Reactive oxygen species: role in the development of cancer and various chronic conditions

Oxygen derived species such as superoxide radical, hydrogen peroxide, singlet oxygen and hydroxyl radical are well known to be cytotoxic and have been implicated in the etiology of a wide array of human diseases, including cancer. Various carcinogens may also partly exert their effect by generating reactive oxygen species (ROS) during their metabolism. Oxidative damage to cellular DNA can lead to mutations and may, therefore, play an important role in the initiation and progression of multistage carcinogenesis. The changes in DNA such as base modification, rearrangement of DNA sequence, miscoding of DNA lesion, gene duplication and the activation of oncogenes may be involved in the initiation of various cancers. Elevated levels of ROS and down regulation of ROS scavengers and antioxidant enzymes are associated with various human diseases including various cancers. ROS are also implicated in diabtes and neurodegenerative diseases. ROS influences central cellular processes such as proliferation a, apoptosis, senescence which are implicated in the development of cancer. Understanding the role of ROS as key mediators in signaling cascades may provide various opportunities for pharmacological intervention

Hepatitis C Virus and Inflammation

Inflammation is often a rapid coordinated response generated in the host against evading microbial infections or tissue injury. Microorganisms like bacteria and viruses instigate inflammation mediated by pro-inflammatory cytokines and activate cascade of signaling events leading to the recruitment of inflammatory cells (neutrophils and macrophages). Although the main function of inflammation is the resolution of infection, several viruses, including the hepatitis C viruses (HCV) have evolved to utilize this host response and make the cellular environments conducive to infection. In majority of infected individuals, HCV causes persistent chronic liver inflammation leading to development of liver cirrhosis and hepatocellular carcinoma. HCV induces reactive oxygen species (ROS) and activates nuclear factor-κB (NF-κB) leading to the activation of cyclooxygenase-2 (Cox-2) that ultimately produces prostaglandin-E2 (PGE2), thus enhancing inflammatory process. Interestingly, HCV further activates NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome (a multiprotein complex) by recruiting adaptor protein apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC) which are involved in activation of caspase-1 leading to production of interleukin-1beta (IL-1β) and interleukin-18 (IL-18). In this chapter we have highlighted the recent advancements in HCV-induced inflammatory responses and discussed potential future directions to understand the role of inflammation during HCV infection

Bisbenzimidazoles: Anticancer Vacuolar (H⁺)-ATPase Inhibitors

Small molecule chemotherapeutic agents such as Imatinib, Gefitinib, and Erlotinib have played a significant role in the treatment of cancer. Although the unprecedented progress has been achieved in cancer treatment with these targeted agents, there is a strong demand for the development of selective and highly efficacious cancer drugs. V-ATPases are emerging as important target for the identification of novel therapeutic agents for cancer. Our screening and drug discovery processes have identified the bisbenzimidazole derivative (RP-15) as a potent anticancer V-ATPase inhibitor. In the present study, bisbenzimidazoles (compound-25, RP-11 and RP-15) have been tested for proton-pump inhibition activity in human hepatoma cell line (Huh7.5). RP-15 displayed comparable proton-pump inhibition activity to the standard Bafilomycin A1. We examined the antiproliferative activity of these analogs in two highly invasive and metastatic inflammatory breast cancer (IBC) cell lines (SUM 149PT and SUM190PT) along with Huh7.5. The compound-25 (SUM190PT: IC50 = 0.43±0.11 μM) and its structural analog RP-11 (SUM190PT: IC50 = 0.49±0.09 μM) have shown significant inhibition toward IBC cell lines. Additionally, RP-11 and RP-15 have demonstrated very good cytotoxicity toward the majority of cancer cell lines in the NCI 60 cell line panel

Relation of IL28B Gene Polymorphism with Biochemical and Histological Features in Hepatitis C Virus-Induced Liver Disease

BACKGROUND/AIMS: Polymorphism at the IL28B gene may modify the course of hepatitis C virus (HCV) chronic infection. Our aim was to study the influence of IL28B rs12979860 gene polymorphism on the biochemistry and pathology of HCV-induced disease in the clinical course from mild chronic hepatitis C to hepatocellular carcinoma. METHODS: We have determined the rs12979860 single nucleotide polymorphism (SNP) upstream IL28B gene in two groups of patients with HCV-induced chronic liver disease: 1) 268 patients (159 men) with biopsy-proven chronic hepatitis C, to analyse its relation with biochemical, virological and histological features; and 2) 134 patients (97 men) with HCV-related hepatocellular carcinoma. The distribution of the analysed SNP in hepatocellular carcinoma patients was compared with that found in untreated chronic hepatitis C patients. All patients were white and most were Spaniards. RESULTS: In multivariate analysis ALT values were higher (P = 0.001) and GGT values were lower (P<0.001) in chronic hepatitis C patients homozygotes for the major rs12979860C allele as compared with carriers of the mutated rs12979860T allele. Steatosis was more frequent (Odds ratio = 1.764, 95% C.I. 1.053-2.955) and severe (P = 0.026) in carriers of the rs12979860T allele. No relation was found between the analysed SNP and METAVIR scores for necroinflammation and fibrosis, and there were no differences in the distribution of the analysed SNP between hepatocellular carcinoma and untreated chronic hepatitis C patients. CONCLUSION: The IL28B rs12979860 polymorphism correlates with the biochemical activity and the presence and severity of liver steatosis in chronic hepatitis C

Hepatitis C Virus Stimulates the Expression of Cyclooxygenase-2 via Oxidative Stress: Role of Prostaglandin E(2) in RNA Replication

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Recently, the activation of cyclooxygenase-2 (Cox-2) has been implicated in the HCV-associated hepatocellular carcinoma. In this study, we focus on the signaling pathway leading to Cox-2 activation induced by HCV gene expression. Here, we demonstrate that the HCV-induced reactive oxygen species and subsequent activation of NF-κB mediate the activation of Cox-2. The HCV-induced Cox-2 was sensitive to antioxidant (pyrrolidine dithiocarbamate), Ca(2+) chelator (BAPTA-AM), and calpain inhibitor (N-acetyl-Leu-Leu-Met-H). The levels of prostaglandin E(2) (PGE(2)), the product of Cox-2 activity, are increased in HCV-expressing cells. Furthermore, HCV-expressing cells treated with the inhibitors of Cox-2 (celecoxib and NS-398) showed significant reduction in PGE(2) levels. We also observed the enhanced phosphorylation of Akt and its downstream substrates glycogen synthase kinase-3β and proapoptotic Bad in the HCV replicon-expressing cells. These phosphorylation events were sensitive to inhibitors of Cox-2 (celecoxib and NS-398) and phosphatidylinositol 3-kinase (LY294002). Our results also suggest a potential role of Cox-2 and PGE(2) in HCV RNA replication. These studies provide insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with viral infection

Interaction between STAT-3 and HNF-3 Leads to the Activation of Liver-Specific Hepatitis B Virus Enhancer 1 Function

The signal transducer and activator of transcription 3 (STAT-3), a member of the STAT family of proteins, binds to a large number of transcriptional control elements and regulates gene expression in response to cytokines. While it binds to its cognate nucleotide sequences, it has been recently shown to directly interact with other transcriptional factors in the absence of DNA. We report here one such novel interaction between STAT-3 and hepatocyte nuclear factor 3 (HNF-3) in the absence of DNA. We have identified a STAT-3 binding site within the core domain of hepatitis B virus (HBV) enhancer 1. The HBV enhancer 1 DNA-STAT-3 protein interaction is shown to be stimulated by interleukin-6 (IL-6) and epidermal growth factor, which leads to an overall stimulation of HBV enhancer 1 function and viral gene expression. Using mobility shift assays and transient transfection schemes, we demonstrate a cooperative interaction between HNF-3 and STAT-3 in mediating the cytokine-mediated HBV enhancer function. Cytokine stimulation of HBV gene expression represents an important regulatory scheme of direct relevance to liver disease pathogenesis associated with HBV infection

Two-step affinity purification of the hepatitis C virus ribonucleoprotein complex

Positive-strand RNA viruses replicate their RNA genome within a ribonucleoprotein (RNP) complex that is associated with cellular membranes. We used a two-step method of purification to isolate hepatitis C virus (HCV) RNP complexes from human hepatoma cell line Huh7, which stably expresses HCV subgenomic replicons. The procedure involved hybridization of replicon-expressing cellular lysates with oligonucleotides tagged with biotin and digoxigenin at their respective termini complementary to subgenomic replicon RNA followed by avidin-agarose enrichment of the mixture and subsequent immunoprecipitation of biotin-eluted material with anti-digoxigenin antibody. The immunoprecipitates were immunoblotted with antisera against HCV nonstructural (NS) proteins. The analysis revealed the association of all the HCV NS proteins (NS3, NS4a, NS4b, NS5a, and NS5b) that are encoded by the subgenomic replicon RNA. The HCV RNP complex migrated in a native polyacrylamide gel with an approximate molecular mass of 450 kD. The association of these viral proteins in the RNP complex reinforces the widely acknowledged notion that RNA viruses accomplish replication within a membranous RNP complex

Activation of TGF-β1 promoter by hepatitis C virus-induced AP-1 and Sp1: role of TGF-β1 in hepatic stellate cell activation and invasion.

Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection