Clinical correlation of cadherin-17 marker with advanced tumor stages and poor prognosis of cholangiocarcinoma.

BACKGROUND AND OBJECTIVES: In this retrospective study, we examined the CA17 tissue expression and analyzed its clinical significance in cholangiocarcinoma (CCA). MATERIALS AND METHODS: Immunohistochemistry was performed to assess CA17 expression on tissue microarrays in a training cohort enrolling 120 CCA patients and a validation cohort comprising 60 CCA patients. Image pro plus was applied to score the staining intensity and expression level of CA17 marker. Kaplan-Meier analysis, Cox's proportional hazards regression, and nomogram were applied to evaluate the prognostic significance of CA17. RESULTS: CA17 cancer biomarker over-expression was significantly observed in CCA compared to their non-tumor counterparts, and positively correlated with aggressive tumor phenotypes, like lymph node metastasis. Meanwhile, patients with high expression of CA17 correlated with worse postoperative overall survival (OS) and recurrence-free survival. Besides, multivariate analysis identified that CA17 expression was an independent prognostic factor for cholangiocarcinoma patients, which indicated that the CA17 could be more efficient than serum CA19-9 in predicting the OS of CCA patients. Notably, the nomogram integrating CA17 expression had better prognostic performance as compared with current TNM staging systems. CONCLUSION: CA17 was an independent adverse prognostic factor for CCA patients' survival, which may serve as a promising prognostic biomarker for CCA patients.

Ubiquitin-specific protease 7 accelerates p14(ARF) degradation by deubiquitinating thyroid hormone receptor-interacting protein 12 and promotes hepatocellular carcinoma progression.

UNLABELLED: The prognosis for hepatocellular carcinoma (HCC) remains dismal in terms of overall survival (OS), and its molecular pathogenesis has not been completely defined. Here, we report that expression of deubiquitylase ubiquitin-specific protease 7 (USP7) is higher in human HCC tissues than in matched peritumoral tissues. Ectopic USP7 expression promotes growth of HCC cells in vivo and in vitro. Mechanistically, USP7 overexpression fosters HCC cell growth by forming a complex with and stabilizing thyroid hormone receptor-interacting protein 12 (TRIP12), which induces constitutive p14(ARF) ubiquitination. Clinically, USP7 overexpression is significantly correlated with a malignant phenotype, including larger tumor size, multiple tumor, poor differentiation, elevated alpha-fetoprotein, and microvascular invasion. Moreover, overexpression of USP7 and/or TRIP12 correlates with shorter OS and higher cumulative recurrence rates of HCC. CONCLUSION: USP7 stabilizes TRIP12 by deubiquitination, thus constitutively inactivating p14(ARF) and promoting HCC progression. This represents a novel marker for predicting prognosis and a potential therapeutic target for HCC.

Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma.

UNLABELLED: It has been reported that tetraspanin CD151 acts as a promoter of metastasis in several tumors and plays an important role in c-Met/hepatocyte growth factor signaling. However, the role of CD151 alone and coexpression of CD151/c-Met in hepatocellular carcinoma (HCC) remains unclear. We found that expression of CD151 was positively related to metastatic potential of HCC cell lines, and modified cells with CD151(high) showed higher secretion of matrix metalloproteinase 9 and aggressiveness in vitro and higher metastatic ability in vivo. Furthermore, HCC patients with vascular invasion, large tumors, multiple tumors, high tumor-node-metastasis stage, and undifferentiated tumor were prone to have higher CD151 expression. The postoperative 3-, 5-, and 7-year overall survival (OS) of patients in HCCs with CD151(high) were significantly lower than those in the CD151(low) group, and correspondingly cumulative recurrence rates in HCCs with CD151(high) were significantly higher than those in the CD151(low) group. Both CD151 and c-Met were remarkably overexpressed in HCCs, compared with adjacent nontumorous and normal liver tissues. Pearson correlation analysis showed a slight correlation between CD151 and c-Met in HCCs. Importantly, the 5- and 7-year OS rates in CD151(high)/c-Met(high) patients were 50.5% and 37.8%, respectively, significantly lower than those of CD151(low)/c-Met(low) patients (63.9% and 54.6%, respectively). Five- and 7-year cumulative recurrence rates in CD151(high)/c-Met(high) patients were 53.3% and 71.9%, respectively, markedly higher than those of CD151(low)/c-Met(low) patients (39.0% and 52.5%, respectively). Multivariate analysis revealed that CD151 and combination of CD151/c-Met were independent prognostic indicators for OS and cumulative recurrence. CONCLUSION: CD151 is positively associated with invasiveness of HCC, and CD151 or combination of CD151/c-Met is a novel marker in predicting the prognosis of HCC and a potential therapeutic target.

Cannabinoid type 2 receptor agonist JWH133 decreases blood pressure of spontaneously hypertensive rats through relieving inflammation in the rostral ventrolateral medulla of the brain.

OBJECTIVE: Neuroinflammation in the rostral ventrolateral medulla (RVLM) has been reported to be associated with hypertension. The upregulation and activation of the cannabinoid type 2 (CB2) receptor may be part of the active process of limiting or downregulating the inflammatory process. This study was designed to determine the role of the CB2 receptor in blood pressure (BP) through relieving neuroinflammation in the RVLM in spontaneously hypertensive rats (SHRs). METHODS: The long-term effects of intracerebroventricular injection of JWH133, a selective CB2 receptor agonist, on BP, heart rate (HR) and renal sympathetic nerve activity (RSNA) in SHR and Wistar-Kyoto (WKY) rats were determined. ELISA was used to measure the levels of proinflammatory cytokines, and western blotting was employed to detect protein expression of the CB2 receptor. Immunofluorescence staining was used to localize the CB2 receptor. Gene silencing of the CB2 receptor was realized by injecting adeno-associated virus (AAV) expressing CB2-specific shRNA (AAV2-r-CB2shRNA) into the RVLM. RESULTS: We found that SHRs exhibited higher levels of basal BP, HR, RSNA and proinflammatory cytokines (TNFα, IL-6 and IL-1ß) than those in WKY rats. The protein level of the CB2 receptor in the RVLM was robustly increased in SHRs. In addition, the CB2 receptor was mainly expressed on microglia cells of SHRs but not in WKY rats. No expression of the CB2 receptor was found on neurons of either WKY rats or SHRs. Furthermore, intracerebroventricular injection of JWH133 (1 mmol/l, 10 µl) for 28 days decreased the BP, HR, RSNA and proinflammatory cytokines significantly in SHRs, but it had no such effects in WKY rats. These effects were abolished by microinjection of 300 nl AAV2-r-CB2shRNA into the RVLM to knock down the CB2 receptor. CONCLUSION: Taken together, our results suggest that exciting the CB2 receptor relieves proinflammatory cytokine levels in the RVLM to decrease the BP, HR and RSNA in SHRs.

Mortalin promotes cell proliferation and epithelial mesenchymal transition of intrahepatic cholangiocarcinoma cells in vitro.

AIMS: The prognosis of patients with intrahepatic cholangiocarcinoma (ICC) remains poor in terms of overall survival (OS) and recurrence rate. Mortalin, a stress chaperone, has been reported to be involved in carcinogenesis and metastasis. However, its role in ICC has not been defined. METHODS: Mortalin expression in tumour samples from patients with ICC was examined by Western blot and immunohistochemistry, and correlation between its expression and clinicopathological features was assessed. In addition, invasion, migration proliferation and apoptosis, and the expression of epithelial-mesenchymal transition (EMT)-related markers in ICC cells were assessed after mortalin depletion. Finally, the prognostic significance of mortalin in patients with ICC was further evaluated by Kaplan-Meier and Cox regression analysis. RESULTS: We provide evidence that expression of mortalin in human ICC tissues is higher than that in matched peritumoural tissues. The interference of mortalin expression inhibited the proliferation and invasion of ICC cells in vitro. Mechanistically, inhibition of mortalin expression in ICC cells upregulated E-cadherin expression and decreased vimentin and snail expression. Clinically, a high level of mortalin in ICC samples was associated with loss of E-cadherin, and increased expression of vimentin and snail. Patients with ICC and high mortalin expression had a shorter OS and a higher recurrence rate. Multivariate analysis revealed that mortalin overexpression was an independent prognostic indicator for patients with ICC. CONCLUSIONS: Mortalin may promote cell proliferation and invasion via induction of EMT of ICC cells. A high level of mortalin may be used as a prognostic biomarker and therapeutic target for patients with ICC.

The membrane-cytoskeleton organizer ezrin is necessary for hepatocellular carcinoma cell growth and invasiveness.

PURPOSE: The change of cell mobility is one of the preconditions of tumor metastasis. Cell skeleton alteration and rearrangement of F-actin was closely related to cell mobility. Ezrin is a membrane-cytoskeleton organizer that can mediate the rearrangement and the function of F-actin. In this paper, we investigated the effect of ezrin on hepatocellular carcinoma cell growth and invasiveness. METHODS: Hepatocellular carcinoma cell lines such as MHCC-1, MHCC97-H, SF7721, SMMC7721, Hep3B, and HepG2 were chosen in this study. We first examined the expression and the distribution of ezrin and F-actin in these cell lines using immunofluorescence, RT-PCR, and the western blot. Next we used small interfering RNA (siRNA) to down-regulate ezrin expression in MHCC-1, MHCC97-H, SF7721, and HepG2 to investigate the role of ezrin in tumor cell growth and invasiveness. RESULTS: Our preliminary results showed that the expression of ezrin and gamma-actin in MHCC-1, MHCC97-H, and SF7721 with higher metastatic potential were obviously up-regulated than those in SMMC7721, Hep3B, and HepG2 with lower potential. No different expression of beta-actin was found in the above tumor cell lines. The outcome of RNAi indicated that decreasing ezrin expression can notably inhibit the proliferation of the four hepatocellular carcinoma cell lines (p < 0.01, n = 10). The proportion of cells in G2-M phase also decreased after RNAi. The number of pseudopods decreased as well after RNAi treatment (p < 0.01, n = 5). The mobility and invasiveness of cancer cells decreased with decreasing ezrin expression tested by transwell assay (p < 0.01, n = 8). CONCLUSION: Ezrin plays an important role in the process of hepatocellular carcinoma cell proliferation, migration, and invasiveness.

[The influence of downregulation of ezrin expression by RNA interference on the growth and metastasis of hepatocellular carcinoma: experiment in vitro].

OBJECTIVE: To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of human hepatocellular carcinoma cell (HCC) lines. METHODS: Human HCC cells of the lines SF/SMMC7721, MHCC97-H, MHCC-1, and HepG2 were cultured. Four pairs of small interfering RNA (siRNA) targeting erzin were designed and transfected into the HCC cells. 48 h after transfection the cell total RNA was extracted and 72 h later the total cell protein was extracted. RT-PCR and Western blotting were used to detect the transfection rates so as to screen the most effective siRNA to be transfected into the HCC cells. HCC cells were collected every day for 7 days to extract the total RNA and protein. Real-time PCR and Western blotting were used to detect the downregulation rate of erzin at different times. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell cycle and apoptosis. Scanning electron microscopy was used to observe the cell pseudopods. Transwell test was used to detect the invasion ability of the transfected HCC cells. RESULTS: Real-time PCR and western-blotting revealed that ezrin siRNA notably down-regulated ezrin expression at both mRNA and protein levels. Down-regulation of ezrin expression distinctly decreased the proliferation rates of these 4 kinds of HCC line. After RNAi treatment the cell proportion in G(2)-M phase decreased from 28.07% to 21.53% in the SF/SMMC7721 cells, from 24.94% to 13.92% in the MHCC97-H cells, from 19.30% to 13.2% in the MHCC-1cells, and from 7.73% to 4.24% in the HepG2 cells. After RNAi treatment, the number of pseudopods decreased from 20.8 +/- 3.0 to 13.2 +/- 2.4 in the SF/SMMC7721: cells (P < 0.05), from 18.4 +/- 2.7 to 14.0 +/- 2.9 in the MHCC97-H cells (P < 0.01), from 22.6 +/- 3.5 to 13.3 +/- 1.9 in the MHCC-1: cells (P < 0.01), and from 31.0 +/- 2.9 to 17.8 +/- 2.3 in the HepG2 cells (P < 0.01); and the motility and invasiveness decreased from 49.9 +/- 7.7 to 31.9 +/- 5.2 in the SF/SMMC7721 cells (P < 0.05), from 58.5 +/- 4.2 to 33.0 +/- 3.3 in the MHCC97-H cells (P < 0.01), from 57.6 +/- 6.1 to 28.3 +/- 3.4 in the MHCC-1 cells (P < 0.01), and from 37.3 +/- 3.0 to 25.3 +/- 2.3 in the HepG2 cells (P < 0.01). The pseudopods of the HCC cells remarkably shortened and decreased in number (for the SF/SMMC7721 cells: t = 4.95, P < 0.05, for the MHCC97-H cells: t = 5.88, P < 0.01, for the MHCC-1 cells: t = 5.56, P < 0.01, and for the HepG2 cells: t = 5.71, P < 0.01) after siRNA interference. CONCLUSION: Ezrin is necessary for HCC proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis.

[The effects of ezrin on the proliferation and the invasiveness of cells of different hepatocellular carcinoma cell lines].

OBJECTIVE: To investigate the different expressions of cytoskeletal organizer ezrin and cytoskeleton protein beta- and gamma-actin in hepatocellular carcinoma (HCC) cell lines with different metastatic potentials and to explore the role of ezrin in cell growth and metastasis in HCC cell lines SF7721 and MHCC97-H. METHODS: Immunofluorescence, RT-PCR and Western blot were used to detect the gene and protein expressions of ezrin and actin in hepatocellular carcinoma cell lines with different metastatic potentials. RNA interference (RNAi) was applied to down-regulate the ezrin expression in SF7721 and MHCC97-H. Changes of the cell growth and metastasis potentials after the RNAi treatment were studied. MTT assay was used to detect cell proliferation changes and Transwell assay was applied to observe the changes of cell motility and invasiveness. RESULTS: Both ezrin and cytoskeleton protein were demonstrated in the cytoplasma of the cells at the same time. The expression of them in cell lines with high metastatic potential, such as SF7721, MHCC-1 and MHCC97-H was obviously higher than in those with low metastatic potentials, such as SMMC-7721, Hep3B and HepG2 (chi2= 13.277, P = 0.010; chi2= 21.815). The mRNA and ezrin and cytoskeleton protein gamma-actin were over-expressed in HCC cell lines with high metastatic potentials. The expressions of beta-actin of cell lines with different metastatic potentials showed no differences. Ezrin protein was successfully down-regulated and the proliferation and the invasiveness of the cells decreased with low ezrin protein level in SF7721 and MHCC97-H. CONCLUSION: Over-expression of ezrin and cytoskeleton protein gamma-actin are associated with the process of metastasis of hepatocellular carcinoma cells. The growth and invasiveness of SF7721 and MHCC97-H cells can be inhibited by down-regulating ezrin expression.

Generation and characterization of a tetraspanin CD151/integrin α6ß1-binding domain competitively binding monoclonal antibody for inhibition of tumor progression in HCC.

Our previous studies revealed that tetraspanin CD151 plays multiple roles in the progression of hepatocellular carcinoma (HCC) by forming a functional complex with integrin α6ß1. Herein, we generated a monoclonal antibody (mAb) that dissociates the CD151/integrin α6ß1 complex, and we evaluated its bioactivity in HCCs. A murine mAb, tetraspanin CD151 (IgG1, called CD151 mAb 9B), was successfully generated against the CD151-integrin α6ß1 binding site of CD151 extracellular domains. Co-immunoprecipitation using CD151 mAb 9B followed by Western blotting detected a 28 kDa protein. Both immunofluorescent and immunohistochemical staining showed a good reactivity of CD151 mAb 9B in the plasma membrane and cytoplasm of HCC cells, as well as in liver cells. In vitro assays demonstrated that CD151 mAb 9B could inhibit neoangiogenesis and both the mobility and the invasiveness of HCC cells. An in vivo assay showed that CD151 mAb 9B inhibited tumor growth potential and HCC cells metastasis. We successfully produced a CD151 mAb 9B targeting the CD151/integrin α6ß1-binding domain, which not only can displayed good reactivity to the CD151 antigen but also prevented tumor progression in HCC.

Anti-cadherin-17 antibody modulates beta-catenin signaling and tumorigenicity of hepatocellular carcinoma.

Cadherin-17 (CDH17) is an oncofetal molecule associated with poor prognostic outcomes of hepatocellular carcinoma (HCC), for which the treatment options are very limited. The present study investigates the therapeutic potential of a monoclonal antibody (Lic5) that targets the CDH17 antigen in HCC. In vitro experiments showed Lic5 could markedly reduce CDH17 expression in a dose-dependent manner, suppress ß-catenin signaling, and induce cleavages of apoptotic enzymes caspase-8 and -9 in HCC cells. Treatment of animals in subcutaneous HCC xenograft model similarly demonstrated significant tumor growth inhibition (TGI) using Lic5 antibody alone (5 mg/kg, i.p., t.i.w.; ca.60-65% TGI vs. vehicle at day 28), or in combination with conventional chemotherapy regimen (cisplatin 1 mg/kg; ca. 85-90% TGI). Strikingly, lung metastasis was markedly suppressed by Lic5 treatments. Immunohistochemical and western blot analyses of xenograft explants revealed inactivation of the Wnt pathway and suppression of Wnt signaling components in HCC tissues. Collectively, anti-CDH17 antibody promises as an effective biologic agent for treating malignant HCC.

[The influence of active calcium ions solution on the ultrastructure of the enamel and cementum of extracted teeth and the evaluation of its cytotoxicity].

PURPOSE: To detect the effect of active calcium ions solution on the ultrastructure of enamel and cementum of the extracted teeth and observe the cytotoxicity of the solution on NIH3T3 cell through cell culture. METHODS: Active calcium ions solution composition was detected with a Quanta200FEG field emission scanning electron microscopy, and the ultrastructure of teeth enamel and cementum was observed before and after the application of calcium ions solution. After diluting the calcium ions solution, MTT assay method was used to observe the impact of active calcium ions solution of different concentrations on the NIH3T3 cells growth. Based on cell relative growth rate, the cytotoxicity grade was rated. RESULTS: Active calcium ions solution was composed of calcium, carbon, oxygen, and chlorine. And there was no change in the enamel and cementum before and after soaking the teeth with active calcium ions solution. The maximum concentration of safe calcium ions solution on cytotoxicity grade was 0.344 mg/mL. CONCLUSIONS: Active calcium ions solution can be used in the oral cavity,but appropriate concentration should be selected.

Proteomic identification of a monoclonal antibody recognizing caveolin-1 in hepatocellular carcinoma with metastatic potential.

A monoclonal antibody, McAb9E (IgG3), was generated against a metastatic HCC cell line, MHCC-1. The antigen was characterized as human Caveolin-1 (Cav-1, 21kDa), with pI of 5.65. The Cav-1 antigen was found significantly over expressed in metastatic HCC cell lines as well as in tumor specimens. The Cav-1 specific McAb may be a useful molecular agent for metastatic HCC.

Hepatic stellate cell-targeted delivery of M6P-HSA-glycyrrhetinic acid attenuates hepatic fibrogenesis in a bile duct ligation rat model.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) play a key role in fibrogenesis. Here, we used mannose-6-phosphate-modified human serum albumin (M6P(26)-HSA) as a selective carrier to deliver antifibrotic drug 18beta-glycyrrhetinic acid (18beta-GA) in experimental fibrosis animals, and tested its effect in injured liver tissues. METHODS: Bile duct ligation (BDL) was performed to induce liver damage in rats. Masson's stain and immunocytochemistry were used to assess hepatic collagen deposits and uptakes of M6P(26)-HSA-GA in HSCs in rat livers. Gene expression profiles of procollagen type I alpha2, smooth muscle actin (SMA), and transforming growth factor-beta1 (TGF-beta1) were analysed by TaqMan and quantitative polymerase chain reaction assays. The depositions of M6P(26)-HSA-GA in the HSC-T6 cell line and primary HSCs were assessed by immunofluorescent staining. RESULTS: Treatment with M6P(26)-HSA-GA at 10 mg/kg (three times/week for 2 weeks), but not the equivalent doses of free 18beta-GA and M6P(26)-HSA carrier alone, could significantly attenuate collagen deposits in BDL rat liver. Masson's stain and TaqMan assay revealed significant modulation of procollagen type I alpha2 in the BDL-injured liver. The depositions of M6P(26)-HSA-GA in HSCs were revealed by immunostaining with HSA and SMA markers. M6P(26)-HSA bound activated HSCs in vitro and the immunoreactivity of M6P(26)-HSA-GA was detected in the cytoplasm and cell surface of HSCs and HSC-T6 cells. The gene transcript levels of SMA and TGF-beta1 were modulated in HSC-T6 cells treated with M6P(26)-HSA-GA. CONCLUSIONS: The M6P(26)-HSA holds promise as a targeting carrier for the liver or HSCs, which may be used to deliver 18beta-GA as a therapeutic agent to treat liver fibrosis.

Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values.

To facilitate the identification of candidate molecular biomarkers that are linked to the pathogenesis of hepatocellular carcinoma (HCC), we investigated protein-expression profiles of 146 tissue specimens including 67 pairs of tumors and adjacent non-tumors resected from HCC patients as well as 12 normal livers by 2-DE. Among the 1800 spots displayed in the liver proteome, a total of 90 protein species were found to be significantly different between the three groups (P < 0.05). Three of the top candidate markers up-regulated in HCC, with high receiver operating characteristic (ROC) curves, were identified by MS/MS analysis and belonged to the chaperone members: heat-shock protein (Hsp)27, Hsp70 and glucose-regulated protein (GRP)78. Over-expression of these chaperone proteins in HCC tissues was confirmed by Western blotting and immunohistochemistry. In correlation with clinico-pathological parameters, expression of Hsp27 was linked to alpha-fetoprotein level (P = 0.007) whereas up-regulation of GRP78 was associated with tumor venous infiltration (P = 0.035). No significant association of Hsp70 with any pathologic features was observed. The present HCC proteome analysis revealed that in response to the stressful cancerous microenvironment, tumor cells strived to increase the expression of chaperone proteins for cyto-protective function and to enhance tumor growth and metastasis.

Proteomic identification of Ku70/Ku80 autoantigen recognized by monoclonal antibody against hepatocellular carcinoma.

A murine monoclonal antibody (mAb), CLD3 (IgG(1),kappa), was generated against hepatocellular carcinoma (HCC). Both immunofluorescence and immunohistochemical assays indicated the reactivity of CLD3 mAb localized at the nucleus and/or cytoplasm of tumorigenic HCC cell lines as well as in liver cancer tissues. By immunoprecipitation and using the matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach, the antigenic specificity of CLD3 was determined to be heterodimeric Ku70 and Ku80 autoantigen, which was confirmed by Western blotting.