Assessment of liver function-related mRNA expression and fluorescence imaging in outflow-obstructed regions in rats.

PURPOSE: The liver function in outflow-obstructed regions is reportedly impaired; however, the functional decrease has not been quantitatively assessed. We therefore evaluated the uptake of indocyanine green (ICG) into hepatocytes and the mRNA expression associated with the liver function in outflow-obstructed regions using rat models. METHODS: A total of 20 rats with the ligation of the right median hepatic vein to induce outflow obstruction were studied. Five rats each were grouped by the time of re-laparotomy, and the fluorescence intensity (FI) values of ICG. The mRNA expression, including that of Albumin, Cytochrome P450 (Cyp) 1a2, Cyp3a1, Cyp7a1, and Gamma-glutamylcysteine synthetase, in outflow-obstructed (mRNAOut-Ob) and non-outflow-obstructed (mRNANon) regions was assessed. RESULTS: Microscopic fluorescence imaging showed that the FI values were significantly lower in outflow-obstructed regions than in non-outflow-obstructed regions at 12 h, 24 h, and 3 days after ligation of the hepatic vein. The mRNAOut-Ob/mRNANon ratios decreased to approximately 30% at 12 h after the outflow obstruction and increased to approximately 70-80% at 7 days. CONCLUSIONS: The liver function in outflow-obstructed regions was impaired in terms of the uptake of ICG and the mRNA expression. Our findings may help estimate the postoperative functional remnant liver volume by considering the decrease in the liver function in outflow-obstructed regions.

The impact of the covalently closed circular DNA level on recurrence of hepatocellular carcinoma after initial hepatectomy: an analysis of patients with resolved hepatitis B virus infection.

BACKGROUND: We assessed whether or not covalently closed circular DNA (cccDNA) levels in the background liver influence the recurrence of hepatocellular carcinoma (HCC) in patients with resolved hepatitis B virus (HBV) infection. METHODS: Among 425 patients who underwent initial hepatectomy for HCC between 2010 and 2018, a retrospective review was performed in 44 with resolved HBV infection. The clinicopathologic characteristics were analyzed for correlation with tumor recurrence. The HBV cccDNA levels were tested via a droplet digital polymerase chain reaction assay. RESULTS: HBV cccDNA was detected in 27 of 44 patients (61%), and the median level was 1.0 copies/1000 ng (range, 0-931.3 copies/1000 ng). Anti-HBc ≥8.9 S/CO was associated with cccDNA detection (odds ratio, 11.08; 95% confidence interval [95% CI], 2.48-49.46; P = 0.002). Twenty-eight patients (64%) developed HCC recurrence after hepatectomy. The overall 3- and 5-year recurrence-free survival rates were 45.7% and 34.3%, respectively.19 HBV cccDNA levels was not significantly associated with HCC recurrence, while the presence of multiple tumors was an independent risk fact or (hazard ratio, 6.53; 95% CI, 2.48-17.19; P < 0.001. CONCLUSION: HBV cccDNA levels did not influence HCC recurrence after hepatectomy. Anti-HBc levels may be used as a surrogate marker for cccDNA.

Host MICA Polymorphism as a Potential Predictive Marker in Response to Chemotherapy for Colorectal Liver Metastases.

BACKGROUND: Understanding the genetic background of a tumor is important to better stratify patient prognosis and select optimal treatment. For colorectal liver metastases (CLM), however, clinically available biomarkers remain limited. METHODS: After a comprehensive sequencing of 578 cancer-related genes in 10 patients exhibiting very good/poor responses to chemotherapy, the A5.1 variant of the MICA gene was selected as a potential biomarker for CLM. The clinical relevance of MICA A5.1 was then investigated in 58 patients who underwent CLM resection after chemotherapy. RESULTS: The A5.1 variant was observed in 16 (27.6%) patients examined using direct DNA sequencing, and a very high concordance rate (56/58, 96.6%) for the MICA variant was confirmed between tumor tissues and normal liver parenchyma. A multivariate analysis of 38 patients with no history of treatment with anti-EGFR antibodies confirmed that MICA A5.1 was significantly correlated with an optimal CT morphologic response (OR 11.67; 95% CI 2.08-65.60; p = 0.005) and tended to be correlated with a tumor viability of < 20% after chemotherapy (OR 5.91; 95% CI 0.97-36.02; p = 0.054). MICA A5.1 was also associated with a decreased risk of progression after CLM resection. CONCLUSION: The MICA A5.1 polymorphism was associated with a better CT morphologic response to chemotherapy and a reduced risk of relapse after CLM resection. Given the high concordance rate in MICA variants between normal liver tissue and CLM, the genetic background of the host could be a new biomarker for CLM.

Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists.

Knockdown of TFAP2E results in rapid G2/M transition in oral squamous cell carcinoma cells.

TFAP2E is a member of the activator protein-2 transcription factor family and acts as a tumor suppressor in several types of cancer. Downregulation of TFAP2E expression is significantly associated with a shorter overall survival period in patients with oral squamous cell carcinoma (OSCC). To evaluate the molecular mechanisms by which TFAP2E suppresses the development or progression of OSCC, the present study investigated the effects of TFAP2E downregulation on OSCC-derived Ca9-22 and HSC-4 cells. The present study demonstrated that small interfering RNA mediated-knockdown of TFAP2E accelerated the proliferation of these OSCC cell lines compared with that in the control group, as determined by the standard water-soluble tetrazolium salt-8 assay. To analyze the cell cycle progression rate, the cell cycle distribution patterns of TFAP2E-knockdown and control cells cultured in the presence of nocodazole, which prevents the completion of mitosis, were analyzed by fluorescence-activated cell sorting at different time points. When analyzing cellular DNA contents, no major differences in cell cycle profiles were observed; however, the rate of increase in cells positive for histone H3 Serine 28 phosphorylation, a standard molecular marker of early M phase, was significantly higher in TFAP2E-knockdown cells than in the control cells. Collectively, these results suggested that TFAP2E may attenuate the proliferation of OSCC cells by regulating G2/M transition.

Real-Time Fluorescence Imaging to Identify Cholangiocarcinoma in the Extrahepatic Biliary Tree Using an Enzyme-Activatable Probe.

Introduction: Complete resection is the only possible treatment for cholangiocarcinoma in the extrahepatic biliary tree (eCCA), although current imaging modalities are limited in their ability to accurately diagnose longitudinal spread. We aimed to develop fluorescence imaging techniques for real-time identification of eCCA using an enzyme-activatable probe, which emits fluorescence immediately after activation by a cancer-specific enzyme. Methods: Using lysates and small tissue fragments collected from surgically resected specimens, we selected the most specific probe for eCCA from among 800 enzyme-activatable probes. The selected probe was directly sprayed onto resected specimens and fluorescence images were acquired; these images were evaluated for diagnostic accuracy. We also comprehensively searched for enzymes that could activate the probe, then compared their expression levels in cancer and non-cancer tissues. Results: Analyses of 19 samples (four cancer lysates, seven non-cancer lysates, and eight bile samples) and 54 tissue fragments (13 cancer tissues and 41 non-cancer tissues) revealed that PM-2MeSiR was the most specific fluorophore for eCCA. Fluorescence images of 7 patients were obtained; these images enabled rapid identification of cancerous regions, which closely matched histopathology findings in 4 patients. Puromycin-sensitive aminopeptidase was identified as the enzyme that might activate the probe, and its expression was upregulated in eCCA. Conclusion: Fluorescence imaging with PM-2MeSiR, which may be activated by puromycin-sensitive aminopeptidase, yielded generally high accuracy. This technique may be useful for real-time identification of the spread of eCCA during surgery and endoscopic examinations.

The management of hepatocellular carcinoma around the world: a comparison of guidelines from 2001 to 2011.

In the past 10 years, many guidelines for hepatocellular carcinoma (HCC) have been published worldwide. To promote standard care for HCC, we systematically evaluated 17 current guidelines for HCC around the world, including 5 guidelines from the USA, 7 from Asia and 5 from Europe, according to the selection criteria of credibility influence and multi-faceted. After a systematic evaluation, we found that these guidelines have both similarities and differences in terms of what organizations or bodies drafted the guidelines and the approach, applicability, content and recent updates of the guidelines as well as in terms of diagnostic and treatment algorithms. The differences could be attributed to various aetiological factors, high-risk patients, health systems, health resources, medical technology, treatment choices and income levels in different countries. Besides, although the full implementation of guidelines could benefit clinicians, patients and authorities, there is still a gap between projected goals and implementation. The factors potentially influencing implementation are what organizations or bodies are drafting guidelines, content and emphasis, modification and consistency of guidelines. Comparative analysis suggested that countries pay close attention to targeted audiences, a basis in evidence, a basis in available resources, applicable patients and systematic evaluation when establishing and implementing domestic guidelines for HCC.

Targeting c-Met as a promising strategy for the treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a severe condition that is found worldwide. Liver transplantation, surgical resection, and local-regional therapy such as transarterial chemoembolization have made great progress and play a dominant role in HCC management. However, the high frequency of tumor recurrence and/or metastasis after those treatments acquires systematic drug intervention. The approval of sorafenib, an agent that targets receptor tyrosine kinases (RTKs), as the first effective drug for systemic treatment of HCC represents a milestone in treatment of this disease. As a typical member of the RTK family, c-Met represents an intriguing target for cancer therapy. However, the role of the c-Met signal transduction pathway is less unambiguous in HCC pathology, giving rise to concerns about the feasibility of utilizing c-Met targeting approaches for HCC treatment. Recently, studies on des-γ-carboxy prothrombin, an abnormal cytokine secreted by HCC cells, by the current authors and other researchers have highlighted the critical role of c-Met signaling in HCC progression. This review takes a second look at the c-Met signal transduction pathway and discusses the possibility of targeting c-Met as a therapeutic strategy for HCC treatment.

Partially MHC-matched donor CD8+ T cells are indispensable for switching to splenocytic chimerism.

BACKGROUND: We have shown that partially major histocompatibility complex (MHC)-matched splenocytes can replace bone marrow (BM) and maintain skin grafts by establishing splenocytic chimeras. Our aim was to identify the population of splenocytes that are indispensible for switching from BM to splenocytic chimerism. METHODS: C3H/B6D2F1 mixed BM chimeras were established in lethally irradiated C3H mice. Skin grafts from C57BL/6 mice were transplanted 30 d later. After an additional 30 d, splenocytes from B6C3F1 mice were transplanted to establish splenocytic BM chimeras using total splenocytes (group A), CD90(+)-depleted splenocytes (group B), CD4(+)-depleted splenocytes (group C), or CD8(+)-depleted splenocytes (group D). RESULTS: In group A, the BM switched to splenocyte-derived BM chimeras. Total B6C3F1 splenocytes created stable splenocyte BM chimeras that permitted long-term retention of skin grafts, without rejection. In groups B and D, the splenocytes failed to replace the recipient BM, and there was no decrease in the number of recipient-derived BM cells compared with group A from d 14 to 28 after splenocyte injection, as was the case for CD8(+) T cells. In group C mice, the recipient BM was slowly and incompletely replaced. CONCLUSIONS: Partially MHC-matched donor CD8(+) T cells are indispensable for generating splenocyte chimeras in BM and maintaining allogeneic skin grafts.

Chimeric acceleration by donor CD4+CD25+T-reg depleted fraction in splenocyte transplantation.

BACKGROUND: We have established a splenocytic chimera model that can induce donor-specific tolerance and reconstitute the recipient immune system by donor splenocytes. AIM: To accelerate such reconstitution, we investigated the role of donor-derived CD4(+)CD25(+) regulatory T-cells (T-reg). METHODS: We established C3H/B6D2F1 mixed bone marrow chimeras in lethally irradiated C3H mice. We transplanted skin grafts from C57BL/6 mice 30 d later. After an additional 30 d, we transplanted the following types of splenocytes from B6C3F1 mice: total splenocytes (group A), CD4(+)CD25(+) T-reg depleted splenocytes (group B), CD8(+)-depleted splenocytes (group C), and CD4(+)-depleted splenocytes (group D). We assessed class I major histocompatibility complex, percentage of chimeric cells in peripheral blood, and survival of skin grafts in each group. RESULTS: Group A and B mice switched to splenocytic chimeras, permitting the long-term survival of skin grafts. The proportions of H-2K(b+)H-2K(k-) cells in group B were significantly lower than those in group A on day 14 (0.47% ± 0.68% versus 9.49% ± 8.30%; P = .01) and day 21 (0.16% ± 0.25% versus 3.35% ± 2.78%; P = .01). The initial increase in the proportion of H-2K(b+)H-2K(k+) double-positive cells in group B was faster than that in group A (from 0.33% ± 0.10% versus. 0.39% ± 0.14% before splenocyte injection to 39.03% ± 30.50% versus 10.73% ± 11.54% on day 7; P = .02). The initial increase in the proportion of CD8(+) T-cells was faster in group B than in group A (from 2.72% ± 0.52% versus 2.49% ± 1.07% before splenocyte injection to 29.61% ± 26.72% versus 4.92% ± 1.56% on day 7; P = .04). CONCLUSIONS: The depletion of CD4(+)CD25(+) T-reg fraction in donor splenocytes can accelerate switching to splenocytic chimera.

Bufalin and cinobufagin induce apoptosis of human hepatocellular carcinoma cells via Fas- and mitochondria-mediated pathways.

Bufadienolides bufalin and cinobufagin are cardiotonic steroids isolated from the skin and parotid venom glands of the toad Bufo bufo gargarizans Cantor. They have been shown to induce a wide spectrum of cancer cell apoptosis. However, the detailed molecular mechanisms of inducing apoptosis in hepatocellular carcinoma (HCC) are still unclear. In the present study, the apoptosis-inducing effect of bufalin and cinobufagin on HCC cell line HepG(2) was investigated. We found bufalin and cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells. This apoptotic induction was associated with an increase in Fas, Bax and Bid expression, a decrease in Bcl-2 expression, disruption of the mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, -8, -9 and -10, and the cleavage of poly(ADP-ribose)polymerase (PARP), which indicated that bufalin and cinobufagin induced apoptosis through both Fas- and mitochondria-mediated pathways. In addition, caspase activation during bufalin- and cinobufagin-induced apoptosis was further confirmed by caspase-3 inhibitor Z-DEVD-FMK, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK and caspase-10 inhibitor Z-AEVD-FMK. The results showed that bufalin- and cinobufagin-induced apoptosis was blocked by these inhibitors and particularly by caspase-10 inhibitor. Taken together, bufalin and cinobufagin induce apoptosis of HepG(2) cells via both Fas- and mitochondria-mediated pathways, and a Fas-mediated caspase-10-dependent pathway might play a crucial role.

Clinical and molecular insights into the hepatocellular carcinoma tumour marker des-γ-carboxyprothrombin.

Des-γ-carboxyprothrombin (DCP) is known as a tumour marker for hepatocellular carcinoma (HCC). Various tumour markers have been developed for serological diagnosis of cancers, including HCC, in order to increase the survival rate of cancer patients. The currently recommended combined testing of DCP and α-fetoprotein (AFP) or Lens culinaris agglutinin-reactive fraction of α-fetoprotein has been established to diagnose HCC. This combined testing using several tumour markers helps to increase the sensitivity of diagnosis of HCC, thus significantly increasing the clinical usefulness of DCP. The excessive production of DCP may be related to worse tumour behaviour, such as the presence of vascular invasion and intrahepatic metastasis of HCC cells. A high level of DCP was suggested to be useful as one of the factors in new recipient selection criteria of liver transplantation. The clinical use of DCP, therefore, might play a vital role in predicting tumour behaviour in patients with HCC. That said, the basic mechanism of DCP production has not been fully clarified. Various factors such as vitamin K(2) and γ-glutamyl carboxylase may contribute to the production of DCP and have a complex relationship. Moreover, recent studies have revealed that DCP functions as a growth factor and might play significant roles in cancer progression. Thus, DCP represents a potential target of drug discovery to establish new chemotherapeutic strategy for HCC. However, various issues have to be resolved to construct a novel therapy for HCC-targeting DCP. Innovation is required to make further progress in examining DCP.

Fluorescence Imaging Using Enzyme-Activatable Probes for Real-Time Identification of Pancreatic Cancer.

INTRODUCTION: Radical resection is the only curative treatment for pancreatic cancer, which is a life-threatening disease. However, it is often not easy to accurately identify the extent of the tumor before and during surgery. Here we describe the development of a novel method to detect pancreatic tumors using a tumor-specific enzyme-activatable fluorescence probe. METHODS: Tumor and non-tumor lysate or small specimen collected from the resected specimen were selected to serve as the most appropriate fluorescence probe to distinguish cancer tissues from noncancerous tissues. The selected probe was sprayed onto the cut surface of the resected specimen of cancer tissue to acquire a fluorescence image. Next, we evaluated the ability of the probe to detect the tumor and calculated the tumor-to-background ratio (TBR) by comparing the fluorescence image with the pathological extent of the tumor. Finally, we searched for a tumor-specific enzyme that optimally activates the selected probe. RESULTS: Using a library comprising 309 unique fluorescence probes, we selected GP-HMRG as the most appropriate activatable fluorescence probe. We obtained eight fluorescence images of resected specimens, among which four approximated the pathological findings of the tumor, which achieved the highest TBR. Finally, dipeptidyl-peptidase IV (DPP-IV) or a DPP-IV-like enzyme was identified as the target enzyme. CONCLUSION: This novel method may enable rapid and real-time visualization of pancreatic cancer through the enzymatic activities of cancer tissues.

Anti-hepatitis B virus activities of cinobufacini and its active components bufalin and cinobufagin in HepG2.2.15 cells.

Cinobufacini (Huachansu) is a Chinese medicine prepared from the skin of Bufo bufo gargarizans Cantor (Bufonidae), which has long been used in traditional Chinese medicine (TCM). The aim of present study was to examine the anti-hepatitis B virus (HBV) activities of cinobufacini and its active components bufalin and cinobufagin in the human HBV-transfected cell line HepG2.2.15. The hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core-related antigen (HBcrAg) concentrations in cell culture medium were determined by chemiluminescent enzyme immunoassay after HepG2.2.15 cells were respectively treated with different concentrations of cinobufacini, bufalin, and cinobufagin for 3 or 6 d. HBV DNA and mRNA were determined using transcription-mediated amplification and real-time polymerase chain reaction (PCR), respectively. On d 3, cinobufacini at a concentration of 1 µg/ml had no activity against HBV virological markers. However, on d 6, cinobufacini at 1 µg/ml effectively inhibited the secretion of HBsAg, HBeAg, and HBcrAg by 29.58, 32.87, and 42.52%. It was more potent than the positive control lamivudine (100 µg/ml). Bufalin and cinobufagin slightly inhibited HBV antigen secretion. Treatment with cinobufacini, bufalin, or cinobufagin had no anti-HBV effect on DNA in cell culture medium. Consistent with the HBV antigen reduction, HBV mRNA expression was markedly inhibited in comparison to the control when HepG2.2.15 cells were treated with cinobufacini, bufalin, or cinobufagin. Results suggested that cinobufacini had more potent activity against HBV antigen secretion than its components bufalin and cinobufagin and this inhibitory role was attributed to the specific inhibition of HBV mRNA expression.

Knockdown of E2F5 induces cell death via the TP53­dependent pathway in breast cancer cells carrying wild­type TP53.

E2F transcription factor 5 (E2F5) is a member of the E2F family of transcription factors, which are involved in regulation of various cellular processes, including cellular proliferation, apoptosis, differentiation and DNA damage response. Previously, we reported that E2F5 was aberrantly overexpressed in estrogen receptor (ER)­negative breast cancer, especially in triple­negative breast cancer (TNBC). In the present study, it was revealed that E2F5 gene silencing caused a significant reduction in the proliferation rate of breast cancer MCF7 (ER­positive luminal­type) and MDA­MB­231 (TNBC­type) cells. Additional experiments demonstrated that E2F5 knockdown triggered cell death of MCF7 cells but not MDA­MB­231 cells. As MCF7 and MDA­MB­231 cells carry wild­type and mutant TP53, respectively, and BT474 (ER­negative, HER2­positive type) carrying mutant TP53 exhibited similar results to MDA­MB­231, the possible effects of E2F5 gene depletion on cell death­related TP53­target gene expression were examined. Real­time RT­qPCR analysis revealed that knockdown of E2F5 in MCF7 cells stimulated cell death­related transcription of TP53­target genes such as BAX, NOXA and PUMA. For MDA­MB­231 and BT474 cells, E2F5 gene silencing revealed marginal effects on the expression of TP53 target genes. In addition, silencing of TP53 abrogated the effect of E2F5 silencing in MCF7 cells. Collectively, the present results indicated that E2F5 participated in the carcinogenesis of breast cancer carrying wild­type TP53 through suppression of TP53, while E2F5 had a pro­proliferative but not anti­apoptotic effect on breast cancer with TP53 mutation.

KL-6 mucin in metastatic liver cancer tissues from primary colorectal carcinoma.

BACKGROUND/AIMS: KL-6 mucin is MUC1 bearing sialylated carbohydrate epitopes recognized by KL-6 antibody. This study aimed to assess subcellular localization of KL-6 mucin in liver metastatic tissues from colorectal carcinoma and hepatocellular carcinoma tissues. METHODOLOGY: Tissue samples were collected from 56 patients with liver metastasis of colorectal carcinoma and 92 patients with hepatocellular carcinoma who underwent a hepatectomy. Tissues were subjected to immunohistochemical analysis using KL-6 antibody. RESULTS: All 56 cases with metastatic liver cancer showed positive staining for KL-6 mucin in cancer tissues but not in non-cancerous tissues. All staining was observed in the circumferential membrane and/or cytoplasm of the cancer cells. In contrast, no staining for KL-6 mucin was observed in either cancerous or non-cancerous tissues of the 92 patients with hepatocellular carcinoma. CONCLUSIONS: KL-6 mucin may be an indicator for liver metastasis of colorectal carcinoma. Additionally, detection of KL-6 mucin may be helpful in distinguishing hepatocellular carcinoma from metastatic liver cancer.

The impact of apurinic-apyrimidinic endonuclease I on hepatocyte immuno-inflammatory factors and cell apoptosis.

To explore the effect of apurinic-apyrimidinic endonuclease I (APE-1) on hepatocyte immune inflammatory factors and cell apoptosis. The gene expression profiles of peripheral blood of patients with or without immune tolerance after liver transplantation were obtained from the Gene Expression Omnibus (GEO). Differentially expressed genes were analyzed with a program in the R language, and the APE-1 gene was identified as a gene related to immune tolerance of liver transplantation. Four APE-1 shRNA vectors were constructed in parallel and verified as correct using plasmid sequencing, real-time PCR, and Western blotting. An APE-1 overexpression vector was similarly constructed and verified as correct. The STRING website predicted the protein-protein interaction network of APE-1. ELISA was used to detect the effects of APE-1 silencing and overexpression on inflammatory cytokines IL-1ß, IL-10, TNFα, and INF-γ in the control group, APE-1-silenced group, and APE-1 overexpression group. Flow cytometry was used to detect apoptosis in each group. Forty differentially expressed genes related to immune tolerance after liver transplantation were screened, and the highly expressed gene APE-1 was selected. The best APE-1 shRNA_1 vector and APE-1 overexpression vector were obtained. APE-1 is predicted to interact with ANP32A, FEN1, HMGB2, LIG1, MUTYH, NTHL1, OGG1, PCNA, POLB, SET, and other proteins. APE-1 silencing resulted in a significant increase in the expression of the inflammatory factors IL-1ß, IL-10, TNFα, and INF-γin L-02 cells. In contrast, the expression of APE-1 led to a significant decrease in the expression of inflammatory factors. APE-1 silencing significantly increased the rate of apoptosis of L-02 cells, and APE-1 overexpression resulted in a significant decrease in the rate of apoptosis of L-02 cells. In conclusion APE-1 affects the expression of inflammatory factors and apoptosis in L-02 cells, so it may be a key gene in immune tolerance of liver transplantation.

A novel liver-specific fluorescent anti-cancer drug delivery system using indocyanine green.

Indocyanine green (ICG) accumulates only in hepatocytes and their malignant counterpart, hepatocellular carcinoma (HCC). We have developed ICG-conjugated anti-cancer drugs and noted their significant accumulation in HCC cells both in vitro and in vivo. ICG-conjugated gemcitabine was less toxic to normal cells and it had superior anti-tumor action compared to gemcitabine alone in a subcutaneous tumor xenograft. ICG conjugation can provide a novel fluorescent drug delivery system for treatment of liver cancer and this system can be used to both diagnose and treat HCC.

Silencing of EPHB2 promotes the epithelial-mesenchymal transition of skin squamous cell carcinoma-derived A431 cells.

Erythropoietin-producing hepatocellular (Eph) receptors and their ligand ephrins serve crucial roles in the interactions among epithelial cells. Eph receptor/ephrin signaling regulates cell functions, including proliferation, differentiation and migration, via these cell-cell interactions. We reported previously that EPHB2, a member of the Eph receptor family, was highly expressed in chemically induced cutaneous squamous cell carcinoma (cSCC) tissues in mice. Although the higher expression level of EPHB2 has been observed in various human cancers, its roles in the development and progression of cancers are still unclear. In the present study, the functional implications of EPHB2 in the acquisition of malignant phenotypes of cSCC cells was investigated. Silencing of EPHB2 in the human cSCC cell line A431 induced epithelial-mesenchymal transition (EMT)-like morphological changes accompanied by a significant upregulation of epithelial-mesenchymal transition-associated genes such as zinc finger E-box binding homeobox 1/2. In addition, silencing of EPHB2 suppressed anchorage-independent cell growth under 3D culture conditions. Consistent with these observations, EPHB2 exhibited higher levels of expression in tumor spheres formed under 3D culture conditions than in cells cultured in adherent form, and the expression pattern of EMT markers indicated that EMT was suppressed in tumor spheres. The results of the present study indicated that EPHB2 serves a pivotal role in promoting the anchorage-independent growth of A431 cells through the suppression of EMT.