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Augmentation of Transport for Cisplatin-Glutathione Adduct in Cisplatin-resistant Cancer Cells¹

Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki 852 [S. G., T. M., Y. U., T. K.]; First Department of Medicine, Hokkaido University School of Medicine, Sapporo 060 [K. Y.]; and Department of Biochemistry, Osaka University School of Medicine, Suita 565 [K. S.], Japan

We studied the outward transport of cisplatin (CDDP)-glutathione (GSH) adduct (DDP-GSH) in CDDP-resistant cancer cells. Incubating the cells in the presence of CDDP resulted in the formation of an adduct with GSH and subsequent transport outside the cells. We used human colonic cancer cells sensitive (HCT8) and resistant (HCT8DDP) to CDDP and human ovarian cancer cells sensitive (A2780) and resistant (A2780DDP) to CDDP as materials.

The concentration of intracellular GSH was higher in the resistant cells (118.7 ± 5.9 nmol/10⁶ HCT8DDP versus 19.0 ± 1.0 nmol/10⁶ HCT8 and 24.1 ± 1.2 nmol/10⁶ A2780DDP versus 9.4 ± 0.5 nmol/10⁶ A2780, respectively). The activity of the GSH-synthesizing enzyme, -glutamylcysteine synthetase (-GCS) was higher in the CDDP-resistant cells (7.1 ± 0.2 milliunits/10⁶ HCT8DDP versus 2.2 ± 0.1 milliunits/10⁶ HCT8 and 2.9 ± 0.1 milliunits/10⁶ A2780DDP versus 1.4 ± 0.1 milliunits/10⁶ A2780, respectively). Furthermore, immunological levels of -GCS and the expression of -GCS mRNA were higher in these CDDP-resistant cells than those in the control cells, in accordance with the change in the concentration of GSH. DDP-GSH transport increased in the CDDP-resistant colonic cancer cells by 219% (324 ± 12 fmol/10⁶ HCT8DDP cells/min versus 148 ± 11 fmol/10⁶ HCT8 cells/min) and the CDDP-resistant ovarian cancer cells by 126% (127 ± 7 fmol/10⁶ A2780DDP cells/min versus 101 ± 8 fmol/10⁶ A2780 cells/min).

DDP-GSH transport was also estimated using inside-out vesicles from these cells. The active transport of DDP-GSH was 243% of HCT8 in HCT8DDP and 121% of A2780 in A2780DDP. These data suggest that the acquisition of CDDP resistance in cancer cells is due partly to the increase in the transport of DDP-GSH outside the cells as well as the increase in the concentration of GSH. Immunological estimation of the membrane proteins against human erythrocyte glutathione S-conjugate-stimulated Mg²⁺-ATPase sera resulted in no apparent cross-reactivity, suggesting that there are several transport systems for DDP-GSH.

¹ This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan.

² To whom requests for reprints should be addressed.

Received 8/ 3/94. Accepted 7/27/95.

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