Tadafumi Asaoka

MicroRNA-155 controls exosome synthesis and promotes gemcitabine resistance in pancreatic ductal adenocarcinoma

The cancer drug gemcitabine (GEM) is a key drug for treating pancreatic ductal adenocarcinoma (PDAC), but PDAC cells develop chemoresistance after long-term administration. Since the tolerance was immediately spread to every PDAC tissue in a patient, it is assumed that some certain efficient mechanisms underlay in the development of chemoresistance. Changes in the levels of particular microRNAs or alterations in intercellular communication play a dominant role in chemoresistance development, and recent data also suggest that exosomes play an important role in this process. In this study, we revealed that the loop conferred chemoresistance in PDAC cells. The loop was as follows; 1, The long-term exposure of GEM increased miR-155 expression in PDAC cells. 2, The increase of miR-155 induced two different functions; exosome secretion and chemoresistance ability via facilitating the anti-apoptotic activity. 3, Exosome deliver the miR-155 into the other PDAC cells and induce the following function. The target therapy to miR-155 or the exosome secretion effectively attenuated the chemoresistance, and these results were validated with both clinical samples and in vivo experiments. This mechanism represents a novel therapeutic target in GEM treatment to PDAC.

Detection of AFP mRNA-expressing cells in the peripheral blood for prediction of HCC recurrence after living donor liver transplantation

The aim of this study was to evaluate the hypothesis that the detection of alpha‐fetoprotein (AFP) mRNA‐expressing cells could be a novel, direct and accurate method for predicting tumor recurrence after living donor liver transplantation (LDLT) in patients with hepatocellular carcinoma (HCC). The test group consisted of 32 patients who underwent LDLT for end‐stage liver disease with HCC. Quantitative real‐time reverse transcription polymerase chain reaction was used for the detection of AFP mRNA‐expressing cells in the peripheral blood. Nine (28.1%) of the 32 patients developed tumor recurrences during the follow‐up period (mean, 27.9 months). The test for the presence of AFP mRNA in the peripheral blood was positive either preoperatively or postoperatively in 11 (34.3%) of the 32 patients, and positive preoperatively in three patients (9.4%). Univariate analysis revealed that a positive preoperative test for peripheral blood AFP mRNA, as well as exceeding Milan criteria and microscopic evidence of vascular invasion were significant predictors for the recurrence of HCC (P = 0.002, 0.049, and 0.001, respectively). Multivariate analysis using Coxs proportional hazards model revealed that a positive preoperative test for peripheral blood AFP mRNA was an independent risk factor for the recurrence of HCC. We concluded that the presence of AFP mRNA‐expressing cells preoperatively could be a useful predictor of the recurrence of HCC in liver transplant patients.

A Histone Deacetylase Inhibitor Suppresses Epithelial-Mesenchymal Transition and Attenuates Chemoresistance in Biliary Tract Cancer

Epithelial-mesenchymal transition (EMT) is involved in the characteristics of malignancy, such as invasion, metastasis, and chemoresistance. In biliary tract cancer (BTC), EMT is induced by transforming growth factor-beta 1 (TGF-β1). The EMT is reversible; therefore, it is conceivable that it could be related to some epigenetic changes. We focused on histone deacetylase (HDAC) inhibitors as regulators of TGF-β1 signaling, and investigated their effect on EMT and chemoresistance. We employed four BTC cell lines (MzChA-1, gemcitabine-resistant MzChA-1, TFK-1, and gemcitabine-resistant TFK-1) and used vorinostat as the HDAC inhibitor. The relative mRNA expression of an epithelial marker (CDH1) and mesenchymal markers (CDH2, vimentin, SNAI1) were measured by qRT-PCR to evaluate factors associated with EMT. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the chemoresistance of each cell line. In addition, NOD/SCID mice were used to evaluate the effect of vorinostat in vivo. In the parent MzChA-1 and TFK-1 cell lines, TGF-β1 induced EMT and chemoresistance; while vorinostat inhibited the EMT and chemoresistance induced by TGF-β1. In gemcitabine-resistant cell lines that highly expressed TGF-β1, vorinostat inhibited EMT and attenuated chemoresistance. We showed that vorinostat inhibits nuclear translocation of SMAD4 which is a signaling factor of TGF-β1, and this is one of the mechanisms by which vorinostat regulates EMT. We also showed that vorinostat attenuates the binding affinity of SMAD4 to the CDH1-related transcription factors SNAI1, SNAI2, ZEB1, ZEB2, and TWIST. Furthermore, combination therapy with vorinostat and gemcitabine improved survival time in the mice xenografted with gemcitabine resistant MzChA-1 cells. In conclusion, vorinostat regulated TGF-β1-induced EMT and chemoresistance through inhibition of SMAD4 nuclear translocation.

Differential transcriptome patterns for acute cellular rejection in recipients with recurrent hepatitis C after liver transplantation

Histopathological evaluation of the liver via biopsy remains the standard procedure for the diagnosis of both acute cellular rejection (ACR) and recurrent hepatitis C (RHC) after liver transplantation. Nevertheless, it is often difficult to diagnose ACR in hepatitis C virus–positive recipients because of changes in common and overlapping with RHC. The aim of this study was to identify potential target genes for ACR in recipients with RHC. We analyzed 22 liver biopsy samples obtained from 21 hepatitis C virus–positive recipients. The clinicopathological diagnosis based on biopsy examination was ACR‐predominant with superimposed RHC in 9 samples (ACR group) and RHC without ACR (non‐ACR group) in 13. Using oligonucleotide microarrays, we compared the transcriptional changes in the 2 groups and selected 2206 genes that were significantly modulated in ACR. We analyzed the regulatory networks in ACR with Ingenuity Pathway Analysis software, and we confirmed with quantitative real‐time polymerase chain reaction the reproducibility of caspase 8, apoptosis‐related cysteine peptidase and bone morphogenetic protein 2 up‐regulation in another group of validation samples, representing 2 genes from the core network as the target genes for ACR. Our results demonstrated novel transcriptome patterns for ACR with concurrent RHC that were distinct from those of recipients with only RHC, suggesting that gene expression profiling may be useful in the diagnosis of ACR in recipients with hepatitis C. Liver Transpl 15:1738–1749, 2009.

Prognostic impact of preoperative NLR and CA19-9 in pancreatic cancer

BACKGROUND Recently, several preoperative proinflammatory markers and nutritional factors such as neutrophil-to-lymphocyte ratio (NLR) and prognostic nutrition index (PNI) have been reported as significant predictor for poor prognosis of various malignant tumors. In this study, we evaluated the prognostic values of these preoperative parameters in patients with resectable pancreatic head cancer. METHODS We retrospectively reviewed consecutive patients who underwent PD for pancreatic head cancer between 2007 and 2012. A total of 46 patients were enrolled in this analysis. Preoperative parameters such as CRP, CA19-9, NLR and PNI at the time of presentation were recorded as well as overall survival. Cancer specific survival was assessed using Kaplan-Meier method. Univariate and multivariate Cox regression models were applied to evaluate the prognostic relevance of preoperative parameters. The correlations between CA19-9 values, NLR and pathological findings, first recurrence site were respectively reviewed. RESULTS In multivariable analysis preoperative high NLR (≧2.7) and high CA19-9 (≧230) were independent prognostic factors for poor survival (P value: 0.03 and 0.025, respectively). Kaplan-Meier survival analysis demonstrated the overall 2-year survival rate in patients with high NLR or high CA19-9 were 37.5% compared with 89.9% in patients with low NLR and low CA19-9. CONCLUSION Preoperative NLR and serum CA19-9 offer significant prognostic information associated with overall survival following PD in the patients with pancreatic head cancer.

Steroid-free living donor liver transplantation in adults: impact on hepatitis C recurrence

Abstract:  Introduction:  Although steroid‐free immunosuppression has been proven to be safe and feasible for liver transplantation, its impact on hepatitis C virus (HCV) recurrence remains unknown. We aimed to clarify the impact of steroid‐free immunosuppression on post‐operative HCV recurrence after living donor liver transplantation (LDLT).

A CD13 inhibitor, ubenimex, synergistically enhances the effects of anticancer drugs in hepatocellular carcinoma.

Cancer stem cells (CSCs) were reported to be involved in resistance to chemo/radiation therapy. We previously reported that CD13 was both a marker of CSCs and a candidate therapeutic target in HCC. In the present study, we explored the antitumor effect of a combined therapy, where ubenimex, a CD13 inhibitor, was combined with conventional anticancer drugs, fluorouracil (5-FU), cisplatin (CDDP), doxorubicin (DXR) and sorafenib (SOR), and we elucidated the mechanism of these combination therapies. We evaluated changes in the expression of CD13 before and after treatment with anticancer drugs and with or without ubenimex in the human HCC cell lines HuH7 and PLC/PRF/5. The interactions between the anticancer drugs and ubenimex were determined with isobologram analyses. We analyzed cell cycle, apoptosis, and intracellular reactive oxygen species (ROS) levels to explore the mechanisms of the combination therapies. In both cell lines, the expression of CD13 increased after a 72-h exposure to each anticancer drug alone (P<0.05), and the expression of CD13 decreased with ubenimex administration (P<0.05). Isobologram analyses indicated that ubenimex had synergistic effects with 5-FU, CDDP and DXR, and an additive effect with SOR. Cell cycle analyses showed that ubenimex decreased the proportion of cells in G0/G1. Ubenimex enhanced the effects of 5-FU, CDDP and DXR by increasing apoptosis and intracellular ROS levels. In combination therapies, ubenimex synergistically enhanced the antitumor effects of 5-FU, CDDP and DXR on cell cycle regulation and apoptosis induction in HCC cell lines. The effects of ubenimex were due to increased intracellular ROS levels.

Intragraft transcriptome level of CXCL9 as biomarker of acute cellular rejection after liver transplantation

BACKGROUND Liver transplantation has been a life-saving and well-established treatment for acute liver failure and various end-stage liver diseases. However, acute cellular rejection (ACR) is one of the key factors that determine long-term graft function and survival after liver transplantation, and there are still no specific biomarkers available to monitor the alloimmune response. The aim of the present study was to identify molecular biomarkers for ACR in liver allograft. METHODS We analyzed the gene expression profile using an oligonucleotide microarray covering 44,000 human probes in 35 liver biopsy samples after living donor liver transplant, which consisted of 13 samples with ACR (ACR group; moderate/mild, 6/7), 13 samples with other dysfunctions (non-ACR group; recurrent hepatitis C / ischemia/reperfusion injury (IRI)/ nonspecific inflammation / small-for-size syndrome, 5/4/3/1), and 9 samples without liver dysfunction (protocol group). We selected 113 informative genes based on microarray results and adopted the network analysis to visualize key modulators in ACR. We selected 6 modulators (CXCL9, GZMB, CCL19, GBP2, LAIR1, and CDC25A) and confirmed the reproducibility in 23 independent biopsy samples and investigated the response to the rejection treatment in sequential samples. RESULTS Network analysis revealed the top three subnetworks, which had NF-κB, MAPK, and IFNG as central hubs. Among selected modulators, intragraft expression levels of CXCL9 mRNA was most upregulated and sensitive to alloimmune status. CONCLUSION Intragraft CXCL9 mRNA has a functionally important role in T-cell activation in liver allograft and serves as biomarker for ACR.

Postoperative hyperbilirubinemia and graft outcome in living donor liver transplantation.

Little information is available on the characteristics and clinical significance of serum bilirubin level early after liver transplantation. The aim of this study was to clarify the risk factors for early graft loss and to assess the significance of postoperative hyperbilirubinemia as a predictor of graft outcome in living donor liver transplantation (LDLT). We retrospectively analyzed perioperative parameters in 68 patients who underwent LDLT. Graft loss within 1 year post‐LDLT was confirmed in 9 patients (13.4%). Univariate analysis of risk factors showed that preoperative Model for End‐Stage Liver Disease score, donor age, postoperative peak serum bilirubin level (p‐BIL) within 28 days after LDLT, and surgical complications were significant determinants of early graft loss (<1 year post‐transplant). Multivariate analysis identified p‐BIL (odds ratio = 1.170, 95% confidence interval = 1.030‐1.329, P = 0.016) as the only independent predictor of early graft loss. The incidence of such loss was high in patients with p‐BIL over 27.0 mg/dL (area under the receiver operating characteristic curve = 0.988). In conclusion, serum bilirubin level is a useful predictor of short‐term (<1 year) graft outcome and for considering retransplantation in a timely fashion. Liver Transpt 13: 1538–1544. 2007.

A novel preoperative predictor of pancreatic fistula using computed tomography after distal pancreatectomy with staple closure

PurposeA thick pancreas has proven to be a conspicuous predictor of pancreatic fistula (PF) following distal pancreatectomy (DP) using staples. Other predictors for this serious surgical complication currently remain obscure. This study sought to identify novel predictors of PF following DP.MethodsOne hundred and twenty-two patients were retrospectively assessed to determine the correlation between PF occurrence and the clinicopathological findings and radiologic data from preoperative computed tomography (CT). CT assessments included the thickness of the pancreas (TP) and pancreatic CT number (pancreatic index; PI), calculated by dividing the pancreatic CT by the splenic CT density.ResultsTwenty-four patients (19.7%) developed a clinically relevant PF. TP was identified as an independent risk factor for PF in multivariate analyses (odds ratio 1.17; P = 0.0095). In subgroup analyses, a lower PI in a thick pancreas was a significant predictor of PF (P = 0.032). The combination of these two prediction parameters, known as the TP-to-PI ratio (TPIR), showed a significantly better prediction ability than TP alone (area under the receiver operating characteristic curve for the incidence of PF, TPIR 0.80 vs. TP 0.69; P = 0.037).ConclusionCombining the CT number with TP substantially improves the prediction ability for the incidence of PF following DP with staple use.