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Purpose: Apicularen A has been shown to cause growth inhibition and apoptosis in several cancer cell lines. However, the mechanisms of apicularen A–induced cell death and in vivo effects remain unclear. In this study, we investigated the molecular mechanisms of apicularen A–induced cell death in HM7 human colon cancer cells in vitro and anticancer activity in vivo.Experimental Design: We tested cytotoxicity with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis with DNA fragmentation assay, mitochondrial membrane potential, and cell cycle with fluorescence-activated cell sorting. Caspase activation was done by fluorometry. Alterations of microtubule structure, tubulin protein, and mRNA level were assessed by immunofluorescence, Western blot, and reverse transcription-PCR. In vivo studies were assessed using nude mice tumor cell growth in xenograft model and liver colonization assay.Results: Apicularen A treatment of HM7 cells inhibited cell growth and this inhibition was partially rescued by z-VAD-fmk. Apicularen A caused accumulation of sub-G1-G0, DNA fragmentation, Fas ligand induction, and activation of caspase-8 and caspase-3, but mitochondrial membrane potential was not changed. Furthermore, β-tubulin protein and mRNA were decreased by apicularen A, but in vitro polymerization of tubulin was not affected. Concurrently, apicularen A–treated cell showed disruption of microtubule architecture. In in vivo studies, apicularen A reduced tumor volume by ∼72% at the end of a 15-day treatment. Moreover, apicularen A reduced liver colonization as much as 95.6% (50 μg/kg/d).Conclusion: Apicularen A induces cell death of HM7 cells through up-regulating Fas ligand and disruption of microtubule architecture with down-regulation of tubulin level. These findings indicate that apicularen A is a promising new microtubule-targeting compound.

Background and aims Yes-associated protein (YAP) plays an important role in hepatocarcinogenesis, although the potential role of YAP in non-neoplastic liver diseases remains largely unknown. We report herein that YAP in Kupffer cells (KCs) enhances the production of proinflammatory cytokines and promotes the development of nonalcoholic steatohepatitis (NASH). Our data show that the expression of YAP is significantly increased in KCs of wild-type mice fed a high-fat diet (HFD). Approach and results We generated mice with macrophage/monocyte-specific deletion of YAP (YAPϕKO ) or Toll-like receptor 4 (TLR4; TLR4ϕKO ), and animals were fed an HFD or treated with lipopolysaccharide (LPS). Our data showed that YAPϕKO mice fed an HFD exhibited lower serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels and less hepatic inflammation when compared to their littermate controls. LPS treatment induced accumulation of YAP in KCs in vitro and in mice, which was prevented by macrophage/monocyte-specific deletion of TLR4 (TLR4ϕKO ). LPS transcriptionally activates YAP through activator protein 1 in macrophages/KCs. LPS-induced YAP further enhances expression of proinflammatory cytokines (including monocyte chemoattractant protein 1, tumor necrosis factor alpha, and interleukin 6) through YAP association with the TEA domain-binding motif in the promoter region of inflammatory cytokines. Forced overexpression of active YAP (YAP5SA) in KCs enhanced the production of proinflammatory cytokines. Treatment of HFD-fed mice with verteporfin inhibited KC activation, reduced liver inflammation, and decreased serum ALT/AST levels. Analyses of liver tissues from NASH patients reveal that YAP is increased in KCs and that level of YAP in human liver tissues is positively correlated with expression of proinflammatory cytokines. Conclusions This study describes an important role of YAP in KCs for regulation of liver inflammation in NASH. Our findings suggest that inhibition of YAP may represent an effective therapeutic strategy for NASH treatment.

Recent studies have demonstrated the implication of long noncoding RNAs (lncRNAs) in a variety of physiological and pathological processes. However, the majority of lncRNAs are functionally unknown. The current study describes that the lncRNA MALAT1 regulates TGF-β/Smad signaling pathway through formation of a lncRNA-protein complex containing Smads, SETD2 and PPM1A. Our data show that this lncRNA-proteins complex facilitates the dephosphorylation of pSmad2/3 by providing the interaction niche for pSmad2/3 and their specific phosphatase PPM1A, thus terminating TGF-β/Smad signaling in hepatic cells. Based on these mechanistic studies, we performed further experiments to determine whether depletion of MALAT1 would augment cellular TGF-β/Smad signaling. We observed that MALAT1 depletion enhanced TGF-β/Smad signaling response, as reflect by amplification of Smad-mediated differentiation of induced pluripotent stem (iPS) cells to hepatocytes. Our experimental results demonstrate an important role of MALAT1 for regulation of TGF-β/Smad signaling in hepatic cells. Given the diverse functions of TGF-β/Smad pathway in various physiological and pathogenic processes, our results described in the current study will have broad implications for further understanding the role of MALAT1 in TGF-β/Smad pathway in human biology and disease.

Although hedgehog (Hh) signaling pathway is inactive in adult healthy liver, it becomes activated during acute and chronic liver injury and, thus, modulates the reparative process and disease progression. We developed a novel mouse model with liver-specific knockout of Smoothened (Smo LKO), and animals were subjected to Fas-induced liver injury in vivo. Results showed that Smo deletion in hepatocytes enhances Fas-induced liver injury. Activation of Hh signaling in hepatocytes in the setting of Fas-induced injury was indicated by the fact that Jo2 treatment enhanced hepatic expression of Ptch1, Smo, and its downstream target Gli1 in control but not Smo LKO mice. Primary hepatocytes from control mice showed increased Hh signaling activation in response to Jo2 treatment in vitro. On the other hand, the Smo KO hepatocytes were devoid of Hh activation and were more susceptible to Jo2-induced apoptosis. The levels of NF-κB and related signaling molecules, including epidermal growth factor receptor and Akt, were lower in Smo KO livers/hepatocytes than in control livers/hepatocytes. Accordingly, hydrodynamic gene delivery of active NK-κB prevented Jo2-induced liver injury in the Smo LKO mice. Our findings provide important evidence that adult hepatocytes become responsive to Hh signaling through up-regulation of Smo in the setting of Fas-induced liver injury and that such alteration leads to activation of NF-κB/epidermal growth factor receptor/Akt, which counteracts Fas-induced hepatocyte apoptosis.

Aberrant expression and regulation of miRNAs have been implicated in multiple stages of tumorigenic processes. The current study was designed to explore the biological function and epigenetic regulation of miR-34a in human cholangiocarcinoma (CCA). Our data show that the expression of miR-34a is decreased significantly in CCA cells compared with non-neoplastic biliary epithelial cells. Forced overexpression of miR-34a in CCA cells inhibited their proliferation and clonogenic capacity in vitro, and suppressed tumor xenograft growth in severe combined immunodeficiency mice. We identified three key components of the Notch pathway, Notch1, Notch2, and Jagged 1, as direct targets of miR-34a. Our further studies show that down-regulation of miR-34a is caused by Enhancer of zeste homolog 2 (EZH2)-mediated H3 lysine 27 trimethylation as well as DNA methylation. Accordingly, treatment with the EZH2 inhibitor, selective S-adenosyl-methionine-competitive small-molecule (GSK126), or the DNA methylation inhibitor, 5-Aza-2′-deoxycytidine, partially restored miR-34a levels in human CCA cells. Immunohistochemical staining and Western blot analyses showed increased EZH2 expression in human CCA tissues and cell lines. We observed that GSK126 significantly reduced CCA cell growth in vitro and intrahepatic metastasis in vivo. Our findings provide novel evidence that miR-34a expression is silenced epigenetically by EZH2 and DNA methylation, which promotes CCA cell growth through activation of the Notch pathway. Consequently, these signaling cascades may represent potential therapeutic targets for effective treatment of human CCA.

Hepatitis C virus (HCV) infection compromises the natural defense mechanisms of the liver leading to a progressive end stage disease such as cirrhosis and hepatocellular carcinoma (HCC). The hepatic stress response generated due to viral replication in the endoplasmic reticulum (ER) undergoes a stepwise transition from adaptive to pro-survival signaling to improve host cell survival and liver disease progression. The minute details of hepatic pro-survival unfolded protein response (UPR) signaling that contribute to HCC development in cirrhosis are unknown. This study shows that the UPR sensor, the protein kinase RNA-like ER kinase (PERK), mediates the pro-survival signaling through nuclear factor erythroid 2-related factor 2 (NRF2)-mediated signal transducer and activator of transcription 3 (STAT3) activation in a persistent HCV infection model of Huh-7.5 liver cells. The NRF2-mediated STAT3 activation in persistently infected HCV cell culture model resulted in the decreased expression of hepatocyte nuclear factor 4 alpha (HNF4A), a major liver-specific transcription factor. The stress-induced inhibition of HNF4A expression resulted in a significant reduction of liver-specific microRNA-122 (miR-122) transcription. It was found that the reversal of hepatic adaptive pro-survival signaling and restoration of miR-122 level was more efficient by interferon (IFN)-based antiviral treatment than direct-acting antivirals (DAAs). To test whether miR-122 levels could be utilized as a biomarker of hepatic adaptive stress response in HCV infection, serum miR-122 level was measured among healthy controls, and chronic HCV patients with or without cirrhosis. Our data show that serum miR-122 expression level remained undetectable in most of the patients with cirrhosis (stage IV fibrosis), suggesting that the pro-survival UPR signaling increases the risk of HCC through STAT3-mediated suppression of miR-122. In conclusion, our data indicate that hepatic pro-survival UPR signaling suppresses the liver-specific HNF4A and its downstream target miR-122 in cirrhosis. These results provide an explanation as to why cirrhosis is a risk factor for the development of HCC in chronic HCV infection.

Background and aims Cholangiocarcinoma (CCA) is a highly malignant epithelial tumor of the biliary tree with poor prognosis. In the current study, we present evidence that the histone-lysine methyltransferase G9a is up-regulated in human CCA and that G9a enhances CCA cell growth and invasiveness through regulation of the Hippo pathway kinase large tumor suppressor 2 (LATS2) and yes-associated protein (YAP) signaling pathway. Approach and results Kaplan-Meier survival analysis revealed that high G9a expression is associated with poor prognosis of CCA patients. In experimental systems, depletion of G9a by small interfering RNA/short hairpin RNA or inhibition of G9a by specific pharmacological inhibitors (UNC0642 and UNC0631) significantly inhibited human CCA cell growth in vitro and in severe combined immunodeficient mice. Increased G9a expression was also observed in mouse CCA induced by hydrodynamic tail vein injection of notch intracellular domain (NICD) and myr-Akt. Administration of the G9a inhibitor UNC0642 to NICD/Akt-injected mice reduced the growth of CCA, in vivo. These findings suggest that G9a inhibition may represent an effective therapeutic strategy for the treatment of CCA. Mechanistically, our data show that G9a-derived dimethylated H3K9 (H3K9me2) silenced the expression of the Hippo pathway kinase LATS2, and this effect led to subsequent activation of oncogenic YAP. Consequently, G9a depletion or inhibition reduced the level of H3K9me2 and restored the expression of LATS2 leading to YAP inhibition. Conclusions Our findings provide evidence for an important role of G9a in cholangiocarcinogenesis through regulation of LATS2-YAP signaling and suggest that this pathway may represent a potential therapeutic target for CCA treatment.

Hedgehog (Hh) signaling plays a critical role in liver development, regeneration, injury repair, and carcinogenesis. Activation of Hh signaling has been observed in patients with nonalcoholic fatty liver diseases (NAFLD); however, the pathobiological function and regulatory mechanism of hepatic Hh signaling in the pathogenesis of NAFLD remain to be further defined. This study was designed to examine the effect and mechanism of hepatic Hh signaling in high-fat diet-induced NAFLD by using pharmacological Smoothened (Smo) inhibitors (GDC-0449 and LED225) and liver-specific Smo knockout mice. Administration of Smo inhibitors to high-fat diet-fed wild-type mice significantly reduced the numbers of activated macrophages and decreased the expression of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, monocyte chemoattractant protein 1, and interleukin-6) as assessed by F4/80 immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, respectively. The Smo inhibitors were noted to have variable effects on hepatic fat accumulation. Liver-specific deletion of Smo also reduced macrophage activation and inhibited proinflammatory cytokine expression, while it did not significantly alter fat accumulation in the liver. Mechanistically, we found that activation of glioma-associated oncogene 1 by Hh signaling in primary hepatocytes increased the production of osteopontin, which subsequently enhanced the macrophage-mediated proinflammatory response through paracrine signaling. Conclusion: Hepatocyte Hh signaling can promote liver inflammation through osteopontin-mediated macrophage activation; this mechanism importantly contributes to the progression of NAFLD. (Hepatology 2016;63:1155–1169)

The biological functions and molecular mechanisms of miR-223 action in liver cells and liver diseases remain unclear. We therefore determined the effect and mechanism of action of miR-233 in Fas-induced hepatocyte apoptosis and liver injury. Wild-type (WT) and miR-223 knockout (KO) mice were treated i.p. with 0.5 μg/g body weight anti-Fas antibody Jo2, and the animals were monitored for survival and the extent of liver injury. Although WT mice died 4 to 6 hours after Jo2 injection (n = 6), all of the miR-223 KO mice (n = 6) survived. In comparison to WT mice, the miR-223 KO mice showed resistance to Fas-induced liver injury, as indicated by less tissue damage under histopathological examination, fewer apoptotic hepatocytes under caspase-3 immunostaining, and less elevation of serum transaminases. miR-223 KO livers showed less caspase-3, caspase-8, and caspase-9 activation and less poly (ADP-ribose) polymerase cleavage compared with WT livers (P

Although altered metabolic pathway is an important diagnostic maker and therapeutic target in cancer, it is poorly understood in cancer stem cells (CSCs). Here we show that the CD133 (+) hepatocellular CSCs have distinct metabolic properties, characterized by more active glycolysis over oxidative phosphorylation, compared to the CD133 (−) cells. Inhibition of PDK4 and LDHA markedly suppresses CD133 (+) stemness characteristics and overcome resistance to sorafenib (current chemotherapeutic agent for hepatocellular cancer). Addition of glucose or lactate to CD133 (−) cells promotes CSC phenotypes, as evidenced by increased CD133 (+) cell population, elevated stemness gene expression and enhanced spheroid formation. Furthermore, the liver-specific miRNA, miR-122, inhibits CSC phenotypes by regulating glycolysis through targeting PDK4. Our findings suggest that enhanced glycolysis is associated with CD133 (+) stem-like characteristics and that metabolic reprogramming through miR-122 or PDK4 may represent a novel therapeutic approach for the treatment of hepatocellular cancer.

The NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl group of prostaglandin E2 (PGE2), converting the pro-inflammatory PGE2 to the anti-inflammatory 15-keto-PGE2 (an endogenous ligand for peroxisome proliferator-activated receptor-gamma [PPAR-γ]). To evaluate the significance of 15-PGDH/15-keto-PGE2 cascade in liver inflammation and tissue injury, we generated transgenic mice with targeted expression of 15-PGDH in the liver (15-PGDH Tg) and the animals were subjected to lipopolysaccharide (LPS)/Galactosamine (GalN)-induced acute liver inflammation and injury. Compared to the wild type mice, the 15-PGDH Tg mice showed lower levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), less liver tissue damage, less hepatic apoptosis/necrosis, less macrophage activation, and lower inflammatory cytokine production. In cultured Kupffer cells, treatment with 15-keto-PGE2 or the conditioned medium (CM) from 15-PGDH Tg hepatocyes inhibited LPS-induced cytokine production, in vitro. Both 15-keto-PGE2 and the CM from15-PGDH Tg hepatocyes also up-regulated the expression of PPAR-γ downstream genes in Kupffer cells. In cultured hepatocytes, 15-keto-PGE2 treatment or 15-PGDH overexpression did not influence TNF-α-induced hepatocyte apoptosis. These findings suggest that 15-PGDH protects against LPS/GalN-induced liver injury and the effect is mediated via 15-keto-PGE2, which activates PPAR-γ in Kupffer cells and thus inhibits their ability to produce inflammatory cytokines. Accordingly, we observed that the PPAR-γ antagonist, GW9662, reversed the effect of 15-keto-PGE2 in Kupffer cell in vitro and restored the susceptibility of 15-PGDH Tg mice to LPS/GalN-induced acute liver injury in vivo. Collectively, our findings suggest that 15-PGDH-derived 15-keto-PGE2 from hepatocytes is able to activate PPAR-γ and inhibit inflammatory cytokine production in Kupffer cells and that this paracrine mechanism negatively regulates LPS-induced necro-inflammatory response in the liver. Therefore, induction of 15-PGDH expression or utilization of 15-keto-PGE2 analogue may have therapeutic benefits for the treatment of endotoxin-associated liver inflammation/injury.

This paper proposes a design of message oriented modeling and analysis tool (MOMAT) for naval combat systems (NCS). NCS is composed of heterogeneous and large-scale components such as communication services and data distribution services (DDS). Each component consists of massive and heterogeneous interfaces and modules that are designed by many developers. Furthermore, MOMAT has to meet the requirements of NCS as follows. First, MOMAT has to be able to control multiple parts such as components, interfaces, messages and publisher/subscriber. Secondly, it should improve the application performance in terms of response time and execution time. Thirdly, the scalability of application functions has to be guaranteed for system integration of NCS with heterogeneous sub-parts. Implementation results show that the MOMAT is suitable for naval combat systems regarding task processing time and database access time.

A stable and persistent Hepatitis C virus (HCV) replication cell culture model was developed to examine clearance of viral replication during long-term treatment using interferon-α (IFN-α), IFN-λ, and ribavirin (RBV). Persistently HCV-infected cell culture exhibited an impaired antiviral response to IFN-α+RBV combination treatment, whereas IFN-λ treatment produced a strong and sustained antiviral response that cleared HCV replication. HCV replication in persistently infected cells induced chronic endoplasmic reticulum (ER) stress and an autophagy response that selectively down-regulated the functional IFN-α receptor-1 chain of type I, but not type II (IFN-γ) or type III (IFN-λ) IFN receptors. Down-regulation of IFN-α receptor-1 resulted in defective JAK–STAT signaling, impaired STAT phosphorylation, and impaired nuclear translocation of STAT. Furthermore, HCV replication impaired RBV uptake, because of reduced expression of the nucleoside transporters ENT1 and CNT1. Silencing ER stress and the autophagy response using chemical inhibitors or siRNA additively inhibited HCV replication and induced viral clearance by the IFN-α+RBV combination treatment. These results indicate that HCV induces ER stress and that the autophagy response selectively impairs type I (but not type III) IFN signaling, which explains why IFN-λ (but not IFN-α) produced a sustained antiviral response against HCV. The results also indicate that inhibition of ER stress and of the autophagy response overcomes IFN-α+RBV resistance mechanisms associated with HCV infection.

MiR-122, a pivotal liver specific miRNA, has been implicated in several liver diseases including hepatocellular carcinoma (HCC) and hepatitis C and B viral infection. This study aimed to explore epigenetic regulation of miR-122 in human hepatocellular carcinoma (HCC) cells and to examine the effect of hepatitis C virus (HCV) and hepatitis B virus (HBV). We performed microRNA microarray analysis and identified miR-122 as the most up-regulated miRNA (6-fold) in human hepatocellular cancer cells treated with 5′aza-2′deoxycytidine (5-Aza-CdR, DNA methylation inhibitor) and 4-phenylbutyric acid (PBA, histone deacetylation inhibitor). Real-time PCR analysis verified significant upregulation of miR-122 by 5′aza and PBA in HCC cells, and to a lesser extent in primary hepatocytes. Peroxisome proliferator activated receptor-gamma (PPARγ) and retinoid X receptor alpha (RXRα) complex was found to be associated with the DR1 and DR2 consensus site in the miR-122 gene promoter which enhanced miR-122 gene transcription. 5-Aza-CdR and PBA treatment increased the association of PPARγ/RXRα, but decreased the association of its co-repressors (N-CoR and SMRT), with the miR-122 DR1 and DR2 motifs. The aforementioned DNA-protein complex also contains SUV39H1, a H3K9 histone methyl transferase, which downregulates miR-122 expression. Our findings establish a novel role of the PPARγ binding complex for epigenetic regulation of miR-122 in human HCC cells. Moreover, we show that hepatitis B virus X protein (HBX) binds PPARγ and inhibits the transcription of miR-122, whereas hepatitis C viral particles exhibited no significant effect; these findings provide mechanistic insight into reduction of miR-122 in patients with HBV but not with HCV infection.

Apicularen A is a novel antitumor agent and strongly induces death in tumor cells. In this study, we synthesized apicularen A acetate, an acetyl derivative of apicularen A, and investigated its antitumor effect and mechanism in HM7 colon cancer cells. Apicularen A acetate induced apoptotic cell death and caspase-3 activation; however, the pan-caspase inhibitor Z-VAD-fmk could not prevent this cell death. Apicularen A acetate induced the loss of mitochondrial membrane potential and the translocation of apoptosis-inducing factor (AIF) from mitochondria. In addition, apicularen A acetate significantly decreased tubulin mRNA and protein levels and induced disruption of microtubule networks. Taken together, these results indicate that the mechanism of apicularen A acetate involves caspase-independent apoptotic cell death and disruption of microtubule architecture.

In this paper, a scheduling algorithm is proposed for military satellite networks to improve QoS(Quality of Service) based on WDRR(Weighted Deficit Round Robin) method. When the packet size that has been queued to be larger, the proposed scheme DWDRR(Dynamic WDRR) method give appropriate additional quantum using EWMA(Exponentially Weighted Moving Average). To demonstrate an usefulness of proposed algorithm using OPNET modeler that built the simulation environment, reliability and real-time availability of the proposed algorithm is analyzed. The simulation results show an availability of proposed scheme in terms of reduce queuing delay and packet drop rate compared and analyzed the existing algorithms WRR(Weighted Round Robin), DRR(Deficit Round Robin) and WDRR with DWDRR.

Docosahexaenoic acid (DHA) induces autophagy-associated apoptotic cell death in wild-type p53 cancer cells via regulation of p53. The present study investigated the effects of DHA on PC3 and DU145 prostate cancer cell lines harboring mutant p53. Results show that, in addition to apoptosis, DHA increased the expression levels of lipidated form LC3B and potently stimulated the autophagic flux, suggesting that DHA induces both autophagy and apoptosis in cancer cells expressing mutant p53. DHA led to the generation of mitochondrial reactive oxygen species (ROS), as shown by the mitochondrial ROS-specific probe mitoSOX. Similarly, pretreatment with the antioxidant N-acetyl-cysteine (NAC) markedly inhibited both the autophagy and the apoptosis triggered by DHA, indicating that mitochondrial ROS mediate the cytotoxicity of DHA in mutant p53 cells. Further, DHA reduced the levels of phospho-Akt and phospho-mTOR in a concentration-dependent manner, while NAC almost completely blocked that effect. Collectively, these findings present a novel mechanism of ROS-regulated apoptosis and autophagy that involves Akt-mTOR signaling in prostate cancer cells with mutant p53 exposed to DHA.

Colon cancer is one of the most common malignancies in the western world and the second leading cause of cancer death in Korea. Epidemiology studies have shown a reduced incidence of colon cancer among populations consuming a large quantity of ω3-polyunsaturated fatty acids (ω3-PUFA) of ma- rine origin. Recently, it has been found that ω 3-PUFA has an antineoplastic effect in several cancers. This study was designed to investigate the mechanism of the anti-invasive effect of ω3-PUFA in colon cancer. ω3-PUFA, docosahexaenoic acids (DHA) and eicosapentaenoic acid ( EPA) treatment resulted in a dose-dependent inhibition of cell growth in SW480 human colon cancer cells. In contrast, arach- idonic acid (AA), a ω6-PUFA, exhibited no significant effect. This action likely involves apoptosis, giv- en that DHA treatment increased apoptotic cells in TUNEL assay. Moreover, invasiveness of SW480 cells was inhibited following treatment of DHA in a dose-dependent manner; in contrast, AA had no effect. The levels of MMP-9 and MMP-2 mRNA decreased after DHA pretreatment. MMP-9 and MMP-2 promoter activities were also inhibited by DHA treatment. The levels of NF-kB and p-IkB pro- tein were down-regulated by DHA pretreatment in a dose dependent manner. In addition, DHA in- hibited NF-kB promoter reporter activities. These findings suggest that ω3-PUFA may inhibit cancer cell invasion by inhibition of MMPs via reduction of NF-kB in colon cancer. In conclusion, ω3-PUFA could be used for chemoprevention and treatment of human colon cancer.

Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.

Purpose: Apicularen A has been shown to cause growth inhibition and apoptosis in several cancer cell lines. However, the mechanisms of apicularen A–induced cell death and in vivo effects remain unclear. In this study, we investigated the molecular mechanisms of apicularen A–induced cell death in HM7 human colon cancer cells in vitro and anticancer activity in vivo. Experimental Design: We tested cytotoxicity with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis with DNA fragmentation assay, mitochondrial membrane potential, and cell cycle with fluorescence-activated cell sorting. Caspase activation was done by fluorometry. Alterations of microtubule structure, tubulin protein, and mRNA level were assessed by immunofluorescence, Western blot, and reverse transcription-PCR. In vivo studies were assessed using nude mice tumor cell growth in xenograft model and liver colonization assay. Results: Apicularen A treatment of HM7 cells inhibited cell growth and this inhibition was partially rescued by z-VAD-fmk. Apicularen A caused accumulation of sub-G1-G0, DNA fragmentation, Fas ligand induction, and activation of caspase-8 and caspase-3, but mitochondrial membrane potential was not changed. Furthermore, β-tubulin protein and mRNA were decreased by apicularen A, but in vitro polymerization of tubulin was not affected. Concurrently, apicularen A–treated cell showed disruption of microtubule architecture. In in vivo studies, apicularen A reduced tumor volume by ∼72% at the end of a 15-day treatment. Moreover, apicularen A reduced liver colonization as much as 95.6% (50 μg/kg/d). Conclusion: Apicularen A induces cell death of HM7 cells through up-regulating Fas ligand and disruption of microtubule architecture with down-regulation of tubulin level. These findings indicate that apicularen A is a promising new microtubule-targeting compound.

Fas-induced apoptosis is involved in diverse liver diseases. Herein, we investigated the effect of Mir155 deletion on Fas-induced liver injury. Wild-type (WT) mice and Mir155 knockout (KO) mice were i.p. administered with the anti-Fas antibody (Jo2) to determine animal survival and the extent of liver injury. After Jo2 injection, the Mir155 KO mice exhibited prolonged survival versus the WT mice ( P Mir155 KO mice showed lower alanine aminotransferase and aspartate aminotransferase levels, less liver tissue damage, fewer apoptotic hepatocytes, and lower liver tissue caspase 3/7, 8, and 9 activities compared with the WT mice, indicating that Mir155 deletion prevents Fas-induced hepatocyte apoptosis and liver injury. Hepatocytes isolated from Mir155 KO mice also showed resistance to Fas-induced apoptosis, in vitro . Higher protein level of myeloid cell leukemia-1 (Mcl-1) was also observed in Mir155 KO hepatocytes compared to WT hepatocytes. A miR-155 binding site was identified in the 3′-untranslated region of Mcl-1 mRNA; Mcl1 was identified as a direct target of miR-155 in hepatocytes. Consistently, pretreatment with a siRNA specific for Mcl1 reversed Mir155 deletion–mediated protection against Jo2-induced liver tissue damage. Finally, restoration of Mir155 expression in Mir155 KO mice abolished the protection against Fas-induced hepatocyte apoptosis. Taken together, these findings demonstrate that deletion of Mir155 prevents Fas-induced hepatocyte apoptosis and liver injury through the up-regulation of Mcl1 .

Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit hepatitis C virus (HCV) replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently HCV-infected cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda 1 (IFN-λ1), a type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, downregulated membrane expression of ENT1 and terminated RBV uptake. In contrast, the autophagy inhibitors hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection. IMPORTANCE The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon-free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has not been established. This study provides a potential mechanism for why RBV antiviral activity is impaired in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy to increase RBV antiviral activity against HCV infection. Therefore, it is anticipated that this work would generate a great deal of interest, not only among virologists but also among the general public.

Prostaglandin E2 (PGE2) is a proinflammatory lipid mediator that promotes cancer growth. The 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes oxidation of the 15(S)-hydroxyl group of PGE2, leading to its inactivation. Therefore, 15-PGDH induction may offer a strategy to treat cancers that are driven by PGE2, such as human cholangiocarcinoma. Here, we report that omega-3 polyunsaturated fatty acids (ω-3 PUFA) upregulate 15-PGDH expression by inhibiting miR-26a and miR-26b, thereby contributing to ω-3 PUFA-induced inhibition of human cholangiocarcinoma cell growth. Treatment of human cholangiocarcinoma cells (CCLP1 and TFK-1) with ω-3 PUFA (DHA) or transfection of these cells with the Fat-1 gene (encoding Caenorhabditis elegans desaturase, which converts ω-6 PUFA to ω-3 PUFA) significantly increased 15-PGDH enzymes levels, but with little effect on the activity of the 15-PGDH gene promoter. Mechanistic investigations revealed that this increase in 15-PGDH levels in cells was mediated by a reduction in the expression of miR-26a and miR-26b, which target 15-PGDH mRNA and inhibit 15-PGDH translation. These findings were extended by the demonstration that overexpressing miR-26a or miR-26b decreased 15-PGDH protein levels, reversed ω-3 PUFA-induced accumulation of 15-PGDH protein, and prevented ω-3 PUFA-induced inhibition of cholangiocarcinoma cell growth. We further observed that ω-3 PUFA suppressed miR-26a and miR-26b by inhibiting c-myc, a transcription factor that regulates miR-26a/b. Accordingly, c-myc overexpression enhanced expression of miR-26a/b and ablated the ability of ω-3 PUFA to inhibit cell growth. Taken together, our results reveal a novel mechanism for ω-3 PUFA-induced expression of 15-PGDH in human cholangiocarcinoma and provide a preclinical rationale for the evaluation of ω-3 PUFA in treatment of this malignancy. Cancer Res; 75(7); 1388–98. ©2015 AACR.

Background Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Methods Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. Results We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. Conclusions These results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

Aberrant expression and regulation of microRNAs (miRNAs) have been found in multiple cancers and their dysregulation importantly contribute to the tumorigenic processes. Among these miRNAs, miRNA-34a (miR-34a) functions as a tumor suppressor and its dysregulation has been reported in several cancers. In this study, we investigated epigenetic regulation and biological function of miR-34a in human cholangiocarcinoma cells. Our data indicate that miR-34a expression is epigenetically silenced in human cholangiocarcinoma cells (CCLP1, SG231, Hucct1 and TFK1) compared to the nonmalignant biliary epithelial cells (H69). Treatment of human cholangiocarcinoma cells with the DNA methylation inhibitor, 5’aza-2’deoxycytidine (5-Aza-CdR), or the EZH2 (enhancer of zeste 2 polycomb repressive complex 2) inhibitor, GSK126, enhanced the expression of miR-34a. In parallel, knockdown of EZH2 by shRNA also significantly decreased miR-34a expression in human cholangiocarcinoma cells. We further showed that DNA methylation and EZH2-mediated histone H3 lysine 27 trimethylation (H3K27Me3) repressed miR-34a gene expression, as determined by methylation-specific PCR and chromatin immunoprecipitation. The DNA methylation and EZH2-mediated H3K27Me3 independently repress miR-34a expression in cholangiocarcinoma cells since treatment of 5-Aza-CdR and GSK126 had no effect on H3K27me3 and DNMT1 levels, respectively. Functional analyses revealed that miR-34a mimic decreased cholangiocarcinoma cell proliferation, colony formation and migration, in vitro. Moreover, we identified Notch1, Notch2 and Jagged 1, which are the major receptors and ligands of the Notch pathway, as miR-34a target genes in cholangiocarcinoma cells. Accordingly, forced overexpression of miR-34a significantly decreased the expression of Notch1, Notch2 and Jagged1, as determined by Western blotting and qRT-PCR analyses. Our results provide novel evidence that miR-34a expression is epigenetically silenced by DNA methylation and EZH2 in human cholangiocarcinoma cells. Thus, epigenetic induction of miR-34a or miR-34a replacement therapy may represent a novel therapeutic approach for the treatment of cholangiocarcinoma. Citation Format: Hyunjoo Kwon, Kyoungsub Song, Chang Han, Jinqing Zhang, Nathan Ungerleider, Lu Yao, Tong Wu. Epigenetic silencing of microRNA-34a promotes cholangiocarcinoma growth by regulating Notch pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 949.

The output of a TGF-β response is highly contextual depending on specific tissue types and diseases. This is especially true in hepatocellular carcinoma (HCC) initiation and progression, where the tumor-initialing and malignant cells are highly influenced by the background liver environments. To delineate the role of TGF-β in the process of hepatocarcinogenesis, we employed a novel tumor-initiating cell transplantation system that avoids the possible effects incurred from genetic manipulation of the background liver. Specifically, TGF-β receptor II (Tgfbr2) flox/flox mice at 14 days of age were given one dose (25μg/g bodyweight) of the hepatic carcinogen diethylnitrosamine (DEN) via intraperitoneal injection. Three months later, the initiated hepatocytes from Tgfbr2 flox/flox mice were isolated and transplanted to the same strain (C57Bl/6) wild type recipient mice. Four weeks after transplantation, the recipient mice received one does of Cre recombinase adenovirus (Ad-Cre) through tail vein injection to delete Tgfbr2 in the transplanted tumor-initiating hepatocytes; separate group of the recipient mice were injected with the control adenovirus (Ad-GFP). The recipient mice were closely monitored for liver tumor burden. We observed that deletion of Tgfbr2 in tumor-initiating hepatocytes by tail vein injection of Ad-Cre led to formation of more and bigger liver tumors (compared to Ad-GFP group). This finding suggests that TGF-β inhibits the malignant potential of tumor-initiating hepatocytes. In parallel, we further analyzed the effect of TGF-β in tumor initiating hepatocytes in vitro. Specifically, tumor-initiating hepatocytes isolated from DEN-treated Tgfbr2 flox/flox mice were infected with Ad-Cre or Ad-GFP prior to TGF-β1 treatment in vitro; RNAs were then isolated for transcriptome sequencing analysis. We identified a group of lncRNAs that were noticeably regulated by TGF-β, including the lncRNA H19. We found that deletion of Tgfbr2 by Ad-Cre in tumor initiating hepatocytes led to a 5-fold increase of H19 expression. We observed that deletion of H19 by siRNA decreased the tumor-initiating cell property, whereas forced overexpression of H19 enhanced it. Chromatin immunoprecipitation assay showed that SOX2 bound to the promoter region of H19 gene. While SOX2 overexpression in tumor-initiating hepatocytes enhanced H19 transcription, SOX2 knockdown reduced it. Furthermore, TGF-β treatment reduced the protein level of SOX2 in tumor-initiating hepatocytes. Taken together, our findings disclose a novel mechanism that importantly regulates hepatocarcinogenesis — TGF-β inhibits H19 expression via SOX2 in tumor-initiating hepatocytes and this effect leads to inhibition of HCC development. Citation Format: jinqiang Zhang, Chang Han, Weina Chen, Kyoungsub Song, Ying Wang, Lu Yao, Nathan Ungerleider, Hyunjoo Kwon, Tong Wu. Loss of TGFβ signaling leads to increased susceptibility to HCC in mice: Evidences of lncRNA H19's effects in HCC promotion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 981.

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here we show that DHA increased both the level of microtubule-associated protein 1 light chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.

This study proposes a dynamic guaranteed time slot (GTS) allocation scheme in order to realize a transmission scheme for periodic data in an industrial environment. Further, a personal area network (PAN) coordinator can share the same GTS slot with several nodes using transmission deadline, network utilization, and transmitting rate. The proposed scheme can re­alize an increase in the overall utilization of the GTS bandwidth. Simulation results demonstrated the improved performance of the proposed scheme.

ω3-polyunsaturated fatty acids (ω3- PUFAs) are known to possess anticancer properties. However, the relationship between ω3-PUFAs and β-catenin, one of the key components of the Wnt signaling pathway, in human pancreatic cancer remains poorly characterized.Human pancreatic cancer cells (SW1990 and PANC-1) were exposed to two ω3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), to investigate the relationship between ω3-PUFAs and the Wnt/β-catenin signaling pathway in vitro. Mouse pancreatic cancer (PANC02) cells were implanted into fat-1 transgenic mice, which express ω3 desaturases and result in elevated levels of ω3-PUFAs endogenously. The tumor size, levels of Wnt/β-catenin signaling molecules and apoptosis levels were analyzed to examine the influence of ω3-PUFAs in vivo.DHA and EPA significantly inhibited cell growth and increased cell death in pancreatic cancer cells. DHA also reduced β-catenin expression, T cell factor/lymphoid-enhancing factor reporter activity and induced β-catenin/Axin/GSK-3β complex formation, a known precursor to β-catenin degradation. Furthermore, Wnt3a, a natural canonical Wnt pathway ligand, reversed DHA-induced growth inhibition in PANC-1 cells. Immunohistochemical analysis showed aberrant upregulation and increased nuclear staining of β-catenin in tumor tissues from pancreatic cancer patients. However, β-catenin levels in tumor tissues from fat-1 transgenic mice were reduced with a significant increase in apoptosis compared with those from control mice.ω3-PUFAs may be an effective therapy for the chemoprevention and treatment of human pancreatic cancer. and IAP.

It has been reported that the SW480 human colon cancer cell line consists of E-type and R-type cells. The long-term tumorigenic potential, invasive and metastatic properties of these subclones have not been characterized. E-type and R-type cells were subcloned using limiting dilution methods from parental SW480 cells. The cell growth rate was determined by MTT colorimetric assay, and colony forming efficiency was analyzed using Matrigel-coated plates. The activity of matrix metalloproteinase (MMP) and of urokinase plasminogen activator (uPA) was assessed by zymography. Invasive and locomotive ability was analyzed using transwell chambers. In situ apoptosis detection of these subclones was also performed. In vivo long-term tumorigenicity and nodal metastasis were evaluated using nude mice. E-type cells produced spontaneously regressive tumors in spite of invasion and lymph node metastasis. In contrast, R-type cells revealed progressively growing tumors without invasion or metastasis. E-type cells exhibited increased apoptosis and invasive and motile ability, as well as strong MMP-9 and -2 activity. Although phorbol 12-myristate 13-acetate treatment induced MMP-9 activity in E-type cells, it had no effect on R-type cells. These findings suggest that E- and R-type cells may have different biological properties in terms of colon cancer progression, regression, invasion and nodal metastasis, and might serve as a useful model for these studies.

The cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guerin (BCG/CWS) is an effective antitumor immunotherapy agent. Here, we demonstrate that BCG/CWS has a radiosensitizing effect on colon cancer cells through the induction of autophagic cell death. Exposure of HCT116 colon cancer cells to BCG/CWS before ionizing radiation (IR) resulted in increased cell death in a caspase-independent manner. Treatment with BCG/CWS plus IR resulted in the induction of autophagy in colon cancer cells. Either the autophagy inhibitor 3-methyladenine or knockdown of beclin 1 or Atg7 significantly reduced tumor cell death induced by BCG/CWS plus IR, whereas the caspase inhibitor z-VAD-fmk failed to do so. BCG/CWS plus IR-mediated autophagy and cell death was mediated predominantly by the generation of reactive oxygen species (ROS). The c-Jun NH(2)-terminal kinase pathway functioned upstream of ROS generation in the induction of autophagy and cell death in HCT116 cells after co-treatment with BCG/CWS and IR. Furthermore, toll-like receptor (TLR) 2, and in part, TLR4, were responsible for BCG/CWS-induced radiosensitization. In vivo studies revealed that BCG/CWS-mediated radiosensitization of HCT116 xenograft growth is accompanied predominantly by autophagy. Our data suggest that BCG/CWS in combination with IR is a promising therapeutic strategy for enhancing radiation therapy in colon cancer cells through the induction of autophagy.

The senescence of human mesenchymal stem cells (hMSCs) causes disruption of tissue and organ maintenance, and is thus an obstacle to stem cell-based therapies for disease. Although some researchers have studied changes in the characteristics of hMSCs (decreases in differentiation ability and self-renewal), comparing young and old ages, the mechanisms of stem cell senescence have not yet been defined. In this study, we developed a growth curve for human bone marrow derived MSCs (hBMSCs) which changes into a hyperbolic state after passage number 7. Senescence associated β-galactosidase (SA β-gal) staining of hBMSCs showed 10% in passage 9 and 45% in passage 11. We detected an increase in endogenous superoxide levels during senescence that correlated with senescence markers (SA β-gal, hyperbolic growth curve). Interestingly, even though endogenous superoxide increased in a replicative senescence model, the expression of APE1/Ref-1, which is sensitive to intracellular redox state, decreased. These effects were confirmed in a stress-induced senescence model by exogenous treatment with H2O2. This change is related to the p53 activity that negatively regulates APE1/Ref-1. p21 expression levels, which represent p53 activity, were transiently increased in passage 9, meaning that they correlated with the expression of APE1/Ref-1. Overexpression of APE1/Ref-1 suppressed superoxide production and decreased SA β-gal in hBMSCs. In conclusion, intracellular superoxide accumulation appears to be the main cause of the senescence of hBMSCs, and overexpression of APE1/Ref-1 can rescue cells from the senescence phenotype. Maintaining characteristics of hBMSCs by regulating intracellular reactive oxygen species production can contribute to tissue regeneration and to improved cell therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARgamma (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARgamma expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver diseases and hepatocelluar carcinomas. Over the past few years, the liver-enriched microRNA-122 (miR-122) has been shown to differentially regulate viral replication of HBV and HCV. It is notable that the level of miR-122 is positively and negatively regulated by HCV and HBV, respectively. Consistent with the well-documented phenomenon that miR-122 promotes HCV accumulation, inhibition of miR-122 has been shown as an effective therapy for the treatment of HCV infection in both chimpanzees and humans. On the other hand, miR-122 is also known to block HBV replication, and HBV has recently been shown to inhibit miR-122 expression; such a reciprocal inhibition between miR-122 and HBV suggests an intriguing possibility that miR-122 replacement may represent a potential therapy for treatment of HBV infection. As HBV and HCV have shared transmission routes, dual infection is not an uncommon scenario, which is associated with more advanced liver disease than either HBV or HCV mono-infection. Thus, there is a clear need to further understand the interaction between HBV and HCV and to delineate the role of miR-122 in HBV/HCV dual infection in order to devise effective therapy. This review summarizes the current understanding of HBV/HCV dual infection, focusing on the pathobiological role and therapeutic potential of miR-122.

To gain insight on the role of transacting factors in the regulatory mechanism of H2B histone gene expression during the differentiation of HL-60 cells by 12-O-tetra-decanoylphorbol 13-acetate (TPA), the binding pattern of nuclear proteins to various elements in the human H2B histone gene upstream region have been investigated with DNase I footprinting and DNA mobility shift assay. The level of H2B histone mRNA rapidly reduced at 24 h in TPA-treated HL-60 cells. The H2B histone mRNA was repressed in proportion to the concentration of TPA. In DNase I footprinting analysis, one nuclear factor (octamer-binding transcription factor, OTF) bound at -42 bp (octamer motif), before and after TPA-induced differentiation of HL-60 cells. One DNA-protein complex (OTF) was formed by DNA mobility shift assay when octamer element was incubated with nuclear extract of undifferentiated HL-60 cells. In DNA mobilith shift assay, OTF vanished, and phosphorylated OTF (p-OTF) newly appeared during TPA-induced differentiation. p-OTF was not detected after pretreatment of the protein kinase C inhibitor, staurosporin, but was not changed after CHX treatment. TPA-induced repression of H2B histone mRNA was also restored after pretreatment of staurosporin. These results suggest that OTF is phosphorylated by protein kinase C during TPA-induced differentiation of HL-60 and the transcriptional repression of the H2B histone gene may be mediated by protein kinase C-dependent phosphorylation of OTF.

Over 80% of the lung cancer patients have non-small cell lung cancer (NSCLC). Although the anticancer mechanisms of omega-3 polyunsaturated fatty acids (≥3-PUFAs) have been reported in several cancers, it is still unclear in lung cancer. In this study, we have identified a novel anticancer effect of docosahexaenoic acid (DHA), a α3-PUFAs, on NSCLC. DHA induced cytotoxicity and morphology change in A549 and H1299 NSCLC cells and was confirmed that apoptosis and autophagy are responsible for the cytotoxicity induced by DHA. DHA increased AMPK activity and inhibited PI3K/Akt signaling as well as mTOR signaling molecules. Knockdown AMPK using small interfering RNAs specific for AMPK and overexpression Akt significantly enhanced the mTOR activity and attenuated the cell death caused by DHA treatment, indicating that DHA induces NSCLC cell death via the AMPK- and Akt-regulated mTOR inactivation. We also confirmed this effect of α3-PUFAs in vivo using Fat-1 transgenic mice that are capable of producing α3-PUFAs. When mouse lung cancer LLC cells were subcutaneously implanted into Fat-1 mice, the growth of tumor was markedly inhibited with decrease the level of p-Akt as well as increases in TUNEL-positive staining cells and autophagic levels, compared to wild-type mice. Taken together, these data suggest that the apoptosis and autophagy induced by DHA may be related to mTOR inhibition through AMPK activation and PI3K/Akt inhibition in NSCLC. Therefore, utilization of DHA may represent a potential effective therapy for the chemoprevention and treatment of human non-small cell lung cancer. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (2011-0013263 and 2011-0006232)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2274. doi:1538-7445.AM2012-2274

It has been reported that miRNAs potently influence cellular function through the regulation of extensive gene expression networks and most tumors are characterized by globally diminished miRNA expression. However, the potential mechanism of microRNA expression change in tumorigenesis remains to be further investigated. In this study, we focus on the epigenetic regulation of miRNA expression in hepatocellular carcinoma. We first performed miRNA microarray analysis in HepG2 cells, which reveals significant changes in miRNA expression profiling with 23 upregulated miRNAs and 15 downregulated miRNAs induced by the treatment with 5′aza-2′deoxycytidine (5′-aza, DNA methylation inhibitor) and 4-phenylbutyric acid (PBA, histone deaceltylation inhibitor). Interestingly, miR-122, the most abundant miRNA in the liver, is upregulated by 5′-aza and PBA, which is validated by qRT-PCR analysis in both HepG2 and Huh7 cells. In addition, miR-122 expression is increased by the overexpression of PPARγ or the treatment of PPARγ agonist (15-keto PGE2 or 15d-PGJ2).We then demonstrate that peroxisome proliferator-activated receptor gamma (PPARγ) and retinoid X receptor alpha (RXRα) bind to conserved DR-1 and DR-2 (peroxisome proliferator-activated response element) in the miR-122 promoter region and these bindings are significantly increased by 5′-aza and PBA with the biotinylated oligonicleotides precipitation assay. However, 5′-aza and PBA inhibit the binding of N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator for retinoid and thyroid hormone receptor) to DR-1 or DR-2. And we found that 5′-aza and PBA inhibit PPARγ/N-CoR/SMRT protein complex formation in HepG2 cells with the immunoprecipitation assay. Furthermore, we observe that SUV39H1(H3K9 methyl transferase) protein is decreased by the co-treatment of 5′aza and PBA; and the suppression of endogenous SUV39H1 expression with SUV39H1siRNA increases miR-122 expression. Taken together, our results suggest that histone acethylation and demethylation lead to the release of PPAR-gamma from PPAR-gamma/N-CoR/SMRT protein complex to activate miR-122 transcription and to the inhibition SUV39H1 protein expression for the elimination of histone methylation to further facilitate miR-122 transcription. In conclusion, Epigenetic silencing is one of the most important mechanisms for diminished miR122 expression in hepatic carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 141. doi:1538-7445.AM2012-141

Lung cancer is leading cause of all cancer deaths. Although omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been reported to inhibit cell growth in several cancers, the anti-cancer mechanism of ω3-PUFAs on lung cancer is still unclear. In this study, we have investigated the mechanism of anti-cancer action of DHA, one of ω3-PUFAs, in A549 human non-small cell lung cancer (NSCLC) cell line. Following treatment of DHA, the viability of A549 cells was decreased in a dose-dependent manner. DHA induced apoptotic cell death as revealed by increased cleaved PARP, TUNEL positive cells and subG1 population. The amounts of PI3K and phospho-Akt proteins were decreased after DHA treatment dose-dependent manner. In addition, DHA decreased the level of phospho-phosphatase and tensin homolog deleted on chromosome ten (p-PTEN) protein, which is an inactive form of PTEN. Moreover, transient transfection of constitutive active Akt cDNA and full length of Akt cDNA into A549 cells partially restored DHA-dependent inhibition of cell growth compared with the cells transfected with kinase dead form of Akt. Taken together, these data suggest that inhibition of PI3K/Akt signaling pathway may be related to anti-cancer action of DHA in A549 human NSCLC cells. Therefore, utilization of DHA may represent a potential effective therapy for the chemoprevention and treatment of human non-small cell lung cancer. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (R13-2007-020-01000-0)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1040. doi:10.1158/1538-7445.AM2011-1040

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Omega 3-polyunsaturated fatty acids (ω3-PUFAs) are known to inhibit proliferation of cancer cells; in contrast, ω6-PUFAs promote the growth of cancer cells. The fat-1 gene of the Caenorhabditis elegans encodes a ω3-desaturase that catalyzes the conversion ω6-PUFAs to ω3-PUFAs and then increases the amount of ω3-PUFAs. The objective of this study is to examine the effect of fat-1 gene expression on HeLa and SiHa human cervical cancer cells. Fat-1 gene stable cell lines (f-HeLa and f-SiHa) were established from HeLa and SiHa cells, respectively. The expression of fat-1 gene significantly inhibited cervical cancer cell proliferation and f-HeLa cells showed an increase in the proportion of cells in G2/M phase comparing with the cells expressing the control vector (c-HeLa). In addition, when treating HeLa cells with docosahexaenoic acid (DHA), an enhanced proportion of cells in the G2/M phase was also observed, indicating that fat-1 gene inhibited cervical cancer cell proliferation by inducing a G2/M phase cell-cycle arrest. Furthermore, transwell migration assay for invasion showed a reduction of cell migration in the f-HeLa cells when compared with the c-HeLa cells. Finally, the growth of f-HeLa cells in vivo was significantly reduced comparing with the c-HeLa cells when inoculated into nude mice. Taken together, these results suggest that the expression of fat-1 gene prevents cervical tumor growth and indicate a cancer therapeutic approach of the ω3-PUFAs. Therefore, a stable cell line of fat-1 gene is useful to study the anti-cancer effects of ω3-PUFAs. [This research was supproted by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ([E00026][1] and R13-2007-020-01000-0)]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 862. doi:10.1158/1538-7445.AM2011-862 [1]: /lookup/external-ref?link_type=GEN&access_num=E00026&atom=%2Fcanres%2F71%2F8_Supplement%2F862.atom

Over 90% of human cervical carcinoma is associated with high risk human papillomavirus (HPV), and a number of biological effects that could contribute to cancer suppression by ω3-polyunsaturated fatty acids (ω3-PUFAs) have been reported. However, the anti-cancer effect of ω3-PUFAs on cervical cancer has been not known yet. In this study, we report inhibitory mechanisms of ω3-PUFAs on cell growth and invasion in cervical cancer. DHA inhibited the cell growth in a dose- and time-dependent manner. In flow cytometry analysis, cell cycle of DHA-treated HeLa cells was arrested in G2/M phase and SubG1 cells also increased. Moreover, apoptotic cell death was confirmed by TUNEL assay, the induction of PARP cleavage and down-regulation of Bcl-2. The invasiveness of cells was significantly inhibited by DHA treatment in vitro transwell assay as well. The MMP-9 and MMP-2 promoter activities were decreased after DHA treatment. Cox-2 and VEGF promoter activities were also inhibited by DHA. Furthermore, DHA decreased the levels of reporter activity of NF-κB, which is transcription factor that regulates MMPs, Cox-2 and VEGF expression. In in vivo experiments, when human papillomavirus type 16 (HPV16)-transformed mouse TC-1 cells were injected into the tail vein of Fat1 mice (Fat1 transgenic mice express a Caenorhabditis elegans ω3-desaturase converting ω6- to ω3-PUFAs endogenously.) and WT (wild type) mice, lung metastasis of TC-1 cells was dramatically inhibited in Fat1 transgenic mice compared to WT mice. Taken together, these findings provide evidence that ω3-PUFAs may inhibit metastasis as well as cell growth and invasion through suppression of MMPs/COX-2/VEGF expression by inhibition of NF-κB expression in cervical cancer cells, indicating that the utilization of ω3-PUFAs may represent a potential effective therapy for the chemoprevention and treatment of human cervical cancer.[This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government(MEST) (#E00026) and by the Infection Signaling Network Research Center (R13-2007-020-01000-0) at Chungnam National University]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4439.

Mutations of Bcr-Abl protein reduce the sensitivity of imatinib treatment for chronic myelogenous leukemia (CML). The resistance against imatinib treatment to CML patients needs to develop another target drug of Bcr-Abl through a novel mechanism. In present study, we investigated the effect of MB12066, small molecule related to quinone derivatives, on k562 cells (CML cell line) and the expression level of Bcr-Abl protein down regulated by MB12066. When k562cells were treated with MB12066, dose-and time-dependent Bcr-Abl degradation was detected, followed by decreasing of procaspase-3 and cleavage of PARP, which represent apoptotic stimuli. Quinone oxidoreductases [NAD(P)H: quinone Oxidoreductase 1(NQO1) and NRH: quinone oxidoreductase2 (NQO2)] are the major enzymes involved in the bioreduction of quinone-containing drugs. We found that MB12066 was a substrate of both NQO1 and NQO2 by examining recycling assay. For the functional study of NQO1 and NQO2 in Bcr-Abl degradation, we used inhibitors for each enzymes, dicumarol, quercetin and imatinib respectively. Interestingly, Bcr-Abl degradation was inhibited by dicumarol, but it was accelerated by quercetin and imatinib. Collectively, these results demonstrate that MB12066 destruct the Bcr-Abl through the NQO1 and NQO2 activity and it may show the possibility that combination therapy with NQO2 inhibitor may be beneficial for treating CML patients. * H.J. Kwon and J.Y. Heo contributed equally to this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 668.