Splashed E-box and AP-1 motifs cooperatively drive regeneration response and shape regeneration abilities.

Injury triggers a genetic program that induces gene expression for regeneration. Recent studies have identified regeneration-response enhancers (RREs); however, it remains unclear whether a common mechanism operates in these RREs. We identified three RREs from the zebrafish fn1b promoter by searching for conserved sequences within the surrounding genomic regions of regeneration-induced genes and performed a transgenic assay for regeneration response. Two regions contained in the transposons displayed RRE activity when combined with the -0.7 kb fn1b promoter. Another non-transposon element functioned as a stand-alone enhancer in combination with a minimum promoter. By searching for transcription factor-binding motifs and validation by transgenic assays, we revealed that the cooperation of E-box and activator protein 1 motifs is necessary and sufficient for regenerative response. Such RREs respond to variety of tissue injuries, including those in the zebrafish heart and Xenopus limb buds. Our findings suggest that the fidelity of regeneration response is ensured by the two signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome has helped shape the regenerative capacities during evolution.

Hepatic expression of sodium-glucose cotransporter 2 (SGLT2) in patients with chronic liver disease.

Sodium-glucose cotransporter 2 (SGLT2) occurs in the proximal renal tubule cells. We investigate the hepatic expression of SGLT2 and its related factors in patients with chronic liver disease. This is a retrospective human study. The liver tissues were biopsied from patients with chronic liver disease (n = 30). The expression levels of SGLT2 were evaluated by immunostaining. Furthermore, the undirected graphical model was used to identify factors associated with hepatic expression levels of SGLT2. The SGLT2 expression was observed in not only the kidney, but also the liver in immunostaining (SGLT2 intensity: kidney 165.8 ± 15.6, liver 114.4 ± 49.0 arbitrary units, P < 0.01) and immunoblotting. There was no significant difference in hepatic expression of SGLT2 in the stratified analysis according to age, sex, BMI, and the severity of the liver disease. In the undirected graphical model, SGLT2 directly interacted with various factors such as sex, fatty change, neutrophil-to-lymphocyte ratio, triglyceride, hemoglobin A1c, creatinine, and albumin (partial correlation coefficient 0.4-0.6 for sex and 0.2-0.4 for others). The expression of SGLT2 was observed in the hepatocytes of patients with chronic liver disease. The undirected graphical model demonstrated the complex interaction of hepatic expression levels of SGLT2 with gender, inflammation, renal function, and lipid/glucose/protein metabolisms.

The Expression of PEDF and its Putative Receptors in Hepatocellular Carcinoma and Background Liver Tissue.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) remains one of the biggest medical issues. Pigment epithelial-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors. PEDF interacts with its two receptors, adipose triglyceride lipase (ATGL) and laminin receptor (LR). MATERIALS AND METHODS: We conducted immunohistochemical staining for PEDF, LR and ATGL in 151 resected HCCs and their background liver tissues. RESULTS: High expression of LR in HCC was associated with high histological grade and portal vein invasion, while high expression of PEDF in HCC was associated with absence of portal vein invasion. High LR expression in background liver was statistically associated with low serum albumin levels and was an independent prognostic factor of worse outcomes. No cases with more than 5% fatty degeneration in the background liver tissue showed high PEDF expression. CONCLUSION: PEDF/LR/ATGL could be potential biomarkers in HCC and various chronic hepatic disorders.

Insights regarding skin regeneration in non-amniote vertebrates: Skin regeneration without scar formation and potential step-up to a higher level of regeneration.

Skin wounds are among the most common injuries in animals and humans. Vertebrate skin is composed of an epidermis and dermis. After a deep skin injury in mammals, the wound heals, but the dermis cannot regenerate. Instead, collagenous scar tissue forms to fill the gap in the dermis, but the scar does not function like the dermis and often causes disfiguration. In contrast, in non-amniote vertebrates, including fish and amphibians, the dermis and skin derivatives are regenerated after a deep skin injury, without a recognizable scar remaining. Furthermore, skin regeneration can be compared with a higher level of organ regeneration represented by limb regeneration in these non-amniotes, as fish, anuran amphibians (frogs and toads), and urodele amphibians (newts and salamanders) have a high capacity for organ regeneration. Comparative studies of skin regeneration together with limb or other organ regeneration could reveal how skin regeneration is stepped up to a higher level of regeneration. The long history of regenerative biology research has revealed that fish, anurans, and urodeles have their own strengths as models for regeneration studies, and excellent model organisms of these non-amniote vertebrates that are suitable for molecular genetic studies are now available. Here, we summarize the advantages of fish, anurans, and urodeles for skin regeneration studies with special reference to three model organisms: zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and Iberian ribbed newt (Pleurodele waltl). All three of these animals quickly cover skin wounds with the epidermis (wound epidermis formation) and regenerate the dermis and skin derivatives as adults. The availability of whole genome sequences, transgenesis, and genome editing with these models enables cell lineage tracing and the use of human disease models in skin regeneration phenomena, for example. Zebrafish present particular advantages in genetics research (e.g., human disease model and Cre-loxP system). Amphibians (X. laevis and P. waltl) have a skin structure (keratinized epidermis) common with humans, and skin regeneration in these animals can be stepped up to limb regeneration, a higher level of regeneration.

Dotinurad: a clinical pharmacokinetic study of a novel, selective urate reabsorption inhibitor in subjects with hepatic impairment.

BACKGROUND: Dotinurad is a novel, selective urate reabsorption inhibitor, which reduces serum uric acid levels by inhibiting the urate transporter 1 (URAT1). We compared the pharmacokinetics (PK), pharmacodynamics (PD), and safety of dotinurad in subjects with hepatic impairment and normal hepatic function. METHODS: This was a multicenter, open-label, single dose study. A total of 24 subjects were divided into four groups: the normal hepatic function group and the mild, moderate, and severe hepatic impairment groups. The primary endpoints were changes in plasma dotinurad levels and PK parameters. RESULTS: The geometric mean ratio of the maximum plasma concentration (Cmax) [two-sided 90% confidence interval (CI)] of dotinurad in in the mild, moderate, and severe hepatic impairment groups relative to that in the normal hepatic function group was 0.840 (0.674-1.047), 0.798 (0.653-0.976), and 0.747 (0.570-0.979), respectively, showing a lower Cmax in the moderate and severe hepatic impairment groups. Following adjustment for body weight, only the moderate hepatic impairment group had a lower Cmax than the normal hepatic function group. No meaningful differences in other PK parameters were observed between the groups. Regarding the PD of dotinurad, the changes in serum uric acid levels after dosing were similar in all groups. As for safety, no noteworthy concerns were raised in relation to any group. CONCLUSION: The study revealed no clinically meaningful influence of hepatic impairment on the PK, PD, or safety of dotinurad. These findings indicate possibility that dotinurad can be used without dose adjustment in patients with hepatic impairment.

Pharmacokinetics and Safety of Single-Dose Esaxerenone in Japanese Subjects with Mild to Moderate Hepatic Impairment.

INTRODUCTION: The mineralocorticoid receptor (MR) blocker esaxerenone is a new treatment for hypertension in Japan and under development for treatment of diabetic nephropathy. Hepatic impairment is known to impact the pharmacokinetics (PKs) of other MR blocking drugs. The aim of the present study was to characterise the PKs and safety of a single oral dose of esaxerenone in Japanese subjects with mild-moderate hepatic impairment. METHODS: In this open-label, parallel-group study, subjects with mild (Child-Pugh grade A) or moderate (grade B) hepatic impairment, and healthy controls with normal hepatic function matched by age and BMI (all groups n = 6), received a single 2.5-mg oral dose of esaxerenone. Plasma concentrations were measured by liquid chromatography-tandem mass spectrometry, and PK parameters were calculated using non-compartmental analysis. RESULTS: Geometric least-squares mean (GLSM) ratios (90% confidence intervals [CIs]) for area under the plasma concentration-time curve (up to the last quantifiable time, up to infinity) in subjects with mild hepatic impairment versus normal hepatic function were 0.837 (0.637, 1.099) and 0.824 (0.622, 1.092), respectively. Corresponding values for moderate hepatic impairment versus normal hepatic function were 1.078 (0.820, 1.415) and 1.098 (0.829, 1.454). GLSM ratios (90% CIs) for peak plasma concentration (Cmax) were 0.959 (0.778, 1.182) for mild hepatic impairment versus normal hepatic function and 0.804 (0.653, 0.992) for moderate hepatic impairment versus normal hepatic function. Time to Cmax and clearance values were comparable between groups. The incidence of adverse events (AEs) was 16.7% in the moderate hepatic impairment and normal hepatic function groups. One serious AE (hepatic encephalopathy) occurred in one subject with moderate hepatic impairment. CONCLUSIONS: Mild to moderate hepatic impairment had no clinically relevant effect on esaxerenone exposure. Esaxerenone dosage adjustment based on PKs is unlikely to be needed in patients with mild to moderate hepatic impairment. TRIAL REGISTRATION: JapicCTI-163339. FUNDING: Daiichi Sankyo Co., Ltd.

Pharmacokinetics and Safety of DS-8500a, an Antidiabetic Drug, in Japanese Subjects with Hepatic or Renal Impairment: A Single-Center, Open-Label, Single-Dose Study.

INTRODUCTION: The pharmacokinetics, safety, and tolerability of DS-8500a (a G protein receptor 119 agonist) up to 100 mg have been investigated in healthy Japanese adults. The objective of this study was to evaluate the effects of hepatic or renal impairment on the pharmacokinetics of a single 25-mg oral dose of DS-8500a. METHODS: This single-center, open-label study enrolled subjects into eight groups according to hepatic function (normal; mild or moderate impairment) and renal function [normal; mild, moderate, or severe impairment; and end-stage renal disease (ESRD)]. Drug concentrations were measured by liquid-chromatography tandem mass spectrometry. Pharmacokinetic parameters were evaluated by non-compartmental analysis. Adverse events (AEs) were evaluated for safety. RESULTS: The hepatic and renal groups enrolled 15 and 30 subjects, respectively. Pharmacokinetic parameters of DS-8500a were comparable between the normal hepatic function and mild hepatic impairment groups, but the mean area under the concentration-time curve (AUC) was 1.37-fold higher, and the half-life was longer in the moderate hepatic impairment group compared with the normal hepatic function group. The maximum concentration (Cmax) and AUC values were 0.704- and 0.609-fold lower, respectively, in the ESRD group compared with the values in the other renal impairment groups; no clear differences in AUC and time to Cmax were observed in the normal function and mild, moderate, and severe renal impairment groups. There was no relationship between apparent total body clearance and estimated glomerular filtration rate. The incidence of AEs was similar among all groups. CONCLUSION: DS-8500a exposure in the mild hepatic impairment and mild to severe renal impairment groups was similar to that in the corresponding normal hepatic and renal function groups, but dose adjustments may be required in those with moderate hepatic impairment and ESRD. TRIAL REGISTRATION: Japic CTI-No. 163135. FUNDING: Daiichi Sankyo Co. Ltd., Tokyo, Japan.

STAT3 deficiency prevents hepatocarcinogenesis and promotes biliary proliferation in thioacetamide-induced liver injury.

AIM: To elucidate the role of STAT3 in hepatocarcinogenesis and biliary ductular proliferation following chronic liver injury. METHODS: We investigated thioacetamide (TAA)-induced liver injury, compensatory hepatocyte proliferation, and hepatocellular carcinoma (HCC) development in hepatic STAT3-deficient mice. In addition, we evaluated TAA-induced biliary ductular proliferation and analyzed the activation of sex determining region Y-box9 (SOX9) and Yes-associated protein (YAP), which regulate the transdifferentiation of hepatocytes to cholangiocytes. RESULTS: Both compensatory hepatocyte proliferation and HCC formation were significantly decreased in hepatic STAT3-deficient mice as compared with control mice. STAT3 deficiency resulted in augmentation of hepatic necrosis and fibrosis. On the other hand, biliary ductular proliferation increased in hepatic STAT3-deficient livers as compared with control livers. SOX9 and YAP were upregulated in hepatic STAT3-deficient hepatocytes. CONCLUSION: STAT3 may regulate hepatocyte proliferation as well as transdifferentiation into cholangiocytes and serve as a therapeutic target for HCC inhibition and biliary regeneration.

Pigment Epithelium-Derived Factor (PEDF) Prevents Hepatic Fat Storage, Inflammation, and Fibrosis in Dietary Steatohepatitis of Mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.

Pancreatic Neuroendocrine Tumors and EMT Behavior Are Driven by the CSC Marker DCLK1.

Doublecortin-like kinase 1 (DCLK1), a marker for intestinal and pancreatic cancer stem cells, is highly expressed in neuroblastomas. This study was conducted to assess DCLK1 expression levels in pancreatic neuroendocrine tumor (PNET) tissues and to explore the roles of this molecule in clinical tissue from multiple PNET patients, cells (BON1, QGP1, and CM) and tumor xenografts. Immunohistochemically, all PNET tissues highly and diffusely expressed DCLK1 as a full-length isoform, identical to that detected in primary liver NETs. A DCLK1-overexpressing PNET cell line (QGP1-DCLK1) exhibited epithelial-mesenchymal transition (EMT)-related gene signatures, and robust upregulation of Slug (SNAI2), N-Cadherin (CDH2), and Vimentin (VIM) was validated by real-time PCR and immunoblotting. QGP1-DCLK1 cells had increased cell migration in a wound-healing assay and formed significantly larger xenograft tumors in nude mice. The factors involved in the formation of the fast-growing tumors included p-FAK (on Tyr925), p-ERK1/2, p-AKT, Paxillin, and Cyclin D1, which upon knockdown or pharmacologic inhibition of DCLK1 abolished the expression of these molecules. In conclusion, robust and ubiquitous expression of DCLK1 was first demonstrated here in human PNET tissue specimens and cells. DCLK1 characterized the PNET cell behavior, inducing p-FAK/SLUG-mediated EMT. These findings suggest the possibility of developing novel therapeutic strategies against PNETs by targeting DCLK1.Implications: Evidence here reveals that human PNETs diffusely and robustly express the cancer stem cell marker DCLK1, which drives SLUG-mediated EMT, and suggests that NETs share biological features for druggable targets with other tumors, including neuroblastoma that also highly expresses DCLK1. Mol Cancer Res; 15(6); 744-52. ©2017 AACR.

High expression of the putative cancer stem cell marker, DCLK1, in rectal neuroendocrine tumors.

Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein, is known to regulate neuronal differentiation, migration and neurogenesis. Recent evidence suggests that the protein is a putative marker for intestinal and pancreatic stem cells, including their cancer stem cell counterparts. The present study conducted immunohistochemical analyses for DCLK1 and the stemness marker, NANOG, in human intestinal neuroendocrine tumors (NETs), as their expression had not been previously investigated in these tumors. Eighteen patients with endoscopically resected rectal NETs were enrolled in the study. The mean age of the patients was 51 years old. The mean diameter of the resected tumors was 5.2 mm, and a histological diagnosis of NET grade G1 was formed for all tumors. Immunohistochemical analysis was performed not only for DCLK1, but also for the known NET markers, synaptophysin, chromogranin A and cluster of differentiation (CD)56. The intensity and distribution of staining were scored on a scale of 0-3 and 0-2, respectively. The sum of the scores was calculated for each specimen. Co-expression of DCLK1 and NANOG was also examined. The mean scores for DCLK1 and synaptophysin were significantly higher than those for chromogranin A (P<0.0001) and CD56 (P<0.01). There were no significant differences in the scores between DCLK1 and synaptophysin or between chromogranin A and CD56. Notably, NANOG was expressed in high quantities in all the tumor tissues studied, showing clear co-expression with DCLK1. In conclusion, DCLK1 may be a novel marker for rectal NET, potentially indicating the presence of the stemness gene product, NANOG.

The synthesis and dynamic structures of multinuclear complexes of large porphyrinoids expanded by phenylene and thienylene spacers.

1,3-Bis(2-pyrryl)benzene was used to prepare dibenziamethyrin, in which two pyrrole units of [24]amethyrin(1.0.0.1.0.0) are replaced by benzene. 1,4-Bis(2-pyrryl)benzene, 2,5-bis(2-pyrryl)thiophene, and 4,4'-bis(2-pyrryl)biphenyl were also used in place of 2,2'-bipyrrole to give expanded analogues of [24]rosarin(1.0.1.0.1.0) and [32]octaphyrin(1.0.1.0.1.0.1.0). These large porphyrinoids can incorporate multiple metal units of Rh(CO)2 and Pd(π-allyl) with considerable deviation of the metal atoms from the dipyrrin planes, evidenced by X-ray crystallography. The coordinated Rh(CO)2 group shuttled between both sides of the macrocycle; the rate was dependent on the spacer, ring size, and number of metal atoms. Variable temperature (1) H NMR spectroscopy showed that the tris-rhodium complexes of the expanded rosarins with 1,4-phenylene or 2,5-thienylene spacers adopt a C3v -symmetric form and a Cs -symmetric form as a result of the Rh(CO)2 groups hopping through the macrocycle cavity. The C3v -symmetric form has a greater dipole moment and, therefore, is favored in solvents of greater polarity. The Rh(CO)2 groups in the tris-rhodium complex of the expanded rosarin with 4,4'-biphenylene spacers hop so fast that an averaged spectral pattern (D3h ) was seen in the (1) H NMR spectrum, even at -60 °C. Expanded octaphyrins with 1,4-phenylene and 2,5-thienylene spacers bind four Rh(CO)2 groups outside the macrocycle cavity to form a D2d -symmetric saddle-shaped structure that did not show any dynamic behavior on the NMR timescale, even at 80 °C. This tetranuclear complex is one of the largest porphyrinoid metal complexes characterized by X-ray crystallography to date.

Pigment epithelium-derived factor (PEDF) inhibits survival and proliferation of VEGF-exposed multiple myeloma cells through its anti-oxidative properties.

Vascular endothelial growth factor (VEGF) has been reported not only to induce angiogenesis within the bone marrow, but also directly stimulate the proliferation and survival of multiple myeloma cells, thus being involved in the development and progression of this second most common hematological malignancy. We, along with others, have found that pigment epithelium-derived factor (PEDF) has anti-angiogenic and anti-vasopermeability properties both in cell culture and animal models by counteracting the biological actions of VEGF. However, effects of PEDF on VEGF-exposed myeloma cells remain unknown. In this study, we examined whether and how PEDF could inhibit the VEGF-induced proliferation and survival of myeloma cells. PEDF, a glutathione peroxidase mimetic, ebselen, or an inhibitor of NADPH oxidase, diphenylene iodonium significantly inhibited the VEGF-induced reactive oxygen species (ROS) generation, increase in anti-apoptotic and growth-promoting factor, myeloid cell leukemia 1 (Mcl-1) expression, and proliferation in U266 myeloma cells. VEGF blocked apoptosis of multiple myeloma cells isolated from patients, which was prevented by PEDF. PEDF also reduced p22phox levels in VEGF-exposed U266 cells. Furthermore, overexpression of dominant-negative human Rac-1 mutant mimicked the effects of PEDF on ROS generation and Mcl-1 expression in U266 cells. Our present study suggests that PEDF could block the VEGF-induced proliferation and survival of multiple myeloma U266 cells through its anti-oxidative properties via suppression of p22phox, one of the membrane components of NADPH oxidase. Suppression of VEGF signaling by PEDF may be a novel therapeutic target for multiple myeloma.

Effects of occupational environmental controls and work management on chromosomal damage in dental technicians in Japan.

Dental technicians use various materials, particularly metal alloys and monomers and polymers based on methylmethacrylate. Environmental hygiene issues surrounding the work of dental technicians have been recognized. Despite the need for observance of occupational environmental controls and work management, compliance among dental laboratories appears to be low. We evaluated the relationship between genotoxic effects in dental technicians and occupational environmental controls and work management. We used cytokinesis-blocked micronuclei (CB-MN) frequencies in peripheral lymphocytes and metal levels in scalp hair. We also assessed nutritional factors related to anti-genotoxic effects using a self-administered brief diet history questionnaire. Study subjects were 54 male dental technicians and 38 male clerical workers. The parameters on the micronuclei (MN) frequency in dental technicians and clerical workers were analyzed by forward stepwise multiple regression analyses. Age (P < 0.01, ß = 0.561), occupation as a dental technician (P < 0.01, ß = 0.636) and aluminum levels in scalp hair (P < 0.05, ß = 0.213) were risk factors that significantly increased MN frequency. The significant parameters on the MN frequency in dental technicians were observance of hand-washing as work management (P < 0.01, ß = -0.304), work period (P < 0.01, ß = 0.509), germanium levels in scalp hair (P < 0.01, ß = -0.314) and workplace (P < 0.05, ß = 0.235). To avoid genotoxic effects, observance of occupational environmental controls and work management is necessary for dental technicians.

Relationship between severity of obsessive-compulsive symptoms and schizotypy in obsessive-compulsive disorder.

PURPOSE: Obsessive-compulsive disorder (OCD) patients exhibit a noninhibition response pattern very similar to that observed in schizotypy patients in cognitive tasks. It has been suggested that the reduced cognitive inhibition observed in both schizotypy and OCD may result in the frequent entry into awareness of unacceptable urges and intrusive thoughts. The aim of this study was to investigate the relationship between the severity of obsession or compulsion and schizotypy in OCD. PATIENTS AND METHODS: Sixty subjects (25 males and 35 females) who were OCD outpatients in the University Hospital at the Kyoto Prefectural University of Medicine during the period 2008-2010 were enrolled in the study. Assessments of these patients were made using the Yale-Brown Obsessive Compulsive Scale (Y-BOCS), the Schizotypal Personality Questionnaire (SPQ), the Hamilton Depression Rating Scale (HAM-D), and the Hamilton Anxiety Rating Scale (HAM-A). The Pearson correlation coefficients between Y-BOCS and SPQ scores were calculated. Furthermore, hierarchical multiple linear regression analyses were conducted to assess whether schizotypy predicted the severity of obsession and compulsion. RESULTS: By calculating the Pearson correlation coefficient, it was found that the Y-BOCS obsession score, not the Y-BOCS compulsion score, was correlated with the SPQ total score. Results of the hierarchical multiple linear regression analysis showed that SPQ total score was a significant predictor of the Y-BOCS obsession score, after accounting for control variables (ie, HAM-D and HAM-A). CONCLUSION: Results of this study showed that the Y-BOCS obsession score, not the Y-BOCS compulsion score, was correlated with the SPQ total score. This finding suggests that OCD patients with an elevated SPQ total score experience a reduction of cognitive inhibition, resulting in the frequent entry into obsession. Future longitudinal studies are recommended to clarify the effect of schizotypy on the clinical course of OCD.

Hepatitis C virus core protein upregulates the expression of vascular endothelial growth factor via the nuclear factor-κB/hypoxia-inducible factor-1α axis under hypoxic conditions.

AIM: Hepatitis C virus (HCV) core protein critically contributes to hepatocarcinogenesis, which is often observed in liver cirrhosis. Since the liver cirrhosis microenvironment is affected by hypoxia, we focused on the possible driving force of HCV core protein on signal relay from hypoxia-inducible factor (HIF)-1α to vascular endothelial growth factor (VEGF). METHODS: Human hepatocellular carcinoma cells stably overexpressing HCV core (Core cells) and NS5A (NS5A cells) were established; empty vector-transfected (EV) cells were used as controls. Hypoxia was induced by oxygen deprivation or by using cobalt chloride (CoCl(2) ). YC-1 was used to inhibit HIF-1α expression. Protein analyses for cultured cells and liver tissues obtained from CoCl(2) -treated HCV core-transgenic (Core-Tg) mice were performed by western blot and/or immunocytochemistry. Cellular mRNA levels were evaluated by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Under hypoxia, the sustained expression of HIF-1α, but not HIF-2α, was profoundly observed in Core cells but, was faint in EV and NS5A cells. Immunocytochemistry revealed increased HIF-1α in the nucleus. HIF-1α mRNA levels were significantly higher in Core cells than in EV cells under both normoxia and hypoxia. The HIF-1α-targeted VEGF and Bcl-xL expressions were increased in Core cells under hypoxia and abolished by YC-1 treatment. Hypoxic liver samples of Core-Tg mice indicated significant increases in both HIF-1α and VEGF expression compared with the wild type. CONCLUSIONS: Hepatitis C virus core protein has the distinct potential to transcriptionally upregulate and sustain HIF-1α expression under hypoxia, thereby contributing to increased VEGF expression, a key regulator in the hypoxic milieu of liver cirrhosis.

PPARγ potentiates anticancer effects of gemcitabine on human pancreatic cancer cells.

In order to improve the prognosis of patients with unresectable pancreatic cancer, there is an urgent need for enhancement of the anticancer effect of gemcitabine (Gem), a first-line drug for the disease. Here, we demonstrated that ligands for peroxisome proliferator-activated receptor γ (PPARγ) such as pioglitazone (Pio) and rosiglitazone potentiated the cytotoxic action of Gem on human pancreatic cancer cells in a dosage-dependent manner. Notably, the synergistic effect was PPARγ-dependent, since the effect was augmented by PPARγ overexpression and was attenuated by both a PPARγ inhibitor (GW9662) and PPARγ-specific siRNA. To further increase the collaborative effect, the histone deacetylase (HDAC) inhibitor valproic acid (VPA), a known potentiator for PPARγ function, was added to the combinatorial treatment, robustly inducing apoptosis mediated by highly expressed death receptors, including Fas/CD95 and DR5. In xenograft tumor experiments in nude mice, Gem plus Pio significantly suppressed tumor growth as compared with the control treatment, while Gem-only treatment did not. Triple treatment with Gem, Pio, and VPA failed to demonstrate a significant antitumor effect when compared with Gem plus Pio in the current setting. Considered together, Gem plus PPARγ ligands, including Pio, may have therapeutic advantage in the treatment of advanced pancreatic cancer. Since Pio is widely used in the treatment of diabetes mellitus, it may become a feasible partner of Gem-based chemotherapy, fine-tuning the strength of the therapy in a dosage-dependent fashion.

N-myc downstream regulated gene1/Cap43 overexpression suppresses tumor growth by hepatic cancer cells through cell cycle arrest at the G0/G1 phase.

N-myc downstream regulated gene-1 (NDRG1)/Cap43 regulates tumor growth and metastasis in various carcinomas. In this study we examined whether and how NDRG1/Cap43 modulates tumor growth by human hepatocellular carcinoma (HCC) cells. NDRG1/Cap43 cDNA was used to transfect HCC cell lines (KIM-1), and stable transfectants overexpressing NDRG1/Cap43 (KIM-1/Cap43) were obtained. Cell cycle analysis showed that KIM-1/Cap43 cells were arrested in the G0/G1 phase. Western blot analysis demonstrated an increase in p21 in KIM-1/Cap43 cells in culture under full confluency as compared with KIM-1/Mock. When KIM-1 cells, which are very low in NDRG1/Cap43 expression, were treated with mimosine, a G0/G1 cell cycle blocker, expression of NDRG1/Cap43 was induced in a dose dependent manner, together with p21 induction and CDK4 reduction. In vivo, KIM-1/Cap43 cells showed markedly decreased tumor growth rates compared with those of KIM-1/Mock. Immunohistochemical staining demonstrated markedly higher p21 labeling index in the KIM-1/Cap43 tumor than KIM-1/Mock tumor, and lower CDK4 and Ki-67 labeling index in the KIM-1/Cap43 than KIM-1/Mock. In order to confirm suppressive effects of NDRG1/Cap43, we further established a stable transfectant expressing NDRG1/Cap43 (HAK-1B/Cap43) using another HCC cell line, HAK-1B. Western blot analysis demonstrated an increase in p21 and a decrease in CDK4 in HAK-1B/Cap43 cells in culture under full confluency as compared with HAK-1B/Mock. HAK-1B/Cap43 also showed decreased tumor growth rates as compared with its control counterpart in vivo. NDRG1/Cap43 overexpression thus induced cell cycle arrest at the G0/G1 phase accompanied by increased p21 and decreased CDK4 expression in HCC cells. NDRG1/Cap43 might play a key role in the cell cycle control of G0/G1 in HCC cells.

The ratio of serum placenta growth factor to soluble vascular endothelial growth factor receptor-1 predicts the prognosis of hepatocellular carcinoma.

Angiogenesis plays crucial roles in development and progression of hepatocellular carcinoma (HCC). Placenta growth factor (PLGF), belonging to vascular endothelial growth factor (VEGF) family, is involved in angiogenesis associated with cancer. Soluble VEGF receptor-1 (sVEGFR1) has been thought to be an intrinsic negative regulator for PLGF. We investigated whether serum PLGF and serum sVEGFR1 is associated with prognosis of HCC. Serum PLGF and sVEGFR1 levels were measured in 145 patients with HCC using enzyme-linked immunosorbent assay. The levels of these factors and the ratio of PLGF to sVEGFR1 were analyzed in relation with clinical parameters. The higher level of sVEGFR1 and the lower ratio of PLGF/sVEGFR1 were significantly associated with poor survival in HCC. Cox regression analysis revealed that the lower ratio of PLGF/sVEGFR1 independently correlated to prognosis of patients with HCC. The ratio of PLGF/sVEGFR1 was independent prognostic indicator for HCC. The ratio of PLGF/sVEGFR1 should be addressed in anti-angiogenic therapy for HCC.

Antiemetic effects of granisetron and dexamethasone combination therapy during cisplatin-containing chemotherapy for head and neck cancer: dexamethasone dosage verification trial.

BACKGROUND: Chemotherapy-induced nausea and vomiting (CINV) remains a significant problem for patients and is associated with a substantial deterioration in quality of life; appropriate use of antiemetic drugs is crucial in maintaining the quality of life in patients undergoing chemotherapy. METHODS: This randomized, crossover trial evaluated the antiemetic efficacy and safety of 8 mg per day (low-dose) and 16 mg per day (standard-dose) dexamethasone, in combination with the 5-HT(3) receptor antagonist granisetron, in 36 patients receiving cisplatin (CDDP)-containing chemotherapy for head and neck cancer. Following chemotherapy, the antinausea/vomiting inhibition rate for each dexamethasone dose was measured. RESULTS: During the 24-h period following administration of chemotherapy (acute phase), the antinausea/vomiting inhibition rates (no nausea and no episodes of vomiting) for 8 mg and 16 mg dexamethasone were comparably high (58.3% and 63.8%, respectively; P = 0.8092). Similar results were seen on days 2-5 following chemotherapy. Efficacy during the acute phase, based on the number of instances of vomiting and degree of nausea, was also comparably high for the two dexamethasone doses (overall efficacy rates were 94.4% and 88.8%, respectively, for 8 mg and 16 mg dexamethasone; P = 0.7637). Both doses maintained an 80% or higher response rate until day 3, and neither dose produced severe side effects. CONCLUSION: The results suggest that granisetron and dexamethasone combination therapy is useful in controlling acute and delayed nausea and vomiting induced by CDDP-containing chemotherapy for head and neck cancer. Furthermore, 8 mg and 16 mg dexamethasone have equivalent antiemetic efficacy.