Shin-ichi Akiyama

Active Efflux System for Cisplatin in Cisplatin‐resistant Human KB Cells

Mutants, KCP‐4 and PC‐5, resistant to an anticancer agent, cisplatin, were selected in multiple steps from human epidermoid KB carcinoma cells and human prostate PC‐3 carcinoma cells, respectively. KCP‐4 and PC‐5 were 63 and 10 fold more resistant to cisplatin than the parental cells, respectively. KCP‐4 cells exhibited increased resistance to cisplatin analogues and were also slightly cross‐resistant to melphalan, cyclophosphamide, mitomycin C and methotrexate. KCP‐4 cells were not cross‐resistant to doxorubicin, daunorubicin, vincristine or CdSO4. The accumulations of cisplatin in KCP‐4 cells and PC‐5 in medium containing 50 μM cisplatin were approximately 20% of those in the parental cells. Revertant analysis suggested that a defect in cisplatin accumulation may be related to cisplatin resistance in PC‐5 cells. The uncoupling agent of oxidative phosphorylation, 2,4‐dinitrophenol, increased the accumulation of cisplatin in KCP‐4 and cisplatin‐resistant human prostate carcinoma PC‐5 cells to nearly the same level as in their parental KB‐3‐1 and human prostate carcinoma PC‐3 cells without 2,4‐dinitrophenol, but did not increase accumulation in KB‐3‐1 and PC‐3 cells. Addition of glucose in the medium inhibited the enhancement of cisplatin accumulation in KCP‐4 cells by 2,4‐dinitrophenol. Enhanced active efflux of cisplatin from KCP‐4 cells was observed. A cell‐cell hybridization test showed that the cisplatin resistance and the accumulation defect behaved as codominant traits. These data suggest that an active efflux system for cisplatin exists in cisplatin‐resistant KCP‐4 cells.

The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinomas

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in several types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and that dThdPase has angiogenic activity. We measured dThdPase activity and the level of thrombomodulin (TM) as a marker for endothelial cells in colorectal carcinomas and adjacent normal tissues from 21 patients, and in adenomas from 13 patients. The average dThdPase activity of colorectal carcinomas (11.58 +/- 6.30 nmol/100 micrograms protein/h) was significantly higher than that of adenomas (8.57 +/- 4.14 nmol/100 micrograms protein/h) or normal tissues (4.89 +/- 3.16 nmol/100 micrograms protein/h). In immunohistochemical study, the expression of dThdPase was observed more frequently in colorectal carcinomas than in adenomas or normal mucosas. The amount of TM in colorectal carcinomas (8.32 +/- 5.07 ng/100 micrograms protein) was significantly higher than that of adenomas (4.51 +/- 4.49 ng/100 micrograms protein) or normal tissues (3.51 +/- 2.78 ng/100 micrograms protein). dThdPase activity in human colorectal carcinomas, adenomas and normal tissues was significantly correlated with the expression of TM in these tissues. These results indicate that the expression levels of both dThdPase and TM in colorectal carcinomas are higher than those in colorectal adenomas and normal tissues and suggest that dThdPase may be involved in angiogenesis in human colorectal carcinomas, adenomas and normal tissues.

Non-P-glycoprotein-mediated multidrug-resistant human KB cells selected in medium containing adriamycin, cepharanthine, and mezerein

Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 μg/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine. The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells. These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpressMDR1. Multidrug-resistance-associated protein (MRP) andMRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells. TheMRP gene was amplified in all the MDR cells that overexpressed MRP mRNA. DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.

Expression of the multidrug‐resistance‐associated protein (MRP) gene in human colorectal, gastric and non‐small‐cell lung carcinomas

MRP has been identified as another multidrug‐resistance (MDR) gene and may be involved in an alternative MDR mechanism in some solid tumors. We investigated the expression of MRP mRNA in multidrug‐resistant KB sublines (KB‐8‐5, KB‐C2, C‐A40 and C‐A120), human non‐small‐cell lung carcinomas (NSCLC), gastric and colorectal carcinomas, and compared it with that in drug‐sensitive human KB cells, MRP gene expression was elevated in 8 of 9 (89%) squamous‐cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous‐cell carcinomas (L13, 18, 19 and 20) was more than 3.6 times higher than in KB‐3‐I cells, and the average MRP mRNA expression level of all squamous‐cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. These results suggested that the MRP is responsible, at least in part, for drug resistance in some squamous‐cell carcinomas of the lung.

Progesterone and its metabolites: the potent inhibitors of the transporting activity of P-glycoprotein in the adrenal gland

P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for the multidrug resistant (MDR) phenotype in various cancer cells. It has been shown that P-gp transports various kinds of anti-cancer agents as well as hydrophobic chemicals. Although P-gp is also expressed in normal human tissues, such as liver, kidney, and adrenal gland, its function and transporting substrates in these tissues are still unknown. In previous work, we demonstrated that some compounds in human plasma modulate the transporting activity of P-gp. We also found that P-gp is expressed at a high level in the bovine adrenal gland and that this tissue contains large amount of compounds which inhibit the transporting activity of P-gp. We purified such compounds from the adrenal gland by monitoring the ability to enhance the accumulation of [3H]vincristine in MDR cells. Two major compounds were purified and identified as progesterone and pregnenolone by nuclear magnetic resonance (NMR) analysis. Progesterone was the most potent and abundant compound that inhibited the transporting activity of P-gp among the compounds extracted from bovine adrenal gland with methanol. We also found that six authentic progesterone metabolites in the 5 beta-metabolic pathway but none in the 5 alpha-metabolic pathway were able to enhance the accumulation of [3H]vincristine in MDR cells and to inhibit [3H]azidopine photolabeling of P-gp in the adrenal gland. These results indicate that some progesterone metabolites can interact with P-gp and that stereoisomerism around carbon 5 of the progesterone metabolites is important for them to be recognized by P-gp.