β-Lyase-Dependent Attenuation of Cisplatin-Mediated Toxicity by Selenocysteine Se-Conjugates in Renal Tubular Cell Lines

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Abstract
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 selenocysteineSe-conjugates,Se-methyl-l-selenocysteine,Se-(2-methoxyphenyl)-l-selenocysteine, andSe-(2-chlorobenzyl)-l-selenocysteine, belonging to three structural classes, against the nephrotoxic effects of cisplatin was investigated. SelenocysteineSe-conjugates have previously been proposed as kidney-selective prodrugs of pharmacologically active selenols because of their active uptake and bioactivation by cysteine conjugate β-lyases in the kidney. To elucidate whether chemoprotection is β-lyase-dependent wild-type LLC-PK1 cells, possessing a very low β-lyase activity, and LLC-PK1 cells stably transfected with full-length cDNA coding for rat kidney cysteine conjugate β-lyase/glutamine transaminase K (R1J) were used. The results indicate that all three selenocysteineSe-conjugates were able to attenuate the cisplatin-induced loss of viability in R1J cells but not in the parental LLC-PK1 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 selenocysteineSe-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 β-lyases, further supporting the role of β-lyase in the observed chemoprotection. The precise molecular mechanism by which selenols, generated by β-lyase, provide protection against cisplatin-induced cytotoxicity, however, remains to be established.