The role of the new Ca2+ antagonist, CV159, in hepatic I/R injury-the evaluation of hepatic organ reducing activity using in vivo and ex vivo EPR.

We investigated the organ-reducing ability of 1,2-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylic acid methyl 6-(5-phenyl-3-pyrazolyloxy) hexyl ester (CV159) that exhibits selective blocking of Ca(2+)/calmodulin and inhibition of Ca(2+) overloading in living organisms (Sprague Dawley rats) using an in vivo and an ex vivo electron paramagnetic imaging technique. Decay rates in CV159-treated rats were significantly higher than those in untreated rats and were almost equal to those in the sham group. Both cytosol and mitochondrial superoxide scavenging activity in CV159-treated rats were significantly higher than those in untreated rats, and cytosol superoxide scavenging activity only was slightly higher than that in the sham group. Faint staining for anti-superoxide dismutase antibody was markedly observed in necrotic lesions in the liver of control group. Alanine aminotransferase level in CV-treated rats were significantly decreased as compared with the levels in untreated rats. Electron microscopy showed a decreased number of damaged mitochondria, whereas mitochondrial damage was significantly reduced in CV-treated animals. We conclude that CV159 retains the organ-reducing activity against radicals in hepatic I/R injury that is mediated by the inhibition of Ca(2+) overloading.

An anticancer drug sensitivity test to determine the effectiveness of UFT as postoperative adjuvant chemotherapy for patients with stage III colorectal cancer.

BACKGROUND: Tissue samples from patients with pathologic ((p)) stage III colorectal cancer were tested for sensitivity to 5-fluorouracil (5-FU). On the basis of the results, patients were divided into 5-FU-sensitive and 5-FU-resistant groups, and both groups were treated with fluoropyrimidine (UFT) as postoperative adjuvant chemotherapy. Recurrence, 5-year survival rates, and 5-year recurrence-free survival rates were compared. METHODS: The anticancer sensitivity test described in this study was carried out using tumor samples obtained surgically from 34 patients with curatively resectable colorectal cancer that had been diagnosed definitively as (p)stage III (IIIa, 23 patients; IIIb, 11 patients). Regardless of tumor sensitivity or resistance to 5-FU, all 34 patients were subsequently treated daily with UFT at 300 mg/day as postoperative adjuvant chemotherapy. Treatment was initiated 3 weeks after surgery and continued for 2 years. RESULTS: Of the 34 patients with (p)stage III colorectal cancer, the tumors of 16 (47%) were 5-FU-sensitive (S group) and 18 (53%) were 5-FU-resistant (R group). The recurrence rates in the S and R groups following postadjuvant therapy with UFT were 25% and 61%, respectively, which is a significantly lower response in the S group (P = .045). The odds ratio of recurrence in the R group vs. the S group was 4.71. The 5-year survival rate was 85.7% in the S group and 46.7% in the R group, but the difference was not significant (P = .066). The 5-year recurrence-free survival rate was significantly higher in the S group than in the R groups (71% vs. 32%, P = .010). According to Cox's multivariate analysis of recurrence-free survival, the sensitivity test was significantly predictive. CONCLUSION: Administration of UFT as postoperative adjuvant therapy to 5-FU-resistant patients had no significant effect on outcome.

A new chemosensitivity assay for ascites tumor cells using a thermoreversible gelation polymer as a culture medium and the observed clinical responses.

BACKGROUND/AIM: Ascites tumor cells from patients with peritonitis carcinomatosa were tested for cis-diamminedichloroplatinum (CDDP) sensitivity. The patients were divided into CDDP-sensitive and resistant groups. Survival and time to progression (TTP) rates were compared. MATERIALS AND METHODS: 18 peritonitis carcinomatosa patients with class V ascites based on cytologic diagnosis were enrolled in this study. Chemosensitivity testing of the ascites tumor cells was done to determine their sensitivity to CDDP using a three-dimensional culture matrix of thermoreversible gelation polymer (TGP). CDDP at a dose calculated to achieve ascitic fluid drug levels equivalent to the IC(50) was given intraperitoneally to 12 CDDP-sensitive patients and 6 CDDP-resistant patients. RESULTS: Both the median survival time and the median TTP were significantly longer in CDDP-sensitive patients than in CDDP-resistant patients (survival time 105 vs. 13 days, p = 0.019; TTP 90 vs. 5 days, p = 0.029). CONCLUSION: The results indicate the potential feasibility of controlling ascites in cancer patients in whom a maximal dose effect can be achieved with a minimal dose of chemotherapy.

Superoxide dismutase as a target enzyme for Fe-porphyrin-induced cell death.

The cell viability of human cancer cell lines treated with [5,10-bis(N-methyl-4-pyridyl)-15,20-diphenyl]porphinatoiron(III) (cis-FeMPy(2)P(2)P) has been estimated. The cis-FeMPy(2)P(2)P is a superoxide dismutase (SOD) mimic in vitro that exhibited a significant toxicity in cancer cell lines. This toxicity is rather due to pro-oxidant properties of the iron-porphyrin in vivo. We have demonstrated that there was the relationship between the LD(50) values calculated from the viability of cancer cell lines treated with cis-FeMPy(2)P(2)P and the SOD activities of the cell lines. Furthermore, the inhibition of SOD by antisense S-oligonucleotide increased the cytotoxic effect of cis-FeMPy(2)P(2)P against cancer cells. These results suggest that SOD is a target enzyme for the cell death induced by cis-FeMPy(2)P(2)P as a new class of anticancer agents.

Repeated hepatic intra-arterial chemotherapy based on results of anticancer drug sensitivity test in patients with synchronous hepatic metastases from colorectal cancer.

OBJECTIVE: We determined whether hepatic intra-arterial infusion of 5-fluorouracil (5-FU) in patients with synchronous hepatic metastases from colorectal cancer, in whom the primary lesion was resectable but hepatic metastatic lesions were non-resectable helped improve survival time when administered on the basis of the results of the anticancer drug sensitivity test. PATIENTS AND METHODS: The study population consisted of 29 patients with synchronous hepatic metastases from colorectal cancer who underwent surgical resection of the primary lesion alone. Of these 29 patients, 21 received hepatic intra-arterial infusion of 5-FU postoperatively after the 5-FU sensitivity test. The remaining 8 patients underwent surgical resection of the primary lesion but neither sensitivity testing nor hepatic intra-arterial chemotherapy. Tissue fragments were cultured, with each concentration of 5-FU in the thermoreversible gelation polymer forming a three-dimensional structure at 37 degrees C. The viability of tumor cells was evaluated according to WST methods; inhibitory concentration of 50% (IC50) values were calculated. We considered cancer tissue to be sensitive to IC50 values that were below twice the peak plasma concentration (120 microg/ml). RESULTS: Of the 21 patients, 10 had sensitivity to 5-FU and 11 had no sensitivity. The response rates were 90.0% and 9.1%, respectively. The median survival times were 38 months and 10 months in these groups, respectively, and 7 months in patients who received no chemotherapy. This finding indicates a significantly longer survival time in the sensitive group, compared with either the insensitive group or the no chemotherapy group (P = .0014 P = .0023). The cumulative survival rate was significantly higher in the sensitive group compared with the insensitive group (P = .0001) CONCLUSIONS: Ultimately, the group with sensitivity to 5-FU showed a significantly longer median survival time than the insensitive group.

Thermoreversible gelation polymer induces the emergence of hepatic stem cells in the partially injured rat liver.

Focal injury of the adult liver causes formation of granulomatous tissue and fibrosis. When thermoreversible gelation polymer (TGP) was applied to such defects of the rat liver, complete recovery of hepatic tissues was observed without granulation. We analyzed the mechanism of the regeneration. TGP is a chemically synthesized biocompatible polymer material whose sol-gel transition is reversible by changing the temperature. Cooled TGP was poured into a penetration lesion of the rat liver. Immunohistochemistry and polymerase chain reaction were carried out using tissues and cultured cells isolated from ductular structures. Immunocytochemical and ultrastructural analyses were also conducted. Seven days after TGP treatment, ductular reactions were observed around the wound and ductules elongated to the injured area. Cells in the structures were alpha-fetoprotein (AFP) positive, albumin+, CK19+, c-Kit+, and Thyl+. Hepatocyte-like cells possessing glycogen appeared around the tips of the ductules from day 9. The defect was completely replaced with hepatocytes by day 28. Cells isolated from the ductules expressed Musashi-1, c-Kit, Thyl, AFP, albumin, transferrin, connexin 43, and CK19. When the cultured cells were covered by TGP, they rapidly proliferated to form colonies, whereas without TGP cells gradually died. Morphologically and ultrastructurally the cells were similar to hepatocytes. They expressed not only albumin and transferrin but TAT, CYP2E1, and CCAAT/enhancer binding protein a. Some cells formed bile canaliculus-like structures. In conclusion, TGP may trigger the initiation of hepatic stem cells in biliary ductules, and stem cell activation may occur even in the regeneration of the normal liver.

The role of HMGB-1 on the development of necrosis during hepatic ischemia and hepatic ischemia/reperfusion injury in mice.

BACKGROUND: High-mobility group 1 (HMGB-1) is a late mediator of endotoxin lethality in mice. The release of HMGB-1 is delayed compared to other proinflammatory cytokines that mediate shock and tissue injury. The purpose of this study was to investigate the role of HMGB-1 levels in response to hepatic ischemia, hepatic I/R injury, and the relationship between changes in HMGB-1 and other cytokines. MATERIALS AND METHODS: Three murine models were employed: our robust model of segmental hepatic warm ischemia (SHWI), a model of partial hepatic ischemia/reperfusion injury (PHIRI), and a model of total hepatic ischemia/reperfusion injury (THIRI). Over a 48-h period following ischemic insult and reperfusion using these models, serum HMGB-1 concentrations, concentrations of HMGB-1 in ischemic and nonischemic lobes, and serum concentrations of TNF-alpha and IL-6 levels were determined in mice. An anti-HMGB-1 antibody treatment was used in SHWI and THIRI to evaluate what aspects of response to ischemia and reperfusion were potentially mediated by HMGB-1. RESULTS: Hepatic HMGB-1 tissue concentrations exhibited biphasic changes in SHWI mice, which were increased in the ischemic lobes relative to nonischemic lobes at 12 h but decreased relative to nonischemic lobes at 24 h after ischemic insult. These results suggested that HMGB-1 was released into the systemic circulation by necrotic cells over the first 12 h but this process may be complete by 24 h postischemia. By 6 to 12 h after SHWI, serum TNF-alpha began to increase significantly and continued to increase for 18 h, followed by a sudden decline. Similarly, serum IL-6 increased over 1-3 h after SHWI and then decreased over the next 6 h. Treatment with an anti-HMGB-1 antibody significantly prolonged survival time in SHWI and THIRI. CONCLUSIONS: HMGB-1 plays a significant role in the response to hepatic ischemia and hepatic ischemia/reperfusion injury. The present study demonstrated a time-dependent production of HMGB-1 following hepatic warm ischemia in mice. The inherent HMGB-1 in ischemic areas was exhausted and HMGB-1 may be released by necrotic cells. HMGB-1 activation is involved in immediate proinflammatory stress response to I/R and anti-HMGB-1 antibody treatment remarkably improved survival. We demonstrated that systemic HMGB-1 accumulation was measured at an earlier phase of the hepatic ischemia and ischemia/reperfusion injury model than LPS-induced endotoxemia.

New chemosensitivity test using a thermo-reversible gelation polymer for recurrent gynecologic cancer patients and a preliminary study of mechanisms of anticancer drug resistance.

OBJECTIVES: TGP (thermo-reversible gelation polymer) is a high molecular compound that has so-gel transmitting temperature of 221C Since solid cancer tissue grows in this polymer three-dimensionally, and fibroblasts scarcely grow in it, TGP is suitable for chemosensitivity assays for solid tumors. In this study, a chemosensitivity test using TGP was applied to recurrent gynecologic cancer patients in order to evaluate its utility and efficacy. In some ovarian cancer cases, expression of anticancer drug resistance-related proteins was also analyzed. METHODS: Recurrent tumor tissues were surgically obtained with informed consent. After these tissues were minced and incubated for 4 days with CDDP, mitomycin C, 5-fluorouracil, paclitaxel, and CPT-11, the sensitivity against these drugs was estimated. Western blotting was performed in 8 recurrent ovarian cancer tissues in order to analyze the expression of Bcl-2, MRP2, BCRP, and GST-pi. RESULTS: The total evaluability rate of this assay was 90.6% (29/32). Sensitive drugs could be determined in 5 of 7 ascites samples (71.4%) and in 2 of 3 intra-tumoral fluid samples (66.7%). The overall clinical response rate of chemotherapy determined by these results was 50.0%. There were significant correlations between the IC50 of CDDP and Bcl-2, BCRP, GST-pi, and between that of 5-FU and MRP 2. CONCLUSIONS: Although this was a preliminary study, the chemosensitivity test using this new material appears to be useful for designing 'made-to order' salvage chemotherapy for pretreated recurrent gynecologic patients. In order to overcome multidrug resistance, the mechanisms of multidrug resistance should be further investigated.

Ductular cell proliferation in islet cell neogenesis induced by incomplete ligation of the pancreatic duct in dogs.

PURPOSE: It has been suggested that islet neogenesis can be induced by incomplete ligation of the pancreatic duct in small animals; however, there has been no report of neogenesis and the proliferation of islets occurring in larger animals. When this procedure was performed in the Vervet monkey, it produced a noticeable increase in duct proliferation, but islet neogenesis was not observed, although the number of monkeys examined was very small. We conducted this study to evaluate whether islet neogenesis and ductular proliferation could be induced in larger animals such as the dog, by partial obstruction of the pancreatic duct. METHODS: Incomplete ligation of the pancreatic duct was induced by tying the pancreas around the ventral side of the head with 2-0 silk and reducing the circumference by about 80% to cause partial obstruction. RESULTS: By 2 weeks after ligation, we saw hyperplasia of the epithelial cells, multilayering of cuboidal cells, and proliferation of ductular cells. The terminal ductules involved in the formation of immunohistochemically insulin-positive islets, and islets, formed adjacent to the alignment of the ductular cells. By 8 weeks after ligation we saw scattered islets, less than 50 micro m in diameter and less than 1 000 microm(2) in area. These cells were immunolabeled for both insulin and cytokeratin, and there was continuity between these insulin-positive cells and terminal ductular cells. Glucagon-positive cells and somatostatin-positive cells were also found adjacent to the alignment of ductular cells. CONCLUSIONS: These results suggest that islets may be differentiated from precursor cells in the pancreatic duct, and that stem cells exist even in adults.

Development of a novel polyimide hollow-fiber oxygenator.

We have developed a membrane oxygenator using a novel asymmetric polyimide hollow fiber. The hollow fibers are prepared using a dry/wet phase-inversion process. The gas transfer rates of O(2) and CO(2) through the hollow fibers are investigated in gas-gas and gas-liquid systems. The polyimide hollow fiber has an asymmetric structure characterized by the presence of macrovoids, and the outer diameter of the hollow fiber is 330 microm. It is found that the polyimide hollow-fiber oxygenator can enhance the gas transfer rates of O(2) and CO(2), and that the hollow fiber provides excellent blood compatibility in vitro and in vivo.

Development of a fluorinated polyimide hollow fiber for medical devices.

We fabricated an asymmetric polyimide hollow fiber for medical devices. A dry/wet phase inversion process was applied to a spinning process to prepare the hollow fiber. The outer diameter was 330 microm with a wall thickness of 70 microm. Transfer rates of O2 and CO2 in the asymmetric polyimide fiber were 6.9 x 10(-3) and 5.5 x 10(-3) cm3 (STP)/(cm2 s cmHg), respectively, which are approximately 10 times higher than those measured in the Menox and Si-polypropylene fibers of the presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber was evaluated in vivo, indicating that polyimide had excellent blood compatibility when compared with silicone-coated fiber. Additionally, we fabricated a novel porous membrane with three-dimensional fine structure from cylindrical microscale pores and examined possibility of a porous membrane for use in hemodialysis.

A new method to prepare multicellular spheroids in cancer cell lines using a thermo-reversible gelation polymer.

The purpose of this study is to utilize the thermo-reversible gelation polymer in which the sol-gel transitting phase is reversibly changed by temperature in a three-dimensional culture system. Human cancer cells have been observed to form multicellular spheroids, whereas fibroblasts slowly develop into small spheroids with the culture medium including this polymer. This polymer has some advantages for use as a culture material, as follows: first, cancer cells grow three-dimensionally in the aqueous solution of this polymer; second, it is easy to harvest cells or spheroids in the aqueous solution of this polymer by simply cooling down the temperature; and third, the culture medium including this polymer is so translucent that the cells or spheroids can be observed through a phase-contrast microscope. We thus conclude that this polymer is a very useful material for three-dimensional cultures.

Biocompatibility of fluorinated poly(organophosphazene).

We investigated neutrophil and platelet adhesion on a fluorinated poly(organophosphazene) in vitro. The results suggested that neutrophil and platelet adhesion on the poly(organophosphazene) only occurred on a few occasions, as observed by SEM. We demonstrated that the fluorinated poly(organophosphazene) showed excellent biocompatibility compared with the poly(organophosphazene) without the fluorinated side groups or PDMS. Additionally, we estimated the competitive plasma protein adsorption to the fluorinated poly(organophosphazene) using a gold-colloid-labeled immunoassay. Interestingly, the fluorinated poly(organophosphazene) film selectively adsorbed albumin when compared with gamma-globulin and fibrinogen, suggesting that a selective albumin adsorption on the film is responsible for the suppression of platelet adhesion.

Biodegradation and biocompatibility of polyorganophosphazene.

We investigated biodegradation and biocompatibility of poly(organophosphazenes). We prepared poly(organophosphazenes) having different side chain groups. The blood compatibility of poly(organophosphazenes) containing fluorinated side groups, poly(bis[trifluoroethoxy]phosphazene) (PbFP) and poly([trifluoroethoxy][ethyl glycinate]phosphazene) (PFGP), without heparinization were evaluated in vitro. The deformation and aggregation of platelets adhered on PbFP and PFGP were not observed and they suppressed platelet activation. Additionally, PbFP and PFGP showed a higher degradation rate, despite their high hydrophobic nature. We found that the high mobility of water in PbFP and PFGP was one of the important factors facilitating their degradation. Their polymer structures were formed in a more open nature, indicating that water easily attacked the backbone of the phosphorus and nitrogen atoms in the poly(organophosphazene). On the other hand, the proliferation of HeLa cells cultured on poly(organophosphazene) was reduced compared with that on the control tissue culture polystyrene.

Selective cell death by water-soluble Fe-porphyrins with superoxide dismutase (SOD) activity.

We investigated the effect on cell death of reactive oxygen species induced by [[5,10 (or 5,15)-bis(N-methyl-4-pyridyl)-15,20 (or 10,20) diphenyl]porphinato]iron (cis-FeMPy(2)P(2)P or trans-FeMPy(2)P(2)P) with SOD activity. The SOD activities of the cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P were measured using stopped-flow kinetic analysis. The cell viability of four cell lines treated with cis-Fe-porphyrin, trans-Fe-porphyrin, mitomycin c (MMC), or cisplatin was estimated by the alamar blue exclusion assay of the modified MTT method. The amount of cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P in the Walker 256 cultured for 24 h was 4.0 and 2.6 fmolcell(-1), respectively, indicating that the plasma membrane permeability of the Fe-porphyrins depended on their structure. Cis-FeMPy(2)P(2)P selectively killed Walker 256 and H-4-II-E as cancer cells but not FR and BRL-3A as normal cells and showed a significant cytotoxicity for the cancer cells compared with trans-FeMPy(2)P(2)P, MMC and cisplatin. We believe that cis-FeMPy(2)P(2)P as an SOD mimic converts intracellular O(2)(*-) to H(2)O(2) and that H(2)O(2) or *OH causes DNA damage and induces cell death. This result suggests that for the SOD mimic, O(2)(*-) may be useful as a target molecule to induce selective cell death between cancer and normal cells and that a metalloporphyrin having SOD activity is a new class of anticancer agents.