Apolipoprotein A-1 Accelerated Liver Regeneration Through Regulating Autophagy Via AMPK-ULK1 Pathway.

BACKGROUND & AIMS: Apolipoprotein A-1 (ApoA-1), the main apolipoprotein of high-density lipoprotein, has been well studied in the area of lipid metabolism and cardiovascular diseases. In this project, we clarify the function and mechanism of ApoA-1 in liver regeneration. METHODS: Seventy percent of partial hepatectomy was applied in male ApoA-1 knockout mice and wild-type mice to investigate the effects of ApoA-1 on liver regeneration. D-4F (ApoA-1 mimetic peptide), autophagy activator, and AMPK activator were used to explore the mechanism of ApoA-1 on liver regeneration. RESULTS: We demonstrated that ApoA-1 levels were highly expressed during the early stage of liver regeneration. ApoA-1 deficiency greatly impaired liver regeneration after hepatectomy. Meanwhile, we found that ApoA-1 deficiency inhibited autophagy during liver regeneration. The activation of autophagy protected against ApoA-1 deficiency in inhibiting liver regeneration. Furthermore, ApoA-1 deficiency impaired autophagy through AMPK-ULK1 pathway, and AMPK activation significantly improved liver regeneration. The administration of D-4F could accelerated liver regeneration after hepatectomy. CONCLUSIONS: These findings suggested that ApoA-1 played an essential role in liver regeneration through promoting autophagy in hepatocytes via AMPK-ULK1 pathway. Our findings enrich the understanding of the underlying mechanism of liver regeneration and provide a potential therapeutic strategy for liver injury.

Apolipoprotein A-1 protected hepatic ischaemia-reperfusion injury through suppressing macrophage pyroptosis via TLR4-NF-κB pathway.

BACKGROUND AND AIMS: Apolipoprotein A-1 (ApoA-1), the major apolipoprotein of high-density lipoprotein, plays anti-atherogenic role in cardiovascular diseases and exerts anti-inflammation effect in various inflammatory and infectious diseases. However, the role and mechanism of ApoA-1 in hepatic ischaemia-reperfusion (I/R) injury is unknown. METHODS: In this study, we measured ApoA-1 expression in human liver grafts after transplantation. Mice partial hepatic I/R injury model was made in ApoA-1 knockout mice, ApoA-1 mimetic peptide D-4F treatment mice and corresponding control mice to examine the effect of ApoA-1 on liver damage, inflammation response and cell death. Primary hepatocytes and macrophages were isolated for in vitro study. RESULTS: The results showed that ApoA-1 expression was down-regulated in human liver grafts after transplantation and mice livers subjected to hepatic I/R injury. ApoA-1 deficiency aggravated liver damage and inflammation response induced by hepatic I/R injury. Interestingly, we found that ApoA-1 deficiency increased pyroptosis instead of apoptosis during acute phase of hepatic I/R injury, which mainly occurred in macrophages rather than hepatocytes. The inhibition of pyroptosis compensated for the adverse impact of ApoA-1 deficiency. Furthermore, the up-regulated pyroptosis process was testified to be mediated by ApoA-1 through TLR4-NF-κB pathway and TLR4 inhibition significantly improved hepatic I/R injury. In addition, we confirmed that D-4F ameliorated hepatic I/R injury. CONCLUSIONS: Our study has identified the protective role of ApoA-1 in hepatic I/R injury through inhibiting pyroptosis in macrophages via TLR4-NF-κB pathway. The effect of ApoA-1 may provide a novel therapeutic approach for hepatic I/R injury.

BUB1B promotes extrahepatic cholangiocarcinoma progression via JNK/c-Jun pathways.

Currently, the controversy regarding the expression profile and function of BUB1B in different malignancies still exist. In this project, we aimed to explore the role and molecular mechanism of BUB1B in the progression of extrahepatic cholangiocarcinoma (ECC). The expression levels of BUB1B in human ECC were evaluated by immunohistochemistry, western blot, and real-time PCR. The role and mechanism of BUB1B in CCA cell proliferation and invasion were investigated in both in vitro and in vivo functional studies. To indicate the clinical significance, a tissue microarray was performed on 113 ECC patients, followed by univariate and multivariate analyses. The expression of BUB1B was increased in both human CCA tissues and CCA cells. Results from loss-of-function and gain-of-function experiments suggested that the inhibition of BUB1B decreased the proliferation and invasiveness of CCA cells in vitro and in vivo, while overexpression of BUB1B achieved the opposite effect. Furthermore, the activation of c-Jun N-terminal kinase-c-Jun (JNK)-c-Jun pathway was regulated by BUB1B. BUB1B regulated the proliferation and invasiveness of CAA cells in a JNK-c-Jun-dependent manner. Clinically, ECC patients with BUB1B high expression had worse overall survival and recurrence-free survival than those with BUB1B low expression. Multivariate analysis identified that BUB1B was an independent predictor for postoperative recurrence and overall survival of ECC patients. In conclusion, BUB1B promoted ECC progression via JNK/c-Jun pathways. These findings suggested that BUB1B could be a potential therapeutic target and a biomarker for predicting prognosis for ECC patients.

Clinical significance and functional role of transmembrane protein 47 (TMEM47) in chemoresistance of hepatocellular carcinoma.

Chemoresistance is the main cause of chemotherapy failure in patients with hepatocellular carcinoma (HCC). The gene encoding transmembrane protein 47 (TMEM47) was previously identified to be significantly upregulated in HCC cell lines with acquired chemoresistance. The aim of the present study was to characterize the clinical significance and function of TMEM47 in HCC chemoresistance. The results demonstrated that the TMEM47 expression levels in the tumors of patients not responding to cisplatin­based transarterial chemoembolization (TACE) treatment was significantly higher compared with those in patients who responded to TACE treatment. Moreover, analyses from clinical samples and HCC cell lines indicated that TMEM47 expression may be upregulated in HCC in response to cisplatin treatment. Furthermore, the TMEM47 mRNA expression levels were positively correlated with the degree of cisplatin resistance of HCC cells. Overexpression of TMEM47 in HCC cells significantly promoted cisplatin resistance. The present study also demonstrated that targeted inhibition of TMEM47 could significantly reduce cisplatin resistance of cisplatin­resistant HCC cells via enhancing caspase­mediated apoptosis. In addition, targeted inhibition of TMEM47 enhanced the sensitivity of cisplatin­resistant cells to cisplatin via suppressing cisplatin­induced activation of the genes involved in drug efflux and metabolism. The present study also validated that TMEM47 expression was significantly correlated with multidrug resistance­associated protein 1 in patients with HCC who received TACE treatment. In conclusion, the findings of the present study demonstrated that TMEM47 may be a useful biomarker for predicting the response to chemotherapy and a potential therapeutic target for overcoming HCC chemoresistance.

FTY720 Suppresses Liver Tumor Growth and Metastasis by Reducing Circulating Regulating T Cells and Enhancing the Anti-Tumor Effect of Rapamycin.

BACKGROUND: In this study, we aimed to study the effect of FTY720 treatment in reducing circulating Tregs level and then suppressing liver tumor metastasis after hepatectomy and I/R injury in animal models. Furthermore, we also investigated the synergistic anti-tumor effect of FTY720 combined with rapamycin on hepatocellular carcinoma. METHODS: The effect of FTY720 on suppressing Tregs mobilization and tumor metastasis after hepatectomy was investigated in an orthotopic liver tumor rat model with hepatectomy and hepatic ischemia/reperfusion (I/R) injury. The synergistic anti-tumor effect of FTY720 combined with rapamycin was further explored both in in vitro functional study and in orthotopic liver tumor mouse model. RESULTS: In rat model, hepatic I/R promoted tumor metastasis and increased circulating Tregs after hepatectomy. The treatment of FTY720 reduced liver tumor metastasis and the number of circulating Tregs. Furthermore, FTY720 enhanced the anti-tumor capacity of rapamycin by inhibiting tumor cell proliferation and migration in vitro and reducing tumor growth in vivo through suppressing hepatic stellate cell activation and tumor angiogenesis. CONCLUSION: FTY720 suppressed liver tumor growth and metastasis by reducing the population of circulating Tregs and enhancing the anti-tumor effect of rapamycin. It was suggested that FTY720 single or combined with rapamycin might provide novel insight for suppressing tumor growth and metastasis for HCC patients.

MiR-30a-5p promotes cholangiocarcinoma cell proliferation through targeting SOCS3.

Background: MicroRNAs (miRNAs) play important roles in the occurrence and development of cancers. In this project, we aimed to explore the role and molecular mechanism of mir-30a-5p in cholangiocarcinoma (CCA). Materials and Methods: The expression profile and clinical significance of miR-30a-5p in CCA patients were investigated in 31 ICC and 52 ECC patients respectively. The role and mechanism of miR-30a-5p in CCA cells were investigated by up-regulating and inhibiting miR-30a-5p expression in vitro functional study. Results: The expression of miR-30a-5p was increased in both CCA tissues and cells. The inhibition of miR-30a-5p decreased cell proliferation and induced cell apoptosis while overexpression of miR-30a-5p achieved the opposite effect. Furthermore, SOCS3 was down-regulated in ICC and ECC tissues and negatively regulated by miR-30a-5p. Dual-luciferase reporter assay revealed that co-transfection of miR-30a-5p significantly inhibited the activity of firefly luciferase reporter carrying the wild-type 3'UTR of SOCS3. The inhibition of SOCS3 could largely rescue the inhibitory effect of miR-30a-5p inhibition on CCA cells proliferation. In clinical, up-regulated miR-30a-5p expression was correlated with large tumor size in both ICC and ECC cohorts. Conclusions: miR-30a-5p promoted CCA cells proliferation through targeting SOCS3. These findings suggested that miR-30a-5p could be a potential therapeutic target.

Surgical Strategies for the Treatment of Bismuth Type I and II Hilar Cholangiocarcinoma: Bile Duct Resection with or Without Hepatectomy?

BACKGROUND: The role of hepatic resection in the treatment of type I and II hilar cholangiocarcinoma (HCCA) remains controversial. In the present study, we aimed to identify whether hepatic resection was necessary for type I and II HCCA. METHODS: A total of 23 patients classified as type I and II HCCA undergoing surgical resection were included in this study. The patients were divided into two groups: bile duct resection (BDR) group (n = 15) and hepatic resection (HR) group (n = 8). Systematic review and meta-analysis were performed to compare the R0 resection and long-term survival between BDR and HR for Bismuth type I and II HCCA. A total of 7 studies with 260 cases were included in this meta-analysis. RESULTS: In our cohort, the R0 resection rate was 73.3% in BDR group and 87.5% in HR group. The HR group had a higher number of postoperative complications than the BDR group (P = 0.002). There was no difference in long-term survival (P = 0.544) and recurrence (P = 0.846) between BDR and HR in Bismuth type I and II HCCA. The meta-analysis showed that HR was associated with better R0 resection rate (RR 4.45, 95% CI 2.34-8.48) and overall survival (HR 2.15, 95% CI 1.34-3.44) compared with BDR group. There was no publication bias and undue influence of any single study. CONCLUSIONS: The meta-analysis showed that HR was associated with better R0 resection rate and overall survival compared with BDR for type I and II HCCA patients. More aggressive surgical strategies should be increasingly considered for the treatment of type I and II HCCA patients.

The Inhibition of Aldose Reductase Accelerates Liver Regeneration through Regulating Energy Metabolism.

OBJECTIVES: Our previous study showed that aldose reductase (AR) played key roles in fatty liver ischemia-reperfusion (IR) injury by regulating inflammatory response and energy metabolism. Here, we aim to investigate the role and mechanism of AR in the regeneration of normal and fatty livers after liver surgery. METHODS: The association of AR expression with liver regeneration was studied in the rat small-for-size liver transplantation model and the mice major hepatectomy and hepatic IR injury model with or without fatty change. The direct role and mechanism of AR in liver regeneration was explored in the AR knockout mouse model. RESULTS: Delayed regeneration was detected in fatty liver after liver surgery in both rat and mouse models. Furthermore, the expression of AR was increased in liver after liver surgery, especially in fatty liver. In a functional study, the knockout of AR promoted liver regeneration at day 2 after major hepatectomy and IR injury. Compared to wild-type groups, the expressions of cyclins were increased in normal and fatty livers of AR knockout mice. AR inhibition increased the expressions of PPAR-α and PPAR-γ in both normal liver and fatty liver groups after major hepatectomy and IR injury. In addition, the knockout of AR promoted the expressions of SDHB, AMPK, SIRT1, and PGC1-α and PPAR. CONCLUSIONS: The knockout of AR promoted the regeneration of normal and fatty livers through regulating energy metabolism. AR may be a new potential therapeutic target to accelerate liver regeneration after surgery.

Preoperative Bilirubin Level Predicts Overall Survival and Tumor Recurrence After Resection for Perihilar Cholangiocarcinoma Patients.

OBJECTIVE: Currently, the correlation between preoperative bilirubin level and overall survival (OS) remains poorly defined in respectable perihilar cholangiocarcinoma (CC). The objectives of the current study were to evaluate the outcomes of perihilar CC after resection and then to analyze factors influencing curative resection, tumor recurrence and OS. METHODS: 115 patients with perihilar CC underwent surgical resection were retrospectively analyzed based on clinic characteristics, operative details, tumor recurrence and long-term survival data. RESULTS: The 1-, 3-, and 5-year OS rates after resection were 75.9%, 36.5%, 21.7%, whereas the corresponding tumor recurrence rates were 29.6%, 70.8%, 85.3%, respectively. Preoperative bilirubin level combined with liver resection, resection margin, vascular invasion and perineural invasion, lymph node metastasis and TNM stage were found to be correlated with OS and tumor recurrence. Multivariate analysis showed that preoperative bilirubin level together with resection margin, perineural invasion, and TNM stage were independent predictors of OS and tumor recurrence. Furthermore, preoperative bilirubin level was related with R0 resection, lymph node metastasis, TNM stage and postoperative liver function recovery. CONCLUSION: Preoperative bilirubin level may effectively reflect the severity of perihilar CC and predict the OS and tumor recurrence after resection for perihilar CC patients.

ApoA-1 accelerates regeneration of small-for-size fatty liver graft after transplantation.

OBJECTIVES: Apolipoprotein A-1 (ApoA-1) is involved in regulating both lipid and energy metabolism, which may play important roles in liver regeneration, especially for the liver with steatosis. We here intended to investigate the role of ApoA-1 in regeneration of small-for-size fatty liver graft and to explore the underlying mechanism. METHODS: The association of ApoA-1 expression with liver regeneration was studied in rat liver transplantation models using small-for-size normal graft or small-for-size fatty graft. The direct role of ApoA-1 in liver regeneration was studied in mouse hepatectomy model in vivo and hepatocytes in vitro. RESULTS: Compared to small-for-size normal graft, decreased expression of ApoA-1 associated with delayed regeneration were detected in small-for-size fatty liver graft after transplantation. In functional study, the expression of ApoA-1 was decreased in hepatocytes with steatosis and was inversely associated with the concentration of oleic acid. The ApoA-1 administration effectively attenuated hepatocytes steatosis and accelerated hepatocytes proliferation. In mouse model, ApoA-1 treatment promoted liver regeneration at day 2 after major hepatectomy. In addition, the treatment of ApoA-1 increased the expressions of PGC-1α and its target genes Tfam, Ucp2 and SDHB. CONCLUSIONS: ApoA-1 may accelerate regeneration of small-for-size fatty liver graft at day 2 after transplantation through regulating mitochondrial function. ApoA-1 may be the potential new therapy of promoting liver regeneration.

Glutathione Peroxidase 3 Delivered by hiPSC-MSCs Ameliorated Hepatic IR Injury via Inhibition of Hepatic Senescence.

Background and Aims: Down-regulation of GPx3 accelerated hepatic senescence, which further caused overwhelming inflammation and severe liver graft injury. MSCs derived from human induced pluripotent stem cells (hiPSC-MSCs) have been developed as more efficient delivery vehicle with the property of injury tropism. Here, we aimed to explore the suppressive role of GPx3 in hepatic IR injury using novel delivery system of hiPSC-MSCs. Methods: The mice IR injury model with partial hepatectomy was established. The engineered hiPSC-MSCs delivering GPx3 was constructed. All the mice were segregated into three groups. hiPSC-MSC-GPx3, hiPSC-MSC-pCDH (vector control) or PBS were injected via portal vein after reperfusion. Liver injury was evaluated by histological and serological test. Hepatic apoptosis was detected by Tunel staining and remnant liver regeneration was assessed by Ki67 staining. The role of hepatic senescence in liver graft injury was evaluated in rat orthotopic liver transplantation model. The suppressive effect of GPx3 on hepatic senescence was examined in mice IR injury model and confirmed in vitro. Hepatic senescence was detected by SA-ß-Gal and P16/ink4a staining. Results: GPx3 can be successfully delivered by hiPSC-MSCs into liver tissues. Histological examination showed that hiPSC-MSC-GPx3 treatment significantly ameliorated hepatic IR injury post-operation. Significantly lower LDH (891.43±98.45 mU/mL, P<0.05) and AST (305.77±36.22 IU/L, P<0.01) were observed in hiPSC-MSC-GPx3 group compared with control groups. Less apoptotic hepatocytes were observed and the remnant liver regeneration was more active in hiPSC-MSC-GPx3 group. In rat orthotopic liver transplantation model, more senescent hepatocytes were observed in small-for-size liver graft, in which GPx3 expression was significantly compromised. In mice IR injury model, hiPSC-MSC-GPx3 significantly suppressed hepatic senescence. In addition, rGPx3 inhibited cellular senescence of liver cells in a dose dependent manner. Four candidate genes (CD44, Nox4, IFNG, SERPERINB2) were identified to be responsible for suppressive effect of GPx3 on hepatic senescence. Conclusion: Engineered hiPSC-MSCs delivering GPx3 ameliorated hepatic IR injury via inhibition of hepatic senescence.

Oval Cells Contribute to Fibrogenesis of Marginal Liver Grafts under Stepwise Regulation of Aldose Reductase and Notch Signaling.

Background and Aims: Expanded donor criteria poses increased risk for late phase complications such as fibrosis that lead to graft dysfunction in liver transplantation. There remains a need to elucidate the precise mechanisms of post-transplant liver damage in order to improve the long-term outcomes of marginal liver grafts. In this study, we aimed to examine the role of oval cells in fibrogenic development of marginal liver grafts and explore the underlying mechanisms. Methods: Using an orthotopic rat liver transplantation model and human post-transplant liver biopsy tissues, the dynamics of oval cells in marginal liver grafts was evaluated by the platform integrating immuno-labeling techniques and ultrastructure examination. Underlying mechanisms were further explored in oval cells and an Aldose reductase (AR) knockout mouse model simulating marginal graft injury. Results: We demonstrated that activation of aldose reductase initiated oval cell proliferation in small-for-size fatty grafts during ductular reaction at the early phase after transplantation. These proliferative oval cells subsequently showed prevailing biliary differentiation and exhibited features of mesenchymal transition including dynamically co-expressing epithelial and mesenchymal markers, developing microstructures for extra-cellular matrix degradation (podosomes) or cell migration (filopodia and blebs), and acquiring the capacity in collagen production. Mechanistic studies further indicated that transition of oval cell-derived biliary cells toward mesenchymal phenotype ensued fibrogenesis in marginal grafts under the regulation of notch signaling pathway. Conclusions: Oval cell activation and their subsequent lineage commitment contribute to post-transplant fibrogenesis of small-for-size fatty liver grafts. Interventions targeting oval cell dynamics may serve as potential strategies to refine current clinical management.

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.

The Impact of Liver Graft Injury on Cancer Recurrence Posttransplantation.

Liver transplantation is the most effective treatment for selected patients with hepatocellular carcinoma. However, cancer recurrence, posttransplantation, remains to be the critical issue that affects the long-term outcome of hepatocellular carcinoma recipients. In addition to tumor biology itself, increasing evidence demonstrates that acute-phase liver graft injury is a result of hepatic ischemia reperfusion injury (which is an inevitable consequence during liver transplantation) and may promote cancer recurrence at late phase posttransplantation. The liver grafts from living donors, donors after cardiac death, and steatotic donors have been considered as promising sources of organs for liver transplantation and are associated with high incidence of liver graft injury. The acute-phase liver graft injury will trigger a series of inflammatory cascades, which may not only activate the cell signaling pathways regulating the tumor cell invasion and migration but also mobilize the circulating progenitor and immune cells to facilitate tumor recurrence and metastasis. The injured liver graft may also provide the favorable microenvironment for tumor cell growth, migration, and invasion through the disturbance of microcirculatory barrier function, induction of hypoxia and angiogenesis. This review aims to summarize the latest findings about the role and mechanisms of liver graft injury resulted from hepatic ischemia reperfusion injury on tumor recurrence posttransplantation, both in clinical and animal cohorts.

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-1ß 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 © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

'Obligate' anaerobic Salmonella strain YB1 suppresses liver tumor growth and metastasis in nude mice.

The antitumor properties of bacteria have been demonstrated over the past decades. However, the efficacy is limited and unclear. Furthermore, systemic infection remains a serious concern in bacteria treatment. In this study, the effect of YB1, a rationally designed 'obligate' anaerobic Salmonella typhimurium strain, on liver tumor growth and metastasis in a nude mouse orthotopic liver tumor model was investigated. The orthotopic liver tumor model was established in nude mice using the hepatocellular carcinoma cell line MHCC-97L. Two weeks after orthotopic liver tumor implantation, YB1, SL7207 and saline were respectively administered through the tail vein of the mice. Longitudinal monitoring of tumor growth and metastasis was performed using Xenogen IVIS, and direct measurements of tumor volume were taken 3 weeks after treatment. In vitro, MHCC-97L and PLC cells were incubated with YB1 or SL7207 under anaerobic conditions. YB1 was observed to invade tumor cells and induce tumor cell apoptosis and death. The results revealed that all mice in the YB1 group were alive 3 weeks after YB1 injection while all mice in the SL7207 group died within 11 days of the SL7207 injection. The body weight decreased by ~9% on day 1 after YB1 injection and but subsequently recovered. Liver tumor growth and metastases were significantly inhibited following YB1 treatment. By contrast to the control group, a large number of Gr1-positive cells were detected on days 1 to 21 following YB1 treatment. Furthermore, YB1 also effectively invaded tumor cells and induced tumor cell apoptosis and death. In conclusion, YB1 suppressed liver tumor growth and metastasis in a nude mice liver tumor model. The potential mechanism may be through enhancing innate immune response and inducing tumor cell apoptosis and cell death.

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.

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, TGFß, 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.