Invasion of carcinoma cells into reconstituted type I collagen gels: visual real-time analysis by time-lapse microscopy.

Stromal-epithelial interactions play a critical role in promoting tumorigenesis and invasion. To obtain detailed information on cancer cell behaviors on the stroma and kinetics of cell migration, which cannot be observed by conventionally-used Boyden chamber assays, this study was aimed at analyzing the cell invasion process in vitro using time-lapse microscopic observation. Serum-free conditions and reconstituted type I collagen gels which provided a basal membrane-stroma-like microenvironment were used to first establish a basal condition. Time-lapse microscopic observation for 30 h of cell invasion into the collagen gel revealed kinetic parameters and individualistic behavior of cancer cells. Of breast cancer MDA-MB-231 or MCF-7 cells and colon cancer LS180 or HT29 cells examined, MDA-MB-231 cells most rapidly disappeared from the collagen gel surface under basal conditions. Estrogen-dependent MCF-7 cells disappeared at a rate approximately two times slower than that of MDA-MB-231 cells under serum- and phenol red-free conditions. By the addition of 10 nM ß-estradiol to the basal medium, MCF-7 cell invasion was facilitated to a rate similar to that of MDA-MB-231 cells. Microscopic analyses of collagen gel-sections demonstrated that most of the MDA-MB-231 and MCF-7 cells remained within 60 µm from the gel top under basal conditions, which is consistent with the observation obtained using Boyden chambers that no cells could cross the collagen I gel barrier unless 1% fetal calf serum was added to basal conditions. In summary, this study demonstrated future applicability of this method to understand the initial phase of cancer cell invasion processes.

Role of renal gamma-glutamyltransferase activity in hepatic utilization of exogenous glutathione.

The importance of renal gamma-glutamyltransferase activity in the hepatic utilization of exogenous glutathione (GSH) was evaluated by injecting GSH (1.67 mmol/kg body wt) i.v. into bilaterally nephrectomized and sham-operated Sprague-Dawley rats in which endogenous hepatic GSH had been decreased (0.20 +/- 0.01 mumol/g liver vs 5.87 +/- 0.26 mumol/g liver in normal controls, mean +/- SD) by diethylmaleate (0.5 ml/kg body wt, i.p.). Hepatic GSH concentration 60 min after GSH administration was lower in the nephrectomized than in the sham-operated rats (0.87 +/- 0.25 mumol/g liver vs 3.08 +/- 0.81 mumol/g liver, P < 0.001), while plasma GSH concentration was higher in the former (4.61 +/- 1.07 mM vs 0.11 +/- 0.06 mM, P < 0.001). In rats with intact kidneys which had been given a gamma-glutamyltransferase inhibitor (acivicin, 25 mumol/kg body wt i.v.) prior to GSH administration, the hepatic GSH concentrations (1.11 +/- 0.49 mumol/g liver) were comparable to those obtained in the nephrectomized rats. When N-acetylcysteine (1.67 mmol/kg body wt, i.v.) was administered instead of GSH, the hepatic GSH concentrations were similar in nephrectomized and sham-operated rats (1.54 +/- 0.23 mumol/g liver vs 2.22 +/- 0.58 mumol/g liver, NS). The gamma-glutamyltransferase activity was much higher in the kidney than in the liver (4460 +/- 830 IU/kg body wt vs 14 +/- 7 IU/kg body wt). These results indicate that the kidney plays an essential role in the hepatic utilization of exogenous GSH through its high gamma-glutamyltransferase activity.

Bile flow in a mutant Sprague-Dawley rat with defective biliary excretion of glutathione.

The Eisai hyperbilirubinemic rat is a mutant strain of Sprague-Dawley origin with hereditary defects in the biliary excretion of bilirubin glucuronide, glutathione, and several other organic anions. The correlation between bile flow and bile acid excretion rates during taurocholate infusion revealed that bile acid-independent flow was smaller in the mutant than in intact Sprague-Dawley rats (19.3 vs 56.0 microliters/kg per min), while bile acid-dependent flow was similar. The correlation between bile flow and glutathione excretion rates in Sprague-Dawley rats with modified hepatic glutathione levels revealed that a certain portion of bile flow was proportional to the biliary excretion of glutathione, with a coefficient of 551 bile per 1 mol glutathione. One-third of bile acid-independent bile flow in intact Sprague-Dawley rats was accounted for by glutathione osmosis, which feature was absent in the mutant rats.

Biliary excretion of cholephilic organic dyes in glutathione-depleted rats.

Sprague-Dawley rats were treated with a combination of buthionine sulfoximine (2.5 mmol/kg body wt) and diethyl maleate (6.2 mmol/kg). After 4 h, the hepatic glutathione was depleted to 0.02 +/- 0.01 mumol/g liver (mean +/- SD, n = 6) (4.73 +/- 0.29 mumol/g in controls (n = 6), p < 0.005). The bile flow rate was lower in treated animals than in controls (39.0 +/- 11.2 vs. 73.7 +/- 13.7 microliters.kg-1.min-1, p < 0.005). Bile concentrations of bile acids, phospholipids, and cholesterol were not changed compared to controls, while glutathione was virtually absent from the bile. A bolus of indocyanine green (6.5 mumol/kg), rose bengal (6.5 mumol/kg), or sulfobromophthalein-glutathione conjugate (20 mumol/kg) was injected i.v. and the biliary excretion was monitored. In glutathione-depleted rats, the excretion of indocyanine green was delayed, and the cumulative excretion in 90 min was 36 +/- 8% (n = 6) of the injected dose (79 +/- 18% in controls (n = 7), p < 0.005). The bile concentration of indocyanine green was lower in the glutathione-depleted rats than in controls, while the other dyes increased. The plasma disappearance curve of indocyanine green was not significantly altered by the treatment, whereas the hepatic retention of indocyanine green 90 min after the injection was significantly increased (58 +/- 12% of the injected dose vs. 8 +/- 2% in controls, p < 0.005). The results indicate that depletion of hepatic glutathione inhibits biliary excretion of indocyanine green.

[Relationship between biliary excretion of conjugated sulfobromophthalein and glutathione disulfide in rats].

Relationship between biliary excretion of sulfobromophthalein conjugated with glutathione (BSP-GSH) and glutathione disulfide (GSSG) was investigated in Sprague-Dawley rats. BSP-GSH solution was infused intravenously at three different rates. After administration of a glutathione-oxidizing agent, diamide, biliary excretion of GSSG increased temporarily and that of BSP-GSH decreased during the same period. A linear correlation was found between the increments in biliary excretion of GSSG and the decrements in that of BSP-GSH only when BSP-GSH was infused at a rate near its biliary transport maximum. The results may indicate competition in vivo between GSSG and BSP-GSH for a common transporter on canalicular membrane.

Relationship between biliary excretion of bilirubin and glutathione disulfide.

The effects of two glutathione-oxidizing agents, t-butyl hydroperoxide and diamide, on biliary excretion of bilirubin and glutathione disulfide were investigated in anesthetized male Sprague-Dawley rats. Bilirubin (unconjugated) was infused at a constant rate of 100 nmol/kg/min through the jugular vein. When biliary excretion of bilirubin was stabilized, either of the glutathione-oxidizing agents was administered via the mesenteric vein. Biliary excretion of glutathione disulfide increased temporarily after the administration and returned to its basal levels within 20 min. The biliary excretion of bilirubin decreased during the same period and returned to the former levels thereafter. Changes in bile flow rates remained within 20% of the basal levels. A linear correlation was found between the increments in the bile concentration of glutathione disulfide and the decrements in that of bilirubin. Furthermore, separate experiments revealed that reduction of hepatocellular glutathione per se had little effect on biliary excretion of bilirubin. The results thus indicate that the reduction of biliary excretion of bilirubin by glutathione-oxidizing agents was due to the increase in biliary excretion of glutathione disulfide, and suggest that a common biliary excretory mechanism is shared, at least partially, by bilirubin and glutathione disulfide in Sprague-Dawley rats.

Effects of glutathione isopropyl ester on bile flow in glutathione-depleted rats.

Combined administration of buthionine sulfoximine (2.5 mmol/kg, i.p.) and diethyl maleate (1.0 ml/kg, i.p.) resulted in a near-complete depletion of hepatic glutathione (0.02 mumol/g liver vs 5.17 mumol/g in saline-treated controls) in male Sprague-Dawley rats. Bile flow was markedly reduced in the rats as compared with the controls and glutathione was not detected in the bile. The linear regression of the correlation between bile flow and endogenous bile-acid excretion rates revealed that no bile acid-independent bile flow was produced in the glutathione-depleted rats. The bile flow was partially restored by an intravenous infusion of glutathione isopropyl ester (1.17 mmol/kg/hr). Glutathione levels were increased in the bile (16 nmol/kg/min) and in the liver (0.55 mumol/g) at the end of the 100 min infusion period of the ester. The increments in bile flow rates were not proportional to the biliary excretion rates of bile acids or glutathione, and the flow rates suddenly increased when glutathione levels in the bile reached an apparent threshold. The increments, not accompanied with an excretion of diethyl maleate-glutathione conjugate, were much greater than expected from the osmotic choleresis of glutathione in the bile. These results indicate that hepatic glutathione above a certain level is required for the formation of a portion of bile flow, and that an intravenous administration of glutathione isopropyl ester is effective in partially restoring the bile formation impaired by glutathione depletion.

The relationship between biliary secretion of bilirubin and glutathione in the rat.

The relationship between biliary secretion of bilirubin and glutathione was investigated by infusing bilirubin solution (200 nmol/min/100 g body wt) into Sprague-Dawley rats and measuring bilirubin and glutathione in the bile. Hepatic glutathione level, when modified between the range of 1-10 mumols/g liver wt, did not affect biliary maximal secretory rate (Tm) of bilirubin (80 nmol/min/100 g body wt). However, when biliary secretion of bilirubin exceeded 10 mM or 30 nmol/min/100 g body wt, biliary secretion of glutathione was markedly impaired while the bile flow remained relatively constant. Thus, bilirubin impaired the biliary secretion of glutathione selectively compared to its effect on bile formation. The results indicate that the mechanisms of biliary secretion of the two physiological substances, bilirubin and glutathione, are closely related.

Inhibition of biliary excretion of indocyanine green by the thiol-oxidizing agent, diazenedicarboxylic acid bis[N,N'-dimethylamide].

Biliary excretion of Indocyanine green (ICG) in Sprague-Dawley rats during constant intravenous infusion of the dye in vivo was inhibited by intraperitoneally administered diazenedicarboxylic acid bis[N,N'-dimethylamide] (diamide, 0.5 mmol/kg body wt), a glutathione-specific thiol-oxidizing agent. Significant inhibition of ICG excretion was observed also when ICG was injected rapidly 90 min after diamide administration. Disappearance of ICG from the plasma was not affected by diamide. Oxidized glutathione in bile increased transiently following diamide administration but returned to the basal level within 30 min. Hepatic concentrations of reduced and oxidized glutathione were not different from those of controls when determined 90 min after diamide administration. The inhibition of ICG excretion was completely prevented by subsequent administration of dithiothreitol (0.5 mmol/kg) 30 min after that of diamide. The results, therefore, suggest that the biliary excretion of ICG was inhibited by secondary changes in the redox status of thiols in hepatocytes caused by a transient increase in oxidized glutathione.

A case of gallbladder carcinoma producing both alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA).

A 73-year-old woman with gallbladder carcinoma infiltrating to the liver presenting high serum values of AFP and CEA was reported. Serum values of AFP and CEA were remarkably high (165,000 ng/ml and 1,070 ng/ml). Immunohistochemically the tumor cells were stained for AFP and CEA by the PAP method. Serum AFP subfraction was analyzed by crossed immunoaffinoelectrophoresis with lentil lectin and concanavalin A, which showed most of the serum AFP bound to both lentil lectin and concanavalin A. As a case of gallbladder carcinoma presenting a high serum value of AFP is rare, it may imply a diagnostic challenge.

A clinical evaluation of transcatheter arterial embolization on hepatocellular carcinoma.

TAE was performed in 49 cases of HCC for clinical assessment of the usefulness. This procedure was aimed to subside intraperitoneal hemorrhage in 8 cases among them and to provide a possible antitumor effect in the remaining 41 cases. The prognosis in the 8 cases of intraperitoneal hemorrhage was 54.9 +/- 41.1 days including one case survived as long as 116 days. The prognosis in the remaining 41 cases was 12.6 +/- 8.3 months in group A, 7.8 +/- 6.1 months in group B and 1.6 +/- 1.3 months in group C according to Child's classification, while it was 2.8 +/- 2.5 months in 1st branch occluded, 9.4 +/- 6.1 months in IInd branch occluded and 19.2 +/- 6.7 months in IIIrd branch occluded group according to the portal vein occlusion due to tumor thrombi suggesting that a more prolonged survival was attained with more favorable degree of Child's classification and less affected portal embolization. The cumulative survival time (by Kaplan-Meier's methods) was 6 months in 89% of the cases examined, 1 year in 59%, 2 years in 34% and 3 years or more in 11%, indicating significantly higher survival as compared to our TAI group. Angiographic re-opening of tumor vessel within 3 months was observed in 46.7%. TAE on HCC was useful both for the purpose of antitumor effect and of hemostasis. The degree of Child's classification and severity of portal occlusion at the initial examination may closely relate to the prognosis. Thus, angiography should be repeated within 3 months following the first TAE at least.