Differences in substrate specificity among glutathione conjugates (GS-X) pump family members: comparison between multidrug resistance-associated protein and a novel transporter expressed on a cisplatin-resistant cell line (KCP-4).

The substrate specificity of primary active transporters expressed on two kinds of human epidermoid KB-3-1 derived cell lines, C-A500 and KCP-4, was examined; the former expresses multidrug resistance-associated protein (MRP1), whereas the latter is resistant to cis-diamminedichloroplatinum (II) (cisplatin). Northern blot analysis indicated that neither P-glycoprotein, MRP1, MRP2 (canalicular multispecific organic anion transporter; cMOAT) nor MRP3 was overexpressed on KCP-4. Membrane vesicles isolated from C-A500 and KCP-4, but not from KB-3-1, exhibited the ATP-dependent uptake of glutathione conjugates (GS-X) such as leukotriene C4 and 2,4-dinitrophenyl-S-glutathione (DNP-SG), indicating the presence of GS-X pumps on these cells. The uptake of these GS-X by membrane vesicles from C-A500 was approximately twice that in the case of KCP-4. Kinetic analysis indicated that the Km and Vmax values for DNP-SG uptake were 2.56 and 1.43 microM, and 570 and 160 pmol/min/mg protein for C-A500 and KCP-4, respectively. In marked contrast, significant ATP-dependent uptake of glutathione-platinum complex was observed only in membrane vesicles from KCP-4, but not those from KB-3-1 and C-A500. The transport properties of estradiol-17beta-D-glucuronide (E(2)17betaG) were also different between the two cell lines. This was reflected in the findings that the ATP-dependent uptake of this conjugated metabolite in membrane vesicles from C-A500 (Km=2.33 microM, Vmax=34 pmol/min/mg protein) was much more extensive than that in the case of KCP-4 (Km=5.5 microM, Vmax=35 pmol/min/mg protein), and that comparable uptake was observed between KCP-4 and KB-3-1. Overall, a clear difference in substrate specificity among GS-X pump family members expressed on resistant tumor cells was demonstrated.     

In vivo cisplatin resistance depending upon canalicular multispecific organic anion transporter (cMOAT).

The in vitro sensitivities to cisplatin of AH66 and AH66F cells, a variant obtained from AH66 cells, were very similar, when assayed in a medium containing 5% fetal bovine serum (FBS), whereas in the in vivo experiments AH66F cells were sensitive and AH66 cells were highly resistant to cisplatin. In this study, we examined the mechanism of the in vivo cisplatin resistance of AH66 cells. The in vitro cisplatin sensitivity of AH66 cells was lowered by changing FBS to 5% ascites fluid (ASF) in the assay medium and the sensitivity in FBS by treatment with buthioninesulfoximine (BSO). The sensitivity of AH66F cells was not changed by these treatments. Moreover, after culture in 5% ASF for 48 h, the accumulation of cisplatin in AH66 cells was decreased and the efflux of cisplatin from the cells was accelerated. The accumulation of cisplatin in AH66 cells in ASF was increased by pretreatment with BSO, sodium azide or probenecid. Then, we examined the expression of the glutathione (GSH) conjugate efflux pump family. Among them, only the expression of canalicular multispecific organic anion transporter (cMOAT) in AH66 cells was decreased by culture in FBS and enhanced by ASF. These results suggest that some substances contained in ASF enhanced the expression of cMOAT in the plasma membrane of AH66 cells and this transporter actively extruded cisplatin-GSH conjugate from the cells. Consequently, AH66 cells afford a cisplatin-resistant tumor in the host.     

Membrane transport and antitumor activity of pirarubicin, and comparison with those of doxorubicin.

We have compared the membrane transport and antitumor activity of pirarubicin with those of doxorubicin in M5076 ovarian sarcoma, which exhibits low sensitivity to doxorubicin. Pirarubicin was rapidly taken up by M5076 cells and the intracellular concentration of pirarubicin reached more than 2.5-fold that of doxorubicin. In terms of the 50% cell growth-inhibitory concentration in vitro, pirarubicin was more effective than doxorubicin. Thus, the intracellular concentration influenced the cytotoxicity of these anthracycline agents. On comparison of the nuclear uptake of pirarubicin and doxorubicin, the nucleus/cell ratio of pirarubicin was found to be about 40%, whereas that of doxorubicin reached more than 80%. As the intranuclear concentration of pirarubicin is dependent on nuclear transport, the increases in not only cell membrane transport, but also nuclear membrane transport contributed to the enhancement of the efficacy of pirarubicin. In M5076 solid tumor-bearing mice, pirarubicin reduced the tumor weight to 60% of the control level, although doxorubicin had no effect. These results were supported by the intracellular uptake of pirarubicin. Moreover, theanine, which inhibited the pirarubicin efflux from M5076 cells, increased by 1.3-fold the pirarubicin concentration in the tumor and enhanced the therapeutic efficacy of pirarubicin 1.7-fold. In conclusion, our results suggest that an increase in the concentration of an anthracycline derivative in tumor cells due to alteration of cell membrane transport results in enhancement of the antitumor activity.     

Clinical evaluation of intermittent hepatic arterial infusion therapy with CDDP and 5-FU for liver metastasis of colorectal cancer

Twenty-seven patients with liver metastasis from colorectal cancer were treated with intermittent intra-arterial infusion chemotherapy for 5 years starting from 1993. Five to ten mg of CDDP, 250 mg of 5-FU and/or 3-6 mg of Leucovorin were administered weekly. In the case of nonresponders, the dose of 5-FU was allowed to increase toward 500 mg. The above schedule was repeated as long as possible. The average number of administrations in 12 out of 27 unresectable patients was 28.7. The response rate was 50% (CR; 4 cases, PR; 2), with 2 NC and 3 PD. Four patients given 500 mg of 5-FU showed some response. The 50% survival period was 466 days, and the 1- and 3-year survival rates were 66.7% and 18.3%, respectively. The average number of administrations in the group of patients who underwent prophylactic treatment and resection of the metastasis was 33.1. During an average observational period 681 days, 7 patients (46.7%) had a recurrence in the liver. The 5-year survival rate was 85.7%. The patients who were treated with 250 mg 5-FU experienced no severe side effects, but one who was given 500 mg 5-FU developed a duodenal ulcer.     

Helicobacter pylori infection enhances glandular stomach carcinogenesis in Mongolian gerbils treated with chemical carcinogens

Helicobacter pylori (Hp) is thought to be a stomach carcinogen from epidemiological findings. To determine the effects of infection with the bacteria on experimental carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. Male MGs were treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by inoculation with Hp or infected with Hp followed by N-methyl-N'-nitro-N-nitrosoguanidine administration. Animals were killed at week 50, and their excised stomachs underwent microbiological and histopathological examinations. In addition, a serological investigation was performed. The incidences of adenocarcinomas were significantly higher in animals treated with 60 or 300 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine for 10 weeks followed by Hp inoculation or Hp followed by 20 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine for 30 weeks than in the respective controls. Moreover, tumour-bearing animals had higher titres of anti-Hp antibodies than tumour-free animals. Of interest was the finding that a dose of 100 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine given to infected gerbils eradicated the Hp in about half the animals, with a concomitant reduction in the promoting effect. No tumours were found in animals infected with Hp without N-methyl-N'-nitro-N-nitrosoguanidine or non-treated gerbils. Hp infection enhances glandular stomach carcinogenesis in MGs treated with N-methyl-N'-nitro-N-nitrosoguanidine. Animals with high titres of anti-Hp antibodies are at greatest risk of developing neoplasms.     

Growth Inhibition and Radiosensitization of Cultured Glioma Cells by Nitric Oxide Generating Agents

The authors examined the effect of nitric oxide (NO) generating agents on the growth and radiosensitivity of cultured glioma cells. Three glioma, rat C6, and human T98G and U87 cell lines were treated with the NO generating agents, S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). These agents released NO in the cell culture media and inhibited the growth of the glioma cells. Growth-inhibition was attenuated by hemoglobin, a known inhibitor of NO, suggesting it is mediated by NO. When C6 and T98G cells were irradiated in the presence of SNAP or SNP at 100µM, radiosensitization was observed. SNAP at 100µM exhibited a sensitizer enhancement ratio (SER) of 1.4 for C6 cells and 1.8 for T98G cells. SNP at 100µM only radiosensitized T98G cells with a SER of 1.9. The effect of SNP on radiosensitization of C6 cells was unclear. We conclude that NO generating agents are potential growth inhibitors and radiosensitizers for malignant glioma cells. NO mediated radiosensitization of glioma cells by NO generating agents may offer a new therapeutic approach for malignant glioma.     

Antiarrhythmic and cardiohemodynamic effects of a novel Ca(2+) channel blocker, AH-1058, assessed in canine arrhythmia models

The antiarrhythmic profile and cardiohemodynamic effect of a novel Ca(2+) channel blocker, 4-(5H-Dibenzo[a, d]cyclohepten-5-ylidene)-1-[(E)-3-(3-methoxy-2-nitro)phenyl-2-p ropeny l]piperidine hydrochloride (AH-1058), were analyzed using the epinephrine-, digitalis- and two-stage coronary ligation-induced canine ventricular arrhythmia models. Intravenous administration of AH-1058 (100 microg/kg) effectively suppressed each of the ventricular arrhythmias accompanied by weak hypotensive effects. The results contrast well with those of a typical Ca(2+) channel blocker, verapamil, which suppresses only the epinephrine-induced ventricular arrhythmia with severe hypotension. These results indicate that AH-1058 may possess a more selective inhibitory action on Ca(2+) channels in the heart than on those in the vessels. Furthermore, the antiarrhythmic actions of AH-1058 were slower in onset and longer-lasting, than those in our previous studies using other antiarrhythmic drugs, including Na(+) and Ca(2+) channel blockers. The antiarrhythmic effects of AH-1058 did not correlate with its plasma concentrations when administered either intravenously or orally. These results suggest that AH-1058 can become a long-acting Ca(2+) channel blocker with unique antiarrhythmic properties, and that AH-1058 may be used in certain pathological processes, for which selective inhibition of the cardiac Ca(2+) channels is essential.     

In vivo Cisplatin Resistance Depending upon Canalicular Multispecific Organic Anion Transporter (cMOAT)

The in vitro sensitivities to cisplatin of AH66 and AH66F cells, a variant obtained from AH66 cells, were very similar, when assayed in a medium containing 5% fetal bovine serum (FBS), whereas in the in vivo experiments AH66F cells were sensitive and AH66 cells were highly resistant to cisplatin. In this study, we examined the mechanism of the in vivo cisplatin resistance of AH66 cells. The in vitro cisplatin sensitivity of AH66 cells was lowered by changing FBS to 5% ascites fluid (ASF) in the assay medium and the sensitivity in FBS by treatment with buthioninesulfoximine (BSO). The sensitivity of AH66F cells was not changed by these treatments. Moreover, after culture in 5% ASF for 48 h, the accumulation of cisplatin in AH66 cells was decreased and the efflux of cisplatin from the cells was accelerated. The accumulation of cisplatin in AH66 cells in ASF was increased by pretreatment with BSO, sodium azide or probenecid. Then, we examined the expression of the glutathione (GSH) conjugate efflux pump family. Among them, only the expression of canalicular multispecific organic anion transporter (cMOAT) in AH66 cells was decreased by culture in FBS and enhanced by ASF. These results suggest that some substances contained in ASF enhanced the expression of cMOAT in the plasma membrane of AH66 cells and this transporter actively extruded cisplatin-GSH conjugate from the cells. Consequently, AH66 cells afford a cisplatin-resistant tumor in the host.