Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolated from salmonid fish in Gangwon Province, Korea.

The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed <60% cumulative mortality in Atlantic salmon and coho salmon. The expression of G genes in the kidney was higher than that in the spleen-infected fish, with the highest expression observed in the kidneys of rainbow trout. The relative expression levels of innate immunity genes were higher in rainbow trout than in Atlantic salmon and coho salmon. The expression level of immunoglobulin M increased until day 7, and the expression of type I interferon was higher in the spleen than in other tissues. The expression of Mx-1 was higher in the kidney and liver than other tissues. These results indicate that IHNV isolates from Gangwon province show host-specific virulence in rainbow trout and that their virulence and replication were higher in JRt-Shizuoka type than in JRt-Nagano type isolates.

Computer simulation approach to the identification of visfatin-derived angiogenic peptides.

Angiogenesis plays an essential role in various normal physiological processes, such as embryogenesis, tissue repair, and skin regeneration. Visfatin is a 52 kDa adipokine secreted by various tissues including adipocytes. It stimulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis. However, there are several issues in developing full-length visfatin as a therapeutic drug due to its high molecular weight. Therefore, the purpose of this study was to develop peptides, based on the active site of visfatin, with similar or superior angiogenic activity using computer simulation techniques.Initially, the active site domain (residues 181∼390) of visfatin was first truncated into small peptides using the overlapping technique. Subsequently, the 114 truncated small peptides were then subjected to molecular docking analysis using two docking programs (HADDOCK and GalaxyPepDock) to generate small peptides with the highest affinity for visfatin. Furthermore, molecular dynamics simulations (MD) were conducted to investigate the stability of the protein-ligand complexes by computing root mean square deviation (RSMD) and root mean square fluctuation(RMSF) plots for the visfatin-peptide complexes. Finally, peptides with the highest affinity were examined for angiogenic activities, such as cell migration, invasion, and tubule formation in human umbilical vein endothelial cells (HUVECs). Through the docking analysis of the 114 truncated peptides, we screened nine peptides with a high affinity for visfatin. Of these, we discovered two peptides (peptide-1: LEYKLHDFGY and peptide-2: EYKLHDFGYRGV) with the highest affinity for visfatin. In an in vitrostudy, these two peptides showed superior angiogenic activity compared to visfatin itself and stimulated mRNA expressions of visfatin and VEGF-A. These results show that the peptides generated by the protein-peptide docking simulation have a more efficient angiogenic activity than the original visfatin.

Cancer Stem-Like Phenotype of Mitochondria Dysfunctional Hep3B Hepatocellular Carcinoma Cell Line.

Mitochondria are major organelles that play various roles in cells, and mitochondrial dysfunction is the main cause of numerous diseases. Mitochondrial dysfunction also occurs in many cancer cells, and these changes are known to affect malignancy. The mitochondria of normal embryonic stem cells (ESCs) exist in an undifferentiated state and do not function properly. We hypothesized that mitochondrial dysfunction in cancer cells caused by the depletion of mitochondrial DNA might be similar to the mitochondrial state of ESCs. We generated mitochondria dysfunctional (ρ0) cells from the Hep3B hepatocellular carcinoma cell line and tested whether these ρ0 cells show cancer stem-like properties, such as self-renewal, chemotherapy resistance, and angiogenesis. Compared with Hep3B cells, the characteristics of each cancer stem-like cell were increased in Hep3B/ρ0 cells. The Hep3B/ρ0 cells formed a continuous and large sphere from a single cell. Additionally, the Hep3B/ρ0 cells showed resistance to the anticancer drug doxorubicin because of the increased expression of ATP-binding cassette Subfamily B Member 1. The Hep3B/ρ0 conditioned medium induced more and thicker blood vessels and increased the mobility and invasiveness of the blood vessel cells. Therefore, our data suggest that mitochondrial dysfunction can transform cancer cells into cancer stem-like cells.

C-terminal truncated HBx reduces doxorubicin cytotoxicity via ABCB1 upregulation in Huh-7 hepatocellular carcinoma cells.

Hepatitis B virus (HBV) encoding the HBV x protein (HBx) is a known causative agent of hepatocellular carcinoma (HCC). Its pathogenic activities in HCC include interference with several signaling pathways associated with cell proliferation and apoptosis. Mutant C-terminal-truncated HBx isoforms are frequently found in human HCC and have been shown to enhance proliferation and invasiveness leading to HCC malignancy. We investigated the molecular mechanism of the reduced doxorubicin cytotoxicity by C-terminal truncated HBx. Cells transfected with C-terminal truncated HBx exhibited reduced cytotoxicity to doxorubicin compared to those transfected with full-length HBx. The doxorubicin resistance of cells expressing C-terminal truncated HBx correlated with upregulation of the ATP binding cassette subfamily B member 1(ABCB1) transporter, resulting in the enhanced efflux of doxorubicin. Inhibiting the activity of ABCB1 and silencing ABCB1 expression by small interfering ribonucleic acid (siRNA) increased the cytotoxicity of doxorubicin. These results indicate that elevated ABCB1 expression induced by C-terminal truncation of HBx was responsible for doxorubicin resistance in HCC. Hence, co-treatment with an ABCB1 inhibitor and an anticancer agent may be effective for the treatment of patients with liver cancer containing the C-terminal truncated HBx. [BMB Reports 2019; 52(5): 330-335].

Mitochondrial dysfunction suppresses p53 expression via calcium-mediated nuclear factor-kB signaling in HCT116 human colorectal carcinoma cells.

Mitochondrial DNA (mtDNA) mutations are often observed in various cancer types. Although the correlation between mitochondrial dysfunction and cancer malignancy has been demonstrated by several studies, further research is required to elucidate the molecular mechanisms underlying accelerated tumor development and progression due to mitochondrial mutations. We generated an mtDNA-depleted cell line, ρ°, via long-term ethidium bromide treatment to define the molecular mechanisms of tumor malignancy induced by mitochondrial dysfunction. Mitochondrial dysfunction in ρ° cells reduced drug-induced cell death and decreased the expression of pro-apoptotic proteins including p53. The p53 expression was reduced by activation of nuclear factor-κB that depended on elevated levels of free calcium in HCT116/ρ° cells. Overall, these data provide a novel mechanism for tumor development and drug resistance due to mitochondrial dysfunction. [BMB Reports 2018; 51(6): 296-301].