CD133+ liver cancer stem cells modulate radioresistance in human hepatocellular carcinoma.

CD133 is a cancer stem-cell (CSC) marker associated with radioresistance and chemoresistance in various cancers. In the present study, CD133-expressing liver cancer cells following radiation exposure showed higher activation of MAPK/PI3K signaling pathway and reduction in reactive oxygen species levels compared to CD133- cells. The in vivo study with a xenograft model showed increased tumor formation in irradiated CD133+ cell-injected nude mice compared to the CD133- group, suggesting that CD133 contributes to radioresistance in HCC. Therefore, CD133-expressing liver cancer cells have anti-apoptotic and radioresistance properties that may be useful to improve anti-cancer treatments, including chemotherapy/radiotherapy of HCC.

Silencing of 14-3-3ζ over-expression in hepatocellular carcinoma inhibits tumor growth and enhances chemosensitivity to cis-diammined dichloridoplatium.

The 14-3-3ζ protein plays a key role in regulation of cellular processes. In the present study, we showed that 14-3-3ζ protein was significantly overexpressed in hepatoma cell lines and human tumorous tissues of hepatocellular carcinoma (HCC) patients. Knockdown with RNA interference in hepatoma cell lines with high 14-3-3ζ expression suppressed tumor cell proliferation via activation of c-Jun N-terminal kinase (JNK) and p38/MAPK. Furthermore, suppression of 14-3-3ζ enhanced the anti-cancer effect of cis-diammined dichloridoplatium (CDDP) in hepatoma cell lines. These results suggest that silencing of 14-3-3ζ may be an attractive target for HCC therapeutic development.

Subgenotype and genetic variability in the precore/core regions of hepatitis B virus in Korean patients with chronic liver disease.

OBJECTIVE: The aim of this study was to determine the prevalence of hepatitis B virus (HBV) subgenotypes, the spectrum of mutations in the precore/core region through phylogenetic analysis, and the relevance of viral characteristics in disease progression in Korean patients. METHODS: 133 patients with chronic HBV infection were enrolled. The precore and core region of HBV was amplified and sequenced. Phylogenetic analysis was performed for subgenotyping and the changes of nucleotides and amino acid were compared in liver disease stages. RESULTS: HBV/C2 subgenotype was predominant in chronic HBV carriers (98.5%), followed by HBV/A2 (0.75%) and HBV/C7 (0.75%). The mutations of the precore region were not different between liver disease stages. However, amino acid changes in the cytotoxic T lymphocyte epitope (p < 0.020), CD4+ T cell epitope (p < 0.027), or B cell epitope (p < 0.029) were significantly higher in liver cirrhosis patients than in chronic hepatitis patients, but not in hepatocellular carcinoma patients. CONCLUSION: HBV/C2 is the most prevalent subgenotype in Korea, and HBV/C7 subgenotype found in the Philippines was first identified in the Korean population. Mutations in immune epitopes within the core gene were significantly associated with disease progression.

[Analysis of gene expression in primary hepatocellular carcinoma using differentially displayed reverse transcriptase polymerase chain reaction].

BACKGROUND/AIMS: The investigation of a specific tumor marker for hepatocellular carcinoma (HCC) is needed to examine the carcinogenesis and to select the patients for treatment options. The aim of this study was to find the genes related to HCC. We also examined the expression level of these genes in cancer cell lines and tissue specimens. METHODS: Three pairs of HCC tissue and non-neoplastic hepatic tissue around the HCC were collected from three patients who underwent resection for HCC. Differential display reverse transcriptase-PCR (DD RT-PCR) using GeneFishingTM PCR was used to detect the differences in the gene expression between in HCC tissue and non-neoplatic tissue. Up- or down-regulated genes in HCC tissue were identified through BLAST searches after cloning and sequencing assays. Real-time RT-PCR assay was employed to detect the expression rate in 11 HCC tissues and human cancer cell lines. RESULTS: Differentially expressed 21 genes were identified, and they were classified as genes involved in protein metabolism, ubiquitin-dependent protein catabolism, carbohydrate metabolism, lipid metabolism, DNA repair, and inflammatory response. CONCLUSIONS: We identified differentially expressed genes in HCC, and these genes may play an important role in the study of hepatocarcinogenesis, development of biomarker, and target therapy for HCC.

Functional characterization of human oncoprotein gankyrin in Zebrafish.

Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2- hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.

[In vitro culture of hepatitis C virus (HCV) using immortalized hepatocyte].

BACKGROUND/AIMS: It is essential to develop an in vitro culture model of primary hepatocytes for the study of hepatocellular function and the pathogenesis of hepatitis C virus (HCV) infection. In this study, we have established the immortalized primary human hepatocyte (IPHH) and performed in vitro culture of HCV derived from human patient. METHODS: Primary human hepatocytes were isolated from surgically resected liver tissue and then were immortalized by transfection with the SV40 large T antigen. The characterization of the IPHH during culture was analyzed by immunocytochemistry, RT-PCR, Western blot, ELISA, and soft agar assay. Next, sera and/or liver tissue homogenates from surgically resected liver tissues of patients with HCV infection were inoculated for the culture of HCV in IPHH. After HCV RNA extraction from IPHH and culture media, positive or negative stranded HCV RNA was examined by specific nest RT-PCR. RESULTS: IPHH expressed liver-associated proteins but did not express alpha-fetoprotein. Also IPHH showed ammonia removal activity. With regard to its malignant potential, colony formation in soft agar assay was not observed. Next, positive and negative stranded HCV RNAs in IPHH infected with patient's sera plus liver tissue homogenates were clearly detected whereas those in IPHH infected with only patient's sera were not detected. CONCLUSIONS: These results demonstrated the phenotypic characteristics of IPHH and the feasibility in vitro culture system of HCV infected human samples. This system might be useful for study of pathogenesis of HCV infection or hepatocyte-based applications.