Junichiro Yamauchi

Heme oxygenase-1 accelerates tumor angiogenesis of human pancreatic cancer

Angiogenesis is necessary for the continued growth of solid tumors, invasion and metastasis. Several studies clearly showed that heme oxygenase-1 (HO-1) plays an important role in angiogenesis. In this study, we used the vital microscope system, transparent skinfold model, lung colonization model and transduced pancreatic cancer cell line (Panc-1)/human heme oxygenase-1 (hHO-1) cells, to precisely analyze, for the first time, the effect of hHO-1 gene on tumor growth, angiogenesis and metastasis. Our results revealed that HO-1 stimulates angiogenesis of pancreatic carcinoma in severe combined immune deficient mice. Overexpression of human hHO-1 after its retroviral transfer into Panc-1 cells did not interfere with tumor growth in vitro. While in vivo the development of tumors was accelerated upon transfection with hHO-1. On the other hand, inhibition of heme oxygenase (HO) activity by stannous mesoporphyrin was able transiently to delay tumor growth in a dose dependent manner. Tumor angiogenesis was markedly increased in Panc-1/hHO-1 compared to mock transfected and wild type. Lectin staining and Ki-67 proliferation index confirmed these results. In addition hHO-1 stimulated in vitro tumor angiogenesis and increased endothelial cell survival. In a lung colonization model, overexpression of hHO-1 increased the occurrence of metastasis, while inhibition of HO activity by stannous mesoporphyrin completely inhibited the occurrence of metastasis. In conclusion, overexpression of HO-1 genes potentiates pancreatic cancer aggressiveness, by increasing tumor growth, angiogenesis and metastasis and that the inhibition of the HO system may be of useful benefit for the future treatment of the disease.

In vivo evaluation of the early events associated with liver metastasis of circulating cancer cells

The mechanism of metastasis formation remains still largely unknown. Many studies underline the importance and complexity of the initial arrest of the circulating tumour cells in the target organ, a key stage in metastasis occurrence. In our study, we evaluated by visual means the metastasis formation using an in vivo microscopy system in a murine model. Moreover, we investigated the involvement of P-selectin in these processes using immunohistochemistry and P-selectin knockout mice. The present study offers direct evidence of distinct pathways for tumour metastasis formation by a lymphoma cell – EL-4 and a solid tumour cell – C26. Off-line analysis of the images and histological data confirmed that mechanical entrapment of the solid tumour cell, which had a bigger diameter than that of the liver sinusoids, promoted metastasis without any detectable involvement of adhesion molecules. On the other hand, we observed that lymphoma cells, in spite of their smaller diameter as compared to the sinusoids, promoted liver metastasis as well, but with the essential participation in their arrest of P-selectin, indicating an adhesion molecule-mediated pathway.

Early microcirculatory derangement in mild and severe pancreatitis models in mice.

Abstract An in vivo microscopic technique was used to clarify the increase in microvascular permeability and enhanced leukocyte–endothelium interaction of pancreatic microcirculation in experimental pancreatitis of differing severity. Using bovine albumin fluorescein isothiocyanate (FITC) and carboxyfluorescein diacetate succinimidyl ester (CFDASE) as tracers, the change in permeability and the behavior of leukocytes in the acinar microcirculation were quantified during the initial 1, 2, 6, and 12 h after the induction of caerulein pancreatitis in mice. Cold stress was added to produce the severe model. It was revealed that the early microcirculatory changes in the pancreas of caerulein pancreatitis included the increased permeability of endothelial lining and an accumulation of extravasated fluid in the perilobular space, which were more severe if cold stress was added. A decrease in flow velocity was also noted 2 h after the onset of severe pancreatitis. Leukocyte adherence to the endothelial cells was not observed during the first 12 h in either model of severity. In contrast, observation of the hepatic microcirculation revealed a significant number of adherent leukocytes 2 h after the induction of severe pancreatitis. These results suggest that during the early course of acute pancreatitis, leukocyte adherence in the pancreatic microcirculation is a secondary event following the increase in pancreatic vascular permeability.

Do peritoneal macrophages play an essential role in the progression of acute pancreatitis in rats

Introduction Macrophages are considered to play an essential role in the events leading to systemic inflammatory response. Some are known to reside in the peritoneal cavity but there are no reports defining the participation of peritoneal macrophages (PMs) in the progression of acute pancreatitis. Aim To clarify the role of PMs in the progression of acute pancreatitis. Methodology Acute pancreatitis was induced in rats from which macrophages other than PMs were greatly depleted, and in rats greatly depleted of macrophages including PMs. Macrophages were depleted by the injection of liposome encapsulated dichloromethylene bisphosphonate. After the induction of acute pancreatitis, local pancreatic inflammation, intraperitoneal inflammation and lung injury were compared between the 2 groups. Results Local pancreatic inflammation did not differ between the 2 groups. However, intraperitoneal inflammation was clearly improved by the depletion of PMs. Serum cytokine level and lung injury were also improved by the depletion of PMs. Conclusion Peritoneal macrophages extend inflammation from the pancreas to the peritoneal cavity and subsequently induce lung injury in acute pancreatitis. Peritoneal macrophages play an essential role in the systemic inflammatory response and the progression of acute pancreatitis in the rat.

Gene therapy for intraperitoneally disseminated pancreatic cancers by Escherichia coli uracil phosphoribosiltransferase (UPRT) gene mediated by restricted replication-competent adenoviral vectors

Although patients with unresectable pancreatic tumors have been treated with 5‐fluorouracil (5FU)‐based combination chemotherapy, the drug resistance of cancer cells presents a crucial therapeutic problem. It was reported that UPRT overcomes 5FU resistance. UPRT catalyzes the synthesis of 5‐fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5‐fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits thymidylate synthetase (TS). We first demonstrated that injecting an E1‐deficient adenoviral vector (Adv) expressing UPRT (AxCAUPRT) followed by 5‐FU treatment resulted in a volume reduction of xenotransplanted human tumors. In examining the therapeutic effect of AxCAUPRT/5‐FU against peritoneal dissemination, we found that non‐selective gene transduction of AxCAUPRT caused severe adverse effects arising from the increase of F‐dUMP in normal intestine. Because the therapeutic gene delivered by a restricted replication‐competent Adv lacking 55 kDa E1B protein (AxE1AdB) is speculated to be expressed selectively in tumors, mice with established tumors were injected with AxE1AdB and E1‐deleted Adv expressing the lacZ reporter gene (AxCAlacZ). The expression of the reporter gene (lacZ) was selectively enhanced in disseminated tumors. The therapeutic advantage of restricted replication competent Adv that expresses UPRT (AxE1AdB‐UPRT) was evaluated in an intraperitoneal disseminated tumor model. To study the anti‐tumor effect of AxE1AdB‐UPRT/5FU, mice with disseminated AsPC‐1 tumors were administered the Adv, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB‐UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. Our results showed that the AxE1AdB‐UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer.

Continuous regional application of protease inhibitor in the treatment of acute pancreatitis

In clinical settings, the effectiveness of protease inhibitors in the treatment of acute pancreatitis has been still controversial. With the concept that sufficient tissue concentration of protease inhibitor in the pancreas has to be included to achieve its potent inhibitory effect, we applied a continuous regional intra-arterial (CRI) application of low-molecular-weight protease inhibitor, nafamostat mesilate (FUT-175), for closed duodenal loop obstruction model in mongrel dogs. The use of CRI application led to a higher concentration of FUT-175 in the pancreatic tissue (4,453 ± 758 ng/g) when compared with that applied intravenously (905 ± 48 ng/g). Consequently, pancreatic parenchyma in CRI application animals was remarkably preserved, as assessed by the lower extent of pancreatic necrosis (12.4 ± 2.6% in CRI vs. 25.6 ± 1.9% in intravenous). Additionally, the elevation of trypsin-like activity in the pancreas was significantly inhibited in CRI animals. Based on these findings, the dose as well as the route of protease inhibitors should be carefully considered to achieve its beneficial effect.

A novel diamino-pyridine derivative prevents excessive leukocyte infiltration in aggravation of acute necrotizing pancreatitis

Leukocyte infiltration in the pancreas is involved in the aggravation of acute pancreatitis from edematous phase into necrotic change, and mild disease into severe disease; however, the mechanism responsible for leukocyte accumulation is not fully understood. This study was designed to clarify the mechanism underlying leukocyte accumulation into the pancreas and to elucidate the therapeutic efficacy of a novel diamino-pyridine derivative, IS-741 on leukocyte-endothelial cell interaction using rat necrotizing pancreatitis model. The number of adherent leukocytes to pancreatic collecting venules assessed by in vivo fluorescence microscopy increased significantly in necrotizing pancreatitis animals in a time-dependent manner. The expression of CD11b on circulating neutrophils determined by flow cytometric analysis was enhanced to approximately 500% after 2 h. IS-741 attenuated the leukocyte adherence significantly, accompanied by a lower up-regulation of CD11b. These findings were further supported by the histological examination that the accumulation of leukocytes in the pancreas was remarkably inhibited by IS-741. These results suggest that the leukocyte accumulation in the early phase of acute necrotizing pancreatitis may be mediated by leukocyte-endothelial cell interaction via leukocyte integrin CD11b/18. IS-741 attenuated the leukocyte endothelial cell interaction as a consequence of its inhibitory effect on CD11b upregulation.

Cytotoxic Effect of Bone Marrow-Derived Dendritic Cells

Some reports suggest that cancer patients have a better prognosis when dendritic cells (DCs) infiltrate their tumors. Although the role of these DCs is not fully understood, we focused on a potential effector function of DCs and examined whether DCs could directly mediate tumor cell cytotoxicity. Murine DCs were propagated from bone marrow stem cells using granulocyte/monocyte colony-stimulating factor and interleukin-4 for 7 days and were sorted by a magnetic-activated cell sorter system. Purified CD11c+ DCs were shown to be cytotoxic against murine tumor cells such as MCA205 fibrosarcoma. The annexin V staining assay showed that MCA205 underwent apoptosis when incubated with DCs for 4h. This cytotoxic effect was not blocked by an anti-Fas ligand antibody but was partially abrogated by the Ca2+ chelator EGTA/MgCl2. These data suggest that DCs are directly cytotoxic to some kinds of tumor cell, and this effect is mediated via both calcium-dependent and calcium-independent pathways.

Strategy for treatment of acute necrotizing pancreatitis: Importance of continuous regional arterial infusion of protease inhibitor and antibiotics in the early phase

A new strategy for the treatment of acute necrotizing pancreatitis (ANP) is reported. In this prospective study, all patients received intensive medical support. Surgery was performed in patients with infected pancreatic necrosis and/or sepsis. Continuous regional arterial infusion (CRAI) of the protease inhibitor, nafamostat mesilate, and the antibiotic imipenem was initiated in patients with ANP referred to our hospital within 7 days of the onset of the disease. Sixty patients with ANP were allocated to three groups: group I, no CRAI (n =16); group II, CRAI of nafamostat only (n=22); and group III, CRAI of nafamostat mesilate and imipenem (n=22). The mortality rate was 43.3% in group I, 13.6% in group II (P<0.05 vs group I), and 13.6% in group III (P<0.05 vs group I). The frequency of infected pancreatic necrosis was 50% in group I, 36.4% in group II, and nil in group III (P<0.01 vs group I and II). Combination of the protease inhibitor and the antibiotic infused intraarterially reduced the mortality rate and the frequency of infected pancreatic necrosis. However, 6 patients in the CRAI groups died of multiple organ failure (MOF), although the pancreatic necrosis was sterile. Massive retroperitoneal necrosis and bleeding was observed in these patients. CRAI is a potent mode of treatment in the early phase of necrotizing pancreatitis and most patients respond to this treatment. However, surgical intervention should be considered when the patient does not respond to CRAI and organ failure progresses, even though the pancreatic necrosis is sterile.