Chan-Yen Kuo

Effects of Hepatitis B Virus X Protein (HBx) on Cell-Growth Inhibition in a CCL13-HBx Stable Cell Line

Objective: The known function of hepatitis B virus X protein (HBx) is to determine the fate of cells by modulating various signaling pathways. In our previous study, we demonstrated that HBx inhibits tumor formation in nude mice injected with CCL13-HBx stable cell lines; however, the mechanism underlying this inhibition is unclear. Methods: To investigate the possible mechanisms underlying HBx involvement in CCL13-HBx cells, gene profiles were initially analyzed by DNA microarray technology and subsequently confirmed by performing semiquantitative RT-PCR and Western blotting. Furthermore, the phenomenon of cell death via apoptosis was detected via DNA fragmentation, TUNEL staining, caspase-3 activity assay, and propidium iodide (PI) staining. Results: The results indicated that HBx induction downregulated Wnt-3 and β-catenin that are involved in cell proliferation. Moreover, HBx induction repressed cell growth and downregulated the expressions of cyclin D1, CDK4, cyclin E, CDK2, and cyclin B1. Furthermore, HBx induction triggered cell death via apoptosis, as determined by DNA fragmentation, TUNEL staining, caspase-3 activity assay, and PI staining. Conclusion: Most importantly, our results indicated that HBx induction in the CCL13-HBx stable cell line downregulated Wnt-3/β-catenin expression and suppressed cell growth by repressing cell proliferation or triggering cell apoptosis.

ABCA1 modulates the oligomerization and Golgi exit of caveolin-1 during HDL-mediated cholesterol efflux in aortic endothelial cells

Previously, the authors have shown that the molecular interaction between caveolin-1 and ATP-binding cassette transporter A1 (ABCA1) is associated with the high-density lipoprotein (HDL)-mediated cholesterol efflux pathway in aortic endothelial cells (ECs). This study analyzed the role ABCA1 plays in caveolin-1-mediated cholesterol efflux in aortic ECs. Knockdown of ABCA1 by siRNA in primary rat aortic ECs after cholesterol treatment did not affect caveolin-1 expression but led to the retention of caveolin-1 in the Golgi apparatus, impaired caveolin-1 oligomerization, and reduced cholesterol efflux. Immunoblotting assay and immunofluorescence microscopy demonstrated that HDL transiently up-regulated ABCA1 expression, induced caveolin-1 oligomerization, and promoted its Golgi exit, thereby enhancing cholesterol efflux. These HDL-induced events, however, were inhibited by down-regulation of ABCA1. It is concluded that HDL up-regulates ABCA1 expression, which in turn modulates the oligomerization and Golgi exit of caveolin-1 to enhance cholesterol efflux in aortic ECs.

A prototype tissue engineered blood vessel using amniotic membrane as scaffold

In this study, we used amniotic membrane (AM), a natural extracellular matrix, as a scaffold for the fabrication of tissue engineered blood vessels (TEBVs). The inner surface of the denuded glutaraldehyde cross-linked AM tube was endothelialized with porcine vascular endothelial cells (ECs) and subjected to a physiological (12 dynecm(-2)) shear stress (SS) for 2 and 4 days. The results showed that after applying SS, an intact EC monolayer was maintained in the lumen surface of the TEBV. The ECs were aligned with their long axis parallel to the blood flow. The immunofluorescent microscopy showed that the intercellular junctional proteins, PECAM-1 and VE-cadherin, were surrounding the EC periphery and were better developed and more abundant in SS-treated TEBVs than the static controls. The Western blot indicated that the expressions of PECAM-1 and VE-cadherin were increased by 72 ± 9% and 67 ± 7%, respectively, after shear stress treatment. The distribution pattern of integrin β1 was mainly at the interface of ECs and AM in static TEBVs but it was extended to the cell-cell junctions after SS treatment. The SS promoted the expression of integrin α(v)β(3) without altering its distribution in TEBV. The results suggest that glutaraldehyde cross-linked AM tube can potentially be used as a scaffold biomaterial for TEBV fabrication. Most importantly, the use of an AM tube shortened the TEBV fabrication.

HBx Inhibits the Growth of CCL13-HBX-Stable Cells via the GSK-3β/β-Catenin Cascade

Objective: The hepatitis B virus X protein (HBx) plays critical roles in cell survival via modulation of signaling pathways. In our previous studies, we reported that HBx inhibited the growth of CCL13-HBx-stable cells (Chang-HBx cells) in vitro and tumor formation in vivo in CCL13-HBx-cell-injected nude mice; however, this inhibition mechanism is unclear. Methods: To investigate the role of HBx in Wnt-3/β-catenin signaling pathways, we focused on the key molecules GSK-3β and β-catenin, and analyzed by Western blotting and immunofluorescence staining. Results: Results indicated that following HBx induction, GSK-3β activity was up-regulated, the expression and accumulation of β-catenin in the nucleus were decreased, and cell proliferation was suppressed. Inhibition of GSK-3β activity by pharmacological inhibitors rescued the expression and accumulation of β-catenin in the nucleus and facilitated cell proliferation and growth following HBx induction. The localization of β-catenin, which is involved in cell proliferation, and mediated by GSK-3β activation was also demonstrated. Conclusion: Our findings suggest that HBx negatively regulated proliferation of CCL13-HBx-stable cells via the GSK-3β/β-catenin cascade.

Hepatitis B virus X protein disrupts stress fiber formation and triggers apoptosis

Cytoskeletal proteins are key participants in the cellular progression to apoptosis. In a previous study we injected nude mice with CCL13-HBx cells and identified in contrast to non-HBx transfected cells a differentially phosphorylated myosin light chain (p-MLC) by two-dimensional PAGE and mass spectrometry of the tumor material. To investigate the role of HBx in myosin light chain kinase (MLCK) signaling pathways, we analyzed the key molecules, p-MLC and MLCK, by western blotting. Immunofluorescence staining analysis showed that HBx disrupted stress fiber formation and that focal adhesion kinase (FAK) and integrin-linked kinase (ILK) were regulated by HBx-mediated phosphatase and tensin homolog (PTEN). We also used pharmacological inhibitors to explore the correlation between cytoskeletal rearrangements and HBx-mediated cell apoptosis via an MLCK and a PTEN-dependent pathway. The results showed that both ML9 and bvp restored the effects caused by HBx induction. Our findings suggest that HBx disrupts stress fiber formation and triggers apoptosis via an MLCK and a PTEN-dependent pathway.

Apoptosis induced by hepatitis B virus X protein in a CCL13-HBx stable cell line

The hepatitis B virus X protein (HBx) critically modulates cell growth by inducing apoptosis or proliferation. We sought to clarify whether HBx-mediated apoptosis in a CCL13 stable cell line (Chang-HBx) with inducible HBx expression proceeds through the extrinsic (death receptor-mediated) and/or intrinsic (mitochondrial-mediated) pathways of apoptosis. We used western blotting, cell viability assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, caspase activity assays, JC-1 staining and DNA fragmentation analysis to study the role of HBx in apoptosis. The expression of the pro-apoptotic proteins Bax and Bad and the release of cytochrome c also increased slightly upon HBx induction. JC-1 staining showed a loss of mitochondrial membrane potential upon HBx induction. Additionally, induction of HBx increased the levels of cleaved caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway) and the common effector caspase-3 as measured by western blotting. This elevation of cleaved caspase-8 or caspase-3 and caspase-9 or caspase-3 decreased in the presence of caspase-8 inhibitor Z-IETD-FMK or caspase-9 inhibitor Z-LEHD-FMK, respectively. Both inhibitors also rescued cell growth, and the caspase-8 inhibitor Z-IETD-FMK prevented apoptotic phenomena including the TUNEL signal. DNA fragmentation analysis showed that these phenomena were not detected in the presence of higher concentration of inhibitors. Our data suggest that HBx induces apoptosis through both extrinsic and intrinsic pathways.

Interaction abolishment between mutant caveolin-1Δ62–100 and ABCA1 reduces HDL-mediated cellular cholesterol efflux

Our previous study shows that caveolin-1 colocalizes and interacts with ATP-binding cassette transporter A1 (ABCA1), which is intimately involved in cellular cholesterol efflux. In this study, we further clarified the region of caveolin-1 that interacts with ABCA1. We also examined the interaction between mutant caveolin-1 and ABCA1 in HDL-mediated cholesterol efflux. We constructed a panel of mutant caveolin-1 proteins and co-transfected them into rat aortic endothelial and human embryonic kidney 293 (HEK293) cells. The co-immunoprecipitation shows that mutant oligomerization domain of caveolin-1, caveolin-1(Δ62-100), is required for the interaction of caveolin-1 with ABCA1. Caveolin-1(Δ62-100) did not colocalize with ABCA1 in the cholesterol-loaded cells after HDL incubation as observed by immunofluorescence confocal microscopy. Concomitantly, caveolin-1(Δ62-100) suppressed HDL-mediated cholesterol efflux. The results suggest that the region of caveolin-1 between amino acids 62 and 100 is an oligomerization domain as well as an attachment site for ABCA1 interaction that regulates HDL-mediated cholesterol efflux.

Functional Characterization of Hepatitis B Virus X Protein Based on the Inhibition of Tumorigenesis in Nude Mice Injected with CCL13-HBx Cells

Objective: This study aimed to determine the effects of HBx on the inhibition of tumorigenesis in nude mice injected with CCL13-HBx cells. Therefore, the characteristics of the induced tumors and the phenomenon of apoptosis were assessed. Methods: The induced tumors were identified using the specific marker of hepatocellular carcinoma (HCC), anti-α-fetoprotein (AFP), and their characteristics were pathologically examined. Apoptosis was detected by DNA fragmentation, and the expression of the proapoptotic proteins p53, Bax, Bad, caspase-3, and caspase-8 and the anti-apoptotic protein Bcl-2 was detected by Western blotting. To identify possible molecules involved in the inhibition of tumorigenesis, extracts of the induced tumors were separated by 2D-PAGE, and the proteins were identified by MS. Results: The tumors of the nude mice injected with CCL13 and CCL13-HBx cells were identified as HCCs. Moreover, HBx was found to suppress tumor growth via apoptosis in the nude mice injected with CCL13-HBx cells. The MS findings revealed that phosphorylated myosin light chain was a candidate molecule involved in the inhibition of tumorigenesis. Conclusion: HBx suppressed tumorigenesis in the nude mice injected with CCL13-HBx cells, which proved to be a good animal model for the in vivo study of the effects of HBx on tumorigenesis.

Delivery of caveolin-1 Gene into Arterial Walls of Rabbits via an Infiltrator Angioplasty Balloon Catheter

Gene therapy is a rapidly developing field with great potential for the treatment of atherosclerosis. Choosing the appropriate system for the precise delivery of genes into targeted plaques is still a challenging task for vascular gene therapists. We investigated the site-specific expression of the caveolin-1 gene within the aortic vessel walls in a rabbit model following local delivery of the gene via an infiltrator angioplasty balloon catheter (IABC). The full-length untagged cDNA encoding human caveolin-1 was fused to the myc reporter gene using a pcDNA 3.1 plasmid. The caveolin-1-myc was then infused into the using a pcDNA 3.1 plasmid. The caveolin-1-myc was then infused into the aorta via IABC twice every three days for two weeks. The site-specific expression of caveolin-1 protein was analyzed by immunohistochemisty at two weeks after the last injection. Immunofluorescence analysis and Western blot indicated that caveolin-1-myc was present in the transfected cells. Caveolin-1 protein was detected in the medial layers of rabbit aorta for up to two weeks after IABC infusion and there was no intimal/medial thickening in the infusion area. IABC appears to be an effective gene delivery system for the treatment of cardiovascular diseases.