Serotonin type-3 receptor antagonists selectively kill melanoma cells through classical apoptosis, microtubule depolymerisation, ERK activation, and NF-κB downregulation.

Malignant melanoma is a highly metastatic tumour, resistant to treatment. Serotonin type-3 (5-HT3) receptor antagonists, such as tropisetron and ondansetron, are well-tolerated antiemetic drugs commonly used to prevent nausea caused by chemotherapy or radiotherapy. We investigated the anticancer effects of these drugs on melanoma cancer cell lines WM-266-4 and B16F10 with or without paclitaxel. We constructed IC50 curves and performed Chou-Talalay analysis, using data obtained with the MTT assay. Flow cytometry and fluorescent microscopy were used to examine characteristics of the cell cycle, cell death and cytoskeleton changes. Protein levels and activation were analysed by western blotting and molecular docking studies carried out. Data were analysed by one way ANOVA and post hoc testing. Ondansetron and tropisetron showed selective concentration-dependent cytotoxicity in melanoma cell lines WM-266-4 and B16F10. The effect in combination with paclitaxel was synergistic. The drugs did not cause cell cycle arrest but did promote characteristics of classical apoptosis, including accumulation of subG1 DNA, cleaved caspase-3, mitochondrial membrane permeability and phosphatidylserine exposure. As well, the cytosolic calcium level in the melanoma cells was enhanced, phosphorylated ERK1/2 induced and NF-κB inhibited. Finally, the formation of microtubules was shown to be impaired in melanoma cells treated with ondansetron or tropisetron. Docking studies were used to predict that these drugs could bind to the colchicine binding site on the tubulin molecule. Antiemetic drugs, already given in combination with chemotherapy, may enhance the cytotoxic effect of chemotherapy, following successful delivery to the tumour site.

Nanobodies identify an activated state of the TRIB2 pseudokinase.

Tribbles proteins (TRIB1-3) are pseudokinases that recruit substrates to the COP1 ubiquitin ligase. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukemia oncogene, because it recruits the C/EBPα transcription factor for ubiquitination by COP1. Here we report identification of nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of the TRIB2-Nb4.103 complex identified the nanobody to bind the N-terminal lobe of TRIB2, enabling specific recognition of TRIB2 in an activated conformation that is similar to the C/EBPα-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilize a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors and nanobody tools for future investigation of TRIB2 function.