Combination of T cell-redirecting bispecific antibody ERY974 and chemotherapy reciprocally enhances efficacy against non-inflamed tumours.

Identifying a strategy with strong efficacy against non-inflamed tumours is vital in cancer immune therapy. ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody that recognizes glypican-3 and CD3. Here we examine the combination effect of ERY974 and chemotherapy (paclitaxel, cisplatin, and capecitabine) in the treatment of non-inflamed tumours in a xenograft model. ERY974 monotherapy shows a minor antitumour effect on non-inflamed NCI-H446 xenografted tumours, as infiltration of ERY974-redirected T cells is limited to the tumour-stromal boundary. However, combination therapy improves efficacy by promoting T cell infiltration into the tumour centre, and increasing ERY974 distribution in the tumour. ERY974 increases capecitabine-induced cytotoxicity by promoting capecitabine conversion to its active form by inducing thymidine phosphorylase expression in non-inflamed MKN45 tumour through ERY974-induced IFNγ and TNFα in T cells. We show that ERY974 with chemotherapy synergistically and reciprocally increases antitumour efficacy, eradicating non-inflamed tumours.

Budding Uninhibited by Benzimidazole-1 Insufficiency Prevents Acute Renal Failure in Severe Sepsis by Maintaining Anticoagulant Functions of Vascular Endothelial Cells.

Severe sepsis is critical to health and can result in acute renal failure (ARF). Tissue factor (TF) and thrombomodulin (TM) play key roles in vascular endothelial functions by helping maintain microcirculation in the kidney. Budding uninhibited by benzimidazole-1 (Bub1) plays a role in Akt and JNK signaling, which control TF and TM, respectively. We hypothesized that Bub1 could control vascular endothelial function in sepsis. The aim of this study was to determine the role of Bub1 in septic ARF. We used Mouse cecum ligation and puncture (CLP) using low Bub1 expressing (Bub1) and wild-type (Bub1) mice in vivo and lipopolysaccharide (LPS) stimulation of human aortic endothelial cell (HAEC) in vitro. Bub1 mice had a higher survival rate after CLP than Bub1. Bub1 mice had more severe ARF after CLP than Bub1 with blood biochemical and pathological analyses. TF expression in Bub1 mice and control HAEC (control) significantly increased in the septic model compared with Bub1 and Bub1 silenced HAEC (siBub1). TM expression in the control significantly decreased after LPS stimulation compared with siBub1. Akt and JNK phosphorylation of siBub1 were attenuated after LPS stimulation. Associations of Bub1 with Akt or JNK after LPS stimulation of HAEC were detected using immunoprecipitation, suggesting that Bub1 is involved in the phosphorylation of Akt and JNK after LPS stimulation. Bub1 insufficiency attenuates TF expression and reduces TM suppression by blocking Akt and JNK phosphorylation, respectively, thus leading to the prevention of ARF and death caused by sepsis.

Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet.

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, details of the liver-specific molecular mechanisms responsible for the NAFLD-NASH-HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis-associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.

BUBR1 Insufficiency in Mice Increases their Sensitivity to Oxidative Stress.

BACKGROUND/AIM: Budding uninhibited by benzimidazole-related 1 (BUBR1) plays an important role in the spindle assembly checkpoint to prevent chromosome missegregation and aneuploidy during mitosis. We previously generated mutant mice that express BUBR1 at only 20% of the normal level (BubR1L/L mice). Here, we examined the effect of low BUBR1 expression on oxidative stress-induced carcinogenesis in mice. MATERIALS AND METHODS: We orally administered either a potassium bromate (KBrO3) solution (2 g/l) or tap water to BubR1L/L and wild-type (BubR1+/+)mice for 16 weeks and examined the subsequent incidence of tumours. RESULTS: KBrO3-treated BubR1L/L mice showed significantly higher mortality than the KBrO3-treated BubR1+/+ and control tap water-treated mice (p=0.0082). Histopathological and immunohistochemical analyses revealed that the spleens of surviving BubR1L/L mice were occupied by non-B-, non-T-cells with high proliferative potential. CONCLUSION: Our results indicate that low BUBR1 expression increases oxidative stress-induced mortality in mice, possibly caused by splenic neoplasms.

BubR1 Insufficiency Impairs Liver Regeneration in Aged Mice after Hepatectomy through Intercalated Disc Abnormality.

A delay in liver regeneration after partial hepatectomy (PHx) leads to acute liver injury, and such delays are frequently observed in aged patients. BubR1 (budding uninhibited by benzimidazole-related 1) controls chromosome mitotic segregation through the spindle assembly checkpoint, and BubR1 down-regulation promotes aging-associated phenotypes. In this study we investigated the effects of BubR1 insufficiency on liver regeneration in mice. Low-BubR1-expressing mutant (BubR1(L/L)) mice had a delayed recovery of the liver weight-to-body weight ratio and increased liver deviation enzyme levels after PHx. Microscopic observation of BubR1(L/L) mouse liver showed an increased number of necrotic hepatocytes and intercalated disc anomalies, resulting in widened inter-hepatocyte and perisinusoidal spaces, smaller hepatocytes and early-stage microvilli atrophy. Up-regulation of desmocollin-1 (DSC1) was observed in wild-type, but not BubR1(L/L), mice after PHx. In addition, knockdown of BubR1 expression caused down-regulation of DSC1 in a human keratinocyte cell line. BubR1 insufficiency results in the impaired liver regeneration through weakened microstructural adaptation against PHx, enhanced transient liver failure and delayed hepatocyte proliferation. Thus, our data suggest that a reduction in BubR1 levels causes failure of liver regeneration through the DSC1 abnormality.

Correlation of HER2 expression with clinicopathological characteristics and prognosis in resectable gastric cancer.

Results from the Trastuzumab for Gastric Cancer (ToGA) trial highlighted the clinical significance of trastuzumab in the treatment of HER2 (Human Epidermal Growth Factor Receptor type 2)-positive gastric cancer. However, whether expression of HER2 is related to prognosis of gastric cancer is still controversial. A total of 360 consecutive patients with gastric cancer who underwent surgical resection in our Department from 1994 to 2007 were analyzed. We performed immunohistochemical analysis of HER2 expression. HER2 expression level was classified into four scores (0, 1+, 2+ and 3+). There were 37 (10%) patients with a score of 3+. A score of 3+ was defined as being HER2-positive. Recurrence-free survival was worse in HER2-positive cases (p=0.045). When the analysis was conducted with intestinal types of cancer, RFS was considerably worse in the HER2-positive group (p=0.011). HER2 expression may have potential as a prognostic factor for intestinal cancer types. Further research is warranted.

Aberrations of BUBR1 and TP53 gene mutually associated with chromosomal instability in human colorectal cancer.

BACKGROUND/AIM: Defects in mitotic checkpoint and p53-dependent pathways associate with chromosomal instability. In the present study, we investigated the interplay between BUBR1 and p53 and their association with genetic instability in colorectal cancer. PATIENTS AND METHODS: 139 colorectal cases were examined for BUBR1, p53 and genetic instability indicators. BUBR1 expression was evaluated by immunohistochemistry and TP53 gene was directly sequenced. DNA ploidy was studied by laser scanning cytometry; MSI and TP53 loss of heterozygosity was also examined. RESULTS: 64% of cases had high BUBR1 expression and were associated with the TP53 mutation. High BUBR1 expression and TP53 mutation associated with DNA aneuploidy and showed an inverse association with MSI. Cases with high BUBR1 expression and TP53 mutation had profound aneuploidy phenotypes and less frequent MSI compared to cases with one or neither aberration. CONCLUSION: Our findings indicated an interplay between BUBR1 and p53 in colorectal cancer. Altered expression of both molecules was associated with chromosomal instability.

Significance of accurate human epidermal growth factor receptor-2 (HER2) evaluation as a new biomarker in gastric cancer.

BACKGROUND: HER2 testing in gastric cancer differs from testing in breast cancer because of inherent differences in tumor biology; gastric cancer more frequently shows HER2 heterogeneity and incomplete membrane staining. The aim of the present study was to evaluate the frequency and accuracy of detection of HER2 expression by application of standard criteria in Japanese patients with gastric cancer. MATERIAL AND METHODS: A total of 198 tumor specimens were assessed for HER2 expression by immunohistochemistry (IHC) using the antibodies HercepTest™ and 4B5. Both hand-operated and automated IHC were performed. RESULTS: HER2 expression differed according to the IHC method and antibodies used. HER2 IHC3+ tumors were identified in 21 (10%) and 7 (3.5%) cases by hand-operated and automated IHC, respectively. CONCLUSION: Among patients with gastric cancer, FISH may be performed in cases of IHC1+ by automated IHC. Further research is required to clarify the relevance of HER2 staining and scoring for the clinical response to HER2-targeted therapy.

Competitive inhibition of SGLT2 by tofogliflozin or phlorizin induces urinary glucose excretion through extending splay in cynomolgus monkeys.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed a glucose lowering effect in type 2 diabetes patients through inducing renal glucose excretion. Detailed analysis of the mechanism of the glucosuric effect of SGLT2 inhibition, however, has been hampered by limitations of clinical study. Here, we investigated the mechanism of urinary glucose excretion using nonhuman primates with SGLT inhibitors tofogliflozin and phlorizin, both in vitro and in vivo. In cells overexpressing cynomolgus monkey SGLT2 (cSGLT2), both tofogliflozin and phlorizin competitively inhibited uptake of the substrate (α-methyl-d-glucopyranoside; AMG). Tofogliflozin was found to be a selective cSGLT2 inhibitor, inhibiting cSGLT2 more strongly than did phlorizin, with selectivity toward cSGLT2 1,000 times that toward cSGLT1; phlorizin was found to be a nonselective cSGLT1/2 inhibitor. In a glucose titration study in cynomolgus monkeys under conditions of controlled plasma drug concentration, both tofogliflozin and phlorizin increased fractional excretion of glucose (FEG) by up to 50% under hyperglycemic conditions. By fitting the titration curve using a newly introduced method that avoids variability in estimating the threshold of renal glucose excretion, we found that tofogliflozin and phlorizin lowered the threshold and extended the splay in a dose-dependent manner without significantly affecting the tubular transport maximum for glucose (TmG). Our results demonstrate the contribution of SGLT2 to renal glucose reabsorption (RGR) in cynomolgus monkeys and demonstrate that competitive inhibition of cSGLT2 exerts a glucosuric effect by mainly extending splay and lowering threshold without affecting TmG.

Mortalin is a prognostic factor of gastric cancer with normal p53 function.

BACKGROUND: Mortalin is a heat-non-inducible member of the heat shock protein 70 family. Mortalin binds to p53 and prevents p53 from entering the nucleus. To understand the significance of mortalin in gastric cancer, we investigated the expression of mortalin and p53. METHODS: Expression of mortalin and p53 was examined by immunohistochemical staining of 182 clinical samples of gastric cancer. RESULTS: Mortalin-positive and aberrant p53-positive tumors were found in 75.2 and 49.5 % of cases, respectively. Mortalin-positive tumors were deeper in invasion and had more lymph node and liver metastases compared with mortalin-negative tumors (P < 0.01, P < 0.05, respectively). Mortalin-positive tumors had worse prognosis compared with mortalin-negative tumors (P = 0.035). Moreover, in tumors with normal p53 function, mortalin-positive tumors had worse prognosis compared with mortalin-negative tumors (P = 0.017). CONCLUSIONS: Mortalin has a great impact on gastric cancer with normal p53. Therefore, mortalin is a target molecule for treatment of gastric cancer, as well as a promising prognostic factor, especially in tumors with normal p53.

Contribution of BubR1 to oxidative stress-induced aneuploidy in p53-deficient cells.

DNA aneuploidy is observed in various human tumors and is associated with the abnormal expression of spindle assembly checkpoint (SAC) proteins. Oxidative stress (OS) causes DNA damage and chromosome instability that may lead to carcinogenesis. OS is also suggested to contribute to an increase in aneuploid cells. However, it is not clear how OS is involved in the regulation of SAC and contributes to carcinogenesis associated with aneuploidy. Here we show that an oxidant (KBrO3) activated the p53 signaling pathway and suppressed the expression of SAC factors, BubR1, and Mad2, in human diploid fibroblast MRC5 cells. This suppression was dependent on functional p53 and reactive oxygen species. In p53 knockdown cells, KBrO3 did not suppress BubR1 and Mad2 expression and increased both binucleated cells and cells with >4N DNA content. BubR1 and not Mad2 downregulation suppressed KBrO3-induced binucleated cells and cells with >4N DNA content in p53 knockdown cells, suggesting that BubR1 contributes to enhanced polyploidization by a mechanism other than its SAC function. In analysis of 182 gastric cancer specimens, we found that BubR1 expression was significantly high when p53 was positively stained, which indicates loss of p53 function (P = 0.0019). Moreover, positive staining of p53 and high expression of BubR1 in tumors were significantly correlated with DNA aneuploidy (P = 0.0065). These observations suggest that p53 deficiency may lead to the failure of BubR1 downregulation by OS and that p53 deficiency and BubR1 accumulation could contribute to gastric carcinogenesis associated with aneuploidy.

Hepatic interferon-gamma-induced protein-10 expression is more strongly associated with liver fibrosis than interleukin-28B single nucleotide polymorphisms in hepatocellular carcinoma resected patients with chronic hepatitis C.

AIM: Single nucleotide polymorphisms (SNP) around IL-28B and interferon (IFN)-stimulated gene (ISG) expression are predictors of response to standard therapy involving IFN for chronic hepatitis C virus (HCV) infection. We analyzed the association between these predictors to improve the prediction of the response to IFN therapy after liver resection for hepatocellular carcinoma (HCC). METHODS: Data were collected from 74 patients with HCV-induced HCC. The IL-28B genotype and hepatic ISG mRNA levels were analyzed to clarify their association, focusing on the progression of liver fibrosis. RESULTS: Fifty patients were identified as having major alleles (rs8099917 TT) and the remaining 24 patients had minor alleles (rs8099917 TG or GG). Hepatic ISG15 expression was lower in the IL-28B major group than that in the IL-28B minor group (P < 0.005). IP-10 expression was similar between the IL-28B major and minor groups (P = 0.44). IP-10 expression was elevated with advancing stages of liver fibrosis in HCV infected patients (P = 0.005). In patients with mild or no fibrosis, the IL-28B major group had lower IP-10 expression than the IL-28B minor group (P = 0.02). However, in patients with advanced fibrosis, IP-10 expression was not different between the IL-28B major and minor groups (P = 0.66). CONCLUSION: Hepatic ISG15 expression is associated with IL-28B polymorphisms, while IP-10 is strongly affected by liver fibrosis.

Needle-free jet injection of DNA and protein vaccine of the far-eastern subtype of tick-borne encephalitis virus induces protective immunity in mice.

Tick-borne encephalitis virus (TBEV) causes severe encephalitis in humans. It is endemic in one area of Japan; however no commercial vaccine is available in that country. In this Japan-based study, the efficacy of subviral particles (SPs) of TBEV administered by needle-free injector was evaluated as a vaccine candidate. Inoculation with SP-encoding DNA by needle-free injector induced neutralizing antibodies more efficiently than when administered by needle and syringe, and mice vaccinated with one dose by needle-free injector survived challenge with a lethal dose of TBEV. These results suggest that SP vaccines delivered by needle-free injector can protect against TBEV infection.

Development of an ELISA system for tick-borne encephalitis virus infection in rodents.

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.