Yuzo Yamamoto

Increased expression of co-chaperone HOP with HSP90 and HSC70 and complex formation in human colonic carcinoma

Co-chaperone HOP (also called stress-inducible protein 1) is a co-chaperone that interacts with the cytosolic 70-kDa heat shock protein (HSP70) and 90-kDa heat shock protein (HSP90) families using different tetratricopeptide repeat domains. HOP plays crucial roles in the productive folding of substrate proteins by controlling the chaperone activities of HSP70 and HSP90. Here, we examined the levels of HOP, HSC70 (cognate of HSP70, also called HSP73), and HSP90 in the tumor tissues from colon cancer patients, in comparison with the non-tumor tissues from the same patients. Expression level of HOP was significantly increased in the tumor tissues (68% of patients, nu2009=u200919). Levels of HSC70 and HSP90 were also increased in the tumor tissues (95% and 74% of patients, respectively), and the HOP level was highly correlated with those of HSP90 (ru2009=u20090.77, pu2009<u20090.001) and HSC70 (ru2009=u20090.68, pu2009<u20090.01). Immunoprecipitation experiments indicated that HOP complexes with HSC70 or HSP90 in the tumor tissues. These data are consistent with increased formation of co-chaperone complexes in colon tumor specimens compared to adjacent normal tissue and could reflect a role for HOP in this process.

Nrf2 Activation Protects the Liver From Ischemia/Reperfusion Injury in Mice

Objective:To investigate the role of Nrf2 in the pathogenesis of hepatic ischemia-reperfusion (I/R) injury. Background:Hepatic I/R injury is a serious complication that leads to liver failure after liver surgery. NF-E2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting cells against oxidative stress. Therefore, it is suggested that Nrf2 activation protects the liver from I/R injury. Methods:Wild-type and Nrf2-deficient mice were treated with 15-deoxy-&Dgr;12,14–prostaglandin J2 (15d-PGJ2), or a vehicle. Subsequently, these mice were subjected to 60-minute hepatic 70% ischemia, followed by reperfusion. Liver and blood samples were collected to evaluate liver injury and mRNA expressions. Results:After hepatic I/R, Nrf2-deficient livers exhibited enhanced tissue damage; impaired GSTm1, NQO1, and GCLc inductions; disturbed redox state; and aggravated tumor necrosis factor &agr; mRNA expression in comparison with wild-type livers. 15d-PGJ2 treatment protected the livers of wild-type mice from I/R injury via increased expressions of GSTm1, NQO1, and GCLc; maintained redox status; and decreased tumor necrosis factor &agr; induction. These effects induced by 15d-PGJ2 were not seen in the livers of Nrf2−/− mice and were not annulled by peroxisome proliferator-activated receptor &ggr; antagonist in Nrf2+/+ mice, suggesting that the protective effect of 15d-PGJ2 is mediated by Nrf2-dependent antioxidant response. Conclusions:Nrf2 plays a critical role in the mechanism of hepatic I/R injury and would be a new therapeutic target for preventing hepatic I/R injury during liver surgery.

Changes in the fatty acid composition of the liver with the administration of N-3 polyunsaturated fatty acids and the effects on warm ischemia/reperfusion injury in the rat liver.

Prostanoids play a pivotal role among the inflammatory mediators associated with I/R injury. The aim of this study was to determine the effects of oral supplementation of n-3 polyunsaturated fatty acids (PUFA)-rich oil on inflammatory reactions and microcirculatory disorders caused by a hepatic warm I/R in rats. The rats were orally supplemented with n-3 PUFA-rich oil, n-6 PUFA-rich oil, or the same volume of water for 7 days. The PUFA concentration in the blood and liver tissues were evaluated, and the effects on I/R injury of the liver were assessed. The n-3 PUFA supplementation elevated the n-3/n-6 ratio in the blood and liver tissues. After reperfusion, thromboxane B2 in the blood and prostaglandin E2 in the liver were significantly suppressed in the n-3 PUFA-treated rats. Hepatic microcirculation was well maintained from the early phase (30 min) of reperfusion, and the serum concentrations of TNF-&agr; and IL-6 were significantly lower in this group. The transaminase blood levels were also suppressed in the n-3 PUFA-treated rats. Expression of COX-2 mRNA was increased in all groups at 2 h after reperfusion but there were no differences among three groups. In conclusion, preoperative n-3/n-6 ratio augmentation in the blood and in the liver can result in a successful alleviation of hepatic I/R injury.ABBREVIATIONS-AA-arachidonic acid; &agr;-LA-&agr;-linolenic acid; ALT-alanine aminotransferase; AST-aspartate aminotransferase; COX-cyclooxygenase; DHA-docosahexaenoic acid; DPA-docosapentaenoic acid; ELISA-enzyme-linked immunosorbent assay; EPA-eicosapentaenoic acid; ICU-intensive care unit; LA-linoleic acid; LDH-lactic dehydrogenase; MPO-myeloperoxidase; PG-prostaglandin; PUFA-polyunsaturated fatty acids; TX-thromboxane

Ante-situm hepatic resection for tumors involving the confluence of hepatic veins and IVC.

BackgroundFor patients with bulky liver tumors that have invaded all three hepatic veins at the hepatic venous confluence to the inferior vena cava (IVC), reconstruction of at least one hepatic vein outlet to the IVC is necessary.MethodsTo effectively treat these tumors, we performed 7 ante-situm liver resections. The ante-situm position provides surgeons with excellent visualization of the anatomical structures that should be maintained in the remnant liver. In addition, replacement of the involved IVC by a ringed expanded polytetrafluoroethylene (ePTFE) graft can be safely completed.ResultsBecause of the far-advanced malignancies of our 7 patients, the survival periods after surgery were limited, but the survival benefits of the procedure were not disappointing. From what has been reported in the literature, the survival periods in our patients appear to justify the employment of the procedure even when compared with survivals in patients who have less complex tumor extensions.ConclusionBecause of the complexity of this procedure, a team of surgeons familiar with liver surgery and liver transplantation in a specialized hepatobiliary center is required. However, we anticipate that this procedure will be feasible and justified for selected patients in whom tumor invasion at the confluence of all three hepatic veins and the IVC is too extensive to treat with other surgical procedures.

Role of preferential cyclooxygenase-2 inhibition by meloxicam in ischemia/reperfusion injury of the rat liver.

Background: Ischemia/reperfusion injury (IRI) is one of the major clinical problems in liver and transplant surgery. Livers subjected to warm ischemia in vivo often show a severe dysfunction and the release of numerous inflammatory cytokines and arachidonic acid metabolites. Cyclooxygenase (COX)-2 is the inducible isoform of an intracellular enzyme that converts arachidonic acid into prostaglandins. The aim of the study was to evaluate the effect of COX-2 inhibition and the role of Kupffer cells in IRI of the liver. Methods: Male Wistar rats [250- 280 g body weight (BW)] were anesthetized and subjected to 30-min warm ischemia of the liver (Pringles maneuver) and 60-min reperfusion after median laparotomy. The I/R group received no additional treatment. In the COX-2 inhibitor (COX-2I) group, the animals received 1 mg/kg BW meloxicam prior to operation. Gadolinium chloride (GdCl3) (10 mg/kg BW) was given 24 h prior to operation in the GdCl3 and GdCl3 + COX-2I groups for the selective depletion of Kupffer cells. The GdCl3 + COX-2I group received both GdCl3 and meloxicam treatment prior to operation. Blood and liver samples were obtained at the end of the experiments for further investigations. Results: After 30 min of warm ischemia in vivo, severe hepatocellular damage was observed in the I/R group. These impairments could be significantly prevented by the selective COX-2 inhibition and the depletion of Kupffer cells. Alanine aminotransferase was significantly reduced upon meloxicam and GdCl3 treatment compared to the I/R group: I/R, 3,240 ± 1,262 U/l versus COX-2I, 973 ± 649 U/l, p < 0.001; I/R versus GdCl3, 1,611 ± 600 U/l, p < 0.05, and I/R versus GdCl3 + COX-2I, 1,511 ± 575 U/l, p < 0.01. Plasma levels of tumor necrosis factor alpha (TNF-α) were significantly reduced in the COX-2I treatment group compared to I/R (3.5 ± 1.5 vs. 16.3 ± 11.7 pg/ml, respectively; p < 0.05). Similarly, the amount of TxB2, a marker for COX-2 metabolism, was significantly reduced in the meloxicam treatment groups compared to the I/R group: I/R, 22,500 ± 5,210 pg/ml versus COX-2I, 1,822 ± 938 pg/ml, p < 0.001, and I/R versus GdCl3 + COX-2I, 1,530 ± 907 pg/ml, p < 0.001. All values are given as mean ± SD (n = 6). Conclusion: These results suggest that the inhibition of COX-2 suppressed the initiation of an inflammatory cascade by attenuating the release of TNF-α, which is an initiator of the inflammatory reaction in hepatic IRI. Therefore, we conclude that preferential inhibition of COX-2 is a possible therapeutic approach against warm IRI of the liver.

The roles of interferon regulatory factors 1 and 2 in the progression of human pancreatic cancer.

Objective Pancreatic cancer is one of the most malignant diseases worldwide. Interferon regulatory factor (IRF) 1 and IRF2 function as a tumor suppressor and oncoprotein, respectively, in several types of cancers. We investigated whether IRF1 and IRF2 are involved in the progression of pancreatic cancer. Methods We examined the expressions of IRF1 and IRF2 in pancreatic cancer specimens and analyzed the association with clinicopathologic features. We evaluated the biological effects of IRF1 and IRF2 using a pancreatic cancer cell line. Results The expression levels of IRF1 and IRF2 were decreased and increased, respectively, in the pancreatic cancer cells compared with those observed in the paired normal areas. A higher expression of IRF1 was associated with better features of tumor differentiation, infiltration depth, tumor size, and survival, whereas that of IRF2 was associated with a worse feature of tumor infiltration depth. Interferon regulatory factor 2–overexpressing PANC-1 cells exhibited an increase in cell growth, less apoptotic features, and chemoresistance to gemcitabine treatment. In contrast, IRF1-overexpressing cells exhibited the opposite characteristics. Conclusions Interferon regulatory factors 1 and 2 may regulate the progression of pancreatic cancer by functioning as an antioncoprotein and oncoprotein, respectively. These molecules may serve as potential targets of therapy.

Meloxicam, a COX-2 inhibitor, ameliorates ischemia/reperfusion injury in non-heart-beating donor livers.

Background/Aims: Chronic organ donor shortage has led to the consideration to expand the donor pool with livers from non-heart-beating donors (NHBD), although a higher risk of graft dys- or nonfunction is associated with these livers. We examined the effects of selective cyclooxygenase-2 (COX-2) inhibition on hepatic warm ischemia (WI) reperfusion (I/R) injury of NHBD. Methods: Male Wistar rats were used as donors and meloxicam (5 mg/kg body weight) was administered into the preservation solution. Livers were excised after 60 min of WI in situ, flushed and preserved for 24 h at 4°C. Reperfusion was carried out in vitro at a constant flow for 45 min. During reperfusion (5, 15, 30 and 45 min), enzyme release of alanine aminotransferase and glutamate lactate dehydrogenase were measured as well as portal venous pressure, bile production and oxygen consumption. The production of malondialdehyde was quantified and TUNEL staining was performed. Quantitative PCR analyzed COX-2 mRNA. COX-2 immunohistochemistry and TxB2 detection completed the measurements. Results: Meloxicam treatment led to better functional recovery concerning liver enzyme release, vascular resistance and metabolic activity over time in all animals. Oxidative stress and apoptosis were considerably reduced. Conclusion: Cold storage using meloxicam resulted in significantly better integrity and function of livers retrieved from NHBD. Selective COX-2 inhibition is a new therapeutic approach achieving improved preservation of grafts from NHBD.

Enhancement of liver regeneration by adenosine triphosphate-sensitive K⁺ channel opener (diazoxide) after partial hepatectomy.

Background Enhancement of liver regeneration is a matter of importance after partial liver transplantation including small-for-size grafting. Mitochondrial adenosine triphosphate (ATP)-sensitive K+ (mitoKATP) channel plays an important role in mitochondrial bioenergetics, which is a prerequisite for liver regeneration. However, the ATP-sensitive K+ (KATP) channel in hepatocytes is incompletely understood. We investigated the KATP channel in hepatocytes and examined the effects of diazoxide, a potent KATP channel opener, on liver regeneration using a rat model. Methods Using rat primary hepatocytes, expression and localization of KATP channel subunits, Kir6.x and sulfonylurea receptor (SUR)x, were studied by polymerase chain reaction, Western blotting, and immunostaining. To investigate the role of KATP channel openers in liver regeneration, we allocated rats into four groups: control (vehicle) (n=24), diazoxide (n=24), vehicle plus channel blocker (n=6), and diazoxide plus channel blocker (n=6) groups. After 70% partial hepatectomy, hepatic tissue ATP levels, liver-to-body weight ratio, and proliferation rate of hepatocytes were examined. Results KATP channel subunits, Kir6.1 and SUR1, were detected on hepatic mitochondria. During liver regeneration, liver-to-body weight ratio, proliferation rate of hepatocytes, and the hepatic ATP level were significantly higher in the diazoxide group than the control group at 2 days after partial hepatectomy. These effects of diazoxide were neutralized by a KATP channel blocker. Conclusions We demonstrated the existence of a mitoKATP channel in hepatocytes composed of Kir6.1 and SUR1. Diazoxide could enhance liver regeneration by keeping a higher ATP content of the liver tissue. These results suggest that diazoxide will sustain the mitochondrial energetics through the mitoKATP channel opening.

Implication of Protein Kinase A for a Hepato-Protective Mechanism of Milrinone Pretreatment

BACKGROUNDnWe have previously reported that an increase of adenosine 3,5-cyclic monophosphate (cAMP) in liver tissue after an administration of milrinone, a phosphodiesterase-3 inhibitor attenuates hepatic warm ischemia-reperfusion injury. The aim of this study was to determine whether cAMP-dependent protein kinase (protein kinase A) activation was involved in the milrinone-induced hepatoprotective effect on an ischemia-reperfusion injury in an in vivo model.nnnMATERIALS AND METHODSnMale Lewis rats were allocated into 3 groups. In Group M, milrinone was administrated before ischemia; in Group I, a protein kinase A inhibitor, adenosine 3,5-cyclic monophosphorothioate, 8-bromo-, Rp-isomer, sodium salt (Rp-8-Br-cAMPS), was injected prior to the administration of milrinone; and in Group C, the control group, there was no pretreatment. After pretreatment, all rats were exposed to a 45-min total hepatic inflow occlusion.nnnRESULTSnAfter milrinone administration, liver cAMP concentrations and protein kinase A activity ratios were elevated. They protected the liver from ischemia-reperfusion injury. Rp-8-Br-cAMPS suppressed protein kinase A activation without affecting cAMP elevating responses to milrinone. Compared with Group C, hepatocellular necrosis, neutrophil infiltration, and congestion were ameliorated, and serum tumor necrosis factor-alpha, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase levels were significantly suppressed in Group M. Rp-8-Br-cAMPS canceled this effect, showing histological damages in Group I as severe as in Group C. The levels of tumor necrosis factor-alpha, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were the same in Groups C and I.nnnCONCLUSIONSnActivation of protein kinase A might play an important role in the mechanism of milrinone-induced ischemic tolerance in the liver.

Liver epithelial cells proliferate under hypoxia and protect the liver from ischemic injury via expression of HIF-1 alpha target genes

BACKGROUNDnThe remnant liver after extended liver resection is susceptible to ischemic injury, resulting in the failure of liver regeneration and liver dysfunction. The present study is aimed to investigate the protective role of the liver epithelial cells (LEC), a liver progenitor cell, on hepatocytes with ischemia inxa0vitro and inxa0vivo.nnnMETHODSnLECs were isolated from rats and cultured under hypoxic conditions (2% O(2)). The cell viability and intracellular ATP levels were measured. The activation of hypoxia-inducible factor-1α (HIF-1α) was assessed by immunofluorescence. The expression of pyruvate dehydrogenase kinase-1 (PDK-1), stromal cell-derived factor-1 (SDF-1), and chemokine receptor 4 (CXCR4) were measured. Hepatocytes were treated with SDF-1 or LEC-conditioned medium under hypoxia, and cell viability was assessed. Finally, hemorrhagic shock was induced in rats with inxa0vivo induction of endogenous LECs, and liver damage was assessed.nnnRESULTSnIn LECs, but not in hepatocytes, cellular viability and intracellular ATP levels were maintained, and nuclear translocation of HIF-1α and expression of pyruvate dehydrogenase kinase-1 mRNA were increased under hypoxic culture conditions. LECs express SDF-1, and CXCR4 expression was increased in hepatocytes under hypoxia. The survival of hepatocytes under hypoxic condition was significantly increased after treatment with SDF-1 or LEC-conditioned medium. The protective effect of conditioned medium was impaired by CXCR4 antagonists. Inxa0vivo induction of endogenous LECs suppressed elevation of serum AST and ALT levels after hemorrhage shock and ischemia-reperfusion.nnnCONCLUSIONnLECs are resistant to hypoxia and have a protective role for hepatocytes against hypoxia. Our results suggest that induction of endogenous LECs protected the liver from lethal insults by paracrine signaling of SDF-1 and differentiation into parenchymal cells.