Compromised AMPK-PGCIα Axis Exacerbated Steatotic Graft Injury by Dysregulating Mitochondrial Homeostasis in Living Donor Liver Transplantation.

OBJECTIVE: To investigate the association of graft steatosis with long-term outcome, and to elucidate the mechanism of steatotic graft injury in adult living donor liver transplantation. SUMMARY OF BACKGROUND DATA: The utilization of steatotic graft expands the donor pool for living donor liver transplantation (LDLT). However, it remains controversial due to its high morbidity and mortality. Elucidating the mechanism of steatotic graft injury is crucial to develop therapeutic strategies targeting at graft injury and to further expand the donor pool. METHODS: Five hundred thirty patients receiving LDLT were prospectively included for risk factor analysis and outcome comparison. Rat orthotopic liver transplantation, in vitro functional experiments and mouse hepatic ischemia/ reperfusion models were established to explore the mechanisms of steatotic graft injury. RESULTS: We identified that graft with >10% steatosis was an independent risk factor for long-term graft loss after LDLT (hazard ratio 2.652, P = 0.001), and was associated with shorter cancer recurrence-free survival and acute phase liver injury. Steatotic graft displayed distinct mitochondrial dysfunction, including membrane, calcium, and energy homeostasis dysregulation. Specifically, the mitochondrial biogenesis was remarkably downregulated in steatotic graft. Inhibition of AMPK-PGC1α axis impaired mitochondrial biogenesis and was lethal to fatty hepatocyte in vitro , whereas reactivation of AMPK promoted PGC1α-mediated mitochondrial biogenesis and attenuated liver injury via restoring mitochondrial function in animal model. Conclusions: We provided a new mechanism that compromised AMPK-PGC1α axis exacerbated steatotic graft injury in LDLT by dysregulating mitochondrial homeostasis through impairment of biogenesis.

Biomarkers and predictive models of early allograft dysfunction in liver transplantation - A systematic review of the literature, meta-analysis, and expert panel recommendations.

BACKGROUND: Prompt identification of early allograft dysfunction (EAD) is critical to reduce morbidity and mortality in liver transplant (LT) recipients. OBJECTIVES: Evaluate the evidence supporting biomarkers that can provide diagnostic and predictive value for EAD. DATA SOURCES: Ovid MEDLINE, Embase, Scopus, Google Scholar, and Cochrane Central. METHODS: Systematic review following PRISMA guidelines and recommendations using the GRADE approach was derived from an international expert panel. Studies that investigated biomarkers or models for predicting EAD in adult LT recipients were included for in-depth evaluation and meta-analysis. Olthoff's criteria were used as the standard reference for the diagnostic accuracy evaluation. PROSPERO ID: CRD42021293838 RESULTS: Ten studies were included for the systematic review. Lactate, lactate clearance, uric acid, Factor V, HMGB-1, CRP to ALB ratio, phosphocholine, total cholesterol, and metabolomic predictive model were identified as potential early EAD predictive biomarkers. The sensitivity ranged between .39 and .92, while the specificity ranged from .63 to .90. Elevated lactate level was most indicative of EAD after adult LT (pooled diagnostic odds ratio of 7.15 (95%CI: 2.38-21.46)). The quality of evidence (QOE) for lactate as indicator was moderate according to the GRADE approach, whereas the QOE for other biomarkers was very low to low likely as consequence of study design characteristics such as single study, small sample size, and large ranges of sensitivity or specificity. CONCLUSIONS: Lactate is an early indicator to predict EAD after LT (Quality of Evidence: Moderate | Grade of Recommendation: Strong). Further multicenter studies and the use of machine perfusion setting should be implemented for validation.

Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma.

BACKGROUND & AIMS: The roles of alternatively activated (M2) macrophages on pro-tumour phenotypes have been well documented in many cancers except hepatocellular carcinoma (HCC). Considering their close relationship with chronic tissue injuries as well as enhanced tumour invasiveness and growth, we aimed to investigate the direct effects of M2 macrophages on HCC. METHODS: M2 macrophages in 95 HCC clinical specimens were quantified using immunohistochemistry and quantitative PCR. The pro-tumour functions and the underlying molecular mechanisms of M2 macrophages in HCC were investigated in vivo and in an in vitro co-culture system. RESULTS: In the clinical study, high M2-specific CD163 (hazard ratio=2.693; p=0.043) and scavenger receptor A (hazard ratio=3.563; p=0.044) levels indicated poor prognosis and correlated with increased tumour nodules and venous infiltration in HCC patients. In an orthotopic model, the liver tumour volume was increased 3.26-fold (1.27 cm3±0.36) after M2 macrophage injection compared with the control (0.39 cm3±0.05) (p=0.032). An increased rate of lung metastasis was also found in the treatment group. In vitro, co-cultivation with M2 macrophages elevated the number of HCC cells (MHCC97L) and migration events by 1.3-fold and 3.2-fold, respectively (p<0.05). Strongly induced by MHCC97L, M2 macrophage-derived CCL22 was proven to enhance tumour migration capacities and correlate with venous infiltration in HCC patients. Increased epithelial-mesenchymal transition (EMT) via Snail activation in MHCC97L was found to be promoted by M2 macrophages and CCL22. CONCLUSIONS: M2 macrophages contribute to poor prognosis in HCC and promote tumour invasiveness through CCL22-induced EMT.

Post-transplant endothelial progenitor cell mobilization via CXCL10/CXCR3 signaling promotes liver tumor growth.

BACKGROUND & AIMS: Patients with hepatocellular carcinoma (HCC) receiving living donor liver transplantation appear to possess significantly higher tumor recurrence than the recipients receiving deceased donor liver transplantation. The underlying mechanism for HCC recurrence after transplantation remains unclear. Here, we aim to investigate the impact of small-for-size liver graft injury on HCC recurrence after transplantation. METHODS: The correlation between tumor recurrence, liver graft injury, CXCL10 expression and endothelial progenitor cell (EPC) mobilization was studied in 115 liver transplant recipients and rat orthotopic liver transplantation (OLT) models. The direct role of CXCL10/CXCR3 signaling on EPC mobilization was investigated in CXCL10(-/-) mice and CXCR3(-/-) mice. The role of EPCs on tumor growth and angiogenesis was further investigated in an orthotopic liver tumor model. RESULTS: Clinically, patients with small-for-size liver grafts (<60% of standard liver weight, SLW) had significantly higher HCC recurrence (p=0.04), accompanied by more circulating EPCs and higher early-phase intragraft and plasma CXCL10 levels, than the recipients with large grafts (≥60% of SLW), which were further validated in rat OLT models. Circulatory EPC mobilization was reduced after liver injury both in CXCL10(-/-) mice and CXCR3(-/-) mice in comparison to wild-type controls. CXCL10 recruited EPCs in dose-dependent and CXCR3-dependent manners in vitro. Early-phase EPC/CXCL10 injection enhanced orthotopic liver tumor growth, angiogenesis and metastasis in nude mice. CONCLUSIONS: Post-transplant enhanced CXCL10/CXCR3 signaling in small-for-size liver grafts directly induced EPC mobilization, differentiation and neovessel formation, which further promotes tumor growth. Targeting CXCL10/CXCR3 signaling may attenuate early-phase liver graft injury and prevent late-phase tumor recurrence/metastasis after transplantation.

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.

A novel oxygen carrier "YQ23" suppresses the liver tumor metastasis by decreasing circulating endothelial progenitor cells and regulatory T cells.

BACKGROUND: Surgical therapies are the first-line treatments for hepatocellular carcinoma (HCC) patients. However, the high incidence of tumor metastasis after liver surgery remains a severe problem. We aim to investigate the roles and the underlying mechanism of YQ23, stabilized non-polymeric diaspirin cross-linked tetrameric hemoglobin, in liver tumor metastasis after major hepatectomy and partial hepatic ischemia reperfusion (I/R) injury. METHODS: An orthotopic liver tumor model in Buffalo rat was established using the hepatocellular carcinoma cell line McA-RH7777. Major hepatectomy for tumor-bearing lobe and partial hepatic I/R injury were performed at two weeks after orthotopic liver tumor implantation. YQ23 (0.2 g/kg) was administered at 1 hour before ischemia and immediately after reperfusion. Blood samples were collected at day 0, 1, 7, 14, 21 and 28 for detection of circulating endothelial progenitor cells (EPCs) and regulatory T cells (Tregs). RESULTS: Our results showed that YQ23 treatment effectively inhibited intrahepatic and lung metastases together with less tumor angiogenesis at 4 weeks after major hepatectomy and partial hepatic I/R injury. The levels of circulating EPCs and Tregs were significantly decreased in YQ23 treatment group. Furthermore, YQ23 treatment also increased liver tissue oxygenation during hepatic I/R injury. Up-regulation of HO1 and down-regulation of CXCR3, TNF-α and IL6 were detected after YQ23 treatment. CONCLUSIONS: YQ23 treatment suppressed liver tumor metastasis after major hepatectomy and partial hepatic I/R injury in a rat liver tumor model through increasing liver oxygen and reducing the populations of circulating EPCs and Tregs.

Postoperative Plasmacytoid Dendritic Cells Secrete IFNα to Promote Recruitment of Myeloid-Derived Suppressor Cells and Drive Hepatocellular Carcinoma Recurrence.

Patients with hepatocellular carcinoma (HCC) confront a high incidence of tumor recurrence after curative surgical resection. Hepatic ischemia-reperfusion injury (IRI) is the major consequence of surgical stress during hepatectomy. Although it has been suggested that hepatic IRI-induced immunosuppression could contribute to tumor relapse after surgery, the underlying mechanisms have not been fully defined. Here, using a multiplex cytokine array, we found that levels of postoperative IFNα serve as an independent risk factor for tumor recurrence in 100 patients with HCC with curative hepatectomy. Plasmacytoid dendritic cells (pDC), the major source of IFNα, were activated after surgery and correlated with poor disease-free survival. Functionally, IFNα was responsible for mobilization of myeloid-derived suppressor cells (MDSC) following hepatic IRI. Conditioned medium from IFNα-treated hepatocytes mediated the migration of MDSCs in vitro. Mechanistically, IFNα upregulated IRF1 to promote hepatocyte expression of CX3CL1, which subsequently recruited CX3CR1+ monocytic MDSCs. Knockdown of Irf1 or Cx3cl1 in hepatocytes significantly inhibited the accumulation of monocytic MDSCs in vivo. Therapeutically, elimination of pDCs, IFNα, or CX3CR1 could restore the tumor-killing activity of CD8+ T cells, hence limiting tumor growth and lung metastasis following hepatic IRI. Taken together, these data suggest that IFNα-producing pDCs drive CX3CR1+ MDSC recruitment via hepatocyte IRF1/CX3CL1 signaling and lead to tumor recurrence after hepatectomy in HCC. Targeting pDCs and the IFNα/CX3CL1/CX3CR1 axis could inhibit surgical stress-induced HCC recurrence by attenuating postoperative immunosuppression. SIGNIFICANCE: IFNα secreted by plasmacytoid dendritic cells drives postoperative immunosuppression and early recurrence of hepatocellular carcinoma, providing new biomarkers and therapeutic targets to improve patient outcomes after surgical resection.

Inhibition of Carnitine Palmitoyltransferase 1A Aggravates Fatty Liver Graft Injury via Promoting Mitochondrial Permeability Transition.

BACKGROUND: Hepatic steatosis is a major risk factor for graft failure due to increased susceptibility of fatty liver to ischemia-reperfusion injury (IRI) during transplantation. Here, we aimed to investigate the role of carnitine palmitoyltransferase 1A (CPT1A) in fatty liver graft injury and to explore the underlying mechanism and therapeutic potential on attenuating hepatic IRI. METHODS: Intragraft CPT1A expression profile and the association with fatty graft injury were investigated in human and rat liver transplantation samples. The underlying mechanism and therapeutic potential of CPT1A activator against IRI were also explored in mouse hepatic ischemia-reperfusion plus major hepatectomy model and in in vitro. RESULTS: CPT1A expression was significantly reduced (P = 0.0019; n = 96) in human fatty liver graft compared with normal one at early phase after transplantation. Low expression of CPT1A was significantly associated with high serum alanine aminotransferase (P = 0.0144) and aspartate aminotransferase (P = 0.0060) levels. The inhibited CPT1A and poor liver function were consistently observed in rat and mouse models with fatty livers. Furthermore, inhibition of CPT1A significantly promoted the translocation of chloride intracellular channel 1 to form chloride ion channel. The dysregulation of chloride ion channel activity subsequently triggered mitochondrial permeability transition (MPT) pore opening, exacerbated cellular oxidative stress, and energy depletion. Importantly, our intravital confocal imaging showed that CPT1A activation attenuated hepatic injury through preventing MPT after reperfusion in fatty mice. CONCLUSIONS: CPT1A inhibition triggered MPT contributed to severe IRI in fatty liver graft. CPT1A restoration may offer therapeutic potential on attenuating hepatic IRI.

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.

Activation of interleukin-6-induced glycoprotein 130/signal transducer and activator of transcription 3 pathway in mesenchymal stem cells enhances hepatic differentiation, proliferation, and liver regeneration.

Adult bone marrow-derived mesenchymal stem cells (MSCs) exist in all living species and are capable of differentiating into different types of specific cells. In this study, we demonstrate the therapeutic effectiveness of rat MSC transplantation in D-galactosamine (GalN)-induced acute liver injury and identified the novel pathways which are involved in hepatic differentiation of MSCs. In vivo, intraportal transplantation with 5 × 10(6) MSCs at 24 hours after GalN administration resulted in significant reduction in serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin compared to the control group. Engrafted MSCs actively proliferated, differentiated, and further enhanced hepatocyte proliferation activity. In vitro, coculture of MSCs with GalN-induced injured hepatocytes showed efficient differentiation and was evidenced by progressive increase in messenger RNA levels of hepatic markers, including albumin, α-fetoprotein, CCAAT-enhancer binding protein α, α-1-antitryspin, and hepatocyte nuclear factor-3ß. Immunofluorescent staining revealed that these cells were positive for albumin, α-fetoprotein, and cytokeratin 18, but not clusters of differentiation 34, cytokeratin 19, or OV6. During hepatic differentiation, signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling were constantly activated, and a gradual down-regulation of ß-catenin expression in messenger RNA and protein levels was detected. Hyper-interleukin-6 fusion protein but not interleukin-6 (IL-6) alone caused reduction in ß-catenin expression associated with the up-regulation of Wnt-5a in MSCs via activating the glycoprotein 130 (gp130)-mediated STAT3 signaling pathway, which indicates the operation of the trans-signaling mechanism. Activation of IL-6/gp130-mediated STAT3 signaling pathway in MSCs triggered wound healing, cell migration, and proliferation. In conclusion, transplantation of MSCs promotes cell proliferation and organ repair, and activation of IL-6/gp130-mediated STAT3 signaling pathway via soluble IL-6 receptor is crucial in hepatic differentiation of MSCs.

Transcriptome Analysis of Acute Phase Liver Graft Injury in Liver Transplantation.

BACKGROUND: Liver transplantation remains the treatment of choice for a selected group of hepatocellular carcinoma (HCC) patients. However, the long-term benefit is greatly hampered by post-transplant HCC recurrence. Our previous studies have identified liver graft injury as an acute phase event leading to post-transplant tumor recurrence. METHODS: To re-examine this acute phase event at the molecular level and in an unbiased way, RNA sequencing (RNA-Seq) was performed on liver graft biopsies obtained from the transplant recipients two hours after portal vein reperfusion with an aim to capture frequently altered pathways that account for post-transplant tumor recurrence. Liver grafts from recurrent recipients (n = 6) were sequenced and compared with those from recipients without recurrence (n = 5). RESULTS: RNA expression profiles comparison pointed to several frequently altered pathways, among which pathways related to cell adhesion molecules were the most involved. Subsequent validation using quantitative polymerase chain reaction confirmed the differential involvement of two cell adhesion molecules HFE (hemochromatosis) and CD274 and their related molecules in the acute phase event. CONCLUSION: This whole transcriptome strategy unravels the molecular landscape of liver graft gene expression alterations, which can identify key pathways and genes that are involved in acute phase liver graft injury that may lead to post-transplant tumor recurrence.

'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.

First detection and complete genome sequence of a phylogenetically distinct human polyomavirus 6 highly prevalent in human bile samples.

Oncovirus-associated malignancies are potentially preventable diseases with major public health significance. Human polyomaviruses (HPyVs) may be associated with oncogenesis or symptomatic illnesses in immunocompromised patients, but the site of viral shedding of most recently discovered HPyVs remains obscure. Using real-time PCR assay using specific primers targeting the HPyV6 large tumor antigen gene, we detected a phylogenetically distinct HPyV6 which was highly prevalent in the bile samples of patients with malignant biliary obstruction (18.8%) and acute gallstone cholangitis (5.5%). The prevalence rate and mean viral load of this HPyV6 were highest in the cholangiocarcinoma subgroup (27.6% and 2.41 × 104copies/ml). These findings were confirmed with another real-time PCR assay using specific primers targeting the HPyV6 viral capsid protein 2 gene. These bile HPyV6 strains may represent a novel clade of HPyV6 as they formed a distinct cluster from the other HPyV6s and exhibited >2% differences in amino acid sequences in their major proteins. While HPyV6 was unlikely the cause of the patients' acute symptoms and liver dysfunction, the virus may be related to immunosuppression in patients with malignancy and/or important in the oncogenesis of cholangiocarcinoma in patients without coinfection with other oncogenic microbes. Further studies to ascertain a causative role of HPyV6 in cholangiocarcinoma should be conducted.

Interferon-gamma inducible protein 10 (IP10) induced cisplatin resistance of HCC after liver transplantation through ER stress signaling pathway.

Tumor recurrence remains an obstacle after liver surgery, especially in living donor liver transplantation (LDLT) for patients with hepatocellular carcinoma (HCC). The acute-phase liver graft injury might potentially induce poor response to chemotherapy in recurrent HCC after liver transplantation. We here intended to explore the mechanism and to identify a therapeutic target to overcome such chemoresistance. The associations among graft injury, overexpression of IP10 and multidrug resistant genes were investigated in a rat liver transplantation model, and further validated in clinical cohort. The role of IP10 on HCC cell proliferation and tumor growth under chemotherapy was studied both in vitro and in vivo. The underlying mechanism was revealed by detecting the activation of endoplasmic reticulum (ER) stress signaling pathways. Moreover, the effect of IP10 neutralizing antibody sensitizing cisplatin treatment was further explored. In rat liver transplantation model, significant up-regulation of IP10 associated with multidrug resistant genes was found in small-for-size liver graft. Clinically, high expression of circulating IP10 was significant correlated with tumor recurrence in HCC patients underwent LDLT. Overexpression of IP10 promoted HCC cell proliferation and tumor growth under cisplatin treatment by activation of ATF6/Grp78 signaling. IP10 neutralizing antibody sensitized cisplatin treatment in nude mice. The overexpression of IP10, which induced by liver graft injury, may lead to cisplatin resistance via ATF6/Grp78 ER stress signaling pathway. IP10 neutralizing antibody could be a potential adjuvant therapy to sensitize cisplatin treatment.

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.

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.

The role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in hepatocellular carcinoma.

AIMS: We previously demonstrated Proline rich tyrosine kinase 2 (Pyk2) plays important roles in regulating tumor progression, migration and invasion in hepatocellular carcinoma (HCC). In this study, we aimed to examine the role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in HCC and to explore its underlying molecular mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Stable transfectants either overexpressing or suppressing Pyk2 were established in different HCC cell lines. MTT, colony formation and Annexin-V assays were employed to examine their in vitro responses to cisplatin. Xenograft ectopic and orthotopic nude mice models were generated to investigate the in vivo responses of them to cisplatin treatment. cDNA microarray was performed to identify Pyk2-induced genes which were further validated by quantitative real-time RT-PCR using clinical HCC samples. In vitro functional study demonstrated that Pyk2-overexpressing HCC transfectants exhibited relatively lower cytotoxicity, higher colony-forming ability and lower apoptosis to cisplatin compared with the control transfectants. Moreover, Pyk2 overexpressing HCC transfectants had a higher survival rate under cisplatin treatment by up-regulation of AKT phosphorylation. In vivo xenograft nude mice model demonstrated that Pyk2-overexpressing transfectants developed higher tolerance to cisplatin treatment together with less tumor necrosis and apoptosis. cDNA microarray analysis revealed that there were more than 4,000 genes differentially expressed upon overexpression of Pyk2. Several upregulated genes were found to be involved in drug resistance and invasion in cancers. Among them, the expression profiles of MDR1, GAGE1, STAT1 and MAP7 were significantly associated with the expression of Pyk2 in clinical HCC samples. CONCLUSIONS: Our results may suggest a new evidence of Pyk2 on promoting cisplatin resistance of HCC cells through preventing cell apoptosis, activation of AKT pathway and upregulation of drug resistant genes.

Suppression of liver tumor growth and metastasis by adiponectin in nude mice through inhibition of tumor angiogenesis and downregulation of Rho kinase/IFN-inducible protein 10/matrix metalloproteinase 9 signaling.

PURPOSE: We aimed to investigate the effects of adiponectin on liver cancer growth and metastasis and explore the underlying mechanisms. EXPERIMENTAL DESIGN: An orthotopic liver tumor nude mice model with distant metastatic potential was applied. Either Ad-adiponectin (1 x 10(8); treatment group) or Ad-luciferase (control group) was injected via portal vein after tumor implantation. Tumor growth and metastasis were monitored by Xenogen In vivo Imaging System. Hepatic stellate cell activation by alpha-smooth muscle actin staining, microvessel density by CD34 staining, macrophage infiltration in tumor tissue, and cell signaling leading to invasion, migration [Rho kinase (ROCK), IFN-inducible protein 10 (IP10), and matrix metalloproteinase 9], and angiogenesis [vascular endothelial growth factor (VEGF) and angiopoietin 1] were also compared. Tumor-nontumor margin was examined under electron microscopy. Direct effects of adiponectin on liver cancer cells and endothelial cells were further investigated by a series of functional studies. RESULTS: Tumor growth was significantly inhibited by adiponectin treatment, accompanied by a lower incidence of lung metastasis. Hepatic stellate cell activation and macrophage infiltration in the liver tumors were suppressed by adiponectin treatment, along with decreased microvessel density. The treatment group had less Ki-67-positive tumor cells and downregulated protein expression of ROCK1, proline-rich tyrosine kinase 2, and VEGF. Tumor vascular endothelial cell damage was found in the treatment group under electron microscopy. In vitro functional study showed that adiponectin not only downregulated the ROCK/IP10/VEGF signaling pathway but also inhibited the formation of lamellipodia, which contribute to cell migration. CONCLUSION: Adiponectin treatment significantly inhibited liver tumor growth and metastasis by suppression of tumor angiogenesis and downregulation of the ROCK/IP10/matrix metalloproteinase 9 pathway.