Lihui Han

Hepatitis B Virus Sensitizes Hepatocytes to TRAIL-Induced Apoptosis through Bax

Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.

Hepatitis B virus core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression

Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in hepatocyte death during HBV infection. We report here that the HBV core protein (HBc) is a potent inhibitor of TRAIL-induced apoptosis. Overexpressing HBc significantly decreased TRAIL-induced apoptosis of human hepatoma cells, whereas knocking-down HBc expression in hepatoma cells transfected with HBV genome enhanced it. When present in the same cell, HBc blocked the pro-apoptotic effect of the HBV X protein (HBx). The resistance of HBc-expressing cells to TRAIL-induced apoptosis was associated with a significant reduction in death receptor 5 (DR5) expression. Upon transfection, HBc significantly repressed the promoter activity of the human DR5 gene. Importantly, HBc gene transfer inhibited hepatocyte death in a mouse model of HBV-induced hepatitis; and in patients with chronic hepatitis, DR5 expression in the liver was significantly reduced. These results indicate that HBc may prevent hepatocytes from TRAIL-induced apoptosis by blocking DR5 expression, which in turn contributes to the development of chronic hepatitis and HCC. They also call into question the potential side effects of HBc-based vaccines.

Acquisition of anoikis resistance reveals a synoikis-like survival style in BEL7402 hepatoma cells.

Resistance to anoikis is a hallmark of human malignancies. Our results showed that hepatoma cells resisted anoikis by non-proliferation, non-apoptosis and cell cycle arrest which were termed synoikis-like. These synoikis-like cells are more resistant to extracellular stimuli and could spontaneously attach and proliferate again under suitable conditions, which indicate a reversible property of these cells. Microarray expression profile reveals the change of molecules involved in the synoikis-like hepatoma cells and our data indicated that ANGPTL4 contributed to anoikis resistance of hepatoma cells. These results demonstrated that hepatoma cells might resist anoikis through a synoikis-like survival style, which may facilitate tumor metastasis.

Prognostic Significance of Preoperative Lymphocyte-Monocyte Ratio in Patients with Resectable Esophageal Squamous Cell Carcinoma

BACKGROUND The interaction between tumor cells and inflammatory cells has not been systematically investigated in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to evaluate whether preoperative the lymphocyte-monocyte ratio (LMR), the neutrophil-lymphocyte ratio (NLR), and the platelet-lymphocyte ratio (PLR) could predict the prognosis of ESCC patients undergoing esophagectomy. MATERIALS AND METHODS Records from 218 patients with histologically diagnosed ESCC who underwent attempted curative surgery from January 2007 to December 2008 were retrospectively reviewed. Besides clinicopathological prognostic factors, we evaluated the prognostic value of the LMR, the NLR, and the PLR using Kaplan-Meier curves and Cox regression models. RESULTS The median follow-up was 38.6 months (range 3-71 months). The cut-off values of 2.57 for the LMR, 2.60 for the NLR and 244 for the PLR were chosen as optimal to discriminate between survival and death by applying receiver operating curve (ROC) analysis. Kaplan-Meier survival analysis of patients with low preoperative LMR demonstrated a significant worse prognosis for DFS (p=0.004) and OS (p=0.002) than those with high preoperative LMR. The high NLR cohort had lower DFS (p=0.004) and OS (p=0.011). Marginally reduced DFS (p=0.068) and lower OS (p=0.039) were found in the high PLR cohort. On multivariate analysis, only preoperative LMR was an independent prognostic factor for both DFS (p=0.009, HR=1.639, 95% CI 1.129-2.381) and OS (p=0.004, HR=1.759, 95% CI 1.201-2.576) in ESCC patients. CONCLUSIONS Preoperative LMR better predicts cancer survival compared with the cellular components of systemic inflammation in patients with ESCC undergoing esophagectomy.

The hepatitis B virus protein MHBs(t) sensitizes hepatoma cells to TRAIL-induced apoptosis through ERK2

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Infectious agents, such as hepatitis B virus (HBV), may play important roles in regulating the sensitivity of hepatocytes to TRAIL. Our previous studies showed that HBx, a protein encoded by the HBV genome, enhanced TRAIL-induced apoptosis through upregulating Bax. We report here that another HBV protein called MHBs(t) (C-terminally truncated middle hepatitis B surface protein) is also a potent regulator of TRAIL-induced apoptosis. Overexpressing MHBs(t) in hepatoma cells enhanced TRAIL-induced apoptosis. Mechanistic studies reveal that MHBs(t) had no effect on Bax or TRAIL receptor expression or procaspase-8 activation, but selectively enhanced the activation of ERK2 (extracellular signal-regulated kinase 2) and the degradation of procaspases-3 and 9. ERK2 activation is required for the MHBs(t) effect because ERK2 inhibition by its inhibitor PD98059 significantly reversed TRAIL-induced apoptosis of MHBs(t)-transfected cells. These results establish that unlike HBx, MHBs(t) enhances TRAIL-induced hepatocyte apoptosis through a novel mechanism that involves ERK2. Therefore, manipulating the ERK2 signaling pathway may provide new therapeutic opportunities to contain hepatic cell death during HBV infection.

Up-regulation of Tropomyosin related kinase B contributes to resistance to detachment-induced apoptosis in hepatoma multicellular aggregations

To elucidate the molecular mechanisms of resistance to detachment-induced apoptosis, we cultured BEL7402 hepatoma cells on plates coated with poly (2-hydroxyethyl methacrylate), which blocked access to the extracellular matrix. When BEL7402 hepatoma cells were suspended, they could self-assemble into aggregations and resist to detachment-induced apoptosis. Expression of TrkB on detached cells was much higher than that of attached ones. Protein structure analysis revealed that TrkB contained adhesion domain, which might contribute to the aggregation formation of hepatoma cells. These aggregations had higher proliferation indices with BDNF treatment. These data demonstrate that TrkB may contribute to metastasis by facilitating formation of multicellular aggregations and induce their resistance to detachment-induced apoptosis.

Prognostic and diagnostic potential of miR-146a in oesophageal squamous cell carcinoma.

Background:Accumulating evidence indicates that dysregulated microRNA-146a (miR-146a) is involved in tumour genesis and cancer progression. We aimed to evaluate its expression level and the potential for the diagnosis and prognosis in oesophageal squamous cell cancer (ESCC).Methods:We examined miR-146a expression in 62 pairs of ESCC cancerous and matched paracancerous tissue, 115 formalin-fixed paraffin-embedded (FFPE) tissue samples and serum samples from 154 ESCC patients and 154 healthy volunteers using quantitative reverse transcription–PCR (qRT–PCR). Kaplan–Meier method, Cox regression and receiver-operating characteristic (ROC) curve analysis were applied to analyse its prognostic and diagnostic value.Results:MicroRNA-146a expression level was significantly decreased in ESCC tissue compared with paracancerous tissue (P<0.001). Its regulation level was negatively associated with T factor and TNM stage. Kaplan–Meier curve revealed that its downregulation level predicted worse overall survival (OS) and progression-free survival (PFS). Both univariate and multivariate analyses identified miR-146a expression as independent prognostic factor for OS and PFS. Serum miR-146a was significantly reduced in ESCC patients than in healthy controls (P<0.001). Area under the curve ROC value, sensitivity and specificity for this marker were 0.863±0.033, 85.7% and 68.6% in the Discovery Group, and 0.891±0.027, 82.1% and 83.3% in the Validation Group.Conclusions:MicroRNA-146a is significantly reduced in cancerous tissue and serum samples of ESCC patients. It is an ideal biomarker for the prognosis and diagnosis of ESCC.

Aggregation formation mediated anoikis resistance of BEL7402 hepatoma cells.

Anoikis resistance is the prerequisite of cancer cells metastasis. Elucidation of the mechanism of anoikis resistance remains a significant challenge. We reported here a model to mimic anoikis resistant process of hepatoma cells in vitro. Experimental results indicated cell to cell aggregation could mediate anoikis resistance of BEL7402 hepatoma cells. Further investigation of these aggregations indicated the biological properties changed greatly after the hepatoma cells lost their anchorage. Aggregation forming process could be separated into three distinct phases according to their biological characteristics, comprising of premature phase, mature phase and postmature phase. Mature phase aggregations have the premium state of cell viability and may mimic the metastatic cells in the circulating system. Biological properties of these three phases aggregations were studied in details including morphological alteration, cell viability and microarray expression profiles. It indicated there was a great upregulation of adhesion molecules during the process of aggregation formation and the cell to cell contact in the aggregation may be mediated independent of calcium involved adhesion pathway. This model might shed light on the anoikis resistance mechanism of hepatoma cells and help to develop new therapies that may target the anoikis resistant hepatoma cells in the metastasis process.

MiR-613: a novel diagnostic and prognostic biomarker for patients with esophageal squamous cell carcinoma

MicroRNA-613 (miR-613) plays important roles in tumorigenesis and cancer progression. We aimed to evaluate its expression level and potential for diagnosis and prognosis in esophageal squamous cell cancer (ESCC). We examined miR-613 expression in 60 pairs of ESCC cancerous and matched paracancerous tissues, serum samples from 75 ESCC patients and 75 healthy volunteers, and 105 formalin-fixed paraffin-embedded (FFPE) tissue samples using quantitative reverse transcription polymerase chain reaction. Receiver-operating characteristic (ROC) curve analysis, Kaplan-Meier method, and Cox regression were applied to analyze its diagnostic and prognostic value. MiR-613 was significantly decreased in ESCC tissue compared with paracancerous tissue (P < 0.001). Moreover, the expression level of miR-613 was significantly reduced with increased T stage of ESCC. Statistically significant difference between ESCC patients and healthy controls in expression level of miR-613 (0.89 ± 0.73 vs. 1.71 ± 1.03, P < 0.001) was found. The area under the ROC curve (AUC) based on serum miR-613 was 0.767 ± 0.040. We also performed analysis on early-stage patients and revealed that the AUC value was 0.728 ± 0.052 (P < 0.001). The Kaplan-Meier curve revealed that the downregulation of miR-613 was related to worse overall survival (OS) and progression-free survival (PFS) of ESCC patients (P = 0.018 and P = 0.035, respectively). Furthermore, the multivariate analysis identified miR-613 to be an independent prognostic factor for OS and PFS (P = 0.031 and P = 0.006, respectively) In conclusion, miR-613 is significantly reduced in cancerous tissue and serum samples of ESCC patients. It can serve as an ideal indicator for the diagnosis and prognosis of ESCC.

Notch2 as a promising prognostic biomarker for oesophageal squamous cell carcinoma

We aimed to examine Notch2 expression in oesophageal squamous cell carcinoma (ESCC) patients and to evaluate its prognostic potential. Immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were utilized to investigate the Notch2 expression status and prognostic value. Furtherly, CCK8 and clonogenic assays were conducted to determine if Notch2 inhibition by shRNA could lead to a decrease in the proliferation and survival of ESCC cells. A notably higher Notch2 expression level was found in ESCC tissues at the mRNA (P < 0.0001) and protein levels (IHC: P = 0.004; western blot: P = 0.021). Log-rank analysis demonstrated that Notch2 overexpression was significantly associated with worse overall survival (OS) (29.1% vs. 49.1%; P = 0.013) and progression-free survival (PFS) (15.3% vs. 34.4%; P = 0.006) rates in ESCC patients. The multivariate analysis revealed Notch2 as an independent prognostic factor for OS and PFS (P = 0.002 and 0.006, resp.). Besides, in vitro assays showed that OD450 values and colony formations were significantly reduced in Notch2-shRNA group (all P < 0.0001). In conclusion, these results show that Notch2 is up-regulated in ESCC tissues and could serve as a promising biomarker for identifying individuals with poor prognostic potential.