Apigenin Targets MicroRNA-155, Enhances SHIP-1 Expression, and Augments Anti-Tumor Responses in Pancreatic Cancer.

Pancreatic cancer (PC) is a deadly disease with a grim prognosis. Pancreatic tumor derived factors (TDF) contribute to the induction of an immunosuppressive tumor microenvironment (TME) that impedes the effectiveness of immunotherapy. PC-induced microRNA-155 (miRNA-155) represses expression of Src homology 2 (SH2) domain-containing Inositol 5'-phosphatase-1 (SHIP-1), a regulator of myeloid cell development and function, thus impacting anti-tumor immunity. We recently reported that the bioflavonoid apigenin (API) increased SHIP-1 expression which correlated with the expansion of tumoricidal macrophages (TAM) and improved anti-tumor immune responses in the TME of mice with PC. We now show that API transcriptionally regulates SHIP-1 expression via the suppression of miRNA-155, impacting anti-tumor immune responses in the bone marrow (BM) and TME of mice with PC. We discovered that API reduced miRNA-155 in the PC milieu, which induced SHIP-1 expression. This promoted the restoration of myelopoiesis and increased anti-tumor immune responses in the TME of heterotopic, orthotopic and transgenic SHIP-1 knockout preclinical mouse models of PC. Our results suggest that manipulating SHIP-1 through miR-155 may assist in augmenting anti-tumor immune responses and aid in the therapeutic intervention of PC.

Role of environmental toxicants in the development of hypertensive and cardiovascular diseases.

The incidence of hypertension with diabetes mellitus (DM) as a co-morbid condition is on the rise worldwide. In 2000, an estimated 972 million adults had hypertension, which is predicted to grow to 1.56 billion by 2025. Hypertension often leads to diabetes mellitus that strongly puts the patients at an increased risk of cardiovascular, kidney, and/or atherosclerotic diseases. Hypertension has been identified as a major risk factor for the development of diabetes; patients with hypertension are at two-to-three-fold higher risk of developing diabetes than patients with normal blood pressure (BP). Causes for the increase in hypertension and diabetes are not well understood, environmental factors (e.g., exposure to environmental toxicants like heavy metals, organic solvents, pesticides, alcohol, and urban lifestyle) have been postulated as one of the reasons contributing to hypertension and cardiovascular diseases (CVD). The mechanism of action(s) of these toxicants in developing hypertension and CVDs is not well defined. Research studies have linked hypertension with the chronic consumption of alcohol and exposure to metals like lead, mercury, and arsenic have also been linked to hypertension and CVD. Workers chronically exposed to styrene have a higher incidence of CVD. Recent studies have demonstrated that exposure to particulate matter (PM) in diesel exhaust and urban air contributes to increased CVD and mortality. In this review, we have imparted the role of environmental toxicants such as heavy metals, organic pollutants, PM, alcohol, and some drugs in hypertension and CVD along with possible mechanisms and limitations in extrapolating animal data to humans.

Role of Flavonoids as Epigenetic Modulators in Cancer Prevention and Therapy.

Epigenetic regulation involves reversible changes in histones and DNA modifications that can be inherited without any changes in the DNA sequence. Dysregulation of normal epigenetic processes can lead to aberrant gene expression as observed in many diseases, notably cancer. Recent insights into the mechanisms of DNA methylation, histone modifications, and non-coding RNAs involved in altered gene expression profiles of tumor cells have caused a paradigm shift in the diagnostic and therapeutic approaches towards cancer. There has been a surge in search for compounds that could modulate the altered epigenetic landscape of tumor cells, and to exploit their therapeutic potential against cancers. Flavonoids are naturally occurring phenol compounds which are abundantly found among phytochemicals and have potentials to modulate epigenetic processes. Knowledge of the precise flavonoid-mediated epigenetic alterations is needed for the development of epigenetics drugs and combinatorial therapeutic approaches against cancers. This review is aimed to comprehensively explore the epigenetic modulations of flavonoids and their anti-tumor activities.

Global Phosphoproteomics Reveal CDK Suppression as a Vulnerability to KRas Addiction in Pancreatic Cancer.

PURPOSE: Among human cancers that harbor mutant (mt) KRas, some, but not all, are dependent on mt KRas. However, little is known about what drives KRas dependency. EXPERIMENTAL DESIGN: Global phosphoproteomics, screening of a chemical library of FDA drugs, and genome-wide CRISPR/Cas9 viability database analysis were used to identify vulnerabilities of KRas dependency. RESULTS: Global phosphoproteomics revealed that KRas dependency is driven by a cyclin-dependent kinase (CDK) network. CRISPR/Cas9 viability database analysis revealed that, in mt KRas-driven pancreatic cancer cells, knocking out the cell-cycle regulators CDK1 or CDK2 or the transcriptional regulators CDK7 or CDK9 was as effective as knocking out KRas. Furthermore, screening of a library of FDA drugs identified AT7519, a CDK1, 2, 7, and 9 inhibitor, as a potent inducer of apoptosis in mt KRas-dependent, but not in mt KRas-independent, human cancer cells. In vivo AT7519 inhibited the phosphorylation of CDK1, 2, 7, and 9 substrates and suppressed growth of xenografts from 5 patients with pancreatic cancer. AT7519 also abrogated mt KRas and mt p53 primary and metastatic pancreatic cancer in three-dimensional (3D) organoids from 2 patients, 3D cocultures from 8 patients, and mouse 3D organoids from pancreatic intraepithelial neoplasia, primary, and metastatic tumors. CONCLUSIONS: A link between CDK hyperactivation and mt KRas dependency was uncovered and pharmacologically exploited to abrogate mt KRas-driven pancreatic cancer in highly relevant models, warranting clinical investigations of AT7519 in patients with pancreatic cancer.

Farnesyl dimethyl chromanol targets colon cancer stem cells and prevents colorectal cancer metastasis.

The activation and growth of tumour-initiating cells with stem-like properties in distant organs characterize colorectal cancer (CRC) growth and metastasis. Thus, inhibition of colon cancer stem cell (CCSC) growth holds promise for CRC growth and metastasis prevention. We and others have shown that farnesyl dimethyl chromanol (FDMC) inhibits cancer cell growth and induces apoptosis in vitro and in vivo. We provide the first demonstration that FDMC inhibits CCSC viability, survival, self-renewal (spheroid formation), pluripotent transcription factors (Nanog, Oct4, and Sox2) expression, organoids formation, and Wnt/ß-catenin signalling, as evidenced by comparisons with vehicle-treated controls. In addition, FDMC inhibits CCSC migration, invasion, inflammation (NF-kB), angiogenesis (vascular endothelial growth factor, VEGF), and metastasis (MMP9), which are critical tumour metastasis processes. Moreover, FDMC induced apoptosis (TUNEL, Annexin V, cleaved caspase 3, and cleaved PARP) in CCSCs and CCSC-derived spheroids and organoids. Finally, in an orthotopic (cecum-injected CCSCs) xenograft metastasis model, we show that FDMC significantly retards CCSC-derived tumour growth (Ki-67); inhibits inflammation (NF-kB), angiogenesis (VEGF and CD31), and ß-catenin signalling; and induces apoptosis (cleaved PARP) in tumour tissues and inhibits liver metastasis. In summary, our results demonstrate that FDMC inhibits the CCSC metastatic phenotype and thereby supports investigating its ability to prevent CRC metastases.

Protein kinase 2 (CK2): a potential regulator of immune cell development and function in cancer.

Protein kinase CK2, formally known as casein kinase II, is ubiquitously expressed and highly conserved serine/threonine or tyrosine kinase enzyme that regulates diverse signaling pathways responsible for cellular processes (i.e., cell proliferation and apoptosis) via interactions with over 500 known substrates. The enzyme's physiological interactions and cellular functions have been widely studied, most notably in the blood and solid malignancies. CK2 has intrinsic role in carcinogenesis as overexpression of CK2 subunits (α, α`, and ß) and deregulation of its activity have been linked to various forms of cancers. CK2 also has extrinsic role in cancer stroma or in the tumor microenvironment (TME) including the immune cells. However, very few research studies have focused on extrinsic role of CK2 in regulating immune responses as a therapeutic alternative for cancer. The following review discusses CK2's regulation of key signaling events [Nuclear factor kappa B (NF-κB), Janus kinase/signal transducer and activators of transcription (JAK/STAT), Hypoxia inducible factor-1alpha (HIF-1α), Cyclooygenase-2 (COX-2), Extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), Notch, Protein kinase B/AKT, Ikaros and Wnt] that can influence the development and function of immune cells in cancer. Potential clinical trials using potent CK2 inhibitors will facilitate and improve the treatment of human malignancies.

Apigenin Increases SHIP-1 Expression, Promotes Tumoricidal Macrophages and Anti-Tumor Immune Responses in Murine Pancreatic Cancer.

Pancreatic cancer (PC) has an extremely poor prognosis due to the expansion of immunosuppressive myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in the inflammatory tumor microenvironment (TME), which halts the recruitment of effector immune cells and renders immunotherapy ineffective. Thus, the identification of new molecular targets that can modulate the immunosuppressive TME is warranted for PC intervention. Src Homology-2 (SH2) domain-containing Inositol 5'-Phosphatase-1 (SHIP-1) is a lipid signaling protein and a regulator of myeloid cell development and function. Herein, we used the bioflavonoid apigenin (API) to reduce inflammation in different PC models. Wild type mice harboring heterotopic or orthotopic PC were treated with API, which induced SHIP-1 expression, reduced inflammatory tumor-derived factors (TDF), increased the proportion of tumoricidal macrophages and enhanced anti-tumor immune responses, resulting in a reduction in tumor burden compared to vehicle-treated PC mice. In contrast, SHIP-1-deficient mice exhibited an increased tumor burden and displayed augmented proportions of pro-tumor macrophages. These results provide further support for the importance of SHIP-1 expression in promoting pro-tumor macrophage development in the pancreatic TME. Our findings suggest that agents augmenting SHIP-1 expression may provide novel therapeutic options for the treatment of PC.

Targeting the IκB Kinase Enhancer and Its Feedback Circuit in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with an overall median 5-year survival rate of 8%. This poor prognosis is because of the development of resistance to chemotherapy and radiation therapy and lack of effective targeted therapies. IκB kinase enhancer (IKBKE) overexpression was previously implicated in chemoresistance. Because IKBKE is frequently elevated in PDAC and IKBKE inhibitors are currently in clinical trials, we evaluated IKBKE as a therapeutic target in this disease. Depletion of IKBKE was found to significantly reduce PDAC cell survival, growth, cancer stem cell renewal, and cell migration and invasion. Notably, IKBKE inhibitor CYT387 and IKBKE knockdown dramatically activated the MAPK pathway. Phospho-RTK array analyses showed that IKBKE inhibition leads to rapid upregulation of ErbB3 and IGF-1R expression, which results in MAPK-ERK pathway activation-thereby limiting the efficacy of IKBKE inhibitors. Furthermore, IKBKE inhibition leads to stabilization of FOXO3a, which is required for RTK upregulation on IKBKE inhibition. Finally, we demonstrated that the IKBKE inhibitors synergize with the MEK inhibitor trametinib to significantly induce cell death and inhibit tumor growth and liver metastasis in an orthotopic PDAC mouse model.

Vitamin E δ-tocotrienol sensitizes human pancreatic cancer cells to TRAIL-induced apoptosis through proteasome-mediated down-regulation of c-FLIPs.

BACKGROUND: Vitamin E δ-tocotrienol (VEDT), a vitamin E compound isolated from sources such as palm fruit and annatto beans, has been reported to have cancer chemopreventive and therapeutic effects. METHODS: We report a novel function of VEDT in augmenting tumor necrosis factor-related apoptosis-inducing ligand- (TRAIL-) induced apoptosis in pancreatic cancer cells. The effects of VEDT were shown by its ability to trigger caspase-8-dependent apoptosis in pancreatic cancer cells. RESULTS: When combined with TRAIL, VEDT significantly augmented TRAIL-induced apoptosis of pancreatic cancer cells. VEDT decreased cellular FLICE inhibitory protein (c-FLIP) levels without consistently modulating the expression of decoy death receptors 1, 2, 3 or death receptors 4 and 5. Enforced expression of c-FLIP substantially attenuated VEDT/TRAIL-induced apoptosis. Thus, c-FLIP reduction plays an important part in mediating VEDT/TRAIL-induced apoptosis. Moreover, VEDT increased c-FLIP ubiquitination and degradation but did not affect its transcription, suggesting that VEDT decreases c-FLIP levels through promoting its degradation. Of note, degradation of c-FLIP and enhanced TRAIL-induced apoptosis in pancreatic cancer cells were observed only with the anticancer bioactive vitamin E compounds δ-, γ-, and ß-tocotrienol but not with the anticancer inactive vitamin E compounds α-tocotrienol and α-, ß-, γ-, and δ-tocopherol. CONCLUSIONS: c-FLIP degradation is a key event for death receptor-induced apoptosis by anticancer bioactive vitamin E compounds in pancreatic cancer cells. Moreover, VEDT augmented TRAIL inhibition of pancreatic tumor growth and induction of apoptosis in vivo. Combination therapy with TRAIL agonists and bioactive vitamin E compounds may offer a novel strategy for pancreatic cancer intervention.

Chemoprevention of Azoxymethane-induced Colon Carcinogenesis by Delta-Tocotrienol.

This study evaluated the preclinical activity of δ-tocotrienol (DT3), a bioactive form of vitamin E, in the inhibition of colorectal cancer growth and development in vitro and in vivo DT3 is the most bioactive isomer of vitamin E in inhibiting growth of colorectal cancer cells. However, it had little effect on the proliferation of normal colon mucosal cells NCM460. In HCT-116 and SW-620 colorectal cancer cells, DT3 (50 µmol/L) significantly inhibited malignant transformation (P < 0.02, P < 0.001), cell migration (P < 0.02, P < 0.05), and invasion (P < 0.05, P < 0.01) compared with vehicle. DT3 inhibited markers for epithelial (E-cadherin) to mesenchymal (vimentin) transition, metastasis (matrix metalloproteinase 9), angiogenesis VEGF, inflammation (NF-κB), and Wnt signaling (ß-catenin) compared with vehicle in colorectal cancer cells. DT3 induced apoptosis selectively in colorectal cancer cells (SW-620 cells, HCT-116 cells, and HT-29) without affecting the normal colon cells. In the azoxymethane-induced colorectal carcinogenesis model in rats, DT3 (200 mg/kg orally twice a day) for 20 weeks significantly inhibited colorectal polyps by 70% and colorectal cancer by almost 99% compared with the vehicle treatment group (P < 0.02, P < 0.001), and the cancer inhibition effect was more potent than sulindac (50%). Taken together, these data demonstrate that DT3 is a potential chemopreventive agent in colorectal cancer, warranting further investigation into its clinical use in the prevention and treatment of colorectal cancer.

δ-Tocotrienol, a natural form of vitamin E, inhibits pancreatic cancer stem-like cells and prevents pancreatic cancer metastasis.

The growth, metastasis, and chemotherapy resistance of pancreatic ductal adenocarcinoma (PDAC) is characterized by the activation and growth of tumor-initiating cells in distant organs that have stem-like properties. Thus, inhibiting growth of these cells may prevent PDAC growth and metastases. We have demonstrated that δ-tocotrienol, a natural form of vitamin E (VEDT), is bioactive against cancer, delays progression, and prevents metastases in transgenic mouse models of PDAC. In this report, we provide the first evidence that VEDT selectively inhibits PDAC stem-like cells. VEDT inhibited the viability, survival, self-renewal, and expression of Oct4 and Sox2 transcription factors in 3 models of PDAC stem-like cells. In addition, VEDT inhibited the migration, invasion, and several biomarkers of epithelial-to-mesenchymal transition and angiogenesis in PDAC cells and tumors. These processes are critical for tumor metastases. Furthermore, in the L3.6pl orthotopic model of PDAC metastases, VEDT significantly inhibited growth and metastases of these cells. Finally, in an orthotopic xenograft model of human PDAC stem-like cells, we showed that VEDT significantly retarded the growth and metastases of gemcitabine-resistant PDAC human stem-like cells. Because VEDT has been shown to be safe and to reach bioactive levels in humans, this work supports investigating VEDT for chemoprevention of PDAC metastases.

Knockdown of CSE1L Gene in Colorectal Cancer Reduces Tumorigenesis in Vitro.

Human cellular apoptosis susceptibility (chromosomal segregation 1-like, CSE1L) gene plays a role in nuclear-to-cytoplasm transport and chromosome segregation during mitosis, cellular proliferation, and apoptosis. CSE1L is involved in colon carcinogenesis. CSE1L gene expression was assessed with three data sets using Affymetrix U133 + gene chips on normal human colonic mucosa (NR), adenomas (ADs), and colorectal carcinoma (CRC). CSE1L protein expression in CRC, AD, and NR from the same patients was measured by immunohistochemistry using a tissue microarray. We evaluated CSE1L expression in CRC cells (HCT116, SW480, and HT29) and its biological functions. CSE1L mRNA was significantly increased in all AD and CRC compared with NR (P < 0.001 and P = 0.02, respectivly). We observed a change in CSE1L staining intensity and cellular localization by immunohistochemistry. CSE1L was significantly increased during the transition from AD to CRC when compared with NR in a CRC tissue microarray (P = 0.01 and P < 0.001). HCT116, SW480, and HT29 cells also expressed CSE1L protein. CSE1L knockdown by shRNA inhibited protein, resulting in decreased cell proliferation, reduced colony formation in soft agar, and induction of apoptosis. CSE1L protein is expressed early and across all stages of CRC development. shRNA knockdown of CSE1L was associated with inhibition of tumorigenesis in CRC cells. CSE1L may represent a potential target for treatment of CRC.

Effects of Antioxidant N-acetylcysteine Against Paraquat-Induced Oxidative Stress in Vital Tissues of Mice.

Paraquat (PQ) is a commonly used herbicide that induces oxidative stress via reactive oxygen species (ROS) generation. This study aimed to investigate the effects of the antioxidant N-acetylcysteine (NAC) against PQ-induced oxidative stress in mice. Male Balb/C mice (24) were randomly divided into 4 groups and treated for 3 weeks: 1) control (saline), 2) NAC (0.5% in diet), 3) PQ (20 mg/kg, IP) and 4) combination (PQ + NAC). Afterwards mice were sacrificed and oxidative stress markers were analyzed. Our data showed no significant change in serum antioxidant capacity. PQ enhanced lipid peroxidation (MDA) levels in liver tissue compared to control whereas NAC decreased MDA levels (p<0.05). NAC significantly increased MDA in brain tissue (p<0.05). PQ significantly depleted glutathione (GSH) levels in liver (p=0.001) and brain tissue (p<0.05) but non-significant GSH depletion in lung tissue. NAC counteracted PQ, showing a moderate increase GSH levels in liver and brain tissues. PQ significantly increased 8-oxodeoxyguanosine (8-OH-dG) levels (p<0.05) in liver tissue compared to control without a significant change in brain tissue. NAC treatment ameliorated PQ-induced oxidative DNA damage in the liver tissue. PQ significantly decreased the relative mtDNA amplification and increased the frequency of lesions in liver and brain tissue (p<0.0001), while NAC restored the DNA polymerase activity in liver tissue but not in brain tissue. In conclusion, PQ induced lipid peroxidation, oxidative nuclear DNA and mtDNA damage in liver tissues and depleted liver and brain GSH levels. NAC supplementation ameliorated the PQ-induced oxidative stress response in liver tissue of mice.

Role of Transcription Factors in Steatohepatitis and Hypertension after Ethanol: The Epicenter of Metabolism.

Chronic alcohol consumption induces multi-organ damage, including alcoholic liver disease (ALD), pancreatitis and hypertension. Ethanol and ethanol metabolic products play a significant role in the manifestation of its toxicity. Ethanol metabolizes to acetaldehyde and produces reduced nicotinamide adenine dinucleotide (NADH) by cytosolic alcohol dehydrogenase. Ethanol metabolism mediated by cytochrome-P450 2E1 causes oxidative stress due to increased production of reactive oxygen species (ROS). Acetaldehyde, increased redox cellular state and ROS activate transcription factors, which in turn activate genes for lipid biosynthesis and offer protection of hepatocytes from alcohol toxicity. Sterol regulatory element binding proteins (SREBPs) and peroxisome proliferator activated-receptors (PPARs) are two key lipogenic transcription factors implicated in the development of fatty liver in alcoholic and non-alcoholic steatohepatitis. SREBP-1 is activated in the livers of chronic ethanol abusers. An increase in ROS activates nuclear factor erythroid-2-related factor-2 (Nrf2) and hypoxia inducible factor (HIF) to provide protection to hepatocytes from ethanol toxicity. Under ethanol exposure, due to increased gut permeability, there is release of gram-negative bacteria-derived lipopolysaccharide (LPS) from intestine causing activation of immune response. In addition, the metabolic product, acetaldehyde, modifies the proteins in hepatocyte, which become antigens inviting auto-immune response. LPS activates macrophages, especially the liver resident macrophages, Kupffer cells. These Kupffer cells and circulating macrophages secrete various cytokines. The level of tumor necrosis factor-α (TNFα), interleukin-1beta (IL-1ß), IL-6, IL-8 and IL-12 have been found elevated among chronic alcoholics. In addition to elevation of these cytokines, the peripheral iron (Fe(2+)) is also mobilized. An increased level of hepatic iron has been observed among alcoholics. Increased ROS, IL-1ß, acetaldehyde, and increased hepatic iron, all activate nuclear factor-kappa B (NF-κB) transcription factor. Resolution of increased reactive oxygen species requires increased expression of genes responsible for dismutation of increased ROS which is partially achieved by IL-6 mediated activation of signal transducers and activators of transcription 3 (STAT3). In addition to these transcription factors, activator protein-1 may also be activated in hepatocytes due to its association with resolution of increased ROS. These transcription factors are central to alcohol-mediated hepatotoxicity.

Inflammation, oxidative stress and renin angiotensin system in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease associated with cardiovascular dysfunction including myocardial infarction, unstable angina, sudden cardiac death, stroke and peripheral thromboses. It has been predicted that atherosclerosis will be the primary cause of death in the world by 2020. Atherogenesis is initiated by endothelial injury due to oxidative stress associated with cardiovascular risk factors including diabetes mellitus, hypertension, cigarette smoking, dyslipidemia, obesity, and metabolic syndrome. The impairment of the endothelium associated with cardiovascular risk factors creates an imbalance between vasodilating and vasoconstricting factors, in particular, an increase in angiotensin II (Ang II) and a decrease in nitric oxide. The renin-angiotensin system (RAS), and its primary mediator Ang II, also have a direct influence on the progression of the atherosclerotic process via effects on endothelial function, inflammation, fibrinolytic balance, and plaque stability. Anti-inflammatory agents [statins, secretory phospholipase A2 inhibitor, lipoprotein-associated phospholipase A2 inhibitor, 5-lipoxygenase activating protein, chemokine motif ligand-2, C-C chemokine motif receptor 2 pathway inhibitors, methotrexate, IL-1 pathway inhibitor and RAS inhibitors (angiotensin-converting enzyme inhibitors)], Ang II receptor blockers and ranin inhibitors may slow inflammatory processes and disease progression. Several studies in human using anti-inflammatory agents and RAS inhibitors revealed vascular benefits and reduced progression of coronary atherosclerosis in patients with stable angina pectoris; decreased vascular inflammatory markers, improved common carotid intima-media thickness and plaque volume in patients with diagnosed atherosclerosis. Recent preclinical studies have demonstrated therapeutic efficacy of vitamin D analogs paricalcitol in ApoE-deficient atherosclerotic mice.

Effect of paricalcitol and enalapril on renal inflammation/oxidative stress in atherosclerosis.

AIM: To investigate the protective effect of paricalcitol and enalapril on renal inflammation and oxidative stress in ApoE-knock out mice. METHODS: Animals treated for 4 mo as group (1) ApoE-knock out plus vehicle, group (2) ApoE-knock out plus paricalcitol (200 ng thrice a week), (3) ApoE-knock out plus enalapril (30 mg/L), (4) ApoE-knock out plus paricalcitol plus enalapril and (5) normal. Blood pressure (BP) was recorded using tail cuff method. The kidneys were isolated for biochemical assays using spectrophotometer and Western blot analyses. RESULTS: ApoE-deficient mice developed high BP (127 ± 3 mmHg) and it was ameliorated by enalapril and enalapril plus paricalcitol treatments but not with paricalcitol alone. Renal malondialdehyde concentrations, p22(phox), manganese-superoxide dismutase, inducible nitric oxide synthase (NOS), monocyte chemoattractant protein-1, tumor necrosis factor-alpha and transforming growth factor-ß1 levels significantly elevated but reduced glutathione, CuZn-SOD and eNOS levels significantly depleted in ApoE-knock out animals compared to normal. Administration of paricalcitol, enalapril and combined together ameliorated the renal inflammation and oxidative stress in ApoE-knock out animals. CONCLUSION: Paricalcitol and enalapril combo treatment ameliorates renal inflammation as well as oxidative stress in atherosclerotic animals.

Prognostic Fifteen-Gene Signature for Early Stage Pancreatic Ductal Adenocarcinoma.

The outcomes of patients treated with surgery for early stage pancreatic ductal adenocarcinoma (PDAC) are variable with median survival ranging from 6 months to more than 5 years. This challenge underscores an unmet need for developing personalized medicine strategies to refine the current treatment decision-making process. To derive a prognostic gene signature for patients with early stage PDAC, a PDAC cohort from Moffitt Cancer Center (n = 63) was used with overall survival (OS) as the primary endpoint. This was further evaluated using an independent microarray cohort dataset (Stratford et al: n = 102). Technical validation was performed by NanoString platform. A prognostic 15-gene signature was developed and showed a statistically significant association with OS in the Moffitt cohort (hazard ratio [HR] = 3.26; p<0.001) and Stratford et al cohort (HR = 2.07; p = 0.02), and was independent of other prognostic variables. Moreover, integration of the signature with the TNM staging system improved risk prediction (p<0.01 in both cohorts). In addition, NanoString validation showed that the signature was robust with a high degree of reproducibility and the association with OS remained significant in the two cohorts. The gene signature could be a potential prognostic tool to allow risk-adapted stratification of PDAC patients into personalized treatment protocols; possibly improving the currently poor clinical outcomes of these patients.

Plasma MicroRNAs as Novel Biomarkers for Patients with Intraductal Papillary Mucinous Neoplasms of the Pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers worldwide, partly because methods are lacking to detect disease at an early, operable stage. Noninvasive PDAC precursors called intraductal papillary mucinous neoplasms (IPMN) exist, and strategies are needed to aid in their proper diagnosis and management. Data support the importance of miRNAs in the progression of IPMNs to malignancy, and we hypothesized that miRNAs may be shed from IPMN tissues and detected in blood. Our primary goals were to measure the abundance of miRNAs in archived preoperative plasma from individuals with pathologically confirmed IPMNs and healthy controls and discover plasma miRNAs that distinguish between IPMN patients and controls and between "malignant" and "benign" IPMNs. Using novel nCounter technology to evaluate 800 miRNAs, we showed that a 30-miRNA signature distinguished 42 IPMN cases from 24 controls [area underneath the curve (AUC) = 74.4; 95% confidence interval (CI), 62.3-86.5, P = 0.002]. The signature contained novel miRNAs and miRNAs previously implicated in pancreatic carcinogenesis that had 2- to 4-fold higher expression in cases than controls. We also generated a 5-miRNA signature that discriminated between 21 malignant (high-grade dysplasia and invasive carcinoma) and 21 benign (low- and moderate-grade dysplasia) IPMNs (AUC = 73.2; 95% CI, 57.6-73.2, P = 0.005), and showed that paired plasma and tissue samples from patients with IPMNs can have distinct miRNA expression profiles. This study suggests feasibility of using new cost-effective technology to develop a miRNA-based blood test to aid in the preoperative identification of malignant IPMNs that warrant resection while sparing individuals with benign IPMNs the morbidity associated with overtreatment.

Triciribine Phosphate Monohydrate, an AKT Inhibitor, Enhances Gemcitabine Activity in Pancreatic Cancer Cells.

BACKGROUND: Pancreatic cancer is a highly lethal cancer due to early metastasis and resistance to current chemotherapeutic agents. Abnormal protein kinase B (AKT) activation is an important mechanism of chemoresistance to gemcitabine, the most widely used agent in pancreatic cancer. MATERIAL AND METHODS: In the study, we tested the hypothesis that combining an AKT inhibitor with gemcitabine would augment anti-tumor activity. We treated human pancreatic cancer MiaPaCa-2 cells with gemcitabine and the AKT inhibitor triciribine, alone and in combination, and evaluated treatment effects using trypan blue assay, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium (MTT) assay, and cell death enzyme-linked immunosorbant assay. Colorimetric data of MTT assay were computationally analyzed for synergism of the combination therapy by CalcuSyn2 (Biosoft, Great Shelford, Cambridge, UK). RESULTS: Both gemcitabine and triciribine inhibited cell growth in a dose-dependent manner. Triciribine synergistically enhanced the cytotoxic activity of gemcitabine. The combination index (CI) provides the synergistic, additive, or antagonistic effects of the two-drug combination. CI at the 50% effective dose at 1:500 ratio of gemcitabine to triciribine was 0.74, indicating the synergistic effect of the drugs. The combination treatment with the non-apoptotic dose of each agent distinctly induced apoptosis, with gemcitabine in combination with triciribine, synergistically inhibiting pancreatic cancer cell growth and inducing apoptosis. CONCLUSION: These findings support the use of triciribine to overcome activated AKT-mediated resistance of pancreatic cancer to gemcitabine.

EGR-1/Bax pathway plays a role in vitamin E δ-tocotrienol-induced apoptosis in pancreatic cancer cells.

The anticancer activity of δ-tocotrienol, a bioactive vitamin E present in whole grain cereals, annatto beans and palm fruit, is strongly dependent on its effect on the induction of apoptosis. δ-Tocotrienol-induced apoptosis is associated with consistent induction in the expression of the proapoptotic protein Bcl-2-associated X protein (Bax). The molecular mechanism by which δ-tocotrienol regulates Bax expression is unknown. We carried out a DNA microarray study that identified δ-tocotrienol induction of the zinc finger transcription factor EGR-1 in pancreatic cancer cells. Here, we provide evidence linking δ-tocotrienol-induced apoptosis in pancreatic cancer cells to EGR-1 regulation of Bax expression. Forced expression of EGR-1 induces Bax expression and apoptosis in pancreatic cancer cells. In contrast, knockdown of δ-tocotrienol-induced EGR-1 by small interfering RNA attenuated δ-tocotrienol-induced Bax expression and reduced δ-tocotrienol-induced apoptosis. Further analyses showed that de novo protein synthesis was not required for δ-tocotrienol-induced EGR-1 expression, suggesting a direct effect of δ-tocotrienol on EGR-1 expression. Furthermore, a chromatin immunoprecipitation assay demonstrated that EGR-1 binds to the Bax gene promoter. Finally, δ-tocotrienol treatment induced Bax expression and activated EGR-1 in the pancreatic neoplastic cells of the PDX-Cre Kras genetically engineered model of pancreatic cancer. Our study provides the first evidence for EGR-1 as a direct target of vitamin E δ-tocotrienol, suggesting that EGR-1 may act as a proapoptotic factor in pancreatic cancer cells via induction of Bax.