Clinical relevance and therapeutic potential of angiopoietin-like protein 4 in hepatocellular carcinoma.

BACKGROUND: Development of novel adjuvant therapy to eradicate tumor angiogenesis and metastasis is a pressing need for patients with advanced hepatocellular carcinoma (HCC). We aimed to investigate the clinical relevance and therapeutic potential of angiopoietin-like 4 (ANGPTL4) in HCC. METHODS: ANGPTL4 mRNA levels in tumor and non-tumor liver tissues of HCC patients were analyzed to investigate its clinical relevance. The mechanisms of deregulation of ANGPTL4 in HCC were studied by copy number variation (CNV) and CpG methylation analyses. The orthotopic liver tumor nude mice model was applied using a human metastatic cell line. ANGPTL4-overexpressing adenovirus (Ad-ANGPTL4) was injected via portal vein to investigate its anti-tumorigenic and anti-metastatic potentials. RESULTS: HCC tissues expressed significantly lower levels of ANGPTL4 mRNA than non-tumor tissues. The copy number of ANGPTL4 gene in tumor tissues was significantly lower than in non-tumor tissues of HCC patients. Higher frequency of methylation of CpG sites of ANGPTL4 promoter was detected in tumor tissues compared to non-tumor tissues. Downregulation of ANGPTL4 mRNA in HCC was significantly associated with advanced tumor stage, presence of venous infiltration, poor differentiation, higher AFP level, appearance of tumor recurrence, and poor postoperative overall and disease-free survivals of HCC patients. Treatment with Ad-ANGPTL4 significantly inhibited the in vivo tumor growth, invasiveness and metastasis by promoting tumoral apoptosis, inhibiting tumoral angiogenesis and motility, and suppressing tumor-favorable microenvironment. Moreover, administration of recombinant ANGPTL4 protein suppressed the motility of HCC cells and altered the secretion profile of cytokines from macrophages. CONCLUSION: ANGPTL4 is a diagnostic and prognostic biomarker for HCC patients and a potential therapeutic agent to suppress HCC growth, angiogenesis and metastasis.

The roles of lipocalin-2 in small-for-size fatty liver graft injury.

OBJECTIVE: To investigate the roles and underlying mechanism of an inflammatory mediator-lipocalin-2 (Lcn2) in small-for-size fatty graft liver injury. BACKGROUND: Understanding of the distinct mechanism regulating small-for-size fatty liver graft injury will be crucial to prevent marginal graft failure during living donor liver transplantation (LDLT). METHODS: The roles of Lcn2 in small fatty graft injury were investigated in orthotopic liver transplantation model rats, human LDLT samples, an in vitro simulated ischemia-reperfusion (IR) model, and a hepatic ischemic reperfusion plus major hepatectomy (IR + H) model in mice. RESULTS: Our result showed that Lcn2 was significantly upregulated together with elevation of chemokine (C-X-C motif) ligand 10 (CXCL10) and activation/infiltration of intragraft macrophages after liver transplantation using small-for-size fatty liver graft compared with that of using small-for-size normal liver graft. Intragraft and plasma levels of Lcn2 were intensified in patients who underwent transplantation with small-for-size fatty graft after LDLT. Lcn2 and CXCL10 were expressed higher in fatty hepatocytes after the simulated IR injury compared with normal hepatocytes. Overexpression of Lcn2 significantly deteriorated IR + H-induced hepatic injury in correlation with upregulation of CXCL10 and augmentation of infiltrated macrophages. On the contrary, hepatic injury of small fatty liver remnant after IR + H operation was attenuated in the Lcn-2 mice because of suppression of CXCL10 expression and diminishment of macrophage infiltration. CONCLUSIONS: Lcn2 is an important regulator in small-for-size fatty liver graft injury and targeting Lcn2 may be feasible for preventing marginal graft failure in LDLT.

Suppression of tumorigenesis and metastasis of hepatocellular carcinoma by shRNA interference targeting on homeoprotein Six1.

We previously demonstrated that the overexpression of homeoprotein Six1 in hepatocellular carcinoma (HCC) patients is associated with venous infiltration, advanced pathologic tumor metastasis (pTNM) stage and poor overall survival rate (Ng et al. Br J Cancer 2006;95:1050-5). In this study, short hairpin RNA (shRNA) interference approach was used to suppress the expression of Six1 in a metastatic HCC cell line MHCC97L. Stable transfectant MHCC97L-shSix1 carrying Six1-specific shRNA plasmid was established to downregulate Six1 expression to about 40% when compared with MHCC97L-Control. In vitro functional assays demonstrated that the growth rate and proliferation ability of MHCC97L-shSix1 cells were markedly decreased. Moreover, significant decrease of cell motility and invasiveness were observed in MHCC97L-shSix1 cells. Data from in vivo xenograft tumorigenesis model demonstrated that the size of tumor in MHCC97L-shSix1 group was dramatically reduced. Experimental and spontaneous metastasis models indicated that targeting Six1 suppression noticeably reduced the pulmonary metastasis in MHCC97L-shSix1 group. To identify Six1-regulated targets, cDNA microarray was employed to compare the expression profiles of MHCC97L-Control and MHCC97L-shSix1 cells. Twenty-eight downregulated and 24 upregulated genes with known functions were identified in MHCC97L-shSix1. The functions of these target genes are involved in diverse biological activities. Our data suggest that Six1 may be involved in regulation of proliferation and invasiveness of HCC; thus targeting suppression of Six1 is a viable option for treating HCC patients.

Molecular signature linked to acute phase injury and tumor invasiveness in small-for-size liver grafts.

OBJECTIVE: We aimed to explore the precise molecular mechanism of early and invasive tumor growth in a small-for-size graft after liver transplantation and to identify the distinct molecular signature linked to acute-phase injury and late-phase tumor invasiveness. SUMMARY BACKGROUND DATA: Acute phase small-for-size liver graft injury plays an important role in tumor recurrence after liver transplantation. For prevention of such recurrence, understanding of its underlying mechanism will be important in developing novel therapeutic strategies. METHODS: An orthotopic rat liver transplantation model was applied using whole grafts and small-for-size (50%) grafts. The recipients were injected with hepatoma cell lines via the portal vein to mimic tumor recurrence after liver transplantation. Tumor invasive properties were compared between the tumor developed from small and whole graft. Gene signatures of acute phase graft injury (days 1 and 3) and late phase tumor recurrence (days 14 and 21) were screened using cDNA microarray analysis and further confirmed by quantitative RT-PCR. The potential gene candidate CXCL10 was singled out for further functional studies to investigate its role in tumor progression. RESULTS: A number of genes linked to inflammatory responses and tumor invasiveness were found over-expressed in small-for-size liver grafts and/or tumors developed in small liver grafts by cDNA microarray screening. Real-time RT-PCR also confirmed that the gene CXCL10 was over-expressed not only in small-for-size graft at the early phase, but also in tumor from small-for-size graft at the late phase after liver transplantation. In vitro functional studies further confirmed that CXCL10 promoted tumor-invasion-related properties and tumor-associated macrophage activation. CONCLUSION: CXCL10 over-expression, the distinct gene signature of acute-phase graft injury and tumor invasiveness in small-for-size liver grafts, may contribute to early tumor recurrence after liver transplantation. CXCL10 and its downstream signals may be potential therapeutic targets in the prevention of tumor recurrence after liver transplantation using small-for-size graft.

Intranasal dexmedetomidine combined with local anesthesia for conscious sedation during breast lumpectomy: A prospective randomized trial.

Breast lumpectomy is usually performed under general or local anesthesia. To the best of our knowledge, whether conscious sedation with intranasal dexmedetomidine and local anesthesia is an effective anesthetic technique has not been studied. Thus, the present study aimed to investigate the effectiveness of conscious sedation with intranasal dexmedetomidine combined with local anesthesia in breast lumpectomy, and to identify its optimal dose. A prospective randomized, double-blinded, placebo-controlled, single-center study was designed, and patients undergoing breast lumpectomies were recruited based on the inclusion and exclusion criteria. All patients were randomly allocated to four groups: i) Local anesthesia with 0.9% intranasal saline (placebo); local anesthesia with ii) 1 µg.kg-1; iii) 1.5 µg.kg-1; or iv) 2 µg.kg-1 intranasal dexmedetomidine. The sedation status, pain relief, vital signs, adverse events, and satisfaction of patient and surgeon were recorded. Patients in the three dexmedetomidine groups were significantly more sedated and experienced less pain compared with the placebo group 45 min after intranasal dexmedetomidine administration and during 30 min in the post-anesthesia care unit. Patients in the 1.5 µg.kg-1 group were more sedated compared with the 1 µg.kg-1 group (without reaching statistical significance), whereas the 1.5 µg.kg-1 group exhibited a similar level of sedation 45 min after intranasal dexmedetomidine administration compared with the 2 µg.kg-1 group. In addition, patients in the 1 and 1.5 µg.kg-1 group experienced no adverse hemodynamic effects. Patient and surgeon satisfaction were greater in the 1.5 µg.kg-1 group compared with the 1 and 2 µg.kg-1 groups. Taken together, the results of the present study suggested that conscious sedation with intranasal dexmedetomidine and local anesthesia may be an effective anesthetic for breast lumpectomy surgery, and that the optimal dose for intranasal dexmedetomidine administration may be 1.5 µg.kg-1, as it resulted in good sedation and patient satisfaction without adverse effects.

Identification of transmembrane protein 98 as a novel chemoresistance-conferring gene in hepatocellular carcinoma.

Chemoresistance is one of the major obstacles in systemic chemotherapy and targeted therapy for patients with advanced hepatocellular carcinoma. To identify novel chemoresistance-associated targets in hepatocellular carcinoma, chemoresistant hepatocellular carcinoma cell lines were established. By comparing the global gene expression profiles between chemoresistant and chemosensitive cell lines, eight novel chemoresistance-associated genes were identified to be significantly associated with the commonly augmented chemoresistance of hepatocellular carcinoma cells. One upregulated candidate named transmembrane protein 98 (TMEM98) was found to be overexpressed in 80 of 118 (67.80%) of patients with hepatocellular carcinoma. TMEM98 mRNA in tumor tissues was significantly higher than nontumor tissues of patients with hepatocellular carcinoma (P < 0.0001). Upregulation of TMEM98 was significantly correlated with advanced tumor stage (P = 0.048), high incidence of early tumor recurrence (P = 0.005), poor overall survival (P = 0.029), and poor disease-free survival (P = 0.011) of patients with hepatocellular carcinoma after hepatectomy. Importantly, upregulation of TMEM98 mRNA in patients with hepatocellular carcinoma who received transarterial chemoembolization (TACE) treatment was significantly higher than in patients without TACE treatment (P = 0.046). Moreover, patients with poor response to TACE treatment had higher degree of TMEM98 upregulation than the responsive patients. In vitro and in vivo studies showed that suppression of TMEM98 in chemoresistant hepatocellular carcinoma cells restored their chemosensitivity, while forced overexpression of TMEM98 enhanced their chemoresistance. The mechanism of TMEM98 in conferring chemoresistance of hepatocellular carcinoma might be possibly through activation of the AKT pathway and deactivation of p53. In conclusion, we identified a panel of novel common chemoresistance-associated genes and demonstrated that TMEM98 is a chemoresistance-conferring gene in hepatocellular carcinoma.

A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is highly malignant and metastatic. Currently, there is no effective chemotherapy for patients with advanced HCC leading to an urgent need to seek for novel therapeutic options. We aimed to investigate the effect of a garlic derivative, S-allylcysteine (SAC), on the proliferation and metastasis of HCC. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro experiments including MTT, colony-forming, wound-healing, invasion, apoptosis and cell cycle assays were performed to examine the anti-proliferative and anti-metastatic effects of SAC on a metastatic HCC cell line MHCC97L. The therapeutic values of SAC single and combined with cisplatin treatments were examined in an in vivo orthotopic xenograft liver tumor model. The result showed that the proliferation rate and colony-forming abilities of MHCC97L cells were suppressed by SAC together with significant suppression of the expressions of proliferation markers, Ki-67 and proliferating cell nuclear antigen (PCNA). Moreover, SAC hindered the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. Furthermore, SAC significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1. In vivo xenograft liver tumor model demonstrated that SAC single or combined with cisplatin treatment inhibited the progression and metastasis of HCC tumor. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the anti-proliferative and anti-metastatic effects of SAC on HCC cells and suggest that SAC may be a potential therapeutic agent for the treatment of HCC patients.