TGF-ß1/SMAD3-driven GLI2 isoform expression contributes to aggressive phenotypes of hepatocellular carcinoma.

Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-ß1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-ß1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-ß/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC.

Genome-wide long noncoding RNA and mRNA expression profiles demonstrate associations between exposure to inorganic elements and the risk of developing hepatocellular carcinoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) are closely associated with the development of hepatocellular carcinoma (HCC). The present study conducted a genome-wide microarray analysis and qPCR validation to obtain comprehensive insights into this issue. METHODS: Thirty male HCC patients with chronic HBV infection were included in the present study. Primary HCC tissue and normal tissue were collected. Double-stranded complementary DNA synthesized from 10 pairs of samples was labeled and hybridized to a microarray chip. Further analyses, such as hierarchical clustering, gene ontology (GO) and pathway analyses, were performed. In addition, qPCR validation was performed on tissue samples and additional serum samples. RESULTS: The microarray analysis identified 946 upregulated and 571 downregulated lncRNAs and 1720 upregulated and 1106 downregulated mRNAs. Among these RNAs, ENST00000583827.1 (fold change: 21) and uc010isf.1 (fold change: 18) were the most over- and underexpressed lncRNAs in the HCC tissues, respectively. For the mRNAs, KIF20A (fold change: 26) and HEPACAM (fold change: 50) were the most over- and underexpressed in the HCC tissues, respectively. The GO analysis demonstrated that the most differentially expressed mRNAs were related to the response of metal ions. The pathway analysis also suggested that the most enriched pathway was mineral absorption. CONCLUSIONS: The subsequent qPCR validation exhibited high consistency with the microarray analysis, except for three lncRNAs. The qPCR analysis also demonstrated that TCONS_00008984 had a 767-fold overexpression level in HCC tissues when compared with normal tissues, and this finding was confirmed in the serum samples; therefore, TCONS_00008984 has the potential to serve as a diagnostic marker or prognostic indicator. The GO and pathway analyses indicated that exposure to inorganic elements may be involved in HCC risk.

Identification of mTOR as a primary resistance factor of the IAP antagonist AT406 in hepatocellular carcinoma cells.

Dysregulation of inhibitor of apoptosis (IAP) proteins (IAPs) in hepatocellular carcinoma (HCC) is often associated with poor prognosis. Here we showed that AT406, an IAP antagonist, was cytotoxic and pro-apoptotic to both established (HepG2, SMMC-7721 lines) and primary HCC cells. Activation of mTOR could be a key resistance factor of AT406 in HCC cells. mTOR inhibition (by OSI-027), kinase-dead mutation or knockdown remarkably enhanced AT406-induced lethality in HCC cells. Reversely, forced-activation of mTOR by adding SC79 or exogenous expressing a constitutively active S6K1 (T389E) attenuated AT406-induced cytotoxicity against HCC cells. We showed that AT406 induced degradation of IAPs (cIAP-1 and XIAP), but didn't affect another anti-apoptosis protein Mcl-1. Co-treatment of OSI-027 caused simultaneous Mcl-1 downregulation to overcome AT406's resistance. Significantly, shRNA knockdown of Mcl-1 remarkably facilitated AT406-induced apoptosis in HCC cells. In vivo, AT406 oral administration suppressed HepG2 tumor growth in nude mice. Its activity was potentiated with co-administration of OSI-027. We conclude that mTOR could be a key resistance factor of AT406 in HCC cells.

Expression pattern and clinicopathologic significance of NKD1 in human primary hepatocellular carcinoma.

It has been reported that NKD1 was an antagonist of the canonical Wnt/ß-catenin pathway. While there is little information regarding NKD1 expression pattern in human hepatocellular carcinoma (HCC). The aim of this study was to investigate the clinicopathologic significance and expression pattern of NKD1 in HCC. NKD1 protein expressions in 69 paired HCC cancer/adjacent non-cancerous tissues were detected by Western blot. Immunohistochemical studies were performed on 58 cases of HCC with integrated clinical information. NKD1 protein expression was divided into normal and low expression group and correlations between NKD1 protein expression and clinicopathologic factors were then evaluated. Western blot results showed that NKD1 protein levels were significantly lower in cancerous tissues compared with corresponding normal tissue (p < 0.05). In addition, we found that the level of NKD1 protein expression in HCC was significantly associated with tumor size (p = 0.011), intra or extra-hepatic metastasis (p = 0.010) and differentiation (p = 0.003). This is to our knowledge the first report investigating NKD1 protein expression pattern in HCC. Our data show that decreased NKD1 protein expression is associated with clinicopathologic factors, and suggest that NKD1 may play an important role in the development of HCC and could serve as a novel biomarker for HCC after further investigation.

Chloride intracellular channel 1 is overexpression in hepatic tumor and correlates with a poor prognosis.

Chloride intracellular channel 1 (CLIC1) is expressed in many human tissues and has been reported to be involved in the regulation of cell cycle, cell proliferation, and differentiation. Its roles in human hepatic tumor, however, remain unclear. The aim of this study was to investigate the clinicopathological significance and expression pattern of CLIC1 in human primary hepatic tumors. We examined the expression pattern of CLIC1 mRNA and protein in hepatic tumors using real-time quantitative RT-PCR and Western blot, respectively. CLIC1 protein and mRNA levels were significantly higher in cancerous tissues compared with corresponding normal tissue. In 85 hepatic tumor tissues, CLIC1 was significantly higher in 69 cases (81.2%), as determined by immunohistochemical staining. Increased CLIC1 expression was correlated with tumor size (p = 0.021), distant metastasis (p = 0.025), pathological TNM (pTNM) stage (p = 0.023), and poor survival (25.11 ± 2.27 vs 45.29 ± 4.28 months, p = 0.001). Our data show that increased CLIC1 protein expression is associated with clinicopathological factors and a poor prognosis of hepatic tumors, and suggest that CLIC1 might represent a valuable prognostic marker for human hepatic tumors.

Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors.

BACKGROUND & AIMS: This study aimed at investigating mutations in the hepatitis B surface protein (HBsAg) in occult hepatitis B virus (HBV) infection (OBI) and their influence on viral antigenicity and phenotype. METHODS: The characteristics of 61 carriers with OBI (OBI group), 153 HBsAg(+) carriers with serum HBsAg ≤ 100 IU/ml (HBsAg-L group) and 54 carriers with serum HBsAg >100 IU/ml (HBsAg-H group) from 38,499 blood donors were investigated. Mutations in the major hydrophilic region (MHR) of the viral sequences were determined. Thirteen representative MHR mutations observed in OBI sequences were antigenically characterized with a panel of monoclonal antibodies (MAbs) and commercial HBsAg immunoassays and functionally characterized in HuH7 cells and hydrodynamically injected mice. RESULTS: Of 61 OBI sequences, 34 (55.7%) harbored MHR mutations, which was significantly higher than the frequency in either the HBsAg-L (34.0%, p=0.003) or the HBsAg-H group (17.1%, p<0.001). Alterations in antigenicity induced by the 13 representative MHR mutations identified in the OBI group were assessed by reacting recombinant HBV mutants with 30 different MAbs targeting various epitopes. Four out of the 13 mutations (C124R, C124Y, K141E, and D144A) strongly decreased the analytical sensitivity of seven commercial HBsAg immunoassays, and 10 (G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R) significantly impaired virion and/or S protein secretion in both HuH7 cells and mice. CONCLUSIONS: MHR mutations alter antigenicity and impair virion secretion, both of which may contribute to HBsAg detection failure in individuals with OBI.

Clinical significance and expression of cyclin kinase subunits 1 and 2 in hepatocellular carcinoma.

BACKGROUND: The mammalian cyclin kinase subunit (Cks) family has two members, Cks1 and Cks2, which were identified based on the protein sequence homology to yeast Cks. Overexpression of Cks1 and Cks2 has been reported to be associated with high aggressiveness and a poor prognosis in various malignancies, including gastric, breast and prostate carcinomas. Yet, whether Cks1 and Cks2 are overexpressed in hepatocellular carcinoma (HCC) remains uncharacterized. AIMS: To investigate whether overexpression of the Cks family is clinically relevant to HCC, and whether expression patterns of Cks1 and Cks2 in HCC have diagnostic and prognostic value. METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blot analyses were used to detect the expression of Cks1 and Cks2 at the mRNA and protein levels respectively. The associations between Cks1 and Cks2 expressions and clinical features, as well as the association between Cks1 or Cks2 and p27(kip1) expressions in HCC, were analysed. RESULTS: Expressions of Cks1 and Cks2 at both mRNA and protein levels were significantly higher in HCC than those in the adjacent noncancerous tissues (including chronic hepatitis and cirrhosis) and normal liver tissues. Overexpressions of Cks1 and Cks2 in HCC were closely associated with poor differentiation features. The expressions of both Cks1 and Cks2 were negatively associated with p27(kip1) at the protein level. CONCLUSIONS: Overexpression of Cks1 and Cks2 is associated with the aggressive tumour behaviours of HCC, and thus has diagnostic and prognostic value. Further efforts are needed to develop novel biomarkers for HCC based on CKs1 and Cks2 expressions.

Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice.

AIM: To determine the effects of allopurinol, an inhibitor of xanthine oxidase, and apocynin, an inhibitor of NADPH oxidase, on oxidant stress and liver injury caused by hepatic ischemia/reperfusion (I/R) procedure in mice. METHODS: Mice were pretreated with a xanthine oxidase inhibitor, allopurinol, or NADPH oxidase (NOX) inhibitor, apocynin before the hepatic I/R procedure. Then treated or untreated mice underwent the hepatic I/R procedure. The effects on hepatic injury and superoxide anions were determined after starting reperfusion. RESULTS: A standard warm hepatic I/R procedure led to a marked increase in superoxide anion production as indicated by a superoxide anion tracer, MCLA. At the same time, the procedure caused profound acute liver injury, as indicated by elevated serum alanine aminotransferase and tumor necrosis factor-alpha levels, reduced liver glutathione levels and elevated malondialdehyde contents, as well as a high apoptotic cell count. All these changes were reversed by the use of apocynin or allopurinol prior to the hepatic I/R procedure. CONCLUSION: Allopurinol and apocynin exerted protective effects on hepatic ischemia/reperfusion injury. The protection is associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting xanthine oxidase and NADPH oxidase activity.

Preventive effect of regional radiotherapy with phosphorus-32 glass microspheres in hepatocellular carcinoma recurrence after hepatectomy.

AIM: To evaluate the preventive effects of phosphorus-32 glass microspheres (P32-GMS) in the recurrence of massive hepatocellular carcinomas (HCCs) after tumor resection. METHODS: Twenty-nine patients with massive HCCs received local P32-GMS implantation after liver tumors were removed, while the other 38 patients with massive HCCs were not treated with P32-GMS after hepatectomies. The radioactivity of the blood, urine and liver were examined. The complications, HCC recurrence and overall survival rates in the patients were analyzed. RESULTS: P32-GMS implanted in the liver did not cause systemic absorption of P32. There were no significant differences of postoperative complications between the patients with and without P32-GMS treatment. The short-term (six months and 1 year) and long-term (2, 3 and over 3 years) recurrence rates in patients who received P32-GMS radiotherapy were significantly decreased, and the overall survival rates in this group were significantly improved. CONCLUSION: P32-GMS implantation in the liver can significantly decrease the postoperative recurrence and improve the overall survival in HCCs patients after hepatectomy. This therapy may provide an innovative method in prevention of HCC recurrence after operation.

[Recombinant HEV caspid protein p239 specifically attached on HepG2 cells and blocked the infection of wild-type HEV on liver cells].

By using Western blot and immunofluorescence assays, the recombinant HEV capsid protein p239 was found specifically attached to the HepG2 cell surface and entered to the cytoplasm with the increase of incubation temperature. Pre-mixture of wild-type HEV with p239 blocked the infectivity of the virus on primary cultured human hepatocytes and HepG2 cells, indicating that p239 and HEV competed the same targeting site on these cells. These data provide evidence that p239 has a similar cell surface structure with wild-type HEV.