Kidney-type glutaminase is a biomarker for the diagnosis and prognosis of hepatocellular carcinoma: a prospective study.

PURPOSE: The pathological diagnosis and prognosis prediction of hepatocellular carcinoma (HCC) is challenging due to the lack of specific biomarkers. This study aimed to validate the diagnostic and prognostic efficiency of Kidney-type glutaminase (GLS1) for HCC in prospective cohorts with a large sample size. METHODS: A total of 1140 HCC patients were enrolled in our prospective clinical trials. Control cases included 114 nontumour tissues. The registered clinical trial (ChiCTR-DDT-14,005,102, chictr.org.cn) was referred to for the exact protocol. GLS1 immunohistochemistry was performed on the whole tumour section. The diagnostic and prognostic performances of GLS1 was evaluated by the receiver operating characteristic curve and Cox regression model. RESULTS: The sensitivity, specificity, positive predictive value, negative predictive value, Youden index, and area under the curve of GLS1 for the diagnosis of HCC were 0.746, 0.842, 0.979, 0.249, 0.588, and 0.814, respectively, which could be increased to 0.846, 0.886, 0.987,0.366, 0.732, and 0.921 when combined with glypican 3 (GPC3) and alpha-fetoprotein (AFP), indicating better diagnostic performance. Further, we developed a nomogram with GPC3 and GLS1 for identifying HCC which showed good discrimination and calibration. GLS1 expression was also related with age, T stage, TNM stage, Edmondson-Steiner grade, microvascular invasion, Ki67, VEGFR2, GPC3, and AFP expression in HCC. GLS1 expression was negatively correlated with disease-free survival (P < 0.001) probability of patients with HCC. CONCLUSIONS: It was validated that GLS1 was a sensitive and specific biomarker for pathological diagnosis of HCC and had prognostic value, thus having practical value for clinical application.

MEX3A promotes development and progression of breast cancer through regulation of PIK3CA.

Breast cancer has been identified as the most common malignant tumors among women and the morbidity of breast cancer is still increasing rapidly. MEX3A possesses important functions in the regulation of mRNAs and may be involved in a variety of human diseases including cancer, whose relationship with breast cancer is still not clear. In this study, MEX3A was identified as a potential promotor in breast cancer, whose expression was strongly higher in breast cancer tissues than normal tissues. The in vitro experiments showed that MEX3A is capable of promoting the development of breast cancer through stimulating cell proliferation, inhibiting cell apoptosis, arresting cell cycle and promoting cell migration. The functions of MEX3A were also verified in vivo. Furthermore, a combination of genechip analysis and Ingenuity pathway analysis (IPA) identified PIK3CA as a potential downstream target of MEX3A, knockdown of which executes similar inhibitory effects on breast cancer and could alleviate MEX3A-induced progression of breast cancer. In conclusion, our study unveiled, as the first time, MEX3A as a tumor promotor for breast cancer, whose function was carried out probably through the regulation of PIK3CA.

Application of a novel artificial perfusate based on oxygen-carrying nanoparticles in normothermic machine perfusion for porcine liver preservation after cardiac death.

OBJECTIVE: To investigate the efficacy of a novel artificial perfusate based on oxygen-carrying perfluoronaphthalene-albumin nanoparticles in normothermic machine perfusion (NMP) for preservation of porcine liver donation after cardiac death. METHODS: Artificial perfusate with perfluoronaphthalene-albumin nanoparticles was prepared at 5% albumin (w/v) and its oxygen carrying capacity was calculated. The livers of 16 Landrace pigs were isolated after 1 h of warm ischemia, and then they were divided into 4 groups and preserved continuously for 24 h with different preservation methods: cold preservation with UW solution (SCS group), NMP preservation by whole blood (blood NMP group), NMP preservation by artificial perfusate without nanoparticles (non-nanoparticles NMP group) and NMP preservation by artificial perfusate containing nanoparticles (nanoparticles NMP group). Hemodynamics, tissue metabolism, biochemical indices of perfusate and bile were monitored every 4 h after the beginning of NMP. Liver tissue samples were collected for histological examination (HE and TUNEL staining) before preservation, 12 h and 24 h after preservation. RESULTS: The oxygen carrying capacity of nanoparticles in 100 mL artificial perfusate was 6.94 µL/mmHg (1 mmHg=0.133 kPa). The hepatic artery and portal vein resistance of nanoparticles NMP group and blood NMP group remained stable during perfusion, and the vascular resistance of nanoparticles NMP group was lower than that of blood NMP group. The concentration of lactic acid in the perfusate decreased to the normal range within 8 h in both nanoparticles NMP group and blood NMP group. There were no significant differences in accumulated bile production, alanine aminotransferase and aspartate aminotransferase in perfusate between nanoparticles NMP group and blood NMP group (all P>0.05). After 24 h perfusion, the histological Suzuki score in blood NMP group and nanoparticles NMP group was lower than that in SCS group and non-nanoparticles NMP group (all P<0.05), and the quantities of TUNEL staining positive cells in blood NMP group and non-nanoparticles NMP group was higher than those in nanoparticles NMP group and SCS group 12 h and 24 h after preservation (all P<0.05). CONCLUSION: Artificial perfusate based on oxygen-carrying nanoparticles can meet the oxygen supply requirements of porcine livers donation after cardiac death during NMP preservation, and it may has superiorities in improving tissue microcirculation and alleviating ischemia-reperfusion injury.

An oxygenated perfluorocarbon emulsion improves liver graft preservation evaluated in DCD livers of male sprague dawley rats.

The application of perfluorocarbons, which can carry large quantities of oxygen, in organ preservation was limited by their poor solubility in water. A stable form of perfluorocarbon dispersed in suitable buffers is urgently needed. Perfluorocarbon emulsion was designed and characterized with respect to size distribution, rheology, stability, and oxygen-carrying capacity. The state of DCD rat donor livers preserved by the oxygenated perfluorocarbon emulsion was studied after ex vivo reperfusion by using biochemistry, pathology, and immunohistochemistry methods. Perfluorocarbon emulsion was successfully prepared by high-pressure homogenization. Optimized perfluorocarbon emulsion showed nanoscale size distribution, good stability, and higher oxygen loading capacity than that of HTK solution or water. The state of preserved livers after cardiac death rat liver was improved significantly after static cold storage for 48 hours in this oxygenated perfluorocarbon emulsion. The ATP content and down-regulation of HIF-1a expression after preservation of the liver graft by the oxygenated perfluorocarbon emulsion suggested the advantage of adequate oxygen supply for adequate time. This perfluorocarbon emulsion reported here might be considered a promising system for oxygenated donor liver storage by attenuation of hypoxia.

Microporous cellulosic scaffold as a spheroid culture system modulates chemotherapeutic responses and stemness in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) treatments are evaluated by two-dimensional (2D) in vitro culture systems, despite their limited ability to predict drug efficacy. The three-dimensional (3D) microporous scaffold provides the possibility of generating more reliable preclinical models to increase the efficacy of cancer treatments. The physical properties of a microporous cellulosic scaffold were evaluated. The cellulosic scaffold was biocompatible and had a highly porous network with appropriate pore size, swelling rate, and stiffness of cancer cell cultures. Cellulosic scaffolds were compared with 2D polystyrene for the culture of HepG2 and Huh7 human HCC cells. Cellulosic scaffolds promoted tumor spheroid formation. Cells cultured on scaffolds were more resistant to chemotherapy drugs and showed upregulation of EpCAM and Oct4. The migration ability of HCC cells cultured on scaffolds was significantly greater than that of cells grown in 2D cultures as evidenced by the downregulation of E-cadherin. In addition, the proportion of CD44+/CD133+ HCC cancer stem cells (CSCs) was significantly greater in cells cultured on scaffolds than in those grown in 2D cultures. These findings suggest that cellulosic scaffolds effectively mimic the in vivo tumor behavior and may serve as a platform for the study of anticancer therapeutics and liver CSCs.

Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions.

Patients with obesity have a high prevalence of non-alcoholic fatty liver disease (NAFLD) and, in parallel, increased susceptibility to fibrosis/cirrhosis/hepatocellular carcinoma (HCC). Herein, we report that a high-fat diet (HFD) can augment glycolysis and then accelerate NAFLD-fibrosis progression by downregulating the expression of geranylgeranyl diphosphate synthase (GGPPS), which is a critical enzyme in the mevalonate pathway. Long-term HFD overloading decreases GGPPS expression in mice, which shifts the fuel preference from fatty acids towards glucose. Liver-specific Ggpps deficiency drives the Warburg effect by impairing mitochondrial function, and then induces hepatic inflammation, thus exacerbating fibrosis. Ggpps deficiency also enhances the hyperfarnesylation of liver kinase B1, and promotes metabolic reprogramming by regulating 5'-AMP-activated protein kinase activity. Clinical data further imply that GGPPS expression can predict the stage of NAFLD and recurrence of NAFLD-associated HCC. We conclude that the level of GGPPS is a susceptibility factor for NAFLD-fibrosis progression, and requires more stringent surveillance to ensure early prediction and precision of treatment of NAFLD-related HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial.

OBJECTIVE: There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need. DESIGN AND RESULTS: A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI -12.59 to -2.50; p=0.0018), respectively. CONCLUSIONS: This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group. TRIAL REGISTRATION: NCT01770431; Post-results.

Hepatic expression of Yin Yang 1 (YY1) is associated with the non-alcoholic fatty liver disease (NAFLD) progression in patients undergoing bariatric surgery.

BACKGROUND: This study is to investigate the association between the hepatic expression of Yin Yang 1 (YY1) and the progression of non-alcoholic fatty liver disease (NAFLD) in patients undergoing bariatric surgery. METHODS: Obese patients undergoing bariatric surgery were included. Liver tissues were subjected to the quantitative real-time PCR, Western blot analysis, and immunohistochemical assay, to determine the expression levels of YY1. RESULTS: Totally 88 patients were included. According to the NAFLD activity score (NAS), these patients were divided into the control (n = 12), steatosis (n = 20), non-defining NASH (n = 38), and NASH (n = 18) groups. Significant differences in the serum glucose, insulin, ALT, AST, and HOMA-IR levels were observed among these different NAFLD groups. Hepatic YY1 expression had correlation with serum glucose, insulin, HOMA-IR, ALT, AST, triglycerides, HDL, and GGT. Immunohistochemical analysis showed that, compared with the control group, the expression levels of YY1 were significantly higher in the non-defining NASH and NASH groups. In addition, multivariate regression model showed that the serum ALT and YY1 levels were strongly associated with the NAFLD activity. CONCLUSIONS: Several factors are associated with NAFLD progression, including the expression of YY1. Our findings contribute to understanding of the pathogenesis of NAFLD. TRIAL REGISTRATION: NCT03296605 , registered on September 28, 2017.

Interleukin-10 secreted by mesenchymal stem cells attenuates acute liver failure through inhibiting pyroptosis.

AIM: Recently, the benefit of mesenchymal stem cells (MSCs) as a cell-based therapy for acute liver failure (ALF) has gained much attention, although the mechanism of action of MSCs in the treatment of ALF remains elusive. Pyroptosis is a novel form of programmed cell death with an intense inflammatory response. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects through inhibiting pyroptosis in ALF. METHODS: Mesenchymal stem cells obtained from C57BL/6 mice were isolated and cultured according to an established protocol. The MSCs were transplanted into mice with D-galactosamine (D-Gal)-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, ShIL-RNA, and MCC950 (NLRP3 inhibitor) were given to the mice to explore the therapeutic mechanism of MSCs. Statistical analyses were carried out with spss version 19.0, and all data were analyzed by independent-samples t-test. RESULTS: Injection of IL-10 or MSC transplantation ameliorated D-Gal-induced increase in alanine aminotransferase, aspartate aminotransferase, total bilirubin, NH3, and inflammatory cytokines. Blockage of IL-10 confirmed the therapeutic significance of this cytokine. CONCLUSION: Pyroptosis was inhibited after IL-10 infusion and inhibition of NLRP3 by MCC950 reversed liver dysfunction.

Glissonian approach combined with major hepatic vein first for laparoscopic anatomic hepatectomy.

BACKGROUND: Laparoscopic anatomic hepatectomy remains challenging because of the complex interior structures of the liver. Our novel strategy includes the Glissonian approach and the major hepatic vein first, which serves to define the external and internal landmarks for laparoscopic anatomic hepatectomy. METHODS: Eleven cases underwent laparoscopic anatomic hepatectomy, including three right hepatectomies, three left hepatectomies, three right posterior hepatectomies, and two mesohepatectomies. The Glissonian approach was used to transect the hepatic pedicles as external demarcation. The major hepatic vein near the hepatic portal was exposed and served as the internal landmark for parenchymal transection. The liver parenchyma below and above the major hepatic vein was transected along the major hepatic vein. Fifty-nine subjects were used to compare the distance between the major hepatic vein and secondary Glisson pedicles among different liver diseases. RESULTS: The average operative time was 327 min with an estimated blood loss of 554.55 mL. Only two patients received three units of packed red blood cells. The others recovered normally and were discharged on postoperative day 7. The distance between right posterior Glissonian pedicle and right hepatic vein was shorter in the patients with cirrhosis than that without cirrhosis, and this distance was even shorter in patients with hepatocellular carcinoma. CONCLUSION: The Glissonian approach with the major hepatic vein first is easy and feasible for laparoscopic anatomic hepatectomy, especially in patients with hepatocellular carcinoma and cirrhosis.

MicroRNA-122 Inhibits Lipid Droplet Formation and Hepatic Triglyceride Accumulation via Yin Yang 1.

BACKGROUND/AIMS: An increase in intracellular lipid droplet formation and hepatic triglyceride (TG) content usually results in nonalcoholic fatty liver disease. However, the mechanisms underlying the regulation of hepatic TG homeostasis remain unclear. METHODS: Oil red O staining and TG measurement were performed to determine the lipid content. miRNA expression was evaluated by quantitative PCR. A luciferase assay was performed to validate the regulation of Yin Yang 1 (YY1) by microRNA (miR)-122. The effects of miR-122 expression on YY1 and its mechanisms involving the farnesoid X receptor and small heterodimer partner (FXR-SHP) pathway were evaluated by quantitative PCR and Western blot analyses. RESULTS: miR-122 was downregulated in free fatty acid (FFA)-induced steatotic hepatocytes, and streptozotocin and high-fat diet (STZ-HFD) induced nonalcoholic steatohepatitis (NASH) in mice. Transfection of hepatocytes with miR-122 mimics before FFA induction inhibited lipid droplet formation and TG accumulation in vitro. These results were verified by overexpressing miR-122 in the livers of STZ-HFD-induced NASH mice. The 3'-untranslated region (3'UTR) of YY1 mRNA is predicted to contain an evolutionarily conserved miR-122 binding site. In silico searches, a luciferase reporter assay and quantitative PCR analysis confirmed that miR-122 directly bound to the YY1 3'UTR to negatively regulate YY1 mRNA in HepG2 and Huh7 cells. The (FXR-SHP) signaling axis, which is downstream of YY1, may play a key role in the mechanism of miR-122-regulated lipid homeostasis. YY1-FXR-SHP signaling, which is negatively regulated by FFA, was enhanced by miR-122 overexpression. This finding was also confirmed by overexpression of miR-122 in the livers of NASH mice. CONCLUSIONS: The present results indicate that miR-122 plays an important role in lipid (particularly TG) accumulation in the liver by reducing YY1 mRNA stability to upregulate FXR-SHP signaling.

Genetically engineered bone marrow-derived mesenchymal stem cells co-expressing IFN-γ and IL-10 inhibit hepatocellular carcinoma by modulating MAPK pathway.

PURPOSE: One of the major challenges in delivering cytokines for the treatment of hepatocellular carcinoma (HCC) is the mode of delivery. This study hypothesized that genetically engineered bone marrow derived mesenchymal stem cells (BMSCs) co-expressing IFN-γ and IL-10 can serve as a potential therapeutic strategy in the treatment of HCC by inhibiting cell proliferation. METHODS: Male Sprague-Dawley rats (n=5, 200-250 g) for BMSCs isolation and Nude/SCID mice (n=35,12-20g) to develop liver cancer xenograft model were used. Mice were subcutaneously injected HepG2 cell suspension on left flank. BMSCs were genetically engineered with the recombinant lentiviral vectors expressing IFN-γ and IL-10. The experiments were performed in 5 groups (phosphate buffered saline/PBS, BMSCs, BMSC-IFN-γ, BMSC-IL-10 and BMSC-IFN-γ-IL-10) and the genetically engineered BMSCs were transplanted into HCC mice. Cell viability was measured by MTT assay followed by the evaluation of the effect of cell-cycle regulators (p21, p27, cyclin D1 and Rb). Protein expression of p38, ERK and JNK was assessed by immunohistochemistry using the cell proliferation marker Ki67. RESULTS: The combination of two cytokines (IFN-γ and IL- 10) engineered into BMSCs resulted in a significant reduction in HepG2 cell viability (*p<0.05 vs PBS-treated and #p<0.05 vs BMSC-treated group). Significantly increased expression of cell cycle inhibitors p21 and p27 in parallel with reduced cyclin D1 expression were observed. Reduced phosphorylation of Rb demonstrated the repression of G1/S progression. BMSC-IFN-γ-IL-10 treatment significantly reduced the tumor growth at the end of 36 days compared to the group treated with PBS or BMSCs alone. This effect was accompanied with the modulation of MAPK pathway with the activation of p38 and JNK, and inactivation of ERK. CONCLUSION: The co-expression of IFN-γ and IL-10 in BMSCs inhibits HCC in vitro and in vivo by modulating cell cycle regulators and MAPK pathway.

Hepatitis B virus-human chimeric transcript HBx-LINE1 promotes hepatic injury via sequestering cellular microRNA-122.

BACKGROUND & AIMS: Chronic hepatitis B virus (HBV) carriers have a high risk to develop hepatocellular carcinoma (HCC) but the underlying mechanism remains unclear. Recent studies suggest that viral-human hybrid RNA transcripts, which play a critical role in promoting HCC progression, may be the molecules responsible for the development of HCC in HBV infected patients. Here we determine whether HBx-LINE1, a hybrid RNA transcript of the human LINE1 and the HBV-encoded X gene generated in tumor cells of HBV-positive HCC, can serve as a molecular sponge for sequestering miR-122 and promoting liver cell abnormal mitosis and mouse hepatic injury. METHODS: Paired tumor and distal normal liver tissue specimens, as well as HBx-LINE1 overexpressing hepatic cells, were used to test the relationship between HBx-LINE1 and miR-122. Levels of HBx-LINE1 and miR-122 were assayed by qRT-PCR and Northern blot. HBx-LINE1-miR-122 binding was analyzed by luciferase reporter assay. Mouse hepatic injury was monitored by tissue staining and serum aspartate transaminase, alanine aminotransferase and total bilirubin measurement. RESULTS: HBx-LINE1 in HBV-positive HCC tissues was inversely correlated with miR-122. Each HBx-LINE1 consists of six miR-122-binding sites, and forced expression of HBx-LINE1 effectively depleted cellular miR-122, promoting hepatic cell epithelial-mesenchymal transition (EMT)-like changes, including ß-catenin signaling activation, E-cadherin reduction and cell migration enhancement. Mice administered with HBx-LINE1 display a significant mouse liver cell abnormal mitosis and hepatic injury. However, all these effects of HBx-LINE1 are completely abolished by miR-122. CONCLUSIONS: Our finding illustrates a previously uncharacterized miR-122-sequestering mechanism by which HBx-LINE1 promotes hepatic cell EMT-like changes and mouse liver injury.

Evaluating Tumor-Associated Activity of Extracellular Sulfatase by Analyzing Naturally Occurring Substrate in Tumor Microenvironment of Hepatocellular Carcinoma.

The progress of cancer is intimately connected with the activity of the extracellular matrix (ECM) enzymes. To evaluate the promoting effect of these enzymes on tumor development in a pathological biocontext, we propose in this work to analyze their natural substrates in the ECM. This strategy is demonstrated by studying heparan sulfate (HS), the substrate of ECM sulfatase, in the development of hepatocellular carcinoma (HCC). An assay is designed to study the abundance and sulfation of HS and to evaluate the interactions between HS and the growth factors, such as fibroblast growth factor 2 (FGF2). Peptides derived from the amyloid peptide and various growth factors are employed to detect HS and evaluate their affinity toward the growth factors, whereas the ruthenium polypyridyl complex is taken as a photocatalyst to achieve a more sensitive signal readout. Applying this method to HepG2 cells, correlated changes between the activity of sulfatase 2 in regulating FGF2-induced cell proliferation and the abundance, degree of sulfation, and growth factor binding of HS can be observed. This method has also been applied to analyze clinical tissue samples of HCC. The results may suggest tumor-progress-related alterations in the above-studied biochemical features of HS. These results may point to the prospect of using this method to facilitate the diagnosis and prognosis of HCC in the future.

Electrochemical Detection and Distribution Analysis of ß-Catenin for the Evaluation of Invasion and Metastasis in Hepatocellular Carcinoma.

Pro-metastatic cell signaling controls the switch to distant metastasis and the final cancer death. In hepatocellular carcinoma (HCC), this death switch is turned on by the multiprotein interactions of ß-catenin with many transcription factors, so a method to assay the bioactivity of ß-catenin to participate in these pro-metastatic protein/protein interactions has been proposed in this work. This method employs cost-effective peptide-based protein targeting ligands, while the electrochemical catalytic cross-linking in this method also "finalize" the noncovalent molecular recognition, so that the robustness can be improved to enable detection of relatively more complex biosamples. In studying clinical samples with the proposed method, the cellular distribution and overall expression of ß-catenin show a parallel with the pathological grade of the sample, particularly, nuclear translocation. The pro-metastatic activation of ß-catenin can also be observed as evidently correlated with higher-grade cases, suggesting the active role of ß-catenin in promoting metastasis. According to these results, the proposed method may have the prospective use as a prognostic tool for evaluating the potential of invasion and metastasis in cancer.

MiR-145 functions as a tumor suppressor via regulating angiopoietin-2 in pancreatic cancer cells.

BACKGROUND: Pancreatic cancer is currently one of the leading causes of cancer deaths without any effective therapies. Mir-145 has been found to be tumor-suppressive in various types of cancers. The aim of this study is to investigate the role of miR-145 in pancreatic cancer cells and explore its underlying mechanism. METHODS: Quantitative real time PCR was used to determine the expression level of miR-145 and angiopoietin-2 (Ang-2) mNRA, and the expression level of Ang-2 protein was measured by western blotting. The anti-cancer activities of miR-145 were tested both in in vitro by using cell invasion and colony formation assay and in vivo by using xenograft assay. The direct action of miR-145 on Ang-2 was predicted by TargetScan and confirmed by luciferase report assay. The vascularization of xenografts were performed by immunohistochemical analysis. RESULTS: The expression level of miR-145 was significantly lower and the expression levels of Ang-2 mRNA and protein was significantly higher in the more aggressive pancreatic cancer cells (MiaPaCa-2 and Panc-1) when compared to that in BxPC3 cells. Overexpression of miR-145 in the BxPC3, MiaPaCa-2 and Panc-1 cells suppressed the cell invasion and colony formation ability, and the expression level of Ang-2 protein in MiaPaCa-2 and Panc-1 cells was also suppressed after pre-miR-145 transfection. Intratumoral delivery of miR-145 inhibited the growth of pancreatic cancer xenografts and angiogenesis in vivo, and also suppressed the expression level of angiopoietin-2 protein. Luciferase report assay showed that Ang-2 is a direct target of miR-145, and down-regulation of angiopoietin-2 by treatment with Ang-2 siRNA in the BxPC3, MiaPaCa-2 and Panc-1 cells suppressed cell invasion and colony formation ability. The reverse transcription PCR results also showed that Tie1 and Tie2 were expressed in BxPC3, MiaPaCa-2 and Panc-1 cells. CONCLUSION: MiR-145 functions as a tumor suppressor in pancreatic cancer cells by targeting Ang-2 for translation repression and thus suppresses pancreatic cancer cell invasion and growth, which suggests that restoring of miR-145 may be a potential therapeutic target for pancreatic cancer.

Factors limiting the extent of absolute bioavailability of pradefovir in rat.

1. Pradefovir was designed as an oral liver target prodrug of 9-(2-phosphonylmethoxyethyl) adenine (PMEA). Liver targeting arises through first pass hepatic metabolism by cytochrome P-450 3A4 (CYP3A4). For CYP3A4 primarily exists in intestines and liver, intestinal metabolism may impair its liver selectivity and oral bioavailability, and then impair its efficacy and safety. It was important to reveal details of the disposition of pradefovir in intestines and liver in a preclinical study. 2. The absolute bioavailability of pradefovir was 4.75% based on the intravenous and oral AUC0-24 h in rats. Pradefovir was stable in intestinal segments and microsomes. The fractions of the dose absorbed from the GI tract were 20.3% and 15.3% from intravenous and oral administration of pradefovir in rats and portal vein-cannulated rat models, respectively. The liver extraction ratio was predicted to be 49.2% from liver microsomes system, based on the monitoring substrate loss rate. Rat intestines' Ussing chamber experiment indicated that P-glycoprotein (P-gp) transporter and paracellular pathway may involve in intestinal transportation. 3. Activation of pradefovir mainly occurs in the liver. Low intestinal absorption was the main reason of low bioavailability of pradefovir in rats. The result was suggestive for the disposition of pradefovir in human intestine and liver.

IL-1ß siRNA adenovirus benefits liver regeneration by improving mesenchymal stem cells survival after acute liver failure.

UNLABELLED:   BACKGROUND: Uncontrolled hapatic inflammatory response is regarded as the primary pathological mechanism of acute liver failure and impairs the regeneration of hepatocytes and stem cell grafts. Interleukin-1 plays a key role for activating immune and inflammatory response. Recently, siRNA has made quite a few progresses in treating inflammatory response. AIM: To assess the effect of IL-1? siRNA adenovirus on MSC and the therapeutic effect of MSC combined with IL-1? siRNA adenovirus in ALF. MATERIAL AND METHODS: We implanted MSC or/and IL-1? siRNA adenovirus via the tail vein, using CCl4-induced ALF in a mice model. Mice were sacrificed at different time points. Blood samples and liver tissues were collected. Hepatic injury, liver regeneration, cytokines (CXCL1, IL-1?, IL-10, IL-6, VEGF and HGF), animal survival and vital MSC were assessed after cell transplantation. RESULTS: MSC combined with IL-1? siRNA reduced the inflammatory levels and prevented liver failure. These animals administrated with MSC and IL-1? siRNA also exhibited improved liver regeneration and increased survival rates. Immunohistochemistry and fluorescence microscopy revealed the number of vital MSC in ALF + MSC + IL-1? siRNA group were significantly more than that in ALF + MSC group. CONCLUSION: IL-1? siRNA adenovirus could enhance MSC ability of tissue regeneration through increasing its survival rate. Accordingly, combination of IL-1? siRNA adenovirus and MSC had a synergistic effect on acute liver failure.

Intraportal mesenchymal stem cell transplantation prevents acute liver failure through promoting cell proliferation and inhibiting apoptosis.

BACKGROUND: Transplantation of mesenchymal stem cells (MSCs) has been regarded as a potential treatment for acute liver failure (ALF), but the optimal route was unknown. The present study aimed to explore the most effective MSCs transplantation route in a swine ALF model. METHODS: The swine ALF model induced by intravenous injection of D-Gal was treated by the transplantation of swine MSCs through four routes including intraportal injection (InP group), hepatic intra-arterial injection (AH group), peripheral intravenous injection (PV group) and intrahepatic injection (IH group). The living conditions and survival time were recorded. Blood samples before and after MSCs transplantation were collected for the analysis of hepatic function. The histology of liver injury was interpreted and scored in terminal samples. Hepatic apoptosis was detected by TUNEL assay. Apoptosis and proliferation related protein expressions including cleaved caspase-3, survivin, AKT, phospho-AKT (Ser473), ERK and phospho-ERK (Tyr204) were analyzed by Western blotting. RESULTS: The average survival time of each group was 10.7+/-1.6 days (InP), 6.0+/-0.9 days (AH), 4.7+/-1.4 days (PV), 4.3+/-0.8 days (IH), respectively, when compared with the average survival time of 3.8+/-0.8 days in the D-Gal group. The survival rates between the InP group and D-Gal group revealed a statistically significant difference (P<0.01). Pathological and biochemical analysis showed that liver damage was the worst in the D-Gal group, while less injury in the InP group. Histopathological scores revealed a significant decrease in the InP group (3.17+/-1.04, P<0.01) and AH group (8.17+/-0.76, P<0.05) as compared with that in the D-Gal group (11.50+/-1.32). The apoptosis rate in the InP group (25.0%+/-3.4%, P<0.01) and AH group (40.5%+/-1.0%, P<0.05) was lower than that in the D-Gal group (70.6%+/-8.5%). The expression of active caspase-3 was inhibited, while the expression of survivin, AKT, phospho-AKT (Ser473), ERK and phospho-ERK (Tyr204) was elevated in the InP group. CONCLUSIONS: Intraportal injection was superior to other pathways for MSC transplantation. Intraportal MSC transplantation could improve liver function, inhibit apoptosis and prolong the survival time of swine with ALF. The transplanted MSCs may participate in liver regeneration via promoting cell proliferation and suppressing apoptosis during the initial stage of ALF.