Edaravone inhibits apoptosis caused by ischemia/reperfusion injury in a porcine hepatectomy model.

AIM: To investigate the effect of E3-methyl-1-phenyl-2-pyrazolin-5-one (Edr) on hepatic ischemia-reperfusion (I/R) injury and liver regeneration in a porcine hepatectomy model. METHODS: One hour ischemia was induced by occluding the vessels and the bile duct of the right and median lobes. A 40% left hepatectomy was performed after reperfusion. Six animals received Edr (3 mg/kg per hour) intravenously and six control animals received saline just before reperfusion. Remnant liver volume, hemodynamics, aspartate aminotransferase (AST), alanine aminotransferase, lactate dehydrogenase and lactic acid, were compared between the groups. The expression of transforming growth factor-ß (TGF-ß1) and toll-like receptor (TRL) mRNA in hepatic tissues was examined using reverse transcription polymerase chain reaction. Apoptosis was demonstrated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. RESULTS: Serum AST (P = 0.029), and toll like receptor 4 level (P = 0.043) were significantly lower after 3 h in animals receiving Edr. In addition, TUNEL staining in Edr-treated pigs showed significantly fewer hepatocytes undergoing apoptosis compared with control pigs. After 1 mo, all factors were non-significantly different between the two groups. CONCLUSION: Edr is considered to reduce hepatic injury in the early stage of I/R injury in a porcine model.

Glycated albumin suppresses glucose-induced insulin secretion by impairing glucose metabolism in rat pancreatic ß-cells.

BACKGROUND: Glycated albumin (GA) is an Amadori product used as a marker of hyperglycemia. In this study, we investigated the effect of GA on insulin secretion from pancreatic ß cells. METHODS: Islets were collected from male Wistar rats by collagenase digestion. Insulin secretion in the presence of non-glycated human albumin (HA) and GA was measured under three different glucose concentrations, 3 mM (G3), 7 mM (G7), and 15 mM (G15), with various stimulators. Insulin secretion was measured with antagonists of inducible nitric oxide synthetase (iNOS), and the expression of iNOS-mRNA was investigated by real-time PCR. RESULTS: Insulin secretion in the presence of HA and GA was 20.9 ± 3.9 and 21.6 ± 5.5 µU/3 islets/h for G3 (P = 0.920), and 154 ± 9.3 and 126.1 ± 7.3 µU/3 islets/h (P = 0.046), for G15, respectively. High extracellular potassium and 10 mM tolbutamide abrogated the inhibition of insulin secretion by GA. Glyceraldehyde, dihydroxyacetone, methylpyruvate, GLP-1, and forskolin, an activator of adenylate cyclase, did not abrogate the inhibition. Real-time PCR showed that GA did not induce iNOS-mRNA expression. Furthermore, an inhibitor of nitric oxide synthetase, aminoguanidine, and NG-nitro-L-arginine methyl ester did not abrogate the inhibition of insulin secretion. CONCLUSION: GA suppresses glucose-induced insulin secretion from rat pancreatic ß-cells through impairment of intracellular glucose metabolism.

MK615 decreases RAGE expression and inhibits TAGE-induced proliferation in hepatocellular carcinoma cells.

AIM: To investigate the proliferative effect of advanced glycation end-products (AGEs) and the role of their cellular receptor (RAGE) on hepatocellular carcinoma (HCC) cells, and the inhibitory effects of MK615, an extract from Japanese apricot, against AGEs were also evaluated. METHODS: Two HCC cell lines, HuH7 and HepG2, were used. Expression of RAGE was investigated by polymerase chain reaction, Western blotting, and flow cytometry (FACS). The effect of MK615 on RAGE expression was also evaluated by FACS. The proliferative effects of a control (unglycated bovine serum albumin), glucose-derived AGEs (Glc-AGE), and glyceraldehyde-derived AGEs (Glycer-AGE), and the anti-proliferative effect of MK615 against AGEs, were evaluated using MTT assays. RESULTS: Expression of RAGE was confirmed at both the mRNA and protein levels in both HuH7 and HepG2. FACS revealed that the level of RAGE expression was higher in HuH7 than in HepG2. Treatment with 0.1 µg/mL MK615 decreased the expression level of RAGE from 24.3% to 3.7% in HuH7 and from 6.2% to 4.8% in HepG2. The growth indices for the control, Glc-AGE, and Glycer-AGE were 1.06 ± 0.08, 0.99 ± 0.04, and 1.38 ± 0.05, respectively, in HuH7 (P = 0.037), and were 1.03 ± 0.04, 1.04 ± 0.03, and 1.07 ± 0.05, respectively, in HepG2 (P > 0.05). When the cells were cultured simultaneously with Glycer-AGE and MK615, MK615 abrogated the proliferative effect of Glycer-AGE in HuH7. CONCLUSION: Only Glycer-AGE has a proliferative effect on HuH7, which expresses a higher level of RAGE. MK615 suppresses the proliferative effect of Glycer-AGE on HuH7 by decreasing the expression of RAGE.

Erythropoietin strongly protects the liver from ischemia-reperfusion injury in a pig model.

BACKGROUND/AIMS: We investigated, for the first time, the protective effect of erythropoietin (EPO) against liver ischemia-reperfusion (I-R) injury in a pig model. METHODOLOGY: Partial hepatic ischemia was maintained for 60 min in a pig. Pigs were allocated to 4 groups (n=5 each): (1) Control group with I-R injury (Vehicle); (2) EPO group with I-R injury, given three injections of EPO at 5000 IU/kg (EPO5000x3); (3) EPO group with I-R injury, given a single injection of EPO at 5000 IU/kg (EPO5000x1); and (4) EPO group with I-R injury, given three injections of EPO at 500 IU/kg (EPO500x3). Liver function tests (AST, ALT, LDH), and TUNEL assay were performed. RESULTS: Three hours after I-R injury, AST levels in the Vehicle, EPO5000x3, EPO5000x1, and EPO500 x3 groups were 1494.2 +/- 711.3 U/L, 307.3 +/- 127.6 UL, 296.5 +/- 9.2 U/L, and 474.6 +/- 242.0 UL, respectively (one-factor ANOVA, p = 0.020). At 3h the ALT and LDH levels in the Vehicle group were significantly higher than those in the EPO5000x3 and EPO5000x1 groups. Apoptotic indices in the Vehicle, EPO500x3, EPO5000x1, and EPO500x3 groups 3 h after I-R injury were 2.40 +/- 0.93, 1.36 +/- 0.12, 1.11 +/- 0.17, and 1.51 +/- 0.33, respectively. The apoptotic indices of the EPO5000x1 and EPO500x3 groups were significantly lower than that of the Vehicle group. CONCLUSIONS: EPO treatment significantly ameliorated liver I-R injury in this pig model. The protective effect was exerted by the inhibition of apoptosis. These results will open the door for the clinical application of EPO in liver surgery.

Rapamycin inhibits growth of cholangiocarcinoma cells.

BACKGROUND/AIMS: The immunosuppressive agent rapamycin is currently being evaluated for its antineoplastic effect. In the present study, the antineoplastic effect of rapamycin against cholangiocarcinoma was studied in vitro. METHODOLOGY: To explore the therapeutic potential of rapamycin, expression of mTOR in four cholangiocarcinoma cell lines--TFK1, HuCCT1, NOZW, and OZ--was evaluated by real-time PCR. The cell lines were then cultured with rapamycin (200 nM), and changes in the expression of Akt, phosphorylated PTEN (pPTEN), and phosphorylated S6 (pS6) were evaluated by western blotting. Finally, the cell lines were cultured with rapamycin (0, 25, 50, 100, 200 nM), gemcitabine (0, 0.5, 1, 2 microM), or both, and the antiproliferative effect was evaluated by MTT assay. RESULTS: All four cholangiocarcinoma cell lines expressed endogenous mTOR-mRNA, the of expression being highest in HuCCT1 (65.8) and lowest in TFK1 (17.6). Western blotting revealed that rapamycin treatment decreased Akt expression significantly in all four cell lines (TFK1; 15.5%, HuCCT1; 6.3%, NOZW; 9.8%, OZ; 19.5%), and also decreased the expression of p-PTEN (TFK1; 10.6%, HuCCT1; 5.4%, NOZ-W; 12.2%, OZ; 12.2%) and pS6 (TFK1; 64.0%, HuCCT1; 73.9%, NOZW; 78.6%, OZ; 47.6%) in all four cell lines. Finally, rapamycin significantly inhibited the growth of all four cell lines in a dose-dependent manner. Gemcitabine inhibited the growth of NOZW and HuCCT1, but its effect was less marked on TFK1 and OZ. Furthermore, a synergistic anti-proliferative effect of rapamycin and gemcitabine was observed in TFK1, NOZW, and OZ, but not in HuCCT1. CONCLUSION: Rapamycin effectively inhibited the growth of the four cholangiocarcinoma cell lines tested, and a synergistic effect with gemcitabine was observed in three of them. Rapamycin offers a new therapeutic strategy to inhibit the growth of cholangiocarcinoma.

Influence of tumor peroxisome proliferator-activated receptor gamma and delta expression on postoperative mortality of patients undergoing colorectal cancer surgery.

PURPOSE: To evaluate the influence of peroxisome proliferator-activated receptor gamma (PPAR gamma) and delta (PPAR delta) expression on postoperative mortality of patients with colorectal cancer (CRC). METHODS: Optimal cutoff values were determined for each relative expression ratio (RER) (RER = PPAR expression of tumor/PPAR expression of normal mucosa) of PPAR, and patients were divided into two groups as follows (PPAR staging): patients with elevated RERs of PPAR gamma (> 2.0) or PPAR delta (> 1.0) were termed Group H, and patients showing none of these elevated RERs of PPARs were termed Group L. Prognostic significance was analyzed by univariate and Kaplan-Meier analyses. RESULTS: In total, 26 CRC patients were studied. Univariate analysis revealed that PPAR gamma (> 2.0/ < or = 2.0) (odds ratio, 11.43; 95% C.I., 1.154-113.1; p = .0373), PPAR delta (> 1.0/ < or = 1.0) (odds ratio, 15.00; 95% C.I., 1.503-149.7; p = .0210) and PPAR staging (H/L) (odds ratio, 63.00; 95% C.I., 4.956-800.8; p = .0014) were significant predictors of postoperative mortality. Kaplan-Meier analysis revealed that the survival curve of patients with CRC was clearly divided by PPAR staging (log rank test, p <.0001). CONCLUSIONS: Evaluation of PPAR gamma and delta expression is useful for predicting postoperative mortality in patients undergoing CRC surgery.

Anti-proliferative effect of interferon-gamma is enhanced by iron chelation in colon cancer cell lines in vitro.

BACKGROUND/AIMS: The receptor of interferon-gamma (IFN-gammaR) consists of IFN-gammaR1 and R2. Resistance to the anti-proliferative effect of IFN-gamma is due to downregulation of IFN-gammaR2. The aim of this study was to investigate whether iron chelation could upregulate IFN-gammaR2 and enhance the anti-proliferative effect of IFN-gamma in colon cancer cell lines. METHODOLOGY: The colon cancer cell lines, SW480, COLO, and WiDr were treated with the iron chelating agent DFO, and the expression of IFN-gammaR1 and IFN-gammaR2 was evaluated by FACS. The anti-proliferative effect of IFN-gamma was investigated by MTT assay, and the proapoptotic effect was investigated by FACS with Annexin-V. RESULTS: FACS demonstrated that DFO increased the expression of IFN-gammaR2, whereas the effect on IFN-gammaR1 expression was less marked. MTT assay showed that cell growth was inhibited by DFO. Addition of DFO and IFN-gamma inhibited further, but inhibition was not observed with IFN-gamma alone. Apoptotic cells were increased by DFO, and further increased with DFO + IFN-gamma together. CONCLUSIONS: Expression of IFN-gammaR2 is restored by iron chelation, and the increased expression of IFN-gammaR2 enhances the anti-proliferative effect of IFN-gamma through induction of apoptosis in colon cancer cells.

Erythropoietin and its derivative protect the intestine from severe ischemia/reperfusion injury in the rat.

OBJECTIVE: To investigate the protective effect of erythropoietin (EPO) and its nonhematopoietic derivative (asialoEPO) against intestinal ischemia/reperfusion (I/R) injury in a rat model. METHODS: The superior mesenteric artery of Wistar rats was clamped for 60 minutes and then released. The rats were divided into 4 groups (n = 15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were administered subcutaneously at 1000 units/kg for 10 minutes before clamping, 30 minutes after the start of clamping, and just before declamping. This treatment was followed by determination of 72-hour survival rates, serum TNF-alpha and IL-6 levels, histologic evaluation of the small intestine, quantification of the number of apoptotic cells, and analysis of the antiapoptotic molecules Bcl-xL and XIAP by Western blotting. RESULTS: The survival rates at 72 hours after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 33%, 75%, and 83%, respectively (P < .05). Blood TNF-alpha and IL-6 were significantly more suppressed in the EPO and AsialoEPO groups than in the Vehicle group at 6 hours after I/R injury. Histologically, injury to villi in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. The number of apoptotic cells in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. Western blotting revealed that EPO and asialoEPO constitutively increased the expression of Bcl-xL. CONCLUSIONS: EPO and asialoEPO exert a strong protective effect against intestinal I/R injury, possibly by inhibiting release of TNF-alpha and IL-6 and decreasing apoptosis.

Protective effect of Sivelestat in a porcine hepatectomy model prepared using an intermittent Pringle method.

The effect of Sivelestat, a neutrophil elastase inhibitor, on hepatic ischemia-reperfusion injury was examined in a pig hepatectomy model. An internal jugular vein-splenic vein bypass was prepared in male pigs and about 40% hepatic resection (left lobe) was performed under 15-min liver ischemia and 5-min intermittent reperfusion. Six animals received Sivelestat (10 mg/kg/h) intravenously and six control animals received physiological saline (10 mg/kg/h) from commencement of laparotomy. Hemodynamics, blood chemistry, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), lactic acid, hyaluronic acid, nitrite/nitrate (NOS), and tumor necrosis factor-alpha (TNF-alpha) were compared between the groups. The effects of Sivelestat on NOS generation and expression of iNOS mRNA and TNF-alpha mRNA were also assessed in J774 cells. Expression of TNF-alpha mRNA in hepatic tissues was examined using RT-PCR. The blood pressure of control animals was significantly lower immediately and 3 h after ischemia-reperfusion, compared with that at commencement of laparotomy, whereas there was no decrease of blood pressure in animals administered Sivelestat. Serum AST (P=0.0045), NOS (P=0.0098), and TNF-alpha (P=0.041) levels were significantly lower 3 h after hepatectomy in animals receiving Sivelestat. Sivelestat inhibited NOS production in J774 cells, but did not inhibit expression of iNOS mRNA or TNF-alpha mRNA. In hepatic tissues, Sivelestat showed a greater tendency to inhibit expression of TNF-alpha mRNA and fewer TUNEL-positive cells were present in the hepatic sinusoidal endothelium after Sivelestat treatment, although these differences were not statistically significant. We conclude that Sivelestat inhibits production of TNF-alpha and NO by inhibiting neutrophil elastase, and thus reduces hepatic injury and stabilizes hemodynamics after ischemia-reperfusion.

MK615 inhibits pancreatic cancer cell growth by dual inhibition of Aurora A and B kinases.

AIM: To investigate the anti-neoplastic effect of MK615, an anti-neoplastic compound isolated from Japanese apricot, against human pancreatic cancer cells in vitro. METHODS: Three human pancreatic cancer cell lines PANC-1, PK-1, and PK45H were cultured with MK615 at concentrations of 600, 300, 150, and 0 microg/mL. Growth inhibition was evaluated by cell proliferation assay, and killing activity was determined by lactate dehydrogenase (LDH) assay. Expression of Aurora A and B kinases was detected by real-time polymerase chain reaction (PCR) and Western blotting. Cell cycle stages were evaluated by flow cytometry. RESULTS: The growth inhibitory rates of MK615 at 150, 300, and 600 microg/mL were 2.3%+/-0.9%, 8.9%+/-3.2% and 67.1%+/-8.1% on PANC1 cells, 1.3%+/-0.3%, 8.7%+/-4.1% and 45.7+/-7.6% on PK1 cells, and 1.2+/-0.8%, 9.1%+/-2.1% and 52.1%+/-5.5% on PK45H cells, respectively (P<0.05). The percentage cytotoxicities of MK615 at 0, 150, 300, and 600 microg/mL were 19.6%+/-1.3%, 26.7%+/-1.8%, 25.5%+/-0.9% and 26.4%+/-0.9% in PANC1 cells, 19.7%+/-1.3%, 24.7%+/-0.8%, 25.9%+/-0.9% and 29.9%+/-1.1% in PK1 cells, and 28.0%+/-0.9%, 31.2%+/-0.9%, 30.4%+/-1.1% and 35.3+/-1.0% in PK45H cells, respectively (P<0.05). Real-time PCR and Western blotting showed that MK615 dually inhibited the expression of Aurora A and B kinases. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase. CONCLUSION: MK615 exerts an anti-neoplastic effect on human pancreatic cancer cells in vitro by dual inhibition of Aurora A and B kinases.

New anti-proliferative agent, MK615, from Japanese apricot "Prunus mume" induces striking autophagy in colon cancer cells in vitro.

AIM: To investigate the anti-neoplastic effects of MK615, an extract from the Japanese apricot (Prunus mume), against colon cancer cells. METHODS: Three colon cancer cell lines, SW480, COLO, and WiDr, were cultured with MK615. Growth inhibition was evaluated by cell proliferation assay and killing activity was determined by lactate dehydrogenase assay. Induction of apoptosis was evaluated by annexin V flow cytometry. Morphological changes were studied by light and electron microscopy, and immunofluorescence staining with Atg8. RESULTS: MK615 inhibited growth and lysed SW480, COLO and WiDr cells in a dose-dependent manner. Annexin V flow cytometry showed that MK615 induced apoptosis after 6 h incubation, at which point the occurrence of apoptotic cells was 68.0%, 65.7% and 64.7% for SW480, COLO, and WiDr cells, respectively. Light and electron microscopy, and immunofluorescence staining with Atg8 revealed that MK615 induced massive cytoplasmic vacuoles (autophagosomes) in all three cell lines. CONCLUSION: MK615 has an anti-neoplastic effect against colon cancer cells. The effect may be exerted by induction of apoptosis and autophagy.

A novel anti-cancer substance, MK615, from ume, a variety of Japanese apricot, inhibits growth of hepatocellular carcinoma cells by suppressing Aurora A kinase activity.

BACKGROUND/AIMS: MK615 is an anti-cancer substance extracted from the Japanese apricot. In the present study, the anti-neoplastic effect of MK615 against hepatocellular carcinoma (HCC) was evaluated in vitro, and its mechanism was elucidated. METHODOLOGY: Two HCC lines, HuH7 and Hep3B, were cultured with MK615 at concentrations of 600, 300, 150, and 0 microg/mL. Growth inhibition was evaluated by MTT assay, and killing activity was determined by LDH assay. Cell cycle stages were evaluated by flow cytometry. Expression of Aurora A kinase (Aurora A) was evaluated by real-time PCR and Western blotting, and inhibition of Aurora A activity was determined by HTscan. RESULTS: MK615 inhibited the growth of, and lysed, HuH7 and Hep3B cells in a dose-dependent manner. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase. Real-time PCR and Western blotting showed that MK615 suppressed the expression of Aurora A. HTscan assay demonstrated that Aurora A activity was specifically inhibited by 34.3%, 32.9%, and 54.3% at 150, 300, and 600 microg/mL MK615, respectively. CONCLUSIONS: MK615 has an anti-cancer effect against HCC lines in vitro, and the effect is exerted through inhibition of Aurora A activity.

Asialoerythropoietin has strong renoprotective effects against ischemia-reperfusion injury in a murine model.

BACKGROUND: The renoprotective effect of erythropoietin (EPO) and the nonhematopoietic EPO, asialoEPO was investigated in a murine ischemia-reperfusion injury (I/R) model. METHODS: I/R was created by clamping the right renal pedicle for 60 min after left nephrectomy. Balb/c mice were divided into four groups (n=15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were given at a dose of 500 IU/kg 30 min before I/R. Plasma creatinine (Cr), survival, and the number of apoptotic cells were analyzed. Protein expression was analyzed by Western blotting. RESULTS: Plasma Cr level was not significantly different at 6 hr after I/R. At 24 hr after I/R, the Cr (mg/dL) levels in Sham, Vehicle, EPO, and asialoEPO were 0.13+/-0.01, 1.24+/-0.70, 0.24+/-0.08, and 0.25+/-0.13, respectively (P<0.05). The numbers of apoptotic cells in these groups were 0.1+/-0.1, 98.9+/-42.6, 3.3+/-0.7, and 2.9+/-1.6, respectively (P<0.05). Western blotting revealed that in kidney tissue of mice treated with EPO and asialoEPO, p38-MAPK and the proapoptotic molecule Bad was decreased, and the antiapoptotic molecules Bcl-xL and XIAP were increased. Survival rates at 7 days after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 21.4%, 23.1%, and 53.8%, respectively (P=0.05). CONCLUSION: EPO and asialoEPO attenuated renal dysfunction caused by I/R in mouse kidney at the same level, but only asialoEPO improved survival.

Hepatocellular carcinoma with chronic B-type hepatitis complicated by autoimmune hemolytic anemia: a case report.

A 57-year-old man consulted a local hospital because of a persistent slight fever. At the age of 37 years he was diagnosed having B-type hepatitis, but left the liver dysfunction untreated. Twenty years later, he was diagnosed having chronic hepatitis B, hepatocellular carcinoma (HCC) and macrocytic anemia, and referred to our hospital for further investigation. A HCC with a maximum diameter of 5.2 cm was detected in segment 8. Results of blood tests included 1.8 mg/dL serum total bilirubin, 0.9 mg/dL bilirubin, less than 10 mg/dL haptoglobin, 7.9 g/dL hemoglobin, 130 fL MCV, and 14.5% reticulocytes. A bone marrow sample showed erythroid hyperplasia. The direct Coombs test gave a positive result. We diagnosed the anemia as autoimmmune hemolytic anemia (AIHA), for which prednisolone could not be administered due to positivity for HBsAg and HBeAg. After preparation of washed blood cells for later transfusion, the patient underwent systematic resection of segment 8. The cut surface of the resected specimen demonstrated an encapsulated yellow-brownish tumor measuring 52 mm multiply 40 mm which was diagnosed pathologicaly as moderately differentiated HCC. On the 9th postoperative day, the patient's temperature rose to 38 centigrade, and exacerbated hemolysis was observed. The maximum total bilirubin value was 5.8 mg/dL and minimum hemoglobin level was 4.6 g/dL. He tolerated this period without blood transfusion. Currently he is being followed up as an outpatient, and shows no signs of HCC recurrence or symptoms of anemia. AIHA associated with HBV infection has been described in only three previous cases, and the present case is the first in which surgery was performed for accompanying HCC.

New antineoplastic agent, MK615, from UME (a Variety of) Japanese apricot inhibits growth of breast cancer cells in vitro.

MK615 is an extract mixture containing hydrophobic substances from Japanese apricot. In this study, the antineoplastic effects of MK615 against breast cancer cells were investigated. Two breast cancer cell lines, MDA-MB-468 (MDA) and MCF7, were cultured with (600, 300, and 150 mug/mL) or without MK615. After 72 hours of incubation, growth inhibition was evaluated by MTT assay. The cells were then cultured with MK615 (300 mug/mL) and morphological changes were studied by light and electron microscopy. Finally, the mechanism of the antineoplastic effect of MK615 was evaluated by cell cycle and apoptosis assay. MK615 inhibited the growth of MDA and MCF7 in a dose-dependent manner. The percentage growth inhibition of MDA at dosages of 600, 300, and 150 mug/mL was 59.2%, 52.4%, and 23.3%, respectively, and that for MCF7 was 83.5%, 52.7%, and 16.6%, respectively. Morphological changes after MK615 treatment included massive vacuolization in the cytoplasm and apoptotic changes in the nucleus. These changes began to be apparent after at least 6 hours of incubation. Cell cycle analysis showed that MK615 increased the proportion of cells in the G2-M phase in both MDA (7.8-11.7%) and MCF7 (8.1-18.7%), and finally both cell lines became apoptotic. The proportion of apoptotic cells increased with incubation time. MK615 effectively inhibits the growth of breast cancer cells in vitro, possibly by cell cycle modification and apotosis induction. MK615 should be further investigated as a promising anti-breast cancer agent.

Deferoxamine enhances anti-proliferative effect of interferon-gamma against hepatocellular carcinoma cells.

BACKGROUND: Interferon-gamma (IFN-gamma) is a multifunctional cytokine, whose anti-proliferative effect is expected to be of therapeutic value against human cancer. However, hepatocellular carcinoma (HCC) shows resistance to the anti-proliferative effect of IFN-gamma, due mainly to down-regulation of IFN-gamma receptor chain 2 (IFN-gammaR2), even though IFN-gamma receptor chain 1 (IFN-gammaR1), the domain that includes the binding site of IFN-gamma, is stably expressed. The aims of this study were to investigate whether iron chelation, blocking of the human insulin-like growth factor-1 receptor (hIGF1R), or both could upregulate IFN-gammaR2 and enhance the anti-proliferative effect of IFN-gamma. METHODS: Two HCC cell lines, HuH7 and SNU449, were treated with the iron-chelating agent deferoxamine (DFO), IFN-gamma, and/or anti-hIGF1R blocking antibody. The expression of IFN-gammaR1 and IFN-gammaR2 was then evaluated by flow cytometry and Western blotting. The anti-proliferative effect of IFN-gamma was investigated by MTT assay, and the pro-apoptotic effect was investigated by annexin-V flow cytometry. RESULTS: DFO and blocking with anti-hIGF1R antibody increased the expression of IFN-gammaR2, but the effect on IFN-gammaR1 expression was less marked. DFO, anti-hIGF1R blocking antibody, or both directly enhanced the anti-proliferative effect of IFN-gamma through increased pro-apoptotic activity. CONCLUSION: The present results indicate that IFN-gamma reinforced by iron modulation could be a promising new therapeutic approach for HCC.

Rapamycin enhances the anti-tumor effect of gemcitabine in pancreatic cancer cells.

BACKGROUND/AIMS: Rapamycin is a potent inhibitor of PI3K/Akt pathway activation and its chemotherapeutic effect against pancreatic cancer has been demonstrated. In the present study, the combined effect of rapamycin with gemcitabine was examined and a screening method for detecting sensitivity to combined effects was investigated. METHODOLOGY: Four pancreatic cancer cell lines, MIA, PK-1, PANK-1, and PK-45H, were cultured with or without rapamycin (200, 100, 50, 25nM), or gemcitabine (2, 1, 0.5, 0.25 microM), or with rapamycin and gemcitabine in combination. Growth inhibition of each cell line in response to the agents was examined using the MTT assay. Data were expressed as percentage inhibition at each concentration. Sensitivity to the combined effect of rapamycin and gemcitabine was investigated with real-time PCR (Akt1, 2, and 3, PTEN, mTOR) and western blotting (Akt, Akt/ Ser473, Akt/ Thr308, PTEN). RESULTS: Rapamycin inhibited the growth of MIA (% inhibition: 36.2+/-9.0, 19.5+/-9.0, 10.8+/-6.8, 8.8+/-5.9), PK-1 (41.3+/-0.1, 33.5+/-0.1, 25.2+/-0.1, 22.6+/-0.1), PANK-1 (27.3+/-0.1, 21.7+/-0.1, 15.9+/-0.1, 14.9+/-0.1), and PK-45H (30.0+/-0.1, 24.4+/-0.1, 23.5+/-0.1, 20.5+/-0.1), whereas gemcitabine inhibited the growth in MIA (37.5+/-11.1, 11.5+/-7.8, 3.5+/-1.7, 0.1+/-0.1) and PK-1 (29.2+/-2.8, 26.7+/-0.8, 26.2+/-1.0, 25.1+/-1.0), but only weakly in PANK-1 (7.5+/-5.2, 1.2+/-0.9, 4.2+/-0.8, 2.7+/-0.6) and PK-45H (16.1+/-5.8, 11.8+/-10.0, 7.9+/-1.8, 10.1+/-1.8). However, gemcitabine administered with rapamycin had an enhanced inhibitory effect in PK-45H (61.3+/-12.0%), and no change in PANK-1, when compared to the inhibition by rapamycin or gemcitabine alone. CONCLUSIONS: As a screening method for assessing the combined effects of rapamycin and gemcitabine, the decrease in expression of Akt/Ser473 and Akt/Thr 308 with rapamycin treatment was promising. Rapamycin enhances the anti-tumor effect of gemcitabine. Detecting a decrease in Akt/ Ser473 and Akt/Thr308 after rapamycin treatment, by western blotting, may be an effective way for assessing the combined effect of rapamycin and gemcitabine.

Total colectomy improves altered distribution of regulatory T cells in patients with ulcerative colitis.

BACKGROUND: It is now generally believed that regulatory T cells (Tregs) play an important role in peripheral tolerance, and a defect in Tregs is considered one of the most important factors in the induction of various kinds of autoimmune disease including ulcerative colitis (UC). Here, we examined the change in frequency of Tregs phenotype in five patients with UC whose condition had not been controllable by conventional conservative therapy and who were scheduled for total colectomy. AIMS: The aim of this study was to elucidate the role of Tregs in the pathogenesis of UC in a clinical setting. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from five patients with UC, and the change in frequency of Tregs was analyzed by flow cytometry before total colectomy and on postoperative days 1, 7, and 20. Tregs were defined as CD4+ CD25+ CD45RA+ T cells, and data (%) were expressed as frequency of Tregs/CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) from healthy blood donors (n = 5) and from patients undergoing other types of major surgery (n = 5) were used as controls. RESULTS: Comparison with normal subjects showed that generation of Tregs was suppressed in UC patients before they underwent total colectomy (0.95 versus 5.06; P = 0.009). The frequency of Tregs increased shortly after total colectomy. The frequency at postoperative days 1, 7, and 20 was 3.81%, 8.13%, and 3.76%, respectively. There were significant differences in the change in frequency in the period before surgery and postoperative day 1, between postoperative days 1 and 7, and between days 7 and 20. CONCLUSIONS: Elimination of targeted antigens residing in the colorectal mucosa by total colectomy improved the suppressed distribution of Tregs in UC patients. The present study provides the first direct clinical evidence that Tregs play a pivotal role in the pathogenesis of UC.

Loss of E-cadherin mRNA and gain of osteopontin mRNA are useful markers for detecting early recurrence of HCV-related hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: We investigated whether expression of E-cadherin (E-cad) and osteopontin (OPN) mRNAs could predict the early recurrence of HCV-associated hepatocellular carcinoma (HCV-HCC) after curative surgery. METHODS: Forty-four HCV-HCC patients who underwent curative hepatectomy were divided into three categories: category 1--recurrence (3 mR+: n = 6) or non-recurrence (3 mR-: n = 38) within 3 months; category 2--recurrence (6 mR+: n = 4) or non-recurrence (6 mR-: n = 34) between 3-6 months; category 3--recurrence (1 yR+: n = 4) or non-recurrence (1 yR-: n = 30) between 6-12 months. Levels of expression of E-cad and OPN mRNAs were analyzed quantitatively by real-time PCR and calculated using the formula: t = (copy of E-cad or OPN/copy of GAPDH) x 1,000. RESULTS: The t-value for E-cad was significantly lower in 3 mR+ than in 3 mR- (0.4 and 28.1, P < 0.05), in 6 mR+ than in 6 mR- (4.0 and 30.6, P < 0.05), and in 1 yR+ than in 1 yR- (9.2 and 32.9, respectively, P < 0.05). The t-value for OPN was higher in 3 mR+ than in 3 mR- (92.6 and 65.7, P = 0.57), in 6 mR+ than in 6 mR- (457.1 and 58.15, respectively, P = 0.24), and in 1 yR+ than in 1 yR- (221.8 and 53.5, respectively, P = 0.06). CONCLUSIONS: Low expression of E-cad mRNA and high expression of OPN mRNA are associated with a higher likelihood of early recurrence of HCV-HCC.

Abnormal expression of Rb pathway-related proteins in salivary gland acinic cell carcinoma.

Salivary gland acinic cell carcinoma (ACC) is a relatively rare neoplasm, and limited information is available regarding its molecular pathogenesis. Because the deregulation of Rb pathway is common to most human tumors, we immunohistochemically investigated the expression of Rb pathway-related proteins, including Rb, Rb proteins phosphorylated at serine 780 and 795 (pRb-S780 and pRb-S795, respectively), cyclin D1, and p16INK4a in 18 cases of ACC. The expression of topoisomerase II-alpha and Ki-67 was also examined to evaluate cell proliferation. All the ACCs exhibited substantial numbers of positive cells against Rb antibody that recognizes both unphosphorylated and phosphorylated Rb proteins. The numbers of positive cells for pRb-S795 and cyclin D1 significantly increased in ACCs as compared with normal salivary glands. Double immunofluorescent staining demonstrated that pRb-S795 was colocalized with cyclin D1 in most tumor cells. However, neither significant change of the expression of Rb protein phosphorylated at serine 780 nor its colocalization with cyclin D1 was observed. The loss of p16INK4a is infrequent, but its expression was correlated with phosphorylated Rb proteins. Our results suggest that serine 795 but not serine 780 is the preferred phosphorylation site induced by cyclin D1. This phosphorylation appeared to be critical for inactivation of Rb-mediated growth suppression and may play an important role in the pathogenesis of ACC.