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 c isplatin [cis-diamminedichloroplatinum(II)] into cells and to act as a modu lator 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+-AT Pase activity by cisplatin using an enzyme partially purified from Ca9-22 c ells derived from a human squamous cell carcinoma of the gingiva. Cisplatin inhibited the Na+,K+-dependent ATP hydrolysis activity, and this inhibitio n depended on both the concentration of cisplatin and the preincubation tim e 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 furthe r tested the effect of cisplatin on the partial reactions of the enzyme, Na +-dependent ATP hydrolysis and K+-dependent p-nitrophenylphosphate hydrolys is activities to determine which step in the reaction sequence of Na+,K+-AT Pase was inhibited. Cisplatin inhibited both activities depending on its co ncentration and the preincubation time, whereas the Na+-dependent ATP hydro lysis activity was inhibited even at lower concentrations. Formation of a p hosphointermediate of Na+,K+-ATPase was also inhibited by cisplatin dependi ng on the concentration and preincubation time. Cisplatin (500 mu M) and 8- fold higher concentration of 2-mercaptoethanol (2-ME; 4 mM) prevented inact ivation of the enzyme by cisplatin, and the Na+,K+-ATPase activity inhibite d 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 o f the enzyme and that the inhibition by cisplatin is arrested by an additio n of thiol group. [(C) 1999 Lippincott Williams & Wilkins.].