Norifumi Ohashi

A Carbohydrate Recognition–Based Drug Delivery and Controlled Release System using Intraperitoneal Macrophages as a Cellular Vehicle

The lymphoid tissue in the omentum, at the so-called milky spots, is known as an initial place for disseminated cancer cells to develop into solid tumors. In the present study, i.p. macrophages significantly took up oligomannose-coated liposomes (OMLs) that were injected into the peritoneal cavity, and then gradually accumulated in the omentum and the other lymphoid tissues within 24 hours of i.p. injection of OMLs. When 5-fluorouracil (5-FU) was encapsulated in the OMLs, >60% of administered 5-FU accumulated in the omentum. Treatment of macrophages at 39 degrees C for 30 minutes led to the release of 5-FU from the macrophages, suggesting that controlled release from macrophages could be achieved by mild hyperthermia. We encased magnetic nanoparticles, which are known to convert electromagnetic energy to heat in the OMLs to achieve in vivo hyperthermia at the site. Using this system in a mouse i.p. metastasis model, we successfully controlled tumor development by coadministration of OML-encased 5-FU and OML-encased magnetic nanoparticles, followed by treatment with an alternating magnetic field. No apparent reduction was seen in tumor growth with the administration of OML-encased magnetic nanoparticles or OML-encased 5-FU alone. Thus, we have established the use of i.p. macrophages as a novel drug delivery system for the control of cancer metastatic to milky spots.

Disruptions of occludin and claudin-5 in brain endothelial cells in vitro and in brains of mice with acute liver failure.

Brain edema in acute liver failure (ALF) remains lethal. The role of vasogenic mechanisms of brain edema has not been explored. We previously demonstrated that matrix metalloproteinase‐9 (MMP‐9) contributes to the pathogenesis of brain edema. Here, we show that MMP‐9 mediates disruptions in tight junction (TJ) proteins in vitro and in brains of mice with ALF. We transfected murine brain endothelial cells (ECs) with MMP‐9 complementary DNA (cDNA) using pc DNA3.1 (+)/Myc‐His A expression vector. Tissue inhibitor of matrix metalloproteinases (TIMP‐1) cDNA transfection or GM6001 was used to inhibit MMP‐9. ALF was induced in mice with azoxymethane. Endogenous overexpression of MMP‐9 in brain ECs resulted in significant degradation of the TJ proteins occludin and claudin‐5. The alterations in TJ proteins correlated with increased permeability to fluorescein isothiocyanate–dextran molecules. The degradation of TJ proteins and the increased permeability were reversed by TIMP‐1 and GM6001. Similar results were found when MMP‐9 was exogenously added to brain ECs. We also found that TJ protein degradation was reversed with GM6001 in the brains of mice with ALF. Conclusion: TJ proteins are significantly perturbed in brains of mice with ALF. These data corroborate the important role of MMP‐9 in the vasogenic mechanism of brain edema in ALF. (HEPATOLOGY 2009.)

Biological Significance of Isolated Tumor Cells and Micrometastasis in Lymph Nodes Evaluated Using a Green Fluorescent Protein–Tagged Human Gastric Cancer Cell Line

Purpose: The biological significance of isolated tumor cells and micrometastasis in lymph node defined by the International Union against Cancer remains essentially unknown because of the lack of appropriate animal models. In the present study, we developed a lymph node micrometastasis model featuring a human gastric cancer cell line tagged with green fluorescent protein gene (GCIY-EGFP), which allows visualization of even isolated tumor cells in the development of metastasis without histologic procedure. Using this model, we investigated the effect of surgery and chemotherapy on the growth of early-phase metastasis formation in the lymph nodes. Experimental Design: The time course of spontaneous inguinal lymph node metastasis after s.c. inoculation of GCIY-EGFP cells into nude mice was examined with fluorescence dissecting microscopy. Then, the effects of surgical removal of the primary tumor with or without anti-asialo GM1 treatment or postoperative chemotherapy on the growth of isolated tumor cells and micrometastasis in the lymph nodes were examined. Results: GCIY-EGFP cells were found to metastasize spontaneously to the inguinal lymph nodes to form isolated tumor cells, micrometastasis, and, finally, develop macroscopic metastasis at 1 to 2, 3 to 5, and 5 weeks postinjection, respectively. When the primary tumors were removed within 2 weeks of inoculation, isolated tumor cells, but not micrometastasis, in the lymph nodes regressed by 4 weeks after surgery in all the mice examined (five of five). This spontaneous regression of isolated tumor cells was completely reversed by anti-asialo GM1 treatment, which could deplete natural killer cells effectively in nude mice. Chemotherapy following resection of the primary tumor at an early stage partially eliminated the remaining micrometastasis in the lymph nodes. Conclusions: These results suggest that isolated tumor cells in the regional lymph nodes regressed by removal of the primary tumor mainly via natural killer cell–mediated antitumor activity and that micrometastasis in the lymph nodes could be effectively eliminated by the postoperative chemotherapy.

Occludin is regulated by epidermal growth factor receptor activation in brain endothelial cells and brains of mice with acute liver failure.

Mechanisms of brain edema in acute liver failure (ALF) are not completely understood. We recently demonstrated that matrix metalloproteinase 9 (MMP‐9) induces significant alterations to occludin in brain endothelial cells in vitro and in brains of mice with experimental ALF (Hepatology 2009;50:1914). In this study we show that MMP‐9‐induced transactivation of epidermal growth factor receptor (EGFR) and p38 MAPK/NFκB (mitogen‐activated protein kinase/nuclear factor‐kappa B) signals participate in regulating brain endothelial occludin level. Mouse brain endothelial bEnd3 cells were exposed to MMP‐9 or p38 MAPK up‐regulation in the presence and absence of EGFR inhibitor, p38 MAPK inhibitor, NFκB inhibitor, and/or appropriate small interfering RNA. Reverse‐transcription polymerase chain reaction (RT‐PCR) and western blotting were used for messenger RNA and protein expression analyses. Immunohistochemical staining and confocal microscopy were used to demonstrate cellular EGFR activation. Intraperitoneal azoxymethane was use to induce ALF in mice. Brains of comatose ALF mice were processed for histological and biochemical analyses. When bEnd3 cells were exposed to MMP‐9, EGFR was significantly transactivated, followed by p38 MAPK activation, I‐kappa B alpha (IκBα) degradation, NFκB activation, and suppression of occludin synthesis and expression. Similar EGFR activation and p38 MAPK/NFκB activation were found in the brains of ALF mice, and these changes were attenuated with GM6001 treatment. Conclusion: EGFR activation with p38 MAPK/NFκB signaling contributes to the regulation of tight junction integrity in ALF. EGFR activation may thus play an important role in vasogenic brain edema in ALF. (HEPATOLOGY 2011;)

Phosphorylation of 4E-BP1 predicts sensitivity to everolimus in gastric cancer cells

We studied the effect of everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) on human gastric cancer cell lines. Cell proliferation in 3 of 8 cell lines was effectively inhibited by everolimus. Basal phosphorylation level of 4E-BP1 (T37/46, T70) was significantly higher in everolimus-sensitive cells than in everolimus-resistant cells. In subcutaneous xenograft model, immunohistochemistry analysis revealed that everolimus-sensitive cells expressed high levels of phospho-4E-BP1 (T37/46). In conclusion, phosphorylation of 4E-BP1 may be a predictive biomarker of everolimus sensitivity in gastric cancer.

Simple and reproducible hepatectomy in the mouse using the clip technique.

AIM To investigate the reliability of massive hepatectomy models by using clip techniques. METHODS We analyzed anatomical findings in 100 mice following massive hepatectomy induced by liver reduction > 70%. The impact of various factors in the different models was also analyzed, including learning curves, operative time, survival curves, and histopathological findings. RESULTS According to anatomical results, models with 75%, 80%, and 90% hepatectomy produced massive hepatectomy. Learning curves and operative times were most optimal with the clip technique. Each hepatectomy performed using the clip technique produced a reasonable survival curve, and there were no differences in histopathological findings between the suture and clip techniques. CONCLUSION Massive hepatectomy by the clip technique is simple and can provide reliable and relevant data.

A Randomized Phase III Trial Exploring the Prognostic Value of Extensive Intraoperative Peritoneal Lavage in Addition to Standard Treatment for Resectable Advanced Gastric Cancer: CCOG 1102 Study

A randomized controlled trial has started in Japan to evaluate the efficacy of extensive intraoperative peritoneal lavage in the treatment of resectable advanced gastric cancer. Patients with T3 or deeper carcinoma of the stomach are intraoperatively randomized to either extensive intraoperative peritoneal lavage + arm or extensive intraoperative peritoneal lavage- arm. A total of 300 patients will be accrued from 20 institutions. The primary endpoint is disease-free survival, and secondary end-points are overall survival, peritoneal recurrence-free survival and incidence of adverse events.

Radical Surgery for Gastric Carcinoma: It is Not an Issue of Whether to Perform D1 or D2. Dissect as Many Lymph Nodes as Possible and You Will Be Rewarded

Abstract In the current review article, evidences on radical surgery for gastric cancer reported in the literature are highlighted. The authors conclude that extended lymphadenectomy offers a statistically significant survival benefit. This benefit is only evident if the operative mortality is less than 2%, as obtained in centers of excellence with a high-volume experience of resection of gastric cancer. Lymphadenectomy should no longer be considered only as a tool for cancer-staging, but also as a beneficial therapeutic measure.

Matrix metalloproteinase-9 contributes to parenchymal hemorrhage and necrosis in the remnant liver after extended hepatectomy in mice.

AIM To investigate the effect of matrix metalloproteinase-9 (MMP-9) on the remnant liver after massive hepatectomy in the mouse. METHODS Age-matched, C57BL/6 wild-type (WT), MMP-9(-/-), and tissue inhibitors of metalloproteinases (TIMP)-1(-/-) mice were used. The mice received 80%-partial hepatectomy (PH). Samples were obtained at 6 h after 80%-PH, and we used histology, immunohistochemical staining, western blotting analysis and zymography to investigate the effect of PH on MMP-9. The role of MMP-9 after PH was investigated using a monoclonal antibody and MMP inhibitor. RESULTS We examined the remnant liver 6 h after 80%-PH and found that MMP-9 deficiency attenuated the formation of hemorrhage and necrosis. There were significantly fewer and smaller hemorrhagic and necrotic lesions in MMP-9(-/-) remnant livers compared with WT and TIMP-1(-/-) livers (P < 0.01), with no difference between WT and TIMP-1(-/-) mice. Serum alanine aminotransaminase levels were significantly lower in MMP-9(-/-) mice compared with those in TIMP-1(-/-) mice (WT: 476 ± 83 IU/L, MMP-9(-/-): 392 ± 30 IU/L, TIMP-1(-/-): 673 ± 73 IU/L, P < 0.01). Western blotting and gelatin zymography demonstrated a lack of MMP-9 expression and activity in MMP-9(-/-) mice, which was in contrast to WT and TIMP-1(-/-) mice. No change in MMP-2 expression was observed in any of the study groups. Similar to MMP-9(-/-) mice, when WT mice were treated with MMP-9 monoclonal antibody or the synthetic inhibitor GM6001, hemorrhagic and necrotic lesions were significantly smaller and fewer than in control mice (P < 0.05). These results suggest that MMP-9 plays an important role in the development of parenchymal hemorrhage and necrosis in the small remnant liver. CONCLUSION Successful MMP-9 inhibition attenuates the formation of hemorrhage and necrosis and might be a potential therapy to ameliorate liver injury after massive hepatectomy.

Matrix metalloproteinase-9 in the initial injury after hepatectomy in mice

AIM To investigate the role of matrix metalloproteinase (MMP)-9 in the pathogenesis of postoperative liver failure (PLF) after extended hepatectomy (EH). METHODS An insufficient volume of the remnant liver (RL) results in higher morbidity and mortality, and a murine model with 80%-hepatectomy was used. All investigations were performed 6 h after EH. Mice were first divided into two groups based on the postoperative course (i.e., the PLF caused or did not), and MMP-9 expression was measured by Western blotting. The source of MMP-9 was then determined by immunohistological stainings. Tissue inhibitor of metalloproteinase (TIMP)-1 is the endogenous inhibitor of MMP-9, and MMP-9 behavior was assessed by the experiments in wild-type, MMP-9(-/-) and TIMP-1(-/-) mice by Western blotting and gelatin zymography. The behavior of neutrophils was also assessed by immunohistological stainings. An anti-MMP-9 monoclonal antibody and a broad-spectrum MMP inhibitor were used to examine the role of MMP-9. RESULTS Symptomatic mice showed more severe PLF (histopathological assessments: 2.97 ± 0.92 vs 0.11 ± 0.08, P < 0.05) and a higher expression of MMP-9 (71085 ± 18274 vs 192856 ± 22263, P < 0.01). Nonnative leukocytes appeared to be the main source of MMP-9, because MMP-9 expression corresponding with CD11b positive-cell was observed in the findings of immunohistological stainings. In the histopathological findings, the PLF was improved in MMP-9(-/-) mice (1.65% ± 0.23% vs 0.65% ± 0.19%, P < 0.01) and it was worse in TIMP-1(-/-) mice (1.65% ± 0.23% vs 1.78% ± 0.31%, P < 0.01). Moreover, neutrophil migration was disturbed in MMP-9(-/-) mice in the immunohistological stainings. Two methods of MMP-9 inhibition revealed reduced PLF, and neutrophil migration was strongly disturbed in MMP-9-blocked mice in the histopathological assessments (9.6 ± 1.9 vs 4.2 ± 1.2, P < 0.05, and 9.9 ± 1.5 vs 5.7 ± 1.1, P < 0.05). CONCLUSION MMP-9 is important for the process of PLF. The initial injury is associated with MMP-9 derived from neutrophils, and MMP-9 blockade reduces PLF. MMP-9 may be a potential target to prevent PLF after EH and to overcome an insufficient RL.