The immunosuppressant tributyltin oxide blocks the mTOR pathway, like rapamycin, albeit by a different mechanism

We treated the thymoma cell line (EL4) with two model immunosuppressants, rapamycin and tributyltin oxide (TBTO), and compared their effects on the expression levels of proteins that are downstream targets of mTOR kinase 1 (mammalian target of rapamycin, known also as mechanistic target of rapamycin): p70 ribosomal S6 kinase1 and 4E-binding protein 1, a repressor of the cap-binding protein eIF4E. In addition, we evaluated the levels of ribosomal protein S6, p-eIF4B, substrates of p70S6 kinase1, matrin 3 and ribonucleotide reductase, subunit RRM2. The levels of these proteins were evaluated in cell lysates by immunoblot. We found that both compounds inhibited the phosphorylation state of p70S6 kinase 1 and its substrates; however, TBTO, in contrast to rapamycin, reduced the level of the total p70S6k1. Besides, we detected a band with a molecular weight of c. 32kDa only in the TBTO-treated lysates. This band was detected with a monoclonal antibody specific for S6k1, suggesting that this band might be a degradation product of the kinase. Further, TBTO and rapamycin differentially affected 4E-binding protein 1; the former compound stimulated its phosphorylation state whereas the latter inhibited it. The two immunosuppressants did not affect the level of ribonucleotide reductase, but TBTO downregulated matrin3, in agreement with a previous report, whereas rapamycin had no effect on the expression level of this latter protein. We conclude that TBTO inhibits, like rapamycin, the p70 S6 kinase 1 pathway, but with a different mechanism. However, in contrast to rapamycin, which inhibits the cap-dependent translation, TBTO increases the phosphorylation of 4E-binding protein1. We report here that tributyltin oxide (TBTO) inhibits, like rapamycin, the p70S6k1 pathway, though by a different mechanism in EL4 cell line. Both TBTO and rapamycin downregulated the phosphorylation state of S6k1 assessed at Ser421/Thr424, but in contrast to rapamycin, TBTO decreased the total S6k1 protein. In addition, TBTO reduced the levels of RPS6 (Ser236) and eIF4B (Ser422), downstream substrates of S6k1. Further, TBTO and rapamycin differentially affected 4E-binding protein 1, a repressor of the cap-binding protein eIF4E.