Abstract 3031: Bufalin regulates Cbl-b levels in HeLa cells through protein synthesis inhibition

Cbl-b is a member of ubiquitin ligases (E3s) family. The major function of CBL family E3s is to negatively regulate signaling through ubiquitination and degradation of receptor tyrosine kinases and kinase associated receptors. Cbl-b plays a crucial role in the regulation of the immune response, as it negatively regulates the costimulatory pathway of CD8 T cells and also suppresses natural killer cell function. Thus Cbl-b down-regulation or inhibition may lead to increased adaptive and innate antitumor immunity and can be used as a treatment strategy for a wide variety of tumors. Bufalin is a major component of Chan Su, traditional Chinese medicine derived from the toad venom. Bufalin is a C-24 steroid with the wide variety of biological activities, including cardiotonic, anesthetic and antitumor activities. Bufalin has been shown to inhibit growth and induce cell cycle arrest and apoptosis of various cancer cells. Low doses of bufalin can be safely used for prolonged periods of time without severe side effects, while high doses can cause cardiac arrhythmia, seizure and coma. The purpose of the current study was to investigate if bufalin regulates the levels of Cbl-b and establish the mechanism of Cbl-b regulation. By Western blotting of HeLa cell lysates we found that bufalin decreases protein levels of Cbl-b in a time- and dose-dependent manner without affecting the levels of Cbl. The greatest decrease was observed with 50 nM bufalin after 24 hours treatment. Doses of bufalin as low as 4 nM were effective in Cbl-b down-regulation. Real-time PCR analysis showed 2.9-fold increase in the amount of Cbl-b mRNA upon 50 nM bufalin treatment for 24 hours, suggesting that Cbl-b regulation by bufalin occurs post-transcriptionally. Proteasomal, but not lysosomal inhibitors treatment attenuated the negative effect of bufalin on Cbl-b levels. Co-treatment of HeLa cells with 50 nM bufalin and 50 μg/ml cycloheximide, protein synthesis inhibitor, did not result in any further decrease in Cbl-b levels compared to cycloheximide treatment alone. By measuring the nascent protein synthesis with Click-It metabolic labeling reagents, bufalin was shown to block protein synthesis of the majority of short-lived proteins. These data taken together indicate that bufalin treatment decreases Cbl-b levels in HeLa cells by blocking protein synthesis. We found that 50 nM bufalin treatment for up to 8 hours increased the phosphorylation of eIF2α, translation initiation factor, at Ser51. Phosphorylation of eIF2α known to block translation might explain the effect of bufalin on Cbl-b levels and other short-lived proteins. To conclude, we found that bufalin increases the phosphorylation of eIF2α, thus leading to the block of translation and decreasing protein levels of Cbl-b in HeLa cells. Our future goal is to test the hypothesis that bufalin activates antitumor immunity through down-regulation of Cbl-b and can thus be used as an antineoplastic agent. Citation Format: Mariya S. Liyasova, Stanley Lipkowitz. Bufalin regulates Cbl-b levels in HeLa cells through protein synthesis inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3031.