Nozomi Kobayashi

Gene-expression profile changes correlated with tumor progression and lymph node metastasis in esophageal cancer

Purpose: The purpose of this research was to identify molecular clues to tumor progression and lymph node metastasis in esophageal cancer and to test their value as predictive markers. Experimental Design: We explored the gene expression profiles in cDNA array data of a 36-tissue training set of esophageal squamous cell carcinoma (ESCC) by using generalized linear model-based regression analysis and a feature subset selection algorithm. By applying the identified optimal feature sets (predictive gene sets), we trained and developed ensemble classifiers consisting of multiple probabilistic neural networks combined with AdaBoosting to predict tumor stages and lymph node metastasis. We validated the classifier abilities with 18 independent cases of ESCC. Results: We identified 71 genes of 1289 cancer-related genes of which the expression correlated with tumor stages. Of the 71 genes, 47 significantly differed between the Tumor-Node-Metastasis pT1/2 and pT3/4 stages. Cell cycle regulators and transcriptional factors possibly promoting the growth of tumor cells were highly expressed in the early stages of ESCC, whereas adhesion molecules and extracellular matrix-related molecules possibly promoting invasiveness increased in the later stages. For lymph node metastasis, we identified 44 genes with predictive values, which included cell adhesion molecules and cell membrane receptors showing higher expression in node-positive cases and cell cycle regulators and intracellular signaling molecules showing higher expression in node-negative cases. The ensemble classifiers trained with the selected features predicted tumor stage and lymph node metastasis in the 18 validation cases with respective accuracies of 94.4% and 88.9%. This demonstrated the reproducibility and predictive value of the identified features. Conclusion: We suggest that these characteristic genes will provide useful information for understanding the malignant nature of ESCC as well as information useful for personalizing the treatments.

A novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates inflammatory colonic injury in mice

BACKGROUND In inflammatory bowel disease (IBD), gut inflammation is associated with the activation of nuclear factor kappa B (NF-κB), a key pro-inflammatory transcription factor. AIM To investigate the therapeutic potential of a novel, specific NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), we examined its effect on IBD using murine experimental colitis models. METHODS The in vitro effect of DHMEQ was evaluated by inflammatory cytokine production and p65 immunostaining using HT-29 and RAW264.7 cells. The in vivo therapeutic effect of DHMEQ was studied in colitis induced by dextran sulphate sodium (DSS) and trinitrobenzenesulphonic acid (TNBS). In these, progression and severity of colitis was mainly assessed by the disease activity index (DAI), histopathology, cellular infiltration, and mRNA expression levels of pro-inflammatory cytokines in the colonic tissues. RESULTS In RAW264.7 cells, DHMEQ significantly inhibited tumour necrosis factor (TNF)-α and interleukin (IL)-6 production induced by LPS in a dose-dependent manner by blocking the nuclear translocation of NF-κB. In addition, DHMEQ inhibited IL-8 production induced by LPS in HT-29 cells. DHMEQ significantly ameliorated DSS colitis as assessed by DAI scores, colonic oedema, and histological scores. Immunohistochemistry revealed that DHMEQ inhibited colonic infiltration of nuclear p65(+) cells, CD4(+) lymphocytes, and F4/80(+) macrophages. mRNA expression levels of the pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-12p40, IL-17, and MCP-1 were also suppressed by DHMEQ administration. Furthermore, DHMEQ significantly ameliorated TNBS colitis as assessed by body-weight changes and histological scores. CONCLUSION DHMEQ ameliorated experimental colitis in mice. These results indicate that DHMEQ appears to be an attractive therapeutic agent for IBD.

Array comparative genomic hybridization analysis revealed four genomic prognostic biomarkers for primary gastric cancers

Unlike the case with some other solid tumors, whole genome array screening has not revealed prognostic genetic aberrations in primary gastric cancer. Comparative genomic hybridization (CGH) using bacterial artificial chromosome (BAC) arrays for 56 primary gastric cancers resulted in identification of four prognostic loci in this study: 6q21 (harboring FOXO3A; previously FKHRL1), 9q32 (UGCG), 17q21.1 approximately q21.2 (CASC3), and 17q21.32 (HOXB3 through HOXB9). If any one of these four loci was deleted, the prognosis of the patient was significantly worse (P = 0.0019). This was true even for advanced tumors (stage IIIA, IIB, or IV, n = 39) (P = 0.0113), whereas only 1 of the 17 patients with less advanced tumors (stage IA, IB, or II; n = 17) died of disease after surgery. Multivariate analysis according to the status of four BACs or pathological stage based on the Japanese Classification of Gastric Carcinoma (stages IA, IB, and II vs. stages IIIA, IIIB, and IV) demonstrated that the BAC clone status was also an independent prognostic factor (P = 0.006). These findings may help predict which patients with malignant potential need more intensive therapy, or may point to new therapeutic approaches especially for advanced tumors. The parameter here termed the integrated genomic prognostic biomarker may therefore be of clinical utility as a prognostic biomarker.

Changes in mutagenicity and acute toxicity of solutions of iodinated X-ray contrast media during chlorination.

In the present study, the effects of chlorination on the mutagenicity (assessed via the Ames assay) and acute toxicity (assessed via a bioluminescence inhibition assay) of solutions containing one of five commonly used iodinated X-ray contrast media (ICM) (iopamidol, iohexol, iopromide, iomeprol, and diatrizoate) were investigated. Of the five ICM tested, only iopamidol was degraded by chlorine. Chlorination of the iopamidol-containing solution induced both mutagenicity and acute toxicity, which increased with chlorination time (up to 96 h). The areas of five out of 54 peaks detected on the LC/MS total ion chromatogram had good correlation (r(2)>0.90) between peak area and observed mutagenicity. To identify possible contributors to the observed mutagenicity, the Ames assay and LC/MS analysis were conducted on samples collected at 48-h chlorination time and extracted under different pH conditions. Of the five peaks, one peak was detected in the sample extracted at pH 7, but this sample was not mutagenic, indicating that the peak was not related to the observed mutagenicity. MS/MS analysis with an orbitrap mass spectrometer of the remaining four peaks revealed that two of the peaks represented the same TP (detected in negative and positive ion modes). Finally, three TPs were identified as suspected contributors to the mutagenicity induced by the iopamidol-containing solution after chlorination: 5-[(1,3-dihydroxypropan-2-yl)carbamoyl]-3-[(3-hydroxypropanoyl)oxy]-2,4-diiodobenzoic acid; N-(1,3-dihydroxypropan-2-yl)-3-(2,3-dioxopropyl)-2,4,6-triiodobenzamide; and 3-[(1,3-dihydroxypropan-2-yl)carbamoyl]-5-[(3-hydroxybutanoyl)oxy]-2,4,6-triiodobenzoic acid. Prediction of the mutagenicity potential of these three TPs with a battery of four quantitative structure-activity relationship models did not contradict our conclusion that these TPs contributed to the observed mutagenicity.

Immunomodulatory effect of nuclear factor-κB inhibition by dehydroxymethylepoxyquinomicin in combination with donor-specific blood transfusion.

Background. Nuclear factor-&kgr;B (NF-&kgr;B) is a key molecule in alloimmune responses, however, its role in tolerance induction is not clear. We have previously reported that dehydroxymethylepoxyquinomycin (DHMEQ), a novel NF-&kgr;B inhibitor, prolongs cardiac allograft survival. In this study, we evaluated the immunomodulatory effects of DHMEQ when combined with a donor-specific blood transfusion (DST), and assessed whether the treatment induces tolerance in a mouse heart transplantation model. Methods. DST (20×106 splenocytes) was given intravenously at day −7. DHMEQ (30 mg/kg/day) was administered intraperitoneally for 14 days after DST. Graft survival and histology were evaluated. The underlying mechanisms of immunomodulation by DST and DHMEQ treatments were investigated by assessing alloimmune responses after transplantation. Results. In fully mismatched H2d-to-H2b heart transplants, DST alone prolonged allograft median survival time to 15 days, whereas when DST was combined with DHMEQ treatment, the graft median survival time was prolonged to 39.5 days. When the donor-recipient strain combination was reversed, that is, H2b-to-H2d, heart transplants were accepted (>150 days survival) in more than 60% of recipients treated with a DST and DHMEQ, whereas control allografts were all rejected within 8 days. The combined therapy markedly inhibited immune responses by both the direct and indirect allorecognition pathways mainly attributed to promotion of activation-induced cell death and Treg generation. Conclusions. Our results demonstrate the distinctive ability of NF-&kgr;B inhibition in combination with donor alloantigen to promote transplantation tolerance through multiple cellular mechanisms.

Fatty acid-binding protein 5 function in hepatocellular carcinoma through induction of epithelial-mesenchymal transition

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with poor prognosis. The correlation between overexpression of fatty acid‐binding protein 5 (FABP5) and malignant potential of tumor growth and metastasis in several cancers has been previously reported. However, the correlation between FABP5 expression and HCC malignant behavior remains unknown. We compared FABP5 expression and patient characteristics in paired HCC and adjacent noncancerous liver tissues from 243 patients who underwent surgical resection of primary HCC. Cell proliferation, invasion, and migration assays were performed in HCC cell lines overexpressing FABP5 or downregulated for FABP5. Tumor growths were monitored in xenograft model, and liver and lung metastasis models were established. In the 243 HCC patients, FABP5‐positive staining (n = 139/243, 57.2%) was associated with poor prognosis and recurrence (P < 0.0001) and showed positive correlation with distant metastasis, tumor size and vascular invasion (P < 0.05). Cell proliferation, invasion, and migration in vitro were enhanced by upregulation of FABP5 and decreased by downregulation of FABP5 in HCC cell lines. Similar results in tumor formation and metastasis were obtained through in vivo analyses. PCR array results revealed upregulation of SNAI1 in FABP5‐overexpressing HepG2 cells. Western blot analysis showed significantly increased expression of E‐cadherin and ZO‐1 and decreased SNAI1 expression and nuclear translocation of β‐catenin by knockdown of FABP5. We revealed a significant role for FABP5 in HCC progression and metastasis through the induction of epithelial‐to‐mesenchymal transition. FABP5 may be a potential novel prognostic biomarker and new therapeutic target for HCC.

α-fetoprotein, vascular endothelial growth factor receptor-1 and early recurrence of hepatoma.

AIM To investigate whether α-fetoprotein (AFP) and vascular endothelial growth factor receptor (VEGFR)-1 correlate with early recurrence of hepatoma/hepatocellular carcinoma (HCC). METHODS From 2000 to 2005, 114 consecutive patients with HCC underwent primary curative hepatectomy. The mean age was 60.7 (8.7) years and 94 patients were male. The median follow-up period was 71.2 mo (range: 43-100 mo). Immediately prior to commencing laparotomy, 5 mL bone marrow was aspirated from the sternum and collected in citrate-coated test tubes. The initial 2 mL of bone marrow aspirate was discarded in each case. AFP mRNA and VEGFR-1 mRNA in the bone marrow and peripheral blood (BM- and PH-AFP mRNA and BM- and PH-VEGFR-1 mRNA, respectively) were measured by real-time quantitative reverse transcription polymerase chain reaction. As normal controls, VEGFR-1 mRNA in the bone marrow and peripheral blood was also measured in 11 living liver donors. These data were evaluated for any correlation with early recurrence, comparing clinical and pathological outcomes. RESULTS The cut-off value of the BM-AFP mRNA and PH-AFP mRNA level in patients with HCC was set at 1.92 × 10(-7) and zero, respectively, based on data from the controls. A total of 34 (29.8%) and six (5.4%) patients were positive for BM-AFP mRNA and PH-AFP mRNA, respectively. The BM-VEGFR-1 mRNA levels in all HCC patients were higher than those in the normal controls, and this was the case also for PH-VEGFR-1mRNA. The 25-percentile values for the BM- and PH-VEGFR-1 mRNA in HCC patients were used as the cut-off values for assigning the patients into two groups based on these transcript levels. The High group for BM- VEGFR-1 mRNA contained 81 (71.1%) HCC cases and the Low group was assigned 33 (28.9%) patients. These numbers for PH-VEGFR-1mRNA were 78 (75.0%) and 26 (25.0%), respectively. HCC recurred in 80 patients; in the remnant liver in 48 cases, in the remnant liver and remote tissue in 20, and in the remote tissue alone in 12. BM-AFP mRNA-positive cases showed a significantly higher rate of early recurrence (within 1 year of surgical treatment) compared with BM-AFP mRNA-negative patients (P = 0.0091). Patients were classified into four groups according to the level/status of their BM-VEGFR-1 and BM-AFP mRNA as follows: group A (n = 23), BM-VEGFR-1/BM-AFP mRNA = low/negative; group B (n = 57) high/negative; group C (n = 10) low/positive; group D (n = 24), high/positive. This classification was found to correlate with a recurrence of this disease within 1 year (P = 0.0228). The disease-free survival curve of group A was significantly better than that of groups B, C or D (P = 0.0437, P = 0.0325, P = 0.0225). No other classification (i.e., PH-VEGF-R1/BM-AFP, BM-VEGF-R1/PH-AFP, and PH-VEGF-R1/PH-AFP mRNA) showed such a correlation. CONCLUSION The evaluation of BM-AFP and BM-VEGFR-1 mRNA in patients with HCC may be a valuable predictor of disease recurrence following curative resection.

Successful penile reconstruction after multimodal therapy in patients with primitive neuroectodermal tumor originating from the penis

We herein present an extremely rare case of primitive neuroectodermal tumor originating in the penis. A 16‐year‐old male adolescent presented with painful penile swelling. Pathological, immunohistochemical and cytogenetical examinations of the specimens obtained from total penectomy confirmed the diagnosis of primitive neuroectodermal tumor. After total penectomy, the patient received adjuvant chemotherapy with ifosfamide‐based regimen for 48 weeks. As a series of therapies, the patient underwent penile reconstruction surgery after completing adjuvant chemotherapy. The patient has not shown any evidence of recurrence for the 7 years after penile reconstruction surgery, and voiding function is completely normal. A favorable outcome was observed by multimodal therapy including aggressive resection for local control, intensive adjuvant chemotherapy, and penile reconstruction with cosmetic and functional success. Similar therapeutic approaches might be selected for children with primary malignant tumors of the penis.

The anti-mesothelin monoclonal antibody amatuximab enhances the anti-tumor effect of gemcitabine against mesothelin-high expressing pancreatic cancer cells in a peritoneal metastasis mouse model

Pancreatic cancer often has a very poor prognosis, even after complete resection. The recurrence of hepatic and peritoneal metastases is an important prognostic factor; therefore, the development of improved adjuvant therapy is urgently required. Mesothelin is a cell surface glycoprotein whose expression is restricted to a variety of cancer types, including pancreatic cancer. This expression pattern makes mesothelin an attractive target for cancer therapy, and several agents targeting mesothelin are currently in clinical trials. Here, we used the chimerized high-affinity anti-mesothelin monoclonal antibody amatuximab to investigate its effect on peritoneal metastasis. We used the AsPC-1 pancreatic cancer cell line engineered to express Gaussia luciferase (Gluc), (AsPC-1-Gluc) for in vivo experiments. Results showed that while amatuximab was not directly cytotoxic on an AsPC-1-Gluc tumor cells in a peritoneal metastasis model, it prevented the formation of tumor growth. In combination therapy with gemcitabine, amatuximab exhibited synergistic killing. Our results suggest that blockade of mesothelin by amatuximab may be a useful strategy for preventing the peritoneal dissemination of pancreatic cancer under an adjuvant setting.

3-[(dodecylthiocarbonyl)methyl]-glutarimide attenuates graft arterial disease by suppressing alloimmune responses and vascular smooth muscle cell proliferation.

Background Graft arterial disease (GAD) is a major cause of late graft loss after organ transplantation. Alloimmune responses and vascular remodeling eventually cause the transplant organ to develop GAD. In this study, we aimed to limit the development of GAD by inhibiting alloimmune responses and vascular smooth muscle cell (VSMC) proliferation with a new compound, 3-[(dodecylthiocarbonyl)methyl]-glutarimide ([DTCM]-glutarimide), in a murine cardiac model of GAD. Methods The hearts from B6.CH-2bm12 mice were transplanted into C57BL/6 mouse recipients to examine the extent of GAD. The recipients were treated with either vehicle or DTCM-glutarimide intraperitoneally (40 mg/kg per day) for 4 weeks. Results The administration of DTCM-glutarimide attenuated GAD formation (luminal occlusion: 37.9 ± 5.9% vs 14.8 ± 5.4%, P < 0.05) by inhibiting the number of graft-infiltrating cells and decreasing alloreactive interferon (IFN)-&ggr; production compared with control mice, as measured by the Enzyme-linked ImmunoSpot assay. In vitro, VSMCs proliferated on stimulation with either basic fibroblast growth factor or IFN-&ggr; and splenocytes after transplantation, but the addition of DTCM-glutarimide resulted in the inhibition of VSMC proliferation. Moreover, DTCM-glutarimide suppressed cyclin D1 expression and inhibited cell cycle progression from G1 to S in VSMCs. Conclusions The compound DTCM-glutarimide suppressed GAD development by inhibiting not only alloimmune responses but also VSMC proliferation in the graft.