Akira Yamanoi

Tetracycline analogues (doxycycline and COL-3) induce caspase-dependent and -independent apoptosis in human colon cancer cells

Tetracycline analogues (TCNAs) possess cytotoxic activities as well as matrix metalloproteinase (MMP) inhibitory properties. Previously, we demonstrated that doxycycline (DOXY) could induce apoptosis in human HT29 colon cancer cells. In present study, the molecular apoptotic mechanisms induced by two kinds of TCNAs, designated as DOXY and COL‐3 (chemically modified tetracycline‐3; 6‐demethyl, 6‐deoxy, 4‐dedimethylamino tetracycline), were evaluated in cultured HT29 cells. Both TCNAs inhibited the proliferation of 6 different colorectal cancer cell lines in a dose‐dependent manner. Especially, COL‐3 had a stronger effect on cancer cells than DOXY. Apoptotic changes were actually observed by 10 μg/ml COL‐3 and 20 μg/ml DOXY in a time‐dependent manner. COL‐3 produced the increase in cytosolic cytochrome c and the loss of mitochondrial membrane potential after 3 hr treatment, and thereafter activated caspases. In case of DOXY, these changes were observed after 24 hr. Bax translocation was not a prerequisite for cytochrome c releasing in COL‐3 treatment. Pretreated pancaspase inhibitor (Z‐VAD‐FMK) reduced COL‐3 and DOXY mediated apoptosis up to 81.3 and 35.3%, as compared with nontreated cells, respectively. These data indicated that TCNAs could induce mitochondria‐mediated apoptosis through both caspase‐dependent and ‐independent pathway. In fact, endonuclease G and apoptosis‐inducing factor were released into cytosol after the treatment of TCNAs, which indicated that caspase‐independent apoptotic pathway is also one of the key mechanisms for the treatment of TCNAs. Taken together, we believe that TCNAs could have strong potentials for clinical application in treating colorectal cancers and improve cancer chemotherapy.

Increased Expression of Fractalkine is Correlated with a Better Prognosis and an Increased Number of Both CD8 + T Cells and Natural Killer Cells in Gastric Adenocarcinoma

BackgroundFractalkine (CX3CL1) is the only CX3C chemokine that can chemoattract natural killer (NK) cells, CD8+ T cells, monocytes, and dendritic cells. Although experimental studies have demonstrated that Fractalkine expression by tumor cells is related to the infiltrating lymphocytes and initiates antitumor immunity, the clinical significance of Fractalkine remains to be elucidated in gastric adenocarcinoma.MethodsTissue sections from 158 patients with curatively resected T2 or T3 gastric adenocarcinoma were immunohistochemically stained for Fractalkine. Furthermore, to evaluate CD8+ T cells and NK cells infiltration, antibodies to CD8 and CD57 protein were respectively used for immunohistochemistry.ResultsA significant direct correlation was observed between the Fractalkine scores and the number of CD8+ T cells and NK cells using the Spearman rank correlation coefficient test (Pxa0=xa0.0080, .0031, respectively). Furthermore, the high Fractalkine expression group (nxa0=xa067) showed a significantly better prognosis than the low Fractalkine expression group (nxa0=xa091) regarding the disease-free survival (Pxa0=xa0.0016). In a multivariate analysis, the Fractalkine expression was identified as one of the independent prognosticators for disease-free survival (risk ratio, 2.5; Pxa0=xa0.0147).ConclusionsThese data suggest that the expression of Fractalkine by tumor cells enhances the recruitment of CD8+ T cells and NK cells and induces both innate and adaptive immunity, thereby yielding a better prognosis in gastric adenocarcinoma. Fractalkine is a new independent predictor of the prognosis and can be a novel candidate for development of a more effective therapeutic strategy for gastric adenocarcinomas.

Expression of hypoxia inducible factor-1alpha during liver regeneration induced by partial hepatectomy in rats.

Abstract: Background/Aims: It has been well established that hypoxia inducible factor‐1α (HIF‐1α) transcribes essential factors in cell preservation, including angiogenesis, during hypoxia. However, the transition of HIF‐1α expression during liver regeneration remains unknown. In this study, a role of HIF‐1α was experimentally elucidated in relation to sinusoidal endothelial reconstruction during liver regeneration.

Clinicopathologic comparisons between estrogen receptor-positive and -negative hepatocellular carcinomas.

During the past 8 years, estrogen receptors (ERs) in the cytosol of hepatocellular carcinoma (HCC) were assayed in 66 unselected patients without preceding treatments on whom radical hepatic resection was performed. Twenty-six patients had ERs of 0.9 to 13.4 fmol/mg protein with a mean dissociation constant of 7.8 x 10-10 M. The remaining 40 patients had no detectable amount of the receptor. There were no substantial differences between the ER-positive and ER-negative groups in preoperative clinical and laboratory data such as sex, age, alcohol abuse, underlying liver disease, and hepatic functions. Large tumors were more common in the ER-negative group and therefore the incidence of major hepatic resection was significantly higher in this group. Histopathologic studies revealed no substantial differences between the two groups. Operative mortality rate was 11.5% in the ER-positive and 12.5% in the ER-negative group. Excluding eight operative deaths, the rate and time of tumor recurrence in the residual liver and long-term survival rate were identical for the two study groups. The current results may indicate that the presence or absence of ERs in human HCC does not correlate to either biologic or pathologic characteristics of this tumor, but the true role of ERs in human HCC remains to be elucidated.

Role of peroxisome proliferator‐activated receptor‐gamma (PPARγ) during liver regeneration in rats

Background and Aim:u2002 Peroxisome proliferator‐activated receptor‐gamma (PPARγ), a member of the nuclear receptor superfamily, is widely expressed in adipocytes and other tissues, including the liver. Several reports have shown that PPARγ activation induced cell‐cycle arrest and apoptosis in tumor cells. We investigated the role of the PPARγ/ligand system and the effect of the PPARγ agonist during liver regeneration.

Hemoperfusion with a high-mobility group box 1 adsorption column can prevent the occurrence of hepatic ischemia-reperfusion injury in rats

Objective:High-mobility group box 1, a ubiquitous nonhistone chromosomal protein, is passively released from necrotic cells and actively secreted by inflammatory cells. Extracellular high-mobility group box 1 has recently been recognized to be a mediator of hepatic ischemia–reperfusion injury; however, the kinetics of high-mobility group box 1 during hepatic ischemia–reperfusion and the role of high-mobility group box 1 in ischemia–reperfusion injury still remain poorly understood. This study was designed to assess the localization and the kinetics of high-mobility group box 1 during hepatic ischemia–reperfusion injury and the effects of high-mobility group box 1 adsorption column in hepatic ischemia-reperfusion injury. Design:A prospective, randomized animal study. Setting:University medical center research laboratory. Subjects:Male Sprague-Dawley rats. Investigation:The animals underwent 70% partial hepatic ischemia for 60 or 90 mins and were then reperfused. To investigate the high-mobility group box 1 levels in the serum and in the liver, the animals were killed at predetermined periods. As a lethal model, global hepatic ischemia–reperfusion was induced by portal triad cross-clamping for 30 mins. Hemoperfusion therapy using a cellulofine sulfate bead column (high-mobility group box 1 adsorption column) was performed during global hepatic ischemia. Measurements and Main Results:During 60 mins of 70% hepatic ischemia, nuclear high-mobility group box 1 was translocated to the cytoplasm in hepatocytes; however, serum high-mobility group box 1was not increased. Immediately after reperfusion, the serum high-mobility group box 1 was significantly increased (p < .05). High-mobility group box 1 mediated ischemia–reperfusion injury in not only liver but also the remote organ, lung. Removal of excess high-mobility group box 1 in blood using an adsorption column significantly improved animal survival (p < .03) and liver and lung injuries. Conclusions:High-mobility group box 1 plays an important role in the systemic as well as local pathogenesis of hepatic ischemia–reperfusion injury. The removal of excessive high-mobility group box 1 with adsorption column was beneficial and promising option in ischemia-related liver injuries.

Reconstruction of the Gastrointestinal Tract byhemi-doublestapling Method for the Esophagus andjejunum Using Eea Orvil in Laparoscopic Total Gastrectomy and Proximal Gastrectomy

We report the method of anastomosis based on a hemi-double stapling technique (hereinafter, HDST) using a trans-oral anvil delivery system (EEA OrVil) for reconstructing the esophagus and lifted jejunum following laparoscopic total gastrectomy or proximal gastric resection. As a basic technique, end-to-side anastomosis was used for the cut-off stump of the esophagus and lifted jejunum. After the gastric lymph node dissection, the esophagus was cut off obliquely to the long axis using an automated stapler. EEA OrVil was orally, and a small hole was created at the tip of the obliquely cut-off stump with scissors to let the valve tip pass through. When it was confirmed that the automated stapler and center rod were made completely linear, the anvil and the main unit were connected with each other and firing was carried out. Then, HDST-based anastomosis was completed. The method may safe laparoscopic anastomosis between the esophagus and reconstructed intestine.

FR-167653, a selective p38 MAPK inhibitor, exerts salutary effect on liver cirrhosis through downregulation of Runx2

Liver cirrhosis remains a difficult-to-treat disease with a substantial morbidity and mortality rate. There is an emerging body of data purporting a pivotal role of the activated p38 mitogen-activated protein kinase (MAPK) in the process of cirrhosis. Several anticirrhotic agents have been developed over the past few years, and most of them exert their effects by indirectly inhibiting the p38 pathway. Effect of a selective p38 inhibitor is yet to be reported. In this study, we evaluated the salutary effect of FR-167653 (FR), a selective p38 inhibitor, in a carbon tetrachloride (CCl4)-induced rat cirrhotic model. Twenty rats were assigned into four groups: Sham, olive oil only; Control, CCl4 in olive oil; FR50, FR 50u2009mg/kg/day and CCl4; and FR100, FR 100u2009mg/kg/day and CCl4. FR dose-dependently inhibited activation of p38 and had an ameliorating effect on cirrhosis formation. Significant dose-dependent reduction in α-smooth muscle actin immunostaining and hydroxyproline content of the liver was noticed in the FR-treated rats. Also densitometric analysis showed a significant reduction in azan-stained area in the FR-treated rats. These fibrotic changes were observed in the myofibroblasts including the hepatic stellate cells and portal fibroblasts. mRNA expression of runt-related protein 2 (Runx2), a profibrogenic transcription factor, was significantly low in FR-treated livers, indicating that Runx2 might be a key downstream regulator of the p38 pathway. A similar reduction in expression of Smad4 and tissue inhibitor of metalloproteinase-1 was noticed in the FR-treated rats. In conclusion, FR treatment exerted a significant beneficial effect in a CCl4-induced rat cirrhotic model. The ameliorating effect of FR could be partially attributable to an inhibition of the Smad4/p38/Runx2 axis in the cirrhotic liver.

Experimental investigation of the role of endothelin-1 in idiopathic portal hypertension.

Background and Aim:u2002 The authors previous report revealed that endothelin‐1 might be released from B lymphocytes in cirrhotic patients with hypersplenism. Other investigators have shown that persistent exposure to environmental contaminants including arsenic might induce idiopathic portal hypertension. The aim of this study was to experimentally identify how endothelin‐1 is involved in the development of idiopathic portal hypertension and which cells produce endothelin‐1 in the spleen.

ATTENUATION OF ISCHEMIA-REPERFUSION INJURY OF THE LIVER IN DOGS BY CYCLOSPORINE A COMPARATIVE STUDY WITH ALLOPURINOL AND METHYLPREDNISOLONE

The effects of pretreatment with cyclosporine, allopurinol, or methylprednisolone on ischemia-reperfusion injury of the liver were investigated. A total of 32 adult mongrel dogs that received one of the pretreatments were divided into four groups and were subjected to 90 min liver ischemia. Serum activities of aspartate aminotransferase (s-AST) and lactate dehydrogenase, (s-LDH) as well as animal survivals were used as indicators of liver injury. The elevation of both s-AST and s-LDH was significantly suppressed by pretreatment with cyclosporine as much as by allopurinol. However a significant improvement in animal survival was obtained only in the cyclosporine-pretreated group. Pretreatment with methylprednisolone did not affect either the activities of s-AST and s-LDH or animal survivals when compared with the control group. These data suggest that cyclosporine is a potent protector against ischemic liver injury—as effective as allopurinol or methylprednisolone. Although the precise mechanism of the effect of cyclosporine on liver ischemia still remains unknown, these observations may be of use in liver transplantation.