[Show abstract][Hide abstract] ABSTRACT: The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (
3-PUFAs) have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA), a
3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC) cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK) activation and inactivated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce
3-PUFAs. Lewis lung cancer (LLC) tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus
3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.
[Show abstract][Hide abstract] ABSTRACT: Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease. This study was performed to explore the potential role of miR-150 in LPS/D-GalN and Fas-induced liver injuries by using wild type and miR-150 knockout (KO) mice. Whereas knockout of miR-150 did not significantly alter LPS/D-GalN-induced animal death and liver injury, it protected against Fas-induced liver injury and mortality. The Jo2-induced increase in serum transaminases, apoptotic hepatocytes, PARP cleavage, as well as caspase-3/7, caspase-8, and caspase-9 activities were significantly attenuated in miR-150 KO mice. The liver tissues from Jo2-treated miR-150 KO mice expressed higher levels of Akt1, Akt2, total Akt, as well as p-Akt(Ser473) compared to the wild type livers. Pretreatment with the Akt inhibitor V reversed Jo2-induced liver injury in miR-150 KO mice. The primary hepatocytes isolated from miR-150 KO mice also showed protection against Fas-induced apoptosis in vitro (characterized by less prominent PARP cleavage, less nuclear fragmentation and less caspase activation) in comparison to hepatocytes from wild type mice. Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3'-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150. Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice. These findings demonstrate that miR-150 deficiency prevents Fas-induced hepatocyte apoptosis and liver injury through regulation of the Akt pathway.
PLoS ONE 07/2015; 10(7):e0132734. DOI:10.1371/journal.pone.0132734 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/ml) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6h. In vitro, HSCs were treated with 100 ng/ml LPS till 24h. The medium was transferred to hepatocytes, and determinations were made at 0-12h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ∼4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ∼10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNβ, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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.
World Journal of Hepatology 03/2015; 7(3):498-506. DOI:10.4254/wjh.v7.i3.498
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
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.
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.
Journal of Virology 10/2014; 89(1). DOI:10.1128/JVI.02492-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: miR-17-92 is an oncogenic miRNA cluster implicated in the development of several cancers; however, it remains unknown whether the miR-17-92 cluster is able to regulate cholangiocarcinogenesis. This study was designed to investigate the biological functions and molecular mechanisms of the miR-17-92 cluster in cholangiocarcinoma. In situ hybridization and quantitative RT-PCR analysis showed that the miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared with the nonneoplastic biliary epithelial cells. Forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation, and invasiveness, in vitro. Overexpression of the miR-17-92 cluster or miR-92a also enhanced cholangiocarcinoma growth in vivo in hairless outbred mice with severe combined immunodeficiency (SHO-Prkdc(scid)Hr(hr)). The tumor-suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells via sequence prediction, 3' untranslated region luciferase activity assay, and Western blot analysis. Accordingly, overexpression of the PTEN open reading frame protein (devoid of 3' untranslated region) prevented miR-92a- or miR-19a-induced cholangiocarcinoma cell growth. Microarray analysis revealed additional targets of the miR-17-92 cluster in human cholangiocarcinoma cells, including APAF-1 and PRDM2. Moreover, we observed that the expression of the miR-17-92 cluster is regulated by IL-6/Stat3, a key oncogenic signaling pathway pivotal in cholangiocarcinogenesis. Taken together, our findings disclose a novel IL-6/Stat3-miR-17-92 cluster-PTEN signaling axis that is crucial for cholangiocarcinogenesis and tumor progression.
American Journal Of Pathology 10/2014; 184(10):2828-39. DOI:10.1016/j.ajpath.2014.06.024 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose
Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients.
Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting.
HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients.
HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.
PLoS ONE 09/2014; 9(9):e108616. DOI:10.1371/journal.pone.0108616 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The role of omega-3 polyunsaturated fatty acids (ω3-PUFAs) in cancer prevention has been demonstrated; however, the exact molecular mechanisms underlying the anticancer activity of ω3-PUFAs are not fully understood. Here, we investigated the relationship between the anticancer action of a specific ω3-PUFA docosahexaenoic acid (DHA), and the conventional mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38 whose dysregulation has been implicated in human cancers.
MTT assays were carried out to determine cell viability of cancer cell lines (PA-1, H1299, D54MG and SiHa) from different origins. Apoptosis was confirmed by TUNEL staining, DNA fragmentation analysis and caspase activity assays. Activities of the conventional MAPKs were monitored by their phosphorylation levels using immunoblotting and immunocytochemistry analysis. Reactive oxygen species (ROS) production was measured by flow cytometry and microscopy using fluorescent probes for general ROS and mitochondrial superoxide.
DHA treatment decreased cell viability and induced apoptotic cell death in all four studied cell lines. DHA-induced apoptosis was coupled to the activation of the conventional MAPKs, and knockdown of ERK/JNK/p38 by small interfering RNAs reduced the apoptosis induced by DHA, indicating that the pro-apoptotic effect of DHA is mediated by MAPKs activation. Further study revealed that the DHA-induced MAPKs activation and apoptosis was associated with mitochondrial ROS overproduction and malfunction, and that ROS inhibition remarkably reversed these effects of DHA.
Together, these results indicate that DHA-induced MAPKs activation is dependent on its capacity to provoke mitochondrial ROS generation, and accounts for its cytotoxic effect in human cancer cells.
BMC Cancer 07/2014; 14(1):481. DOI:10.1186/1471-2407-14-481 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: miRNAs have recently been implicated in hepatocarcinogenesis, although the actions and mechanisms of individual miRNAs remain incompletely understood. We examined the biological functions and molecular mechanisms of miR-185 in hepatocellular carcinoma (HCC). The expression of miR-185 decreased in human HCC tissues compared with the nonneoplastic liver parenchyma. Quantitative RT-PCR showed a reduction of miR-185 in human HCC cells compared with primary hepatocytes. miR-185 overexpression in human HCC cells inhibited cell proliferation and invasion in vitro and prevented tumor growth in SCID mice. miR-185 overexpression inhibited DNMT1 3' untranslated region luciferase reporter activity in HCC cells; this effect was abolished when the miR-185 binding site was mutated. miR-185 mimic or overexpression decreased the level of DNMT1 protein in HCC cells. These findings establish DNMT1 as a bona fide target of miR-185 in HCC cells. The role of DNMT1 in miR-185-induced inhibition of HCC growth was further supported by the fact that DNMT1 overexpression prevented miR-185-induced inhibition of HCC cell proliferation/invasion. miR-185 mimic or overexpression reduced PTEN promoter DNA methylation and enhanced PTEN expression, leading to the inhibition of Akt phosphorylation; these effects were partially reversed by DNMT1 overexpression. These results provide novel evidence that miR-185 inhibits HCC cell growth by targeting DNMT1, leading to PTEN induction and Akt inhibition. Thus, reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC treatment.
American Journal Of Pathology 06/2014; DOI:10.1016/j.ajpath.2014.05.004 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
miRNAs are a group of small, noncoding RNAs that modulate the translation of genes by binding to specific target sites in the target mRNA. This study investigated the biologic function and molecular mechanism of miR-21 in human cholangiocarcinoma. In situ hybridization analysis of human cholangiocarcinoma specimens showed increased miR-21 in cholangiocarcinoma tissue compared with the noncancerous biliary epithelium. Lentiviral transduction of miR-21 enhanced human cholangiocarcinoma cell growth and clonogenic efficiency in vitro, whereas inhibition of miR-21 decreased these parameters. Overexpression of miR-21 also promoted cholangiocarcinoma growth using an in vivo xenograft model system. The NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH/HPGD), a key enzyme that converts the protumorigenic prostaglandin E2 (PGE2) to its biologically inactive metabolite, was identified as a direct target of miR-21 in cholangiocarcinoma cells. In parallel, cyclooxygenase-2 (COX2) overexpression and PGE2 treatment increased miR-21 levels and enhanced miR-21 promoter activity in human cholangiocarcinoma cells.
Cholangiocarcinogenesis and tumor progression are regulated by a novel interplay between COX-2/PGE2 and miR-21 signaling, which converges at 15-PGDH.
Molecular Cancer Research 04/2014; 12(6). DOI:10.1158/1541-7786.MCR-13-0419 · 4.38 Impact Factor