Host gene expression modulated by Zika virus infection of human-293 cells.

The HEK-293 cell line was created in 1977 by transformation of primary human embryonic kidney cells with sheared adenovirus type 5 DNA. A previous study determined that the HEK-293 cells have neuronal markers rather than kidney markers. In this study, we tested the hypothesis whether Zika virus (ZIKV), a neurotropic virus, is able to infect and replicate in the HEK-293 cells. We show that the HEK-293 cells infected with ZIKV support viral replication as shown by indirect immunofluorescence (IFA) and quantitative reverse transcriptase-PCR (qRT-PCR). We performed RNA-seq analysis on the ZIKV-infected and the control uninfected HEK-293 cells and find 659 genes that are differentially transcribed in ZIKV-infected HEK-293 cells as compared to uninfected cells. The results show that the top 10 differentially transcribed and upregulated genes are involved in antiviral and inflammatory responses. Seven upregulated genes, IFNL1, DDX58, CXCL10, ISG15, KCNJ15, IFNIH1, and IFIT2, were validated by qRT-PCR. Altogether, our findings show that ZIKV infection alters host gene expression by affecting their antiviral and inflammatory responses.

Inhibition of dengue virus by curcuminoids.

The dengue virus is considered to be a globally important human pathogen prevalent in tropical and subtropical regions of the world. According to a recent estimate, the disease burden due to DENV infections is ∼390 million infections per year globally in ∼100 countries including the southern US, Puerto Rico and Hawaii, resulting in nearly ∼25,000 deaths mostly among children. Despite the significant morbidity and mortality that results from DENV infections, there is currently no effective chemotherapeutic treatment for DENV infections. We identified curcumin as an inhibitor of DENV2 NS2B/NS3protease in a previous high-throughput screening (HTS) campaign. We synthesized four analogues of curcumin (curcuminoids) and tested the in vitro protease inhibition activity and inhibition of replication by cell-based assays. The results revealed that curcumin is a weak inhibitor of the viral protease. However, the analogues exhibited more potent inhibition of DENV infectivity in plaque assays suggesting that the cellular pathway(s) required for viral replication and/or assembly are targeted by these compounds. Further analysis shows that inhibition of genes involved in lipid biosynthesis, and of actin polymerization by curcuminoids, are likely to be involved as their mode of action in DENV2-infected cells. Three of the curcumin derivatives possess good selectivity indices (SI) (>10) when compared to the parent curcumin.

Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

PTEN and p53 abnormalities are indicative and predictive factors for endometrial carcinoma.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) and p53 alterations were expected to be diversely involved in endometrial carcinogenesis. Patients (n=92) with endometrial carcinoma (EC) were analyzed, and PTEN and p53 were immunostained in the tissue sections. Tumor histology, grade of differentiation, presence of endometrial hyperplasia, staining status of PTEN and p53 and clinical information were examined. There were 37 cases (40%) negative for PTEN staining, which suggests lost or reduced PTEN function. Loss of PTEN staining was significantly related to the advanced staging in the grade 1 (G1) and grade 2 (G2) endometrioid adenocarcinoma group (p=0.026). Also, 18 cases (20%) showed positive staining for p53. p53 staining was largely found in grade 3 (G3) endometrioid adenocarcinoma and other phenotypes of EC. In the G1 and G2 group, all 29 cases with reduced PTEN staining showed p53-negative staining (p=0.025). In the G3 and others group, 6 of 8 cases with reduced PTEN staining showed p53-positive staining. p53-positive staining was associated with a high probability of tumor recurrence in the G1 and G2 group (p=0.0234). In contrast, in the G3 and others group, p53-positive cases had a low probability of tumor recurrence (p=0.0473). Both PTEN and p53 staining may be good indicators of clinical stage and probability of tumor recurrence in EC. Reciprocal abnormality of p53 or PTEN occurred at an early phase of carcinogenesis, however simultaneous abnormality of p53 and PTEN often occurred at the a late phase of carcinogenesis. Thus, immunohistochemistry for PTEN and p53 in biopsy specimens of EC can provide supportive information for determining a treatment plan.

Apoptotic pathway related to oval cell proliferation.

BACKGROUND AND AIM: Oval cells, liver stem cell-derived cells, are generated from the liver periportal region and spread into the parenchyma by an autocrine signaling pathway. The mechanism behind how oval cells take their place among packed silent hepatocytes, however, is not well understood. We hypothesized that apoptosis involves a decrease in hepatocytes surrounding oval cells. METHODS: Male Fisher rats were treated using the AAF/PH protocol to induce oval cells in the liver. Apoptosis was assessed by measuring the activity of caspase-3, -8 and -9, and apoptosis-related molecules such as caspase-3, Fas, Fas-L and Bax were also assessed by immunohistochemical analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). Apoptosis was confirmed by TUNEL staining. Regarding antiapoptotic factors, nuclear factor-kappaB (NF-kappaB) DNA binding activity and proliferating cell nuclear antigen (PCNA) expression were examined. RESULTS: NF-kappaB elevated at the early stage of oval cell proliferation. Conversely, caspase activity increased after NF-kappaB elevation. The mRNA of caspase-3, Fas, Fas-L and Bax was induced during and after AAF/PH treatment. Immunohistochemically, oval cells lacked the expression of these proteins, whereas the hepatocytes, particularly those surrounding oval cells, expressed strongly. CONCLUSIONS: The present study suggests that the apoptosis in hepatocytes through both extrinsic and intrinsic pathways mediates oval cell proliferation.

Inhibitory effects of etodolac, a selective COX-2 inhibitor, on the occurrence of tumors in colitis-induced tumorigenesis model in rats.

Ulcerative colitis (UC)-associated neoplasia is one of the complications seen in patients with long-standing UC. Based on many epidemiological studies, colitis is assumed to promote colon tumorigenesis. Tumorigenesis is known to be suppressed in rodents and humans by selective cyclooxygenase-2 inhibitors. However, whether these drugs would serve as protective agents against UC-associated neoplasia remains unclear. Therefore, using a colitis-induced tumorigenesis rat model, we investigated the effects of etodolac, a selective cyclooxygenase-2 inhibitor, on tumorigenesis. The following 4 groups were examined: group A, administered trinitrobenzene sulfonic acid and 1,2-dimethylhydrazine; group B, in addition to the treatment in group A, also received etodolac; group C, administered etodolac alone; and group D, did not receive any agent throughout the study and served as an untreated control. The rats were sacrificed 163 days after the start of experiment, and the number of aberrant crypt foci and tumors in the intestine were counted using a stereoscopic microscope following methylene blue staining. The mean number of aberrant crypt foci was 52.4 in group A, 18.9 in group B, 0 in group C and 0.5 in group D. A total of 9 tumors were observed in group A alone, with none in the remaining groups. The numbers of aberrant crypt foci and tumors in group B were significantly lower than in group A. Etodolac, a selective cyclooxygenase-2 inhibitor, suppresses the occurrence of aberrant crypt foci and tumors in colitis-induced tumorigenesis in rats.

IFN-alpha prevents the growth of pre-neoplastic lesions and inhibits the development of hepatocellular carcinoma in the rat.

Interferon (IFN)-alpha treatment is a common therapy for chronic viral hepatitis and contributes to preventing hepatocarcinogenesis. However, it is not clear whether IFN-alpha directly inhibits the clonal expansion of pre-neoplastic hepatocytes. To clarify the mechanism by which IFN-alpha prevents hepatocarcinogenesis, we examined the effect of IFN-alpha in a chemically induced hepatocarcinogenesis model initiated by diethylnitrosamine (DEN) and promoted by 2-acetylaminofluorene (2-AAF) and partial hepatectomy, in which hepatocellular carcinoma (HCC) arises through pre-neoplastic foci without inflammation or fibrosis. The protocols of IFN-alpha administration were started simultaneously with chemical initiation and lasted for either 4 or 40 weeks. The pre-neoplastic foci and neoplastic HCC were evaluated at 4 or 40 weeks after chemical initiation, respectively. The effects of IFN-alpha were assessed by the expression of tumor-related genes and cell cycle-related genes in the pre-neoplastic foci, using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). As a result of IFN-alpha treatment, the numbers and average volume of pre-neoplastic foci were reduced. The proliferating cell nuclear antigen index and the expression of G(1) cyclins were also reduced in the pre-neoplastic foci in the IFN-treated group. The expression of p21, which is an inhibitor of cyclin-kinase complexes was higher in the foci of the IFN-treated group, while p53 expression was not altered in this group, compared with the control group. IFN-alpha also suppressed the tumor development at 40 weeks after initiation. And in the long-term IFN-alpha-treated group, both the tumor numbers and average tumor size were markedly more reduced than those in the short-term-treated group. Therefore, it was demonstrated that longer treatment with IFN-alpha was more effective, compared with shorter treatment. In conclusion, it was shown that IFN-alpha directly prevented and delayed hepatocarcinogenesis through the suppression of pre-neoplastic cell proliferation and that it may partially depend on p21 induction through a p53-independent pathway.