Seiki Tashiro

Tumor-stroma interaction of human pancreatic cancer: acquired resistance to anticancer drugs and proliferation regulation is dependent on extracellular matrix proteins.

Introduction Pancreatic cancer is one of the major causes of cancer-related deaths in industrialized countries. It is known that pancreatic cancer is resistant to chemotherapy and that cancer cells are surrounded by extracellular matrix (ECM) proteins including collagen I, collagen IV, fibronectin, and laminin. Aims To examine the role of ECM proteins in acquired resistance to anticancer drugs and proliferation regulation in pancreatic cancers. Methodology and Results We used an in vitro model of ECM-induced chemoresistance and cell proliferation of cancer cell lines (MIA PaCa-2, PANC-1, and Capan-1) with 3 different malignancy grades and found that resistance to cytotoxic drugs and proliferation regulation was dependent on ECM proteins. Pancreatic cancer cell lines, especially MIA PaCa-2 cells, adhering to any of the ECM proteins showed decreased cytotoxicity of anticancer drugs, except for gemcitabine. PANC-1 and Capan-1 cells adhering to fibronectin, collagen I, and collagen IV proliferated more than the controls. Conclusion ECM proteins have important roles in acquired resistance to anticancer drugs and cell proliferation regulation of pancreatic cancer cells. Therefore, the expression of ECM proteins in pancreatic cancer specimens could provide valuable information to aid anticancer drug cytotoxicity, and gemcitabine would be useful for treatment of patients with pancreatic cancer.

A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol

BACKGROUND/AIMS A stress-inducible heat shock protein 70 (HSP70) is one of the best-known endogenous factors protecting cell injury under various pathological conditions. The aim of this study was to examine anti-apoptotic actions of a non-toxic HSP70 inducer, geranylgeranylacetone (GGA), on hepatocytes exposed to hydrogen peroxide (H2O2) or ethanol. METHODS Primary cultures of rat hepatocytes were treated with different concentrations of GGA and exposed to 0.5 mM H202 or 100 mM ethanol. The heat shock response was assessed by measuring the activation of heat shock factor 1 (HSF1), HSP70 mRNA expression, and accumulations of HSP70, HSP90, and HSP27. Apoptosis was evaluated by DNA fragmentation. RESULTS Pretreatment with 1 microM GGA for 2 h enhanced nuclear translocation and phosphorylation of HSF1, HSF1-DNA binding, HSP70 mRNA expression, and its accumulation, when the cells were exposed to H202 or ethanol. In association with this accelerated response, GGA suppressed the insult-induced activation of c-Jun N-terminal kinases, caspase 9, and caspase 3-like proteases, leading to significant inhibition of apoptosis. CONCLUSIONS GGA exerted anti-apoptotic actions, at least in part, by priming hepatocytes for enhanced HSP70 induction. Our results suggest that GGA may have a potential benefit for the treatment of alcoholic and ischemia-reperfusion liver injuries.

Role of bile in intestinal barrier function and its inhibitory effect on bacterial translocation in obstructive jaundice in rats

BACKGROUND Our previous study using genetically labeled Escherichia coli strain JNW14 revealed that obstructive jaundice promotes bacterial translocation in rats and that the absence of bile in the intestinal tract is considered to be a factor inducing bacterial translocation. The aim of this study was to investigate the role of bile and bile acids in intestinal barrier function against bacterial translocation. MATERIALS AND METHODS Eight-week-old male specific-pathogen-free Wistar rats were subjected to ligation of their common bile ducts (CBDL). The CBDL rats were treated with bacitracin, neomycin sulfate, and streptomycin sulfate, and the intestinal tract was colonized with E. coli strain JNW14, which was genetically labeled with resistant markers against the above three antibiotics, to monitor the bacterial translocation. The rats were then administered saline, cholic acid (20 mg/100 g BW), taurocholic acid (TCA: 5-50 mg/100 BW), or bile (1.5-6 mL/day) via a duodenal catheter. The degree of bacterial translocation of E. coli strain JNW14 to the mesenteric lymph nodes was compared. Histopathological examination of the terminal ileum and intestinal permeability test using phenolsulfonphthalein was also performed. RESULTS Both cholic acid and TCA showed no inhibitory effect on bacterial translocation at any of the doses tested in CBDL rats, although TCA significantly decreased the numbers of E. coli strain JNW14 in the cecum. However, bile administration reduced the numbers of E. coli strain JNW14 in the cecum and mesenteric lymph nodes in CBDL rats although the inhibitory effect was weak. The integrity and permeability of the intestinal mucosa were kept at normal levels by bile administration in CBDL rats whereas the morphological changes, such as villous atrophy, villous edema, and lacteal canal dilatation, were observed in other CBDL rats. CONCLUSION Bile plays an important role in maintaining the intestinal barrier function to prevent the invasion of enteric bacteria to the underlying tissues, suggesting that the intestinal administration of bile to patients with obstructive jaundice is a useful way to reduce infectious complications by inhibiting bacterial translocation from the intestine to other organs.

Geranylgeranylacetone suppresses inflammatory responses and improves survival after massive hepatectomy in rats

Overproduction of heat shock protein 70 (HSP70) in the liver protects hepatocytes under various pathologic conditions. In this study we examined the effects of a nontoxic HSP70 inducer, geranylgeranylacetone (GGA), on acute hepatic failure after 95% hepatectomy in rats. When GGA (100 mg/kg) or vehicle was intragastrically administered to rats 4 hours before 95% hepatectomy, all 25 rats pretreated with vehicle died within 60 hours after the operation, whereas 10 of 25 rats pretreated with GGA survived. During the 24-hour postoperative period, GGA significantly suppressed the release of aspartate or alanine aminotransferase and elevation of the serum interleukin-6 level, and completely inhibited an increase in the serum level of tumor necrosis factor-alpha. Histologic examinations showed that GGA prevented hemorrhagic necrosis, which was observed in vehicle-treated livers more than 12 hours after the operation. During the 24-hour postoperative period, HSP70 induction was absent in remnant livers of vehicletreated rats. In contrast, GGA stimulated the HSP70 mRNA expression and HSP70 accumulation within 4 hours, and viable hepatocytes contained abundant HSP70 in their nuclei. Our results suggest that GGA may prevent acute liver failure after massive hepatectomy, at least in part, by enhancing HSP70 induction in the remnant liver.

Mechanism of liver regeneration after liver resection and portal vein embolization (ligation) is different

Whether or not liver regeneration after portal branch embolization (PE) (ligation, PVL) in the non-embolized (ligated) lobe is by the same mechanism as regeneration in the remnant lobe after liver resection has been reviewed. Portal vein branch embolization and heat shock protein are then discussed. Tumor growth accelerated in the remnant liver after hepatectomy. In contrast, PE or PVL resulted in marked contralateral hepatic hypertrophy and significant reduction of tumor growth in the non-embolized (non-ligated) lobes. Follistatin administration significantly increased liver regeneration after hepatectomy in rats. In contrast, regeneration of non-ligated lobes after PVL was not accelerated by exogenous follistatin. Tumor growth also was not accelerated. The liver regeneration rate peaked at 48-72 h in the nonligated lobe after PVL, a delay of 24 h compared with the remnant liver after hepatectomy. In the postoperative early stage, the expression of activin betaA, betaC, and betaE mRNAs was stronger in PVL than in hepatectomy. At 72 h the expression of activin receptor type IIA mRNA reached a peak in hepatectomy, but was significantly lower in PVL. Thus, regulation of activin signaling through receptors is one of the factors determining liver regeneration after hepatectomy and PVL. These serial experimental results imply that the mechanism of liver regeneration after portal branch ligation (embolization) is different from that after hepatectomy. Heat shock protein was induced in the liver experimentally by intermittent ischemic preconditioning and could play some beneficial role in the recovery of liver function after hepatectomy, even in cirrhotic patients. When heat shock protein following right portal vein embolization in both the embolized and non-embolized hepatic lobes was investigated in clinical cases, a two to fourfold increase in HSP70 was induced in the non-embolized lobe compared with the embolized lobe. Oral administration of geranylgeranylacetone (a non-toxic HSP inducer) suppressed inflammatory responses and improved survival after 95% hepatectomy by induction of HSP70 in rats.

Daily Pattern of Energy Metabolism in Cirrhosis

The daily pattern of energy expenditure and the oxidation rates of carbohydrates, fats, and protein were evaluated by indirect calorimetry in 18 control subjects (Group 1) and 34 cirrhotic patients who were divided into Groups 2a and 2b showing indocyanin green retention rates at 15 min of <30% and 30% or more, respectively. The ratio of resting energy expenditure to basal energy expenditure (%REE) was higher in the cirrhotic patients than in the controls at 8:30 AM and 2:30 PM. The oxidation rates of carbohydrates and fats under fasting conditions in Group 2b patients were respectively lower, and higher than in Group 1 and 2a patients. After the subjects ate, glucose became the substrate preferentially metabolized, and the proportion of fat metabolized was reduced from 82.9+/-5.1% to 43.9+/-21.9% and from 70.7+/-14.1% to 46.8+/-13.9% in the patients with advanced and less advanced cirrhosis, respectively, and from 59.4+/-27.2% to 48.4+/-18.5% in the controls. The fasting concentrations of non-esterified fatty acids in Group 2b were also significantly higher than those in the Group 1 and Group 2a patients. After eating, these concentrations fell and reached similar levels in the patients and controls. These data indicated that the patients with cirrhosis developed the catabolic state of starvation in the morning because of a lack of glycogen stores. Therefore, frequent meal supplementation to prevent early-onset starvation and energy deficiency may be advisable in such patients to maintain a well-nourished condition.

Effect of intrahepatic omental implantation on angiogenesis in rat liver with hepatic artery ligation.

Abstract Despite early surgical intervention, the results of treating hepatic artery thrombosis remain poor. The use of omental flaps is well documented for its angiogenic potential in promoting neovasculatization in ischemic tissues. This experimental study evaluated the formation of new blood-vessels after omental implantation (OI) in rats after ligation of the hepatic artery. Wistar rats were used and divided into the following groups: I OI with HAL, II OI without HAL, III hepatic artery ligation (HAL). For angiography, measurements were made of liver tissue blood flow by the laser Doppler method and of hepatic artery flow by the colored microsphere method (CMS), and immunohistochemical study was done for vascular endothelial growth factor (VEGF). The rats were killed at 1, 3, 7 or 30 days after laparotomy. Relative arterial hepatic blood flow in the implanted lobe of group I, as determined by CMS, reached 50% of control values 7 days after surgery. Angiography and microscopic studies of the excised liver revealed distinct angiogenesis surrounding the omental implant in the liver for 7 days postoperatively. The formation of new blood vessels after OI was not observed in livers without HAL. Omental implantation appears to be useful in preventing organ anoxia after hepatic artery thrombosis.

Promotion of bacterial translocation by major liver resection in obstructive jaundice in rats colonised predominantly with indigenous Escherichia coli

The influence of major liver resection in obstructive jaundice on bacterial translocation was evaluated in rats that were colonised predominantly with a genetically labelled strain of Escherichia coli. The strain, JNW14, originally isolated from rat faeces, was labelled with bacitracin, neomycin and streptomycin resistance markers. Fifty-two specific-pathogen-free male Wistar rats were divided into three experimental groups and were treated as follows: group 1 (n = 8), sham ligation of common bile duct; group 2 (n = 7), common bile duct ligation (CBDL); and group 3 (n = 37), 70% hepatectomy 7 days after CBDL. The rats were treated with the above antibiotics and then given E. coli strain JNW14 in their drinking water. Translocation of E. coli JNW14 from the gastrointestinal tract to the mesenteric lymph nodes (MLNs), lungs, liver, spleen and portal vein was evaluated in each group. In group 3 (CBDL plus hepatectomy), the incidence of translocation of E. coli JNW14 to the liver and spleen after hepatectomy was significantly higher than in groups 1 and 2. This result indicates that major liver resection in obstructive jaundice promotes bacterial translocation to systemic organs. Furthermore, the numbers of viable E. coli JNW14 in the MLNs in the lung culture-positive rats were significantly higher than those in the lung culture-negative rats, suggesting that lymphatic-thoracic duct systemic circulation is a major route of bacterial translocation.

Effect of donor-specific splenocytes via portal vein and FK506 in rat small bowel transplantation.

BACKGROUND To investigate the role of the liver in immune responses after small bowel transplantation, donor-specific splenocytes were infused perioperatively, via the portal vein, in a rat heterotopic small bowel transplant model. METHODS Heterotopic small bowel transplantation between the fully allogenic Brown Norway (BN) (RT1n) and Lewis (RT1[1]) strain rats were performed. We prepared donor splenocytes from BN or third-party WKA (RT1k) rat spleens for Lewis hosts and injected the splenocytes perioperatively via the host portal vein or the systemic vein. The hosts were treated with a short course of the immunosuppressive agent, FK506 (0.5 mg/kg, 0-3 days postoperatively), following the experimental protocols. RESULTS Untreated Lewis hosts rejected BN small bowel grafts at 5.4+/-0.9 days (n=8). BN splenocytes given alone caused fatal graft-versus-host disease in six of eight animals, and two others died from graft rejection. FK506 alone did not significantly prolong graft survival (6.3+/-1.0 days, n=10). However, BN splenocytes injected via the portal vein, combined with FK506, prolonged graft survival to 12.7+/-2.1 days (n=12, P < 0.01) and 10 of 12 rats survived more than 70 days. This was donor antigen specific. BN splenocytes administered systemically caused fatal graft-versus-host disease in all recipients, and FK506 did not ameliorate this. Histologic findings of graft rejection were remarkably mild in the recipients of the combined therapy, compared with the recipients that were given FK506 alone. Down-regulation of one-way mixed lymphocyte reaction to BN splenocytes was observed in the splenocytes of the tolerant hosts. CONCLUSIONS Combined administration of donor splenocytes and FK506 reduced allograft rejection and prolonged survival in this rat model of small bowel transplantation.

Clinical relevance of antibiotic-induced endotoxin release in patients undergoing hepatic resection.

It has been proved that antibiotics binding to penicillin-binding protein 3 (PBP3) are associated with the greater release of endotoxin than those that bind to PBP2 in both in vitro and animal models. The aim of this study is to evaluate the potential clinical implications of antibiotic-induced endotoxin release after hepatic resection. Forty-five patients who underwent hepatic resection in our clinic were enrolled. The patients were divided into two groups. Group A (n = 26): antibiotics that bind primarily to PBP3, including cefmetazole (CMZ), latamoxef (LMOX), flomoxef (FMOX), were used. Group B (n = 19); antibiotics that bind to both PBP2 and PBP3, including cefazolin (CEZ), cefoperazone (CPZ), cefotiam (CTM). Postoperative complications, liver functional tests, and chemical mediators [endotoxin, interleukins (IL-6, IL-8), tumor necrosis factor alpha (TNFalpha), granulocyte colony-stimulating factor (G-CSF), hepatotrophic growth factor (HGF) were examined after hepatic resection. There were no significant differences in the backgrounds of the two groups. Eight patients in each group developed postoperative complications; in particular, 9 of 13 patients with biliary tract carcinoma developed postoperative complications. No significant elevation of peripheral blood endotoxin was noted by the endospecy method, in any of the patients, although six died following sepsis. Pre- and postoperative levels of cytokines showed no significant difference between the two groups. Our data suggest that clinical antibiotic-induced endotoxin release would not occur after hepatic resection regardless of the antibiotic, probably owing to continuous scavenging of endotoxin from peripheral blood.