Inhibition of Na+,K+-ATPase by cisplatin and its recovery by 2-mercaptoethanol in human squamous cell carcinoma cells.
Na+,K+-ATPase (EC 3.6.1.37) is assumed to be involved in the transport of cisplatin [cis-diamminedichloroplatinum(II)] into cells and to act as a modulator of 5-fluorouracil (5-FU) in combination therapy of cisplatin and 5-FU. Whereas inhibition of Na+,K+-ATPase activity by cisplatin is expected to have effects on both anti-cancer therapy and nephrotoxicity, the inhibition mechanism remains to be elucidated. We studied the inhibition of Na+,K+-ATPase activity by cisplatin using an enzyme partially purified from Ca9-22 cells derived from a human squamous cell carcinoma of the gingiva. Cisplatin inhibited the Na+,K+-dependent ATP hydrolysis activity, and this inhibition depended on both the concentration of cisplatin and the preincubation time with cisplatin. The time-dependent inhibition was thought to be caused by a slow change of cisplatin from the inactive to the active form. We further tested the effect of cisplatin on the partial reactions of the enzyme, Na+-dependent ATP hydrolysis and K+-dependent pnitrophenylphosphate hydrolysis activities to determine which step in the reaction sequence of Na+,K+-ATPase was inhibited. Cisplatin inhibited both activities depending on its concentration and the preincubation time, whereas the Na+-dependent ATP hydrolysis activity was inhibited even at lower concentrations. Formation of a phosphointermediate of Na+,K+-ATPase was also inhibited by cisplatin depending on the concentration and preincubation time. Cisplatin (500 microM) and 8-fold higher concentration of 2-mercaptoethanol (2-ME; 4 mM) prevented inactivation of the enzyme by cisplatin, and the Na+,K+-ATPase activity inhibited by pretreatment with cisplatin was also recovered almost completely by 2-ME. These results suggest that the active form of cisplatin inhibits the Na+,K+-ATPase activity by inhibiting the formation of a phosphointermediate of the enzyme and that the inhibition by cisplatin is arrested by an addition of thiol group.
Sakakibara
N
N
Department of Oral Surgery I, Hokkaido University School of Dentistry, Supporo, Japan. iku@den.hokudai.ac.jp
Suzuki
K
K
Kaneta
H
H
Yoshimura
Y
Y
Deyama
Y
Y
Matsumoto
A
A
Fukuda
H
H
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000201:enzymology*; D002945:Cisplatin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005887:Gingival Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D006801:Humans; D008623:Mercaptoethanol / Q000494:pharmacology*; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D008623:Mercaptoethanol; D000254:Sodium-Potassium-Exchanging ATPase; D002945:Cisplatin