Beta-lyase-dependent attenuation of cisplatin-mediated toxicity by selenocysteine Se-conjugates in renal tubular cell lines

Cisplatin [cis-diamminedichloroplatinum(II)] is a widely used antitumor drug with dose-limiting nephrotoxic side effects due to selective toxicity to the proximal tubule. In the present study, the chemoprotective potential of three selenocysteine Se-conjugates, Se-methyl-L-selenocysteine, Se-(2-methoxyphenyl)-L-selenocysteine, and Se-(2-chlorobenzyl)-L-selenocysteine, belonging to three structural classes, against the nephrotoxic effects of cisplatin was investigated. Selenocysteine Se-conjugates have previously been proposed as kidney-selective prodrugs of pharmacologically active selenols because of their active uptake and bioactivation by cysteine conjugate beta-lyases in the kidney. To elucidate whether chemoprotection is beta-lyase-dependent wild-type LLC-PK(1) cells, possessing a very low beta-lyase activity, and LLC-PK(1) cells stably transfected with full-length cDNA coding for rat kidney cysteine conjugate beta-lyase/glutamine transaminase K (R1J) were used. The results indicate that all three selenocysteine Se-conjugates were able to attenuate the cisplatin-induced loss of viability in R1J cells but not in the parental LLC-PK(1) cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and neutral red uptake. In addition, cisplatin-induced reactive oxygen species (ROS) production was determined using 2',7'-dichlorodihydrofluorescein diacetate. The selenocysteine Se-conjugates were able to decrease ROS levels after cisplatin exposure in both cell types. However, this ROS-protective effect was more profound in R1J cells. Se-Methyl-L-selenocysteine provided the strongest protection. The protective activity against cisplatin-induced cytotoxicity and ROS generation was blocked by aminooxyacetic acid, a selective inhibitor of pyridoxal 5'-phosphate-dependent cysteine conjugate beta-lyases, further supporting the role of beta-lyase in the observed chemoprotection. The precise molecular mechanism by which selenols, generated by beta-lyase, provide protection against cisplatin-induced cytotoxicity, however, remains to be established.