Srikanta Dash

Proteomics computational analyses suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class II fusion proteins.

Class II fusion proteins encoded by tick-borne encephalitis virus (TBEV), dengue virus, and Semliki Forest virus have a fusion peptide located at the end of a rod-like molecule comprised of three antiparallel beta sheet domains. Proteomics computational analyses suggest that hepatitis C virus (HCV) envelope glycoprotein E1 and pestivirus envelope glycoprotein E2 are truncated class II fusion proteins. Similarities were also detected between the receptor-binding portion of TBEV E and HCV E2, and between TBEV small membrane protein precursor prM and pestivirus E1. The proposed models of Flaviviridae envelope proteins can facilitate drug and vaccine development.

Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model.

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Epigenetic regulation of MicroRNA‐122 by peroxisome proliferator activated receptor‐gamma and hepatitis b virus X protein in hepatocellular carcinoma cells

MicroRNA‐122 (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 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 HCC cells treated with 5′aza‐2′deoxycytidine (5‐Aza‐CdR, DNA methylation inhibitor) and 4‐phenylbutyric acid (PBA, histone deacetylation inhibitor). Real‐time polymerase chain reaction (PCR) analysis verified significant up‐regulation 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 corepressors (N‐CoR and SMRT), with the miR‐122 DR1 and DR2 motifs. The aforementioned DNA‐protein complex also contains SUV39H1, an H3K9 histone methyl transferase, which down‐regulates miR‐122 expression. Conclusions: These 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 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. (Hepatology 2013;58:1681–1692)

Integrin subunits alpha5 and alpha6 regulate cell cycle by modulating the chk1 and Rb/E2F pathways to affect breast cancer metastasis

BackgroundAlthough integrins have been implicated in the progression of breast cancer, the exact mechanism whereby they exert this regulation is clearly not understood. To understand the role of integrins in breast cancer, we examined the expression levels of several integrins in mouse breast cancer cell lines by flow cytometry and the data were validated by Western and RT-PCR analysis. The importance of integrins in cell migration and cell invasion was examined by in vitro assays. Further the effect of integrins on metastasis was investigated by in vivo experimental metastasis assays using mouse models.ResultsIntegrin α5 subunit is highly expressed in the nonmetastatic cell line 67NR and is significantly low in the highly invasive cell line 4T1. In contrast, expression levels of integrin α6 subunit are high in 4T1 cells and low in 67NR cells. In vitro data indicated that overexpression of α5 subunit and knockdown of α6 integrin subunit inhibited cell proliferation, migration, and invasion. Our in vivo findings indicated that overexpression of integrin α5 subunit and knockdown of α6 subunit decreased the pulmonary metastasis property of 4T1 cells. Our data also indicated that overexpression of alpha 5 integrin subunit and suppression of alpha6 integrin subunit inhibited cells entering into S phase by up-regulating p27, which results in downregulation of cyclinE/CDK2 complexes, This suggests that these integrins regulate cell growth through their effects on cell-cycle-regulated proteins. We also found that modulation of these integrins upregulates E2F, which may induce the expression of chk1 to regulate cdc25A/cyclin E/CDK2/Rb in a feedback loop mechanism.ConclusionThis study indicates that Integrin α5 subunit functions as a potential metastasis suppressor, while α6 subunit functions as a metastasis promoter. The modulation of integrins reduces cdc25 A, another possible mechanism for downregulation of CDK2. Taken together we demonstrate a link between integrins and the chk1-cdc25-cyclin E/CDK2-Rb pathway.

Interferons α, β, γ each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation

Abstract: Interferon (IFN)‐α is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN‐α, ‐β and ‐γ inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5′ untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full‐length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES‐dependent mechanisms (pCITE‐GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN‐α, ‐β and ‐γ inhibit replication of sub‐genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV‐ and EMCV‐IRES sequences present in the dicistronic HCV sub‐genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES‐mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication.

Hepatitis C Virus Infection Impairs IRF-7 Translocation and Alpha Interferon Synthesis in Immortalized Human Hepatocytes

ABSTRACT Hepatitis C virus (HCV) establishes chronic infection in a significant number of infected humans, although the mechanisms for chronicity remain largely unknown. We have previously shown that HCV infection in immortalized human hepatocytes (IHH) induces beta interferon (IFN-β) expression (T. Kanda, R. Steele, R. Ray, and R. B. Ray, J. Virol. 81:12375-12381, 2007). However, the regulation of the downstream signaling pathway for IFN-α production by HCV is not clearly understood. In this study, the regulation of the IFN signaling pathway following HCV genotype 1a (clone H77) or genotype 2a (clone JFH1) infection of IHH was examined. HCV infection upregulated expression of total STAT1 but failed to induce phosphorylation and efficient nuclear translocation. Subsequent study revealed that HCV infection induces IFN-stimulated response element activation, as evidenced by upregulation of 2′,5′-oligoadenylate synthetase 1. However, nuclear translocation of IRF-7 was impaired following HCV infection. In HCV-infected IHH, IFN-α expression initially increased (up to 24 h) and then decreased at later time points, and IFN-α-inducible protein 27 was not induced. Interestingly, HCV infection blocked IRF-7 nuclear translocation upon poly(I-C) or IFN-α treatment of IHH. Together, our data suggest that HCV infection enhances STAT1 expression but impairs nuclear translocation of IRF-7 and its downstream molecules. These impairments in the IFN-α signaling pathway may, in part, be responsible for establishment of chronic HCV infection.

HCV Infection Selectively Impairs Type I but Not Type III IFN Signaling

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.

Hybrid Paclitaxel and Gold Nanorod-Loaded Human Serum Albumin Nanoparticles for Simultaneous Chemotherapeutic and Photothermal Therapy on 4T1 Breast Cancer Cells

The use of human serum albumin nanoparticles (HSAPs) as a drug carrier system for cancer treatment has proven successful through current marketable clinical formulations. Despite this success, there is a current lack of multifunctional HSAPs, which offer combinational therapies of more than one proven technique. Gold nanorods (AuNRs) have also shown medicinal promise due to their photothermal therapy capabilities. In this study, a desolvation and cross-linking approach was employed to successfully encapsulate gold nanorods into HSAPs simultaneously with the chemotherapeutic drug paclitaxel (PAC); forming PAC-AuNR-HSAPs with desirable overall particle sizes of 299 ± 6 nm. The loading efficiency of paclitaxel into PAC-AuNR-HSAPs reached up to 3 μg PAC/mg HSA. The PAC-AuNR-HSAPs experienced photothermal heating; with the bulk particle solution reaching up to 46 °C after 15 min of near-IR laser exposure. This heat increase marked the successful attainment of the temperature necessary to cause severe cellular hyperthermia and necrosis. The encasement strategy facilitated a colloidal hybrid treatment system capable of enhanced permeability and retention effects, photothermal ablation of cancer cells, and release of the active paclitaxel of up to 188 ng (from PAC-AuNR-HSAPs created with 30 mg HSA) in a single 15 min irradiation session. When treated with PAC-AuNR-HSAPs containing 20 μg PAC/mL particle solution, 4T1 mouse breast cancer cells experienced ∼82% cell death without irradiation and ∼94% cell death after just one irradiation session. The results for PAC-AuNR-HSAPs were better than that of free PAC, which only killed ∼77% of the cells without irradiation and ∼80% with irradiation. The hybrid particle system also lends itself to future customizable external functionalities via conjugated targeting ligands, such as antibodies. Internal entrapment of patient tailored medication combinations are also possible with this combination treatment platform, which may result in improved quality of life for those undergoing treatment.

Cellular delivery of PEGylated PLGA nanoparticles

Objectives  The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide‐co‐gycolide (PLGA) nanoparticles by breast cancer cells.

Development and optimization of nanosomal formulations for siRNA delivery to the liver

The objective of this study is to develop an effective siRNA delivery system for successful delivery to the liver for the treatment of HCV. Nanosize liposomes (nanosomes) have been prepared using a mixture of cholesterol and DOTAP. A functional siRNA was encapsulated into nanosomes following condensation with protamine sulfate. The delivery of siRNA was optimized in an in vitro cell culture system. The efficacy of the formulations was evaluated by measuring functional gene silencing and cytotoxicity. Encapsulation of siRNA ≥ 7.4 nM resulted in successful delivery of siRNA to nearly 100% of cells. The formulations containing lipid-to-siRNA ratio ≥ 10.56:1 instantly cleared approximately 85% of HCV while maintaining cell viability at about 90%. The formulations were sonicated to further reduce the particle size. The size of these formulations was decreased up to 100 nm. However, there were no significant changes observed in zeta potential, or in siRNA encapsulation and integrity following sonication. The sonicated formulations also showed higher liver hepatocytes deposition and gene silencing properties. This study therefore provides a novel approach of siRNA delivery to liver hepatocytes, which can also be applied to treat HCV in chronic liver diseases.