibition of NF-kappaB. Tq as a novel inhibitor of proinflammatory pathways provides a promising strategy that combines anti-inflammatory and proapoptotic modes of action.

Effects of thymoquinone in the expression of mucin 4 in pancreatic cancer cells: Implications for the development of novel cancer therapies

Pancreatic cancer is one of the most lethal cancers in the world, as it continues to be resistant to any therapeutic approaches. The high molecular weight glycoprotein mucin 4 (MUC4) is aberrantly expressed in pancreatic cancer and contributes to the regulation of differentiation, proliferation, metastasis, and the chemoresistance of pancreatic cancer cells. The absence of its expression in the normal pancreatic ductal cells makes MUC4 a promising target for novel cancer therapeutics. Natural products have been widely investigated as potential candidates in cancer therapies, and thymoquinone (TQ), extracted from the seeds of Nigella sativa, has shown excellent antineoplastic properties in some systems. In the present study, we evaluated the effect of TQ on pancreatic cancer cells and specifically investigated its effect on MUC4 expression. The MUC4-expressing pancreatic cancer cells FG/COLO357 and CD18/HPAF were incubated with TQ, and in vitro functional assays were done. The results obtained indicate that treatment with TQ downregulated MUC4 expression through the proteasomal pathway and induced apoptosis in pancreatic cancer cells by the activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase pathways. In agreement with previous studies, the decrease in MUC4 expression correlated with an increase in apoptosis, decreased motility, and decreased migration of pancreatic cancer cells. MUC4 transient silencing studies showed that c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase pathways are activated in pancreatic cancer cells, indicating that the activation of these pathways by TQ is directly related to the MUC4 downregulation induced by the drug. Overall, TQ has potential for the development of novel therapies against pancreatic cancer. Mol Cancer Ther; 9(5); 1419–31. ©2010 AACR.

Angiotensin II induces vascular endothelial growth factor in pancreatic cancer cells through an angiotensin II type 1 receptor and ERK1/2 signaling.

Vascular endothelial growth factor (VEGF) is a crucial pro-angiogenic component in pancreatic ductal adenocarcinoma (PDA), and its high expression levels have been correlated with poor prognosis and early postoperative recurrence. We have recently shown that high levels of angiotensin II (AngII) type 1 receptor (AT1R) correlate and colocalize with VEGF in invasive PDA and that AngII induces VEGF expression in PDA cell lines. In this study, we explored the signaling mechanisms involved in the AngII-mediated VEGF induction and correlated AT1R and VEGF expression in noninvasive precursor lesions. An AT1R antagonist significantly (p < 0.05) inhibited the AngII-mediated induction of VEGF messenger RNA and protein in all PDA cell lines. AngII-VEGF induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a mitogen-activated protein kinase signaling mechanism. AngII activated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the AngII-induced VEGF synthesis. Immunohistochemical analysis of precursor lesions showed increased expression of AT1R in most ductal cells undergoing metaplasia. Pancreatic intraepithelial neoplasms showed more intense AT1R staining when compared to intraductal papillary mucinous neoplasms, which showed heterogeneous immunoreactivity. VEGF followed the same distribution pattern of AT1R in both lesions. AT1R expression in the premalignant pancreatic lesions suggests its involvement in tumor progression and angiogenesis. Our mechanistic findings provide the first insight into an AngII-initiated signaling pathway that regulates PDA angiogenesis. An AT1R-mediated VEGF induction suggests the possibility of AT1R blockade as a novel therapeutic strategy to control angiogenesis in PDA.

Blocking angiotensin II Type 1 receptor triggers apoptotic cell death in human pancreatic cancer cells.

Objectives: Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with an annual mortality rate close to its annual incidence. We recently demonstrated that angiotensin II (AngII) type 1 receptor (AT1R) might be involved in PDA angiogenesis. This study evaluated the antiproliferative and proapoptotic effects of an AT1R blocker, losartan, in PDA cells with different p53 mutation status. Methods: Cell cycle was analyzed by flow cytometric analysis of DNA content; apoptosis by annexin V-fluorescein isothiocyanate (V-FITC) and terminal deoxytransferase (TdT)-mediated dUTP nick-end labeling staining; messenger RNA and protein by real-time polymerase chain reaction and Western blotting; caspase-3 activity by colorimetric assay; and promoter activity by luciferase assay. Results: Losartan dose-dependently decreased cell survival and increased their preG1 accumulation. It also increased p53, p21, p27, and Bax and reduced Bcl-2 and Bcl-xl expression. In wtp53 cells, losartan increased p53 transcription and activated caspase-3 in both cell lines. However, its proapoptotic effects in mtp53 cells were mainly caspase-3-dependent. Conclusion: Our data describe the involvement of AT1R in PDA cell apoptotic machinery and provide the first evidences that losartan stimulates the proapoptotic signaling pathways regardless of the p53 mutation status. As loss of p53 function is frequently observed in PDA patients, our data suggest AT1R blockade as a novel therapeutic strategy to control PDA growth.

Circadian Gene Expression and Clinicopathologic Correlates in Pancreatic Cancer

IntroductionThe circadian rhythm is responsible for physiologic homeostasis, behavior, and components of multiple metabolic processes. Disruption of the circadian rhythm is associated with cancer development, and several circadian clock genes have been implicated in loss of cell cycle control, impaired DNA damage repair, and subsequent tumor formation. Here, we investigated the expression profiles of several circadian clock genes in pancreatic ductal adenocarcinoma (PDA).MethodsQuantitative real-time polymerase chain reaction was used to examine the circadian clock genes (brain-muscle-like (Bmal)-ARNTL, circadian locomotor output cycles kaput (Clock), cryptochrome 1 (Cry1), cryptochrome 2 (Cry2), casein kinase 1ε (CK1ε), period 1 (Per1), period 2 (Per2), period 3 (Per3), timeless (Tim), and timeless-interacting protein (Tipin)) in PDA, as well as matching adjacent and benign tissue. Logistic regression models with robust variance were used to analyze the gene expression levels, and Kaplan–Meier survival curves were generated based on gene expression.ResultsIn the tumor tissue of PDA patients, compared to their matched adjacent tissue, expression levels of all circadian genes were lower, with statistical significance for Per1, Per2, Per3, Cry1, Cry2, Tipin, Tim, CK1ε, Bmal-ARNTL, and Clock (p < 0.025). PDA tumors also expressed significantly lower levels of the circadian genes when compared to benign lesions for Per1, Per2, Per3, Cry2, Tipin, and CK1ε. A significant association between low levels of expression in the tumors and reduced survival was found with Per1, Per2, Per3, Cry2, Tipin, CK1ε, Clock, and Bmal-ARNTL.ConclusionsOur results reveal for the first time a dysregulated transcription of several circadian genes in PDA. Elevation of the gene levels in the benign and matched adjacent tissues may be indicative of their role during the process of tumorigenesis. The potential of using circadian genes as predictive markers of the outcomes and survival and distinguishing PDA from benign pancreas must be studied in larger populations to validate and demonstrate their eventual clinical utility.

Induction of osteopontin expression by nicotine and cigarette smoke in the pancreas and pancreatic ductal adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with etiological association with cigarette smoking. Nicotine, an important component of cigarettes, exists at high concentrations in the bloodstream of smokers. Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype and activates signaling pathways that induce cell survival, proliferation, invasion, and metastasis. Here, we investigated the potential molecular basis of nicotines role in PDA through studying its effect on OPN. Nicotine significantly (p < 0.02) increased OPN mRNA and protein secretion in PDA cells through activation of the OPN gene promoter. The OPN mRNA induction was inhibited by the nicotinic acetylcholine receptor antagonist, mechamylamine. Further, the tyrosine kinase inhibitor genistein inhibited the nicotine‐mediated induction of OPN, suggesting that mitogen activated protein kinase signaling mechanism is involved. Nicotine activated the phosphorylation of ERK1/2, but not p38 or c‐Jun NH2‐terminal MAP kinases. Inhibition of ERK1/2 activation reduced the nicotine‐induced OPN synthesis. Rats exposed to cigarette smoke showed a dose‐dependent increase in pancreatic OPN that paralleled the rise of pancreatic and plasma nicotine levels. Analysis of cancer tissue from invasive PDA patients, the majority of whom were smokers, showed the presence of significant amounts of OPN in the malignant ducts and the surrounding pancreatic acini. Our data suggest that nicotine may contribute to PDA pathogenesis through upregulation of OPN. They provide the first insight into a nicotine‐initiated signal transduction pathway that regulates OPN as a possible tumorigenic mechanism in PDA.

Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer

BACKGROUND Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. METHODS We analyzed paired PDA-normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA. RESULTS COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. CONCLUSION We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.

Caveolin-1 promotes pancreatic cancer cell differentiation and restores membranous E-cadherin via suppression of the epithelial-mesenchymal transition

Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we over-expressed Cav-1 in a pancreatic cancer cell line, Panc 10.05, that does not normally express Cav-1. Here, we show that Cav-1 expression in pancreatic cancer cells induces an epithelial phenotype and promotes cell-cell contact, with increased expression of plasma membrane bound E-cadherin and beta-catenin. Mechanistically, Cav-1 induces Snail downregulation and decreased activation of AKT, MAPK and TGF-beta-Smad signaling pathways. In vitro, Cav-1 expression reduces cell migration and invasion, and attenuates doxorubicin-chemoresistance of pancreatic cancer cells. Importantly, in vivo studies revealed that Cav-1 expression greatly suppresses tumor formation in a xenograft model. Most interestingly, Panc/Cav-1 tumors displayed organized nests of differentiated cells that were totally absent in control tumors. Confirming our in vitro results, these nests of differentiated cells showed reexpression of E-cadherin and beta-catenin at the cell membrane. Thus, we provide evidence that Cav-1 functions as a crucial modulator of EMT and cell differentiation in pancreatic cancer.

Creating a tumor-resistant microenvironment: Cell-mediated delivery of TNFα completely prevents breast cancer tumor formation in vivo

Here, we provide the necessary proof of concept, that it is possible to metabolically create a non-permissive or “hostile” stromal microenvironment, which actively prevents tumor engraftment in vivo. We developed a novel genetically engineered fibroblast cell line that completely prevents tumor formation in mice, with a 100% protection rate. No host side effects were apparent. This could represent a viable cellular strategy for preventing and treating a variety of human cancers. More specifically, we examined the autocrine and paracrine effects of the cellular delivery of TNFα on breast cancer tumor growth and cancer metabolism. For this purpose, we recombinantly overexpressed TNFα in human breast cancer cells (MDA-MB-231) or human immortalized fibroblasts (hTERT-BJ1). Our results directly show that TNFα functions as a potent tumor suppressor. Remarkably, TNFα-expressing breast cancer cells were viable, without any significant increases in their basal apoptotic rate. However, after 4 weeks post-implantation, TNFα-expressing breast cancer cells failed to form any tumors in xenografted mice (0 tumors/10 injections), ultimately conferring 100% protection against tumorigenesis. Similarly, TNFα-overexpressing fibroblasts were also viable, without any increases in apoptosis. Significantly, complete tumor suppression was obtained by co-injecting TNFα expressing stromal fibroblasts with human breast cancer cells, indicating that paracrine cell-mediated delivery of TNFα can also prevent tumor engraftment and growth (0 tumors/10 injections). Mechanistically, TNFα induced autophagy and mitochondrial dysfunction in both epithelial cancer cells and stromal fibroblasts, preventing energy transfer from the tumor microenvironment, likely “starving” the cancer cells to death. In addition, via qRT-PCR analysis of MDA-MB-231 cells, we observed that TNFα mediated the upregulation of gene transcripts associated with inflammation and senescence [IL-1-β, IL-6, IL-8, MCP-1, COX-2, p21(WAF1/CIP1)] and downregulated known tumor-promoting genes (collagen VI and MMP2). Recombinant overexpression of TNFα receptor(s) in MDA-MB-231 cells also significantly reduced tumor growth, but was not as effective as the TNFα ligand itself in preventing tumor growth. Thus, we propose that stromal cell-mediated delivery of TNFα to human tumors [using transfected fibroblasts or mesenchymal stem cells (hMSCs)] may be a novel and effective strategy for the prevention and treatment of human cancers.