Yuen Yuen Ma

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.

FTY720 Suppresses Liver Tumor Metastasis by Reducing the Population of Circulating Endothelial Progenitor Cells

Background Surgical procedures such as liver resection and liver transplantation are the first-line treatments for hepatocellular carcinoma (HCC) patients. However, the high incidence of tumor recurrence and metastasis after liver surgery remains a major problem. Recent studies have shown that hepatic ischemia-reperfusion (I/R) injury and endothelial progenitor cells (EPCs) contribute to tumor growth and metastasis. We aim to investigate the mechanism of FTY720, which was originally applied as an immunomodulator, on suppression of liver tumor metastasis after liver resection and partial hepatic I/R injury. Methodology/Principal Findings An orthotopic liver tumor model in Buffalo rat was established using the hepatocellular carcinoma cell line McA-RH7777. Two weeks after orthotopic liver tumor implantation, the rats underwent liver resection for tumor-bearing lobe and partial hepatic I/R injury. FTY720 (2 mg/kg) was administered through the inferior caval vein before and after I/R injury. Blood samples were taken at days 0, 1, 3, 7, 14, 21 and 28 for detection of circulating EPCs (CD133+CD34+). Our results showed that intrahepatic and lung metastases were significantly inhibited together with less tumor angiogenesis by FTY720 treatment. The number of circulating EPCs was also significantly decreased by FTY720 treatment from day 7 to day 28. Hepatic gene expressions of CXCL10, VEGF, CXCR3, CXCR4 induced by hepatic I/R injury were down-regulated in the treatment group. Conclusions/Significance FTY720 suppressed liver tumor metastasis after liver resection marred by hepatic I/R injury in a rat liver tumor model by attenuating hepatic I/R injury and reducing circulating EPCs.

Regulatory B cells accelerate hepatocellular carcinoma progression via CD40/CD154 signaling pathway

Human hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a poor prognosis of limited survival. The role of regulatory B cell (Breg), a new important B cell subset, in HCC progression remains unclear. We firstly found that the percentage of B cells at tumor margin was significantly higher than that in tumor and non-tumor regions. Especially, increased intrahepatic B cells at tumor margin were positively associated with tumor invasive features and more tumor recurrence. Besides, HCC patients had a significantly higher percentage of circulating Bregs than healthy people. Increased circulating Bregs were correlated with advanced tumor staging, tumor multiplicity and venous infiltration. Next, we firstly revealed that human Bregs promoted HCC tumor growth independent of Tregs in SCID mice. The migration of Bregs from blood into tumor was also confirmed in mice. Finally, we further explored the molecular mechanism of Bregs promoting proliferation and migration of HCC cells in vitro. Bregs promoted HCC growth and invasiveness by directly interacting with liver cancer cells through the CD40/CD154 signaling pathway.

CXCL10/CXCR3 signaling mobilized-regulatory T cells promote liver tumor recurrence after transplantation

BACKGROUND & AIMS Liver graft injury and tumor recurrence are the major challenges of liver transplantation for the patients with hepatocellular carcinoma (HCC). Here, we aimed to explore the role and mechanism of liver graft injury mobilizing regulatory T cells (Tregs), which lead to late phase tumor recurrence after liver transplantation. METHODS The correlation among tumor recurrence, liver graft injury and Tregs mobilization were studied in 257 liver transplant recipients with HCC and orthotopic rat liver transplantation models. The direct roles of CXCL10/CXCR3 signaling on Tregs mobilization and tumor recurrence were investigated in CXCL10-/- and CXCR3-/- mice models with hepatic IR injury. RESULTS Clinically, patients received the graft with graft weight ratio (GWR) <60% had higher HCC recurrence after liver transplantation than the recipients with GWR ⩾60% graft. More circulating Tregs and higher intragraft TLR4/CXCL10/CXCR3 levels were detected in recipients with GWR <60% graft. These results were further validated in rat transplantation model. Foxp3+ cells and expressions of TLR4, CXCL10, TGFβ, CTLA-4 and CD274 were increased in rat liver tumor tissues from small-for-size graft group. In mouse model, the mobilization and recruitment of Tregs were decreased in TLR4-/-, CXCL10-/- and CXCR3-/- mice compared to wild-type mice. Moreover, less CXCR3+ Tregs were recruited into liver in CXCL10-/- mice after hepatic IR injury. The knockout of CXCL10 and depletion of Tregs inhibited tumor recurrence after hepatic IR injury. CONCLUSION CXCL10/CXCR3 signaling upregulated at liver graft injury directly induced the mobilization and intragraft recruitment of Tregs, which further promoted HCC recurrence after transplantation. LAY SUMMARY There were positive correlation among tumor recurrence, circulating Tregs and liver graft injury after human transplantation for HCC patients. The knockout of CXCL10 decreased hepatic recruitment of CXCR3+ Tregs and late phase tumor recurrence after hepatic IR injury.

The inhibition of aldose reductase attenuates hepatic ischemia-reperfusion injury through reducing inflammatory response

Objective:We aim to investigate the role of aldose reductase (AR) in hepatic ischemia-reperfusion injury (IRI) of normal and fatty livers and to explore the underlying mechanisms. Background:Hepatic IRI is a typical inflammatory response during liver surgery. It contributes to liver graft failure or nonfunction after transplantation. Increasing evidence implicates that AR plays a key role in a number of inflammatory diseases. However, the role of AR in hepatic IRI is still unknown. Methods:Intragraft AR expression profile and the association with liver graft injury were investigated in both human and rat liver transplantation using normal or fatty graft. The direct role of AR in hepatic IRI was studied in the AR knockout mice IRI model with or without fatty liver. They were further validated by the simulated IRI in vitro model using fatty LO2 cells with or without AR inhibitor zopolrestat and primary peritoneal macrophages isolated from AR knockout and wild-type mice. Gene expression of inflammatory cytokines/chemokines, the infiltration of macrophages/neutrophils, and NF-&kgr;B pathway activation were compared among different groups. Results:AR was overexpressed in liver graft after human and rat liver transplantation and correlated with consequent liver injuries. The knockout of AR significantly attenuated hepatic sinusoidal damage and apoptosis in both normal and fatty livers after IRI. The expression of proinflammatory cytokines/chemokines and neutrophil chemoattractants, infiltration of macrophage and neutrophil, and activation of inflammation-associated NF-&kgr;B and JNK pathway were downregulated in AR knockout mice. Furthermore, the inhibition of AR effectively suppressed macrophage migration and decreased lipopolysaccharide (LPS)-induced production of proinflammatory cytokines/chemokines in isolated macrophages. Conclusions:The deficiency of AR attenuated hepatic IRI in both normal and fatty livers by reducing liver inflammatory responses.

Early-phase circulating miRNAs predict tumor recurrence and survival of hepatocellular carcinoma patients after liver transplantation

Post-liver transplantation tumor recurrence is a major challenge for hepatocellular carcinoma (HCC) recipients. We aimed to identify early-phase circulating microRNAs after liver transplantation for predicting tumor recurrence and survival of HCC recipients. Circulating microRNA profiles at early-phase (2-hour after portal vein reperfusion) after liver transplantation were compared between HCC recipients with (n=4) and without tumor recurrence (n=8) by microarray analyses. Candidate microRNAs were validated in 62 HCC recipients by quantitative RT-PCR. The prognostic values of microRNAs for tumor recurrence and survival were examined. Simulated in vitro ischemia-reperfusion injury models were employed to characterize the possible mechanism of up-regulation of circulating microRNAs. Our results showed that up-regulation of circulating miR-148a, miR-1246 or miR-1290 at early-phase was significantly associated with HCC recurrence after liver transplantation. Among them, miR-148a (p=0.030) and miR-1246 (p=0.009) were significant predictors of HCC recurrence. MiR-1246 was an independent predictor of overall (p=0.023) and disease-free survival (p=0.020) of HCC recipients. The level of early-phase circulating miR-1246 was positively correlated with serum AST and ALT levels in HCC recipients after liver transplantation. The expression of hepatic miR-1246 was positively correlated with TNFα mRNA. In vitro experiments indicated that injury-induced activation and differentiation of macrophages significantly elevated the expression and secretion of miR-1246. In conclusion, early-phase circulating miR-1246 is an indicator of hepatic injury and a novel prognostic biomarker for tumor recurrence and survival of HCC recipients after liver transplantation.

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. Mol Cancer Ther; 13(5); 1285–97. ©2014 AACR.

NLRP3 inflammasome induced liver graft injury through activation of telomere‐independent RAP1/KC axis

Acute‐phase inflammation plays a critical role in liver graft injury. Inflammasomes, multi‐molecular complexes in the cytoplasm, are responsible for initiating inflammation. Here, we aimed to explore the role of inflammasomes in liver graft injury and further to investigate the regulatory mechanism. In a clinical liver transplant cohort, we found that intragraft expression of nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3 (NLRP3) inflammasomes was significantly up‐regulated post‐transplantation. Importantly, overexpression of NLRP3 was strongly associated with poor liver function characterized by high levels of ALT, AST, and urea, as well as neutrophil infiltration after transplantation. The significant correlation between NLRP3 and IL‐1β mRNA levels led us to focus on one of the associated upstream regulators, telomere‐independent repressor activator protein 1 (RAP1), which was further proved to be co‐localized with NLRP3 in neutrophils. In the liver of a mouse model (hepatic ischaemia/reperfusion and hepatectomy model) and isolated neutrophils from RAP1−/− mice, the expression levels of NLRP3 and keratinocyte chemoattractant (KC) were significantly down‐regulated in contrast to those in wild types. The levels of ALT and AST, as well as the neutrophil infiltration, were also decreased by RAP1 deficiency. In our clinical validation, intragraft KC expression was associated with NLRP3 and co‐localized with RAP1 in neutrophils. Furthermore, NLRP3 inflammasomes were up‐regulated by recombinant KC in the isolated neutrophils and liver of the mouse model. Our data demonstrated that NLRP3 inflammasomes, activated by the RAP1/KC axis, played a critical role in initiating inflammation during the early stage of liver graft injury. Targeting RAP1/KC/NLRP3 inflammasomes may offer a new therapeutic strategy against liver graft injury. Copyright

The clinical significance and potential therapeutic role of GPx3 in tumor recurrence after liver transplantation

Background and Aims: Our previous study showed that small-for-size liver graft may provide favorable micro-environment for tumor growth. GPx3, an anti-oxidant, not only attenuates oxidative stress, but also suppresses liver tumor growth in our recent study. Here, we aimed to characterize the clinical significance and explore the functional role of GPx3 in HCC recurrence after liver transplantation. Methods: To explore the association between GPx3 expression and HCC invasiveness, a rat orthotopic liver transplantation model with tumor development was established. To investigate the clinical relevance of GPx3, 105 HCC patients who underwent liver transplantation were recruited. The suppressive role of GPx3 in HCC cells was studied using wound healing, Matrigel invasion assay and lung metastasis model. The real-time intravital imaging system was applied to directly visualize the tumor cells invasion in a living animal. The underlying mechanism was further explored. Results: GPx3 was identified as a down-regulated protein in small-for-size liver graft and significantly associated with invasive phenotype of tumor growth in a rat model. Plasma GPx3 was significantly lower in small-for-size graft group post-transplantation (day1: 33 vs 1147; day3: 3209 vs 4459; day7: 303 vs 2506; mU/mL, P<0.05) in rat model. Clinically, the plasma GPx3 was significantly lower in the recipients with HCC recurrence post-transplantation (day1: 4.16 vs 8.99 µg/mL, P<0.001; day7: 3.86 vs 9.99 µg/mL, P<0.001). Furthermore, lower plasma GPx3 was identified as an independent predictor (HR=4.528, P=0.046) for poor overall survival post-transplantation. Over-expression of GPx3 significantly suppressed migration, invasiveness and metastasis of HCC cells. Real-time intravital imaging showed that GPx3 significantly suppressed HCC invasiveness in a live animal. GPx3 suppressed the tumor invasiveness through inhibition of JNK-cJun-MMP2 pathway. Conclusion: GPx3 may possess prognostic and therapeutic value for HCC patients after liver transplantation.

A hemoglobin-based oxygen carrier sensitized Cisplatin based chemotherapy in hepatocellular carcinoma

Background and Objective Our previous study showed that liver graft injury not only promotes tumor recurrence, but also induces chemoresistance in recurrent HCC after liver transplantation. Recently, we found that the hemoglobin-based oxygen carrier“YQ23” significantly ameliorates hepatic IR injury and prevent tumor recurrence. Here, we intended to explore the novel therapeutic strategy using oxygen carrier “YQ23”to sensitize chemotherapy in HCC. Methods To investigate the role of YQ23 combined with Cisplatin, the proliferation of HCC cells was examined under combined treatment by MTT and colony formation. To explore the effect of YQ23 on sensitization of Cisplatin based chemotherapy, the orthotopic liver cancer model was established. To characterize the delivery of YQ23 in tumor tissue, the intravital imaging system was applied for longitudinal observation in ectopic liver cancer model. The distribution of YQ23 was examined by IVIS spectrum. Results YQ23 significantly suppressed the proliferation of HCC cells under Cisplatin treatment in a dose and time dependent manner. Moreover, YQ23 administration significantly sensitized Cisplatin based chemotherapy in orthotopic liver cancer model. Down-regulation of DHFR may be one of the reasons for YQ23 sensitizing Cisplatin based chemotherapy. Real-time intravital imaging showed that YQ23 accumulated in the tumor tissue and maintained as long as 3 days in ectopic liver cancer model. The IVIS spectrum examination showed that YQ23 distributed mainly at liver and bladder within the first 36 hours after administration in orthotopic liver cancer model. Conclusion YQ23 treatment may be a potential therapeutic strategy to sensitize chemotherapy in HCC.