Circulating Tumor Cells with Stem-Like Phenotypes for Diagnosis, Prognosis, and Therapeutic Response Evaluation in Hepatocellular Carcinoma.

Background: In the present study, we assessed the clinical value of circulating tumor cells (CTC) with stem-like phenotypes for diagnosis, prognosis, and surveillance in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) by an optimized qPCR-based detection platform.Methods: Differing subsets of CTCs were investigated, and a multimarker diagnostic CTC panel was constructed in a multicenter patient study with independent validation (total n = 1,006), including healthy individuals and patients with chronic hepatitis B infection (CHB), liver cirrhosis (LC), benign hepatic lesion (BHL), and HBV-related HCC, with area under the receiver operating characteristic curve (AUC-ROC) reflecting diagnostic accuracy. The role of the CTC panel in treatment response surveillance and its prognostic significance were further investigated.Results: The AUC of the CTC panel was 0.88 in the training set [sensitivity = 72.5%, specificity = 95.0%, positive predictive value (PPV) = 92.4, negative predictive value (NPV) = 77.8] and 0.93 in the validation set (sensitivity = 82.1%, specificity = 94.2%, PPV = 89.9, NPV = 89.3). This panel performed equally well in detecting early-stage and α-fetoprotein-negative HCC, as well as differentiating HCC from CHB, LC, and BHL. The CTC load was decreased significantly after tumor resection, and patients with persistently high CTC load showed a propensity of tumor recurrence after surgery. The prognostic significance of the CTC panel in predicting tumor recurrence was further confirmed [training: HR = 2.692; 95% confidence interval (CI), 1.617-4.483; P < 0.001; and validation: HR = 3.127; 95% CI, 1.360-7.190; P = 0.007].Conclusions: Our CTC panel showed high sensitivity and specificity in HCC diagnosis and could be a real-time parameter for risk prediction and treatment monitoring, enabling early decision-making to tailor effective antitumor strategies. Clin Cancer Res; 24(9); 2203-13. ©2018 AACR.

Protective effects of curcumin against hepatic fibrosis induced by carbon tetrachloride: modulation of high-mobility group box 1, Toll-like receptor 4 and 2 expression.

The aim of the study was to investigate the effect of curcumin on the liver fibrosis induced by carbon tetrachloride (CCl(4)) in rats, and to elucidate its underlying molecular mechanisms. Rats were administered with CCl(4) together with or without curcumin for 6 weeks. Hepatic damage was evaluated by analysis of liver function tests in serum. Hepatic histopathology and collagen content were employed to quantify liver fibrosis; and activated hepatic stellate cells were assessed. Moreover, the mRNA and protein expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, high-mobility group box 1 (HMGB1), Toll like receptor (TLR) 2 and TLR4 were determined by quantitative real time PCR, Western blot or immunohistochemistry. Treatment with curcumin significantly attenuated CCl(4)-induce liver injury, hepatic inflammation and reduced the levels of proinflammatory mediators (TNF-α, IL-6 and MCP-1). Moreover, curcumin significantly inhibited extracellular matrix deposition, reduced the number of activated stellate cells, and decreased the levels of HMGB1, TLR4 and TLR2 expression in the rat model of fibrogenesis. These results suggest that curcumin could be an effective agent for preventing liver fibrosis and its mechanism may in part be a consequence of the reduction TLR2, TLR4 and HMGB1 expression.

Overexpression of Smad ubiquitin regulatory factor 2 suppresses transforming growth factor-ß mediated liver fibrosis.

OBJECTIVE: To determine the function of Smad ubiquitin regulatory factor 2 (Smurf2) on the development of liver fibrosis and cirrhosis. METHODS: In vivo Smurf2 expression in fibrotic and cirrhotic rat and human liver tissues were measured using reverse transcription-polymerase chain reaction, Western blot (WB) and immunohistochemistry. In vitro Smurf2 levels were determined in LX-2 cell line with or without transforming growth factor (TGF)-ß1 treatment; I, III, IV collagen and laminin levels were determined by ELISA. The recombinant plasmid pcDNA3.1-Smurf2 was transfected into LX-2 cells, and WB and ELISA were utilized to analyze the expression of TGF-ß receptor type I (TßRI), Smad7, collagens and laminin with or without proteasome inhibitor MG-132. Coimmunoprecipitation was utilized to characterize the interactions among these factors and the ubiquitination levels. pcDNA3.1-Smad7 vector was transfected and subsequent examinations were conducted just as Smurf2. RESULTS: Smurf2 levels were elevated in the early period of fibrotic rat liver and TGF-ß1-treated LX-2 cells but were reduced in the cirrhotic livers. Smurf2 overexpression in LX-2 cells reduced TßRI and Smad7 levels, which was accompanied by decreased collagen and laminin levels. Coimmunoprecipitation demonstrated that Smurf2 interacted with TßRI and Smad7, which increased TßRI and Smad7 ubiquitin levels. Smad7 overexpression reduced the TßRI level and was accompanied by decreased collagen and laminin levels. MG-132 could antagonize these effects. CONCLUSION: Smurf2 interacts with Smad7 to suppress TGF-ß-mediated liver fibrosis through the ubiquitin-dependent degradation of TßRI during the early period of liver fibrosis.

Abnormal expression of Smurf2 during the process of rat liver fibrosis.

OBJECTIVE: Liver fibrosis is a prelude of liver cirrhosis. Currently the molecular mechanism of liver fibrosis is not clear. The purpose of this study is to screen the abnormally expressed genes of liver fibrosis and to illustrate the changes of Smurf2 expression in the process of liver fibrosis. METHODS: A liver fibrosis model was established in rats by injection of tetrachlormethane (CCl(4)). A cDNA microarray analysis was performed on the liver at mid-stage of fibrosis. Thereafter, a semi-quantitative RT-PCR, Western blot analysis and immunohistochemistry test were performed for determining Smurf2, Smad2 and SnoN at week 1, 2, 4 and 8 of establishing the liver fibrosis model. RESULTS: Smurf2, FGG, PTAFR, CYP2D6, among others, increased in the fibrosis liver and a semi-quantitative RT-PCR confirmed the reliability of the cDNA microarray analysis. Smurf2 in the liver fibrosis model group was at the same level as that of control group at week 1, but decreased at week 2 and 8 and increased at the week 4. Smad2 increased at week 2 and 8 but increased at week 4. However, Smad2 mRNA increased to the same level at week 4 as that at week 2 and 8. The decrease of Smad2 at week 4 may be due to the enhancement of ubiquitination and proteolytic degradation of Smad2 by the increase of Smurf2. SnoN decreased at week 4 and 8 because of the ubiquitination and degradation caused by Smurf2. The decrease of SnoN may explain the progress of liver fibrosis in spite of the decrease of Smad2 at week 4. CONCLUSION: This study screened the abnormally expressed genes of liver fibrosis and illustrated the changes of Smurf2, Smad2 and SnoN during the process of liver fibrosis.