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Lanthanum carbonate is used to prevent hyperphosphatemia in dialysis patients with chronic renal failure and generally recognized as poorly absorbed by the gastrointestinal tract. However, some clinical cases of lanthanum deposition in the stomach have been shown. In addition, few endoscopic images of lanthanum deposition have been reported, particularly with respect to early-stage gastric cancer. A 64-year-old man with 22 years history of dialysis was treated with lanthanum carbonate for 3 years. With screening esophagogastroduodenoscopy, he was diagnosed with intramucosal gastric cancer, surrounded by the specific endoscopic images of the lanthanum deposition, and underwent endoscopic submucosal dissection. Histopathologically, massive accumulations of macrophages containing fine, granular, eosinophilic materials were observed in the lamina propria. These eosinophilic depositions were present in the lamina propria of the non-tumor region, but not in that of the tumor region. The histological features were further identified as lanthanum phosphate deposition by scanning electron microscopy. This case indicates the clinical significance of lanthanum deposition associated with gastric cancer. It is further required to evaluate more endoscopic images of the gastric mucosa, especially intramucosal cancer, with lanthanum deposition.

Inflammatory pseudotumor (IPT) is a rare benign mass characterized by infiltration of inflammatory cells and proliferation of fibrous tissues. Consistent with increasing knowledge about IgG4-related disease (RD), it has been implicated in the etiology of hepatic IPT, which is pathologically classified into two categories with respect to the proportion of IgG4-positive plasma cells: fibrohistiocytic- and lymphoplasmacytic-type. A 66-year-old man was admitted for treatment of cholecystocholangitis. Incidentally, abdominal computed tomography (CT) revealed an ambiguous low-density mass within segment 4 (S4) of the liver. Magnetic resonance imaging (MRI) showed the typical images of hepatic IPT within S4. Together with CT and MRI imaging, we suspected hepatic IPT, and had the opportunity to biopsy the S4 lesion during surgery for cholecystitis. Histopathological examination of liver tissue showed diffuse fibrous tissues, dense lymphoplasmacytic infiltration, and obliterative phlebitis with no evidence of malignancy. Despite infiltration of IgG4-positive plasma cells, these histological findings corresponded with fibrohistiocytic-type hepatic IPT. Similarly, in the resected gallbladder, relatively abundant IgG4-positive cells were observed, but not entirely consistent with IgG4-RD criteria. Although IgG4 immunostaining can be useful for the classification of hepatic IPT, the present histological tissues were borderline condition defined by IgG4-RD criteria. This rare case of hepatic IPT suggests a future focus on the borderline histological features of IgG4-RD.

Jumonji domain-containing protein 2A (JMJD2A) of the JMJD2 family of histone lysine demethylases has been implicated in tumorigenesis. However, its expression and role in gastric cancer (GC) drug resistance remain unknown. Here, we investigated the role of JMJD2A in GC chemotherapeutic susceptibility and its clinical relevance in GC. We selected 12 relevant genes from previously identified gene signatures that can predict GC susceptibility to docetaxel, cisplatin, and S-1 (DCS) therapy. Each gene was knocked down using siRNA in GC cell lines, and cell viability assays were performed. JMJD2A expression in GC cell lines and tissues was assessed using qRT-PCR and immunohistochemistry, respectively. A JMJD2A downstream target related to drug susceptibility was examined using whole-gene expression array and immunoprecipitation. Among the 12 candidate genes, down-regulation of JMJD2A showed the maximum effect on GC susceptibility to anti-cancer drugs and increased the IC50 values for 5-FU, cisplatin, and docetaxel 15.3-, 2.7-, and 4.0-fold, respectively. JMJD2A was universally expressed in 12 GC cell lines, and its overexpression in GC tissue was positively correlated with tumor regression in 34 DCS-treated patients. A whole-gene expression array of JMJD2A-knockdown GC cells demonstrated a significant decrease in the expression of pro-apoptotic coiled-coil domain containing 8 (CCDC8), a downstream target of JMJD2A. Direct interaction between CCDC8 and JMJD2A was verified using immunoprecipitation. CCDC8 inhibition restored drug resistance to docetaxel, cisplatin, and S-1. Our results indicate that JMJD2A is a novel epigenetic factor affecting GC chemotherapeutic susceptibility, and JMJD2A/CCDC8 is a potential GC therapeutic target.

Background/Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive invasion, early metastasis, and resistance to chemotherapy, leading to a poor prognosis. To clarify the molecular mechanism of these malignant characteristics, we performed a genome-wide microRNA (miRNA) array analysis utilizing micro-cancer tissues from patients with unresectable PDAC (stage IV), obtained by endoscopic ultrasound-fine needle aspiration (EUS-FNA). Methods: The expression profiles of 2,042 miRNAs were determined using micro-cancer tissues from 13 patients with unresectable PDAC obtained by EUS-FNA. The relationship between individual miRNA levels and overall survival (OS) was analyzed. Possible target genes for miRNAs were bioinformatically analyzed using the online database miRDB. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and PK-8 were transfected with miRNA mimic or small interfering RNA, and cell invasion, epithelial-mesenchymal transition (EMT), and apoptosis markers were examined. miRNA and mRNA expressions were examined by quantitative polymerase chain reaction. Results: Of 2,042 miRNAs, the 10 that exhibited the lowest correlation coefficient (p ≤ 0.005) between miRNA expression level and OS among the patients were identified. The miRDB and expression analysis in cancer cell lines for the 10 miRNAs identified miR-296-5p and miR-1207-5p as biomarkers predictive of shorter survival (p < 0.0005). Bioinformative target gene analysis and transfection experiments with miRNA mimics showed that Bcl2-related ovarian killer (BOK), a pro-apoptotic gene, is a target for miR296-5p in pancreatic cancer cells; transfection of miR-296-5p mimic into PANC-1, MIA PaCa-2, and PK-8 cells resulted in significant suppression of BOK mRNA and protein expression. These transfectants showed significantly higher invasion capability compared with control cells, and knock down of BOK in pancreatic cancer cells similarly enhanced invasion capability. Transfectants of miR-296-5p mimic also exhibited aberrant expression of EMT markers, including vimentin and N-cadherin. Moreover, these transfectants showed a significantly lower apoptosis rate in response to 5-fluorouracil and gemcitabine with a decrease of BOK expression, suggesting a role of miR-296-5p in drug resistance. Conclusion: These results suggest that miR-296-5p is a useful biomarker for a poor prognosis in patients with PDAC, and that the miR-296-5p/BOK signaling axis plays an important role in cell invasion, drug resistance, and EMT in PDACs.

Cisplatin (CDDP) is widely used as an anti-cancer platinum agent but its therapeutic efficacy is limited by acquired drug resistance. To develop a new therapeutic strategy that could overcome this resistance, it is important to characterize CDDP-resistant cancer cells. Here we established human lung cancer A549 cell-derived low- and high-grade CDDP-resistant sublines, termed ACR4 and ACR20 cells, by stepwise increasing CDDP concentrations up to 4 and 20 μM, respectively. ACR4 and ACR20 cells showed 6- and 16-fold higher resistance to CDDP than A549 cells, respectively. Cell migration, invasion, and sphere formation were significantly decreased, whereas expression of the stem cell marker CD44v was increased in order of A549, ACR4, and ACR20 cells. The expression of the cystine-glutamate transporter xCT, which is encoded by SLC7A11, was upregulated because of the increased cell surface expression of CD44v in ACR20 cells. Treatment with the xCT inhibitor salazosulfapyridine and knockdown of SLC7A11 mRNA by a specific siRNA significantly improved sensitivity to CDDP in A549, ACR4, and ACR20 cells. Thus, our results suggest that CD44v overexpression is not involved in cancer stem cell properties but increases xCT expression, which leads to the acquisition of CDDP-resistance. This mechanism may contribute to the development of a new therapeutic strategy that can overcome resistance.

Background Exosomes, small-membrane vesicles, are secreted by cells and include several types of proteins and nucleic acids. Exosomes transfer cellular information derived from donor cells and are involved in various physiological and pathological events, such as organ-specific metastasis. Elucidating the exosome uptake mechanisms is important for understanding the progression processes of organ-specific metastasis. However, whether the exosomes secreted by the donor cells are selectively or non-selectively incorporated into the recipient cells is unknown. Methods In this study, three human carcinoma cell lines, A549 (lung), HCT116 and COLO205 (colon), were used. The exosome isolation efficiency was compared between three methods: ultracentrifugation, ExoQuick-TC and Total Exosome Isolation kits. Recipient cells were treated with Pitstop 2, an inhibitor of clathrin-dependent endocytosis, or genistein, an inhibitor of caveolae-dependent endocytosis, and then incubated with DiO-labeled exosomes. Results Among the three methods examined, ultracentrifugation was the most efficient and reproducible. Exosomes derived from a donor cell line are incorporated into the three cell lines, but the exosome uptake capability was different depending on the recipient cell type and did not depend on the donor cell type. Exosome uptake in COLO205 was inhibited by Pitstop 2 and genistein. Exosome uptake in HCT116 was inhibited by Pitstop 2, but not genistein, while that in A549 cells was not inhibited by these inhibitors. Taken together, these results suggest that the exosomes secreted by donor cells are non-selectively incorporated into recipient cells and that the exosome uptake mechanism is different depending on the recipient cells. Conclusions Different recipient cells’ exosome uptake capabilities may be involved in organ-specific metastasis.

The mechanism of resistance to sorafenib in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the miRNAs responsible for resistance. Drug sensitivity, migration/invasion capabilities, and epithelial-mesenchymal transition (EMT) properties were analyzed by biochemical methods. The clinical relevance of the target genes to survival in HCC patients were assessed using a public database. Four miRNAs were significantly upregulated in PLC/PRF5-R1/-R2 compared with PLC/PRF5. Among them, miR-125b-5p mimic-transfected PLC/PRF5 cells (PLC/PRF5-miR125b) and showed a significantly higher IC50 for sorafenib compared with controls, while the other miRNA mimics did not. PLC/PRF5-miR125b showed lower E-cadherin and higher Snail and vimentin expression—findings similar to those for PLC/PRF5-R2—which suggests the induction of EMT in those cells. PLC/PRF5-miR125b exhibited significantly higher migration and invasion capabilities and induced sorafenib resistance in an in vivo mouse model. Bioinformatic analysis revealed ataxin-1 as a target gene of miR-125b-5p. PLC/PRF5 cells transfected with ataxin-1 siRNA showed a significantly higher IC50, higher migration/invasion capability, higher cancer stem cell population, and an EMT phenotype. Median overall survival in the low-ataxin-1 patient group was significantly shorter than in the high-ataxin-1 group. In conclusion, miR-125b-5p suppressed ataxin-1 and consequently induced Snail-mediated EMT and stemness, leading to a poor prognosis in HCC patients., The mechanism of resistance to multikinase inhibitors in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the responsible miRNAs and target genes involved in the mechanism of resistance. Four miRNAs were significantly upregulated. Among them, we found that miR-125-5p induced sorafenib resistance in HCC cells and in a mouse model. We also revealed that miR-125-5p suppressed ataxin-1 as a target gene and consequently induced Snail-mediated epithelial-mesenchymal transition (EMT) and cancer stemness. Moreover, we demonstrated that ataxin-1 expression has an impact on the prognosis of patients with HCCs. In the future, by comparing the expression status of miR-125b-5p/ataxin-1 and the effect of sorafenib in the clinical setting, it is expected that miR-125b-5p will be established as an effective drug selection marker for treatment selection in patients with HCC.

Background: Indigo naturalis (IND) is an herbal medicine that has been used as an anti-inflammatory agent to treat diseases including dermatitis and inflammatory bowel disease in China. However, the mechanism by which IND exerts its immunomodulatory effect is not well understood. Methods: A murine model of dermatitis and inflammatory bowel disease, both induced by oxazolone (OXA), was treated with IND. The severity of dermatitis was evaluated based on ear thickness measurements and histological scoring. The severity of colitis was evaluated by measuring body weight, histological scoring, and endoscopic scoring. The expression of inflammatory cytokines in ear and colon tissue was evaluated using real-time PCR. 16S rRNA DNA sequencing of feces from OXA-induced colitis mice was performed before and after IND treatment. The effects of IND on OXA-induced colitis were also evaluated after depleting the gut flora with antibiotics to test whether alteration of the gut flora by IND influenced the course of intestinal inflammation in this model. Results: IND treatment ameliorated OXA dermatitis with a reduction in IL-4 and eosinophil recruitment. However, OXA colitis was significantly aggravated in spite of a reduction in intestinal IL-13, a pivotal cytokine in the induction of the colitis. It was found that IND dramatically altered the gut flora and IND no longer exacerbated colitis when colitis was induced after gut flora depletion. Conclusions: Our data suggest that IND could modify the inflammatory immune response in multiple ways, either directly (i.e., modification of the allergic immune cell activity) or indirectly (i.e., alteration of commensal compositions).

Background/Aims: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor lesion of colon cancer. Although the relevance of DNA hypermethylation in the SSA/P-cancer sequence is well documented, the role of DNA hypomethylation is unknown. We investigated the biological relevance of DNA hypomethylation in the SSA/P-cancer sequence by using 3-dimensional organoids of SSA/P. Methods: We first analyzed hypomethylated genes using datasets from our previous DNA methylation array analysis on 7 SSA/P and 2 cancer in SSA/P specimens. Expression levels of hypomethylated genes in SSA/P specimens were determined by RT-PCR and immunohistochemistry. We established 3-dimensional SSA/P organoids and performed knockdown experiments using a lentiviral shRNA vector. DNA hypomethylation at CpG sites of the gene was quantitated by MassARRAY analysis. Results: The mean number of hypomethylated genes in SSA/P and cancer in SSA/P was 41.6 ± 27.5 and 214 ± 19.8, respectively, showing a stepwise increment in hypomethylation during the SSA/P-cancer sequence. S100P, S100α2, PKP3, and MUC2 were most commonly hypomethylated in SSA/P specimens. The mRNA and protein expression levels of S100P, S100α2, and MUC2 were significantly elevated in SSA/P compared with normal colon tissues, as revealed by RT-PCR and immunohistochemistry, respectively. Among these, mRNA and protein levels were highest for S100P. Knockdown of the S100P gene using a lentiviral shRNA vector in 3-dimensional SSA/P organoids inhibited cell growth by >50% (p < 0.01). The mean diameter of SSA/P organoids with S100P gene knockdown was significantly smaller compared with control organoids. MassARRAY analysis of DNA hypomethylation in the S100P gene revealed significant hypomethylation at specific CpG sites in intron 1, exon 1, and the 5′-flanking promoter region. Conclusion: These results suggest that DNA hypomethylation, including S100P hypomethylation, is supposedly associated with the SSA/P-cancer sequence. S100P overexpression via DNA hypomethylation plays an important role in promoting cell growth in the SSA/P-cancer sequence.

Nonalcoholic steatohepatitis (NASH) progresses from nonalcoholic fatty liver disease (NAFLD), however, efficacious drugs for NASH treatment are lacking. Sodium alginate (SA), a soluble dietary fiber extracted from brown algae, could protect the small intestine from enterobacterial invasion. NASH pathogenesis has been suggested to be associated with enterobacterial invasion, so we examined the effect of SA on methionine- and choline-deficient (MCD) diet-induced steatohepatitis in mice (the most widely-used model of NASH). The mice (n = 31) were divided into three groups (mice fed with regular chow, MCD diet, and MCD diet premixed with 5% SA) for 4 and 8 weeks. The MCD diet increased lipid accumulation and inflammation in the liver, the NAFLD Activity Score and hepatic mRNA expression of tumor necrosis factor- and collagen 11, and induced macrophage infiltration. Villus shortening, disruption of zonula occludens-1 localization and depletion of mucus production were observed in the small intestine of the MCD-group mice. SA administration improved lipid accumulation and inflammation in the liver, and impaired barrier function in the small intestine. Collectively, these results suggest that SA is useful for NASH treatment because it can prevent hepatic inflammation and fatty degeneration by maintaining intestinal barrier function.

The liver and the small intestine are closely related in the processes of drug absorption, metabolism and excretion via the enterohepatic circulation. Small intestinal ulcers are a serious adverse effect commonly occurring in patients taking nonsteroidal anti-inflammatory drugs. However, the influence of small intestinal ulcers on drug metabolism has not been established. This study examined the expressional changes of cytochrome P450 (CYP) in the liver using an indomethacin-induced small intestinal ulcer rat model and in cultured cells. After the administration of indomethacin to rats, ulcers were observed in the small intestine and expression of CYP3A1, the major isoform of hepatic CYP, was significantly down-regulated in the liver, accompanied by increased expression of inducible nitric oxide synthase, tumor necrosis factor α, interleukin (IL)-1β and IL-6, in the small intestine and the liver. The indomethacin-induced small intestinal ulceration, the increase in inflammatory mediators in the small intestine and the liver, and the down-regulation of CYP3A1 expression in the liver were inhibited by co-administration of ampicillin, an antibacterial agent. In the human hepatic HepG2 cell line, IL-1β, IL-6 and NOC-18, an NO donor, caused down-regulation of CYP3A4, the major isoform of human CYP3A. Thus, this study suggests that after indomethacin treatment small intestinal ulcers cause the down-regulation of CYP3A1 in the rat liver through an increase in ulcer-derived inflammatory mediators. Copyright © 2016 John Wiley & Sons, Ltd.

Recent advances in diagnostic technologies have revealed that nonsteroidal anti-inflammatory drugs (NSAIDs) can cause serious mucosal injury in the upper and lower gastrointestinal tract (including the small intestine). A drug to treat NSAID-induced small-intestinal injury (SII) is lacking. Sodium alginate is a soluble dietary fiber extracted from brown seaweed and its solution has been used as a hemostatic agent to treat gastrointestinal bleeding due to gastric ulcers. Whether sodium alginate has therapeutic effects on NSAID-induced SII and its mechanism of action are not known. Here, we investigated if administration of two forms (high-molecular-weight (HMW) and low-molecular-weight (LMW)) of sodium alginate could ameliorate indomethacin-induced SII. Pretreatment with HMW sodium alginate or LMW sodium alginate before indomethacin administration improved ulceration and the resultant intestinal shortening was associated with reduced histological severity of mucosal injury and ameliorated mRNA expression of inflammation-related molecules in the small intestine. We found that mRNAs of secretory Muc2 and membrane-associated Muc1, Muc3 and Muc4 were expressed in the small intestine. mRNA expression of Muc1-4 was increased in indomethacin-induced SII, and these increases were prevented by sodium alginate. Thus, administration of sodium alginate could be a therapeutic approach to prevent indomethacin-induced SII.

Streptozotocin administration to mice (STZ-mice) induces type I diabetes and hepatocellular carcinoma (HCC). We attempted to elucidate the carcinogenic mechanism and the miRNA expression status in the liver and blood during the precancerous state. Serum and liver tissues were collected from STZ-mice and non-treated mice (CTL-mice) at 6, 10, and 12 W. The exosome enriched fraction extracted from serum was used. Hepatic histological examination and hepatic and exosomal miRNA expression analysis were serially performed using next-generation sequencing (NGS). Human miRNA expression analysis of chronic hepatitis liver tissue and exosomes, which were collected before starting the antiviral treatment, were also performed. No inflammation or fibrosis was found in the liver of CTL-mice during the observation period. In STZ-mice, regeneration and inflammation of hepatocytes was found at 6 W and nodules of atypical hepatocytes were found at 10 and 12 W. In the liver tissue, during 6–12 W, the expression levels of let-7f-5p, miR-143-3p, 148a-3p, 191-5p, 192-5p, 21a-5p, 22-3p, 26a-5p, and 92a-3p was significantly increased in STZ-mice, and anti-oncogenes of their target gene candidates were down-regulated. miR-122-5p was also significantly down-regulated in STZ-mice. Fifteen exosomal miRNAs were upregulated in STZ-mice. Six miRNAs (let-7f-5p, miR-10b-5p, 143-3p, 191-5p, 21a-5p, and 26a-5p) were upregulated, similarly to human HCC cases. From the precancerous state, aberrant expression of hepatic miRNAs has already occurred, and then, it can promote carcinogenesis. In exosomes, the expression pattern of common miRNAs between mice and humans before carcinogenesis was observed and can be expected to be developed as a cancer predictive marker.

Background Hepatocellular carcinoma (HCC) can develop in patients who are negative for the hepatitis B surface antigen (HBsAg) in serum but positive for hepatitis B virus (HBV) DNA in the liver, referred to as occult HBV infection (OBI). Previous reports showed that HBV variants in OBI-related HCC are different from those in HBsAg-positive HCC. In the present study, HBV quasispecies based on the pre-S/S gene in OBI-related HCC patients were examined by high throughput sequencing and compared with those in HBsAg-positive HCC. Methods Nineteen tissue samples (9 OBI-related and 10 HBsAg-positive non-cancerous tissues) were collected at the time of surgery at Kobe University Hospital. The quasispecies with more than 1% variation in the pre-S/S region were isolated and analysed by ultra-deep sequencing. Results There were no significant differences in the major HBV populations, which exhibit more than 20% variation within the entire pre-S/S region, between OBI-related HCC and HBsAg-positive HCC. However, the prevalences of major populations with pre-S2 region mutations and of minor populations with polymerized human serum albumin-binding domain mutations were significantly higher in OBI-related HCC than in HBsAg-positive HCC. Moreover, the major variant populations associated with the B-cell epitope, located within the pre-S1 region, and the a determinant domain, located in the S region, were detected frequently in HBsAg-positive HCC. The minor populations of variants harbouring the W4R, L30S, Q118R/Stop, N123D and S124F/P mutations in the pre-S region and the L21F/S and L42F/S mutations in the S region were detected more frequently in OBI-related HCC than in HBsAg-positive HCC. Conclusions Ultra-deep sequencing revealed that the B-cell epitope domain in the pre-S1 region and alpha determinant domain in the S region were variable in HBsAg-positive HCC, although the quasispecies associated with the pre-S2 region were highly prevalent in OBI-related HCC. Trial registration Ref: R000034382/UMIN000030113; Retrospectively registered 25 November 2017.

Quasispecies of hepatitis B virus (HBV) with variations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) can evolve during infection, allowing HBV to evade neutralizing antibodies. These escape variants may contribute to chronic infections. In this study, we looked for MHR variants in HBV quasispecies using ultradeep sequencing and evaluated the relationship between these variants and clinical manifestations in infected patients. We enrolled 30 Indonesian patients with hepatitis B infection (11 with chronic hepatitis and 19 with advanced liver disease). The most common subgenotype/subtype of HBV was B3/adw (97%). The HBsAg titer was lower in patients with advanced liver disease than that in patients with chronic hepatitis. The MHR variants were grouped based on the percentage of the viral population affected: major, ≥20% of the total population; intermediate, 5% to

Aim Hepatocellular carcinoma (HCC) develops in up to 5% of patients after the successful treatment of chronic hepatitis C virus (HCV) infection using interferon therapy. The aim of this study was to characterize miRNA expression in liver tissues from patients who achieved a sustained viral response (SVR). Methods Seventy-one patients with resected HCC were enrolled into the present study: 61 HCC from patients with continuously infected HCV (HCV-HCC) and 10 from patients who had achieved SVR (SVR-HCC). We also included non-tumor tissues (SVR-NT) from four patients with SVR-HCC, and liver tissue (SVR-CH) from four SVR patients without HCC. Total RNA was extracted from liver samples. The miRNA expression patterns were analyzed using microarrays. In addition, target gene expression was quantified after miRNA overexpression in HEK293 cells. Results We could discriminate between SVR-HCC and HCV-HCC with 75.36% accuracy using the expression pattern of six specific miRNA. The expression levels of 37 miRNA were significantly lower in HCV-HCC than in SVR-HCC, whereas the expression of 25 miRNA was significantly higher in HCV-HCC than SVR-HCC (P

Aims Cisplatin (CDDP) is a platinum-based drug that is widely used in cancer chemotherapy, but the development of resistance in tumor cells is a major weakness of these treatments. Several mechanisms have been proposed to explain cisplatin resistance, and disruption of certain cellular pathways could modulate drug sensitivity to cisplatin. A lower level of cross-resistance to cisplatin leads to better outcomes in clinical use. Main methods Cross-resistance was assessed using cisplatin resistant lung cancer cell line A549/CDDP. Cell cycle analysis was used to examine the effect of cisplatin on cell signaling pathways regulating G2/M transition in cisplatin resistant cells. Key findings A549/CDDP cells exhibited cross-resistance to carboplatin, but not oxaliplatin, which is often found in platinum analogues. Flow cytometry showed that nocodazole treatment caused a G2/M block in both A549/CDDP cells and cisplatin susceptible cells. However, A549/CDDP cells escaped the G2/M block following exposure to cisplatin. Activation of the Cdc2/CyclinB complex is required for transition from G2 to M phase, and the inactive form of phosphorylated Cdc2 is activated by Cdc25C dephosphorylation of Tyr15. In the cisplatin-treated susceptible cells, the levels of phosphorylated Cdc2 and Cdc25C were markedly decreased, leading to a loss of Cdc2 activity and G2/M arrest. In A549/CDDP cells, however, Cdc2 activity was supported by the expression of Cdc2 and Cdc25C after the addition of cisplatin, which resulted in G2/M progression. Significance The resistance phenotype of G2/M progression has been correlated with dysregulation of Cdc2 in a human lung cancer cell line selected for cisplatin.

Hepatitis B virus (HBV) polymerase gene is targeted by nucleos(t)ide analogues (NUC), but it is unclear how HBV quasispecies of whole genome changes during early period of NUC treatment. To understand the unknown region of drug sensitivity and treatment resistance, HBV quasispecies of whole genome during early period of NUC treatment was examined using ultra‑deep sequencing. Eleven patients with chronic HBV infection who received NUC treatment were enrolled in the current study. Viral DNA was extracted from serum samples before and early period of NUC treatment. Polymerase chain reaction analysis was subsequently performed on the DNA products. The viral quasispecies of the entire genome was analyzed by ultra‑deep sequencing. The regions and positions corresponding to the changes in the quasispecies were investigated before and early period of NUC treatment. The secondary structure changes were predicted by mutations/substitutions detected using Lasergene Protean v14.1 software. The frequency of quasispecies variants increased significantly in the polymerase domain from before to early period of NUC treatment (3.08±1.28 vs. 3.51±1.47%, P

BackgroundChildren with cyanotic heart disease develop secondary erythrocytosis and thrombocytopenia via unknown mechanisms. Mature erythrocyte microRNAs may reflect clinical pathologies and cell differentiation processes pre-enucleation. This study evaluated erythrocyte microRNAs in children with cyanotic heart disease.MethodsErythrocyte microRNAs from children with cyanotic and acyanotic heart disease and without cardiac disease were quantified with Ion PGM System (n=10 per group). Differential expression was confirmed by quantitative PCR (qPCR; n=20 per group).ResultsMir-486-3p, mir-486-5p, and mir-155-5p increased in patients with cyanotic heart disease compared with those without heart disease: fold differences (95% confidence interval): mir-486-3p: 1.92 (1.14-3.23), P=0.011; mir-486-5p: 2.27 (1.41-3.65), P

Background/Aims: Gastric xanthomas are frequently observed in the stomach as small yellowish plaques or nodules. A close relationship among Helicobacter pylori infection, atrophic gastritis, and xanthomas has been reported. We assessed the clinicopathological features of gastric cancer with or without xanthomas. Methods: A total of 91 patients who were diagnosed as having early gastric cancer were enrolled. We evaluated the gastritis status using scores for gastritis and atrophy, positivity of H. pylori infection, the prevalence rate of xanthomas, and the clinicopathological features of gastric cancer. Results: Gastric xanthomas were observed in 72.5% of early gastric cancer cases. Scores for gastritis and atrophy were significantly higher in the xanthoma-positive group than those in the xanthoma-negative group. A higher prevalence of differentiated-type adenocarcinoma was found in the xanthoma-positive group. Among the cases with multiple gastric xanthomas, the prevalence of males was significantly higher than that of females. Conclusion: A high prevalence rate of gastric xanthomas in gastric cancer cases was shown. Xanthomas were highly associated with age, the severities of gastritis and atrophy, and differentiated-type adenocarcinoma. Regardless of the eradication of H. pylori, xanthomas may be useful predictive markers for the development of differentiated-type adenocarcinoma.

Background: Non-steroidal anti-inflammatory drugs induce the serious side effect of small intestinal ulcerations (SIUs), but little information is available regarding the consequences to drug metabolism and absorption. Aim: We examined the existence of secondary hepatic inflammation in rats with indomethacin (INM)-induced SIUs and assessed its relationship to the cytochrome P450 (CYP) and P-glycoprotein (mdr1a), the major drug-metabolizing factors in the small intestine and the liver. Methods: Gene expression of the CYP family of enzymes and mdr1a was measured with quantitative real-time polymerase chain reaction (qPCR). Vancomycin (VCM), a poorly absorbed drug, was administered intraduodenally to rats with SIUs. Results: INM induced SIUs predominantly in the lower region of the small intestine with high expression of inflammatory markers. Liver dysfunction was also observed, which suggested a secondary inflammatory response in rats with SIUs. In the liver of rats with SIUs, the expression of CYP2C11, CYP2E1, and CYP3A1 was significantly decreased, and loss of CYP3A protein was observed. Although previous studies have shown a direct effect of INM on CYP3A activity, we could not confirm any change in hepatic CY3A4 expression (major isoform of human CYP3A) in vitro. The plasma VCM concentration was increased in rats with SIUs due to partial absorption from the mucosal injury, but not in normal mucosa. Conclusions: INM-induced SIUs had a subtle effect on intestinal CYP expression, but had an apparent action on hepatic CYP, which was influenced, at least in part, by the secondary inflammation. Furthermore, drug absorption was increased in rats with SIUs.

Serine/arginine-rich splicing factor 3 (SRSF3) is a member of the SR protein family and plays wide-ranging roles in gene expression. The human SRSF3 gene generates two alternative splice transcripts, a major mRNA isoform ( SRSF3-FL) encoding functional full-length protein and a premature termination codon (PTC)-containing isoform ( SRSF3-PTC). The latter is degraded through nonsense-mediated mRNA decay (NMD). Treatment of a human colon cancer cell line (HCT116) with 100 μM sodium arsenite increased SRSF3-PTC mRNA levels without changing SRSF3-FL mRNA levels. A chemiluminescence-based NMD reporter assay system demonstrated that arsenite treatment inhibited NMD activity and increased SRSF3-PTC mRNA levels in the cytoplasm, facilitating translation of a truncated SRSF3 protein (SRSF3-TR) from SRSF3-PTC mRNA. SRSF3-TR lacked two-thirds of the Arg/Ser-rich (RS) domain whose phosphorylation state is known to be crucial for subcellular distribution. SRSF3-FL was localized in the nucleus, while overexpressed SRSF3-TR was diffusely distributed in the cytoplasm and the nucleus. A part of SRSF3-TR was also associated with stress granules in the cytoplasm. Interestingly, treatment of HCT116 cells with a small interference RNA specifically targeting SRSF3-PTC mRNA significantly attenuated arsenite-stimulated induction of c-JUN protein, its binding activity to the AP-1 binding site (−126 to 120 bp) in the interleukin (IL)-8 gene promoter, and AP-1 promoter activity, resulting in significant reduction of arsenite-stimulated IL-8 production. Our results suggest that SRSF3-TR may function as a positive regulator of oxidative stress-initiated inflammatory responses in colon cancer cells.

Background Jumonji domain-containing protein 2A (JMJD2A), belonging to the JMJD2 family of histone lysine demethylases, has been implicated in tumorigenesis. However, its expression profile and role in drug resistance of gastric cancer remains unknown. Previous studies show that docetaxel, cisplatin, and S-1 (DCS) therapy has a high response rate in patients with metastatic gastric cancer, but acquired drug resistance is often observed. In this study, we investigated the role of JMJD2A in drug susceptibility of DCS therapy in patients with gastric cancer, and its clinical relevance in gastric cancer. Methods siRNA-mediated downregulation of 14 relevant genes from previously identified gene signatures was performed to identify functional factor to modulate drug susceptibility in DCS therapy. In 34 clinical tissues with metastatic gastric cancer, we examined whether JMJD2A expression predicted tumor regression rate in patients. Furthermore, the downstream effects of JMJD2A on drug susceptibility were analyzed by whole-gene expression array and immunoprecipitation. Results After specific silencing of 14 candidate genes, inhibition of JMJD2A induced significant drug resistance in three anti-cancer drugs; IC50values for 5-FU, cisplatin, and docetaxel were 15.3-, 2.7-, and 4.0-fold increased, respectively. Overexpressed JMJD2A was universally expressed in 12 gastric cancer cell lines, positively correlated with tumor regression rate in DCS therapy. JMJD2A is physically associated with histone lysine demethylation. By analysis of downstream effect of JMJD2A, cooperation of Coiled-coil domain containing 8 (CCDC8) was identified, using whole-gene expression analysis. Direct interaction of CCDC8 and JMJD2A was verified by immunoprecipitation. Of note, inhibition of CCDC8 also induced significant drug resistance in docetaxel, cisplatin, and S-1. Conclusions JMJD2A sensitizes gastric cancer to combination chemotherapy of S-1, cisplatin, and docetaxel by cooperating CCDC8, and JMJD2A/CCDC8 would be a potential biomarker and therapeutic target. Legal entity responsible for the study The authors. Funding JSPS KAKENHI. Disclosure All authors have declared no conflicts of interest.

Non-steroidal anti-inflammatory drugs (NSAIDs)-induced small intestinal injury is a serious clinical event with recent advances of diagnostic technologies, but a successful therapeutic method to treat such injuries is still lacking. Licorice, a traditional herbal medicine, and its derivatives have been widely used for the treatment of a variety of diseases due to their extensive biological actions. However, it is unknown whether these derivatives have an effect on NSAIDs-induced small intestinal damage. Previously, the anti-inflammatory effects of three compounds extracted from the licorice root, glycyrrhizin, 18β-glycyrrhetinic acid, and dipotassium glycyrrhizinate, were compared in vitro cell culture. The most prominent inhibitory effect on the tumor necrosis factor-α (TNF-α) production was observed with the administration of 18β-glycyrrhetinic acid as an active metabolite of glycyrrhizin. In this study, a complex compound of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin was examined to improve the oral bioavailability. After administration of this complex to indomethacin treated mice, a significantly high plasma concentration of 18β-glycyrrhetinic acid was detected using the tandem mass spectrometry coupled with the HPLC. Furthermore, the complex form of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin reduced mRNA expressions of TNF-α, interleukin (IL)-1β, and IL-6, which was histologically confirmed in the improvement of indomethacin-induced small intestinal damage. These results suggest that the complex of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin has the potential therapeutic value for preventing the adverse effects of indomethacin-induced small intestinal injury.

Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.

Serine/arginine-rich splicing factor 3 (SRSF3), a member of the SRSF family, plays a wide-ranging role in gene expression. The human SRSF3 gene generates a major mRNA isoform encoding a functional, full-length protein and a PTC-containing isoform (SRSF3-PTC). The latter is expected to be degraded through the nonsense-mediated mRNA decay system. However, it was reported that SRSF3-PTC mRNA was produced under stressful conditions and translated into a truncated SRSF3 protein (SRSF3-TR). To disclose unknown functions of SRSF3-TR, we established Flp-In-293 cells stably expressing SRSF3-TR. The SRSF3-TR-expressing cells increased mRNA and protein levels of positive regulators for G1 to S phase transition (cyclin D1, cyclin D3, CDC25A, and E2F1) and accelerated their growth. c-Jun is required for progression through the G1 phase, the mechanism by which involves transcriptional control of the cyclin D1 gene. We also found that the JUN promoter activity was significantly increased in the Flp-In-293 cells stably expressing SRSF3-TR, compared with mock-transfected control cells. The SRSF3-TR-expressing cells increased c-Jun and Sp-1 levels, which are important for the positive autoregulation and basal transcription of JUN, respectively. Our results suggest that stress-inducible SRSF3-TR may participate in the acceleration of cell growth through facilitating c-Jun-mediated G1 progression under stressful conditions.

Objectives: The aim of this study was to identify biomarkers for predicting the efficacy of docetaxel, cisplatin, and S-1 (DCS) therapy for advanced gastric cancer using microarrays of biopsy specimens before chemotherapy. Methods: Nineteen samples were taken from 19 patients with unresectable metastatic gastric cancer who received DCS as a first-line therapy. Laser capture microdissection was performed, and total cellular RNA was extracted from each microdissected sample. Whole-gene expression was analyzed by microarray, and the difference in mRNA expression observed with the microarrays was confirmed by quantitative real-time PCR. Immunohistochemical staining was performed using clinical tissue sections obtained by endoscopic biopsy. Results: Eleven patients were identified as early responders and 8 patients as nonresponders to DCS therapy. Twenty-nine genes showed significant differences in relative expression ratios between tumor and normal tissues. A classifier set of 29 genes had high accuracy (94.7%) for distinguishing gene expression between 11 early responders and 8 nonresponders. Decreasing the size of the classifier set to 4 genes (PDGFB, PCGF3, CISH, and ANXA5) increased the accuracy to 100%. Expression levels by real-time PCR for validation were well correlated with those 4 genes in microarrays. Conclusion: The genes identified may serve as efficient biomarkers for personalized cancer-targeted therapy.

Background Helicobacter pylori infection is associated with risk for chronic gastritis (CG), gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC). The H. pylori Cag type IV secretion system (TFSS) translocates the virulence factor cytotoxin-associated gene A protein into host cells and plays an important role in initiating gastric carcinogenesis. The CagL and CagI proteins are components of the TFSS. The Arg-Gly-Asp (RGD) motif of CagL, and the six most distal C-terminal amino acids (Ser-Lys-Ile-Ile-Val-Lys, and Ser-Lys-Val-Ile-Val-Lys) of CagL and CagI are essential for TFSS adhesion to host cells. Additionally, the CagL variant Tyr58Glu59 was previously shown to be associated with GC patients. Results We isolated 43 H. pylori isolates from 17 CG, 8 GU, 8 DU, and 10 GC patients in Southeast Asia. Total DNAs were extracted and sequenced with MiSeq. H. pylori strain ATCC 26695, which was isolated from CG patients, was used as a reference. We examined the full sequences of H. pylori cagL and cagI using whole-genome sequencing (WGS), and analyzed whether single nucleotide variants and amino acid changes (AACs) correlated with adverse clinical outcomes. Three isolates were excluded from the analysis due to cagPAI rearrangements. CagL RGD motifs were conserved in 39 isolates (97.5%). CagL-Glu59 and Ile234 in the C-terminal motif were more common in 10 H. pylori isolates from GC patients (p

A nucleos(t)ide analog (NA) is the common antiviral drug available for directly treating hepatitis B virus (HBV) infection. However, its application has led to the emergence of NA-resistant mutations mostly in a conserved region of the reverse transcriptase domain of HBV polymerase. Harboring NA-resistant mutations decreases drug effectiveness and increases the frequency of end-stage liver disease. The invention of next-generation sequencing that can generate thousands of sequences from viral complex mixtures provides opportunities to detect minor changes and early viral evolution under drug stress. The present study used ultra-deep sequencing to evaluate discrepant quasispecies in the reverse transcriptase domain of HBV including NA-resistant hotspots between seven treatment-naïve Indonesian patients infected with HBV and five at the early phase of treatment. The most common sub-genotype was HBV B3 (83.34%). The substitution rate of variants determined among amino acids with a ratio of ≥ 1% changes was higher among the population in conserved regions (23.19% vs. 4.59%, P = 0.001) and in the inter-reverse transcriptase domain (23.95% vs. 2.94%, P = 0.002) in treatment naïve, than in treated patients. Nine hotspots of antiviral resistance were identified in both groups, and the mean frequency of changes in all patients was < 1%. The known rtM204I mutation was the most frequent in both groups. The lower rate of variants in HBV quasispecies in patients undergoing treatment could be associated with virus elimination and the extinction of sensitive species by NA therapy. The present findings imply that HBV quasispecies dynamically change during treatment.

The liver and the small intestine are closely related in the processes of drug absorption, metabolism and excretion via the enterohepatic circulation. Small intestinal ulcers are a serious adverse effect commonly occurring in patients taking nonsteroidal anti-inflammatory drugs. However, the influence of small intestinal ulcers on drug metabolism has not been established. This study examined the expressional changes of cytochrome P450 (CYP) in the liver using an indomethacin-induced small intestinal ulcer rat model and in cultured cells. After the administration of indomethacin to rats, ulcers were observed in the small intestine and expression of CYP3A1, the major isoform of hepatic CYP, was significantly down-regulated in the liver, accompanied by increased expression of inducible nitric oxide synthase, tumor necrosis factor α, interleukin (IL)-1β and IL-6, in the small intestine and the liver. The indomethacin-induced small intestinal ulceration, the increase in inflammatory mediators in the small intestine and the liver, and the down-regulation of CYP3A1 expression in the liver were inhibited by co-administration of ampicillin, an antibacterial agent. In the human hepatic HepG2 cell line, IL-1β, IL-6 and NOC-18, an NO donor, caused down-regulation of CYP3A4, the major isoform of human CYP3A. Thus, this study suggests that after indomethacin treatment small intestinal ulcers cause the down-regulation of CYP3A1 in the rat liver through an increase in ulcer-derived inflammatory mediators. Copyright © 2016 John WileySons, Ltd.

Stress-induced production of proinflammatory cytokines in the brain and periphery is associated with mental distress. In this study, we measured changes in levels of salivary cortisol and 50 circulating immune mediators in 28 4th-grade medical students (19 males and 9 females) 7 weeks before, 1 day before, immediately after, and 1 week after an authorized nationwide examination for promotion. Repeated measures ANOVA with multiple testing correction and post hoc tests revealed that the examination significantly increased levels of proinflammatory cytokines (granulocyte colony-stimulating factor, interferon-γ, interleukin (IL)-1β, and tumor necrosis factor-α), Th2 cytokines (IL-4, IL-5, and IL-13), and β-nerve growth factor in association with significant decreases in salivary cortisol levels and anxiety after the examination. These mediators may have a negative impact on the mental state of healthy young adults exposed to naturalistic stressors.

Background: The Toll-like receptor (TLR) 4 signalling pathway has been shown to have oncogenic effects in vitro and in vivo. To demonstrate the role of TLR4 signalling in colon tumourigenesis, we examined the expression of TLR4 and myeloid differentiation factor 88 (MyD88) in colorectal cancer (CRC). Methods: The expression of TLR4 and MyD88 in 108 CRC samples, 15 adenomas, and 15 normal mucosae was evaluated by immunohistochemistry, and the correlations between their immunoscores and clinicopathological variables, including disease-free survival (DFS) and overall survival (OS), were analysed. Results: Compared with normal mucosae and adenomas, 20% cancers displayed high expression of TLR4, and 23% cancers showed high expression of MyD88. The high expression of TLR4 and MyD88 was significantly correlated with liver metastasis (P=0.0001, P=0.0054). In univariate analysis, the high expression of TLR4 was significantly associated with shorter OS (hazard ratio (HR): 2.17; 95% confidence interval (95% CI): 1.15–4.07; P=0.015). The high expression of MyD88 expression was significantly associated with poor DFS and OS (HR: 2.33; 95% CI: 1.31–4.13; P=0.0038 and HR: 3.03; 95% CI: 1.67–5.48; P=0.0002). The high combined expression of TLR4 and MyD88 was also significantly associated with poor DFS and OS (HR: 2.25; 95% CI: 1.27–3.99; P=0.0053 and HR: 2.97; 95% CI: 1.64–5.38; P=0.0003). Multivariate analysis showed that high expressions of TLR4 (OS: adjusted HR: 1.88; 95% CI: 0.99–3.55; P=0.0298) and MyD88 (DFS: adjusted HR: 1.93; 95% CI: 1.01–3.67; P=0.0441; OS: adjusted HR: 2.25; 95% CI: 1.17–4.33; P=0.0112) were independent prognostic factors of OS. Furthermore, high co-expression of TLR4/MyD88 was strongly associated with both poor DFS and OS. Conclusion: Our findings suggest that high expression of TLR4 and MyD88 is associated with liver metastasis and is an independent predictor of poor prognosis in patients with CRC.

Disrupted-in-schizophrenia 1 (DISC1), a known genetic risk factor for schizophrenia (SZ) and major depressive disorder (MDD), interacts with several proteins and some of them are reported to be genetically associated with SZ. Pericentrin (PCNT) also interacts with DISC1 and recently single-nucleotide polymorphisms (SNPs) within the PCNT gene have been found to show significant associations with SZ and MDD. In this study, case-controlled association analysis was performed to determine if the PCNT gene is implicated in SZ. Nine SNPs were analyzed in 1,477 individuals (726 patients with SZ and 751 healthy controls). No significant difference was observed between the controls and the patients in allelic frequencies or genotypic distributions of eight SNPs. Although allelic distribution of rs11702684 was different between the two groups (P = 0.042), the difference did not reach statistical significance after permutation correction for multiple comparisons. In the haplotypic analysis, we could not find any significant association in our subjects, either. This gene may not play a major role independently in the etiology of SZ in the Japanese population.

Caloric restriction (CR) is an effective method for prevention of age-associated diseases as well as overweight and obesity; however, there is controversy regarding the effects of dieting regimens on behavior. In this study, we investigated two different dieting regimens: repeated fasting and refeeding (RFR) and daily feeding of half the amount of food consumed by RFR mice (CR). CR and RFR mice had an approximate 20% reduction in food intake compared with control mice. Open field, light-dark transition, elevated plus maze, and forced swimming tests indicated that CR, but not RFR, reduced anxiety- and depressive-like behaviors, with a reduction peak on day 8. Using a mouse whole genome microarray, we analyzed gene expression in the prefrontal cortex, amygdala, and hypothalamus. In addition to the CR-responsive genes commonly modified by RFR and CR, each regimen differentially changed the expression of distinct genes in each region. The most profound change was observed in the amygdalas of CR mice: 884 genes were specifically upregulated. Ingenuity pathway analysis revealed that these 884 genes significantly modified nine canonical pathways in the amygdala. α-Adrenergic and dopamine receptor signalings were the two top-scoring pathways. Quantitative RT-PCR confirmed the upregulation of six genes in these pathways. Western blotting confirmed that CR specifically increased dopamine- and cAMP-regulated phosphoprotein (Darpp-32), a key regulator of dopamine receptor signaling, in the amygdala. Our results suggest that CR may change behavior through altered gene expression.

The tra2beta gene encoding an alternative splicing regulator, transformer 2-beta (Tra2beta), generates five alternative splice variant transcripts (tra2beta1-5). Functionally active, full-length Tra2beta is encoded by tra2beta1 isoform. Expression and physiological significance of the other isoforms, particularly tra2beta4, are not fully understood. Rat gastric mucosa constitutively expressed tra2beta1 isoform and specifically generated tra2beta4 isoform that includes premature termination codon-containing exon 2, when exposed to restraint and water immersion stress. Treatment of a gastric cancer cell line (AGS) with arsenite (100 microM) preferentially generated tra2beta4 isoform and caused translocation of Tra2beta from the nucleus to the cytoplasm in association with enhanced phosphorylation during the initial 4-6 h (acute phase). Following the acute phase, AGS cells continued upregulated tra2beta1 mRNA expression, and higher amounts of Tra2beta were reaccumulated in their nuclei. Treatment with small interference RNAs targeting up-frameshift-1 or transfection of a plasmid containing tra2beta1 cDNA did not induce tra2beta4 isoform expression and did not modify the arsenite-induced expression of this isoform, suggesting that neither the nonsense-mediated mRNA decay nor the autoregulatory control by excess amounts of Tra2beta participated in the tra2beta4 isoform generation. Knockdown of Tra2beta facilitated skipping of the central variable region of the CD44 gene and suppressed cell growth. In contrast, overexpression of Tra2beta stimulated combinatorial inclusion of multiple variable exons in the region and cell growth. The similar skipping and inclusion of the variable region were observed in arsenite-treated cells. Our results suggest that Tra2beta may regulate cellular oxidative response by changing alternative splicing of distinct genes including CD44.

The phosphodiesterase 4B (PDE4B) interacts with disrupted-in-schizophrenia 1 (DISC1), which is a known genetic risk factor for schizophrenia, bipolar disorder and major depressive disorder (MDD). PDE4B is also important in the regulation of cAMP signaling, a second messenger implicated in learning, memory, and mood. In this study, we determined mRNA expression levels of the PDE4B gene in the peripheral blood leukocytes of patients with MDD and control subjects (n = 33, each). Next we performed two-stage case-controlled association analyses (first set; case = 174, controls = 348; second set; case = 481, controls = 812) in the Japanese population to determine if the PDE4B gene is implicated in MDD. In the leukocytes, a significantly higher expression of the PDE4B mRNA was observed in the drug-naive MDD patients compared with control subjects (P

Prediction of the disease status is one of the most important objectives of genetic studies. To select the genes with strong evidence of the association with type 2 diabetes mellitus, we validated the associations of the seven candidate loci extracted in our earlier study by genotyping the samples in two independent sample panels. However, except for KCNQ1, the association of none of the remaining seven loci was replicated. We then selected 11 genes, KCNQ1, TCF7L2, CDKAL1, CDKN2A/B, IGF2BP2, SLC30A8, HHEX, GCKR, HNF1B, KCNJ11 and PPARG, whose associations with diabetes have already been reported and replicated either in the literature or in this study in the Japanese population. As no evidence of the gene-gene interaction for any pair of the 11 loci was shown, we constructed a prediction model for the disease using the logistic regression analysis by incorporating the number of the risk alleles for the 11 genes, as well as age, sex and body mass index as independent variables. Cumulative risk assessment showed that the addition of one risk allele resulted in an average increase in the odds for the disease of 1.29 (95% CI=1.25-1.33, P=5.4 x 10(-53)). The area under the receiver operating characteristic curve, an estimate of the power of the prediction model, was 0.72, thereby indicating that our prediction model for type 2 diabetes may not be so useful but has some value. Incorporation of data from additional risk loci is most likely to increase the predictive power.

Psychosocial factors are important determinants of disease manifestations, treatment efficacy, and prognosis of functional and inflammatory bowel disorders. Isolation of C57BL/6J mice from their 4 brothers growing in the same cage reduced goblet cells and MUC2 expression with a peak on day 8 in the rectum, but not in the colon. Gene expression analysis using a whole mouse genome microarray showed that the stress induced a 10-fold larger change in the gene expression in the rectum (722 genes) than in the colon (72 genes). The Ingenuity Pathway Analysis (IPA) application organized the rectum-specific 711 genes into stress response-related pathways. Nuclear factor-kappaB-related cytokine networks constructed with IPA showed selective up-regulation of interleukin (IL)-18 mRNA expression, which was also confirmed by real-time polymerase chain reaction. The stress produced active forms of caspase 1, IL-18, and a negative regulator for goblet cells, Notch 1, only in the rectum. IL-18-knockout mouse rectum had significantly increased goblet cells and MUC2 mucin, compared with wild-type mouse rectum. The absence of IL-18 completely blocked the stress-induced changes in gene expression and the goblet cell responses in the rectum. Thus, IL-18 may be a crucial determinant for the vulnerability of the rectum to psychosocial stress.