A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system

Amblyomin-X is a recombinant protein with cytotoxic activity on tumor cells featuring little or no activity on normal cells. The aim of this study was to evaluate the antitumoral activity of amblyomin-X (in vivo and ex vivo) and identify its molecular targets. To that end, global gene expression as well as cytotoxicity, cell cycle modulation and ubiquitin proteasome system function were evaluated in pancreas adenocarcinoma and melanoma human cell lines after treatment with Amblyomin-X. Several genes related to the cell cycle were altered corroborating the G0/G1 phase alteration observed. PSMB2, that encodes a proteasome subunit, was differentially expressed, in agreement with trypsin-like proteasomal decreased activity and increased pool of poli-ubiquitinylated proteins. Altogether, alterations following Amblyomin-X exposure are in agreement with the observed cell death by apoptosis. Furthermore, In vivo treatment with Amblyomin-X induced regression of tumoral mass in mouse murine melanoma model (B16F10) and decreased the number of metastasis events. In conclusion, our results indicate that Amblyomin-X selectively acts on tumoral cells most likely by targeting the ubiquitin-proteasome system. Global measurements of gene expression from pancreatic (Mia-PaCa-2) and melanoma (SK-Mel-28) tumor cell lines exposed to Amblyomin-X were obtained using human Whole Genome Code Link Bioarrays (GE Healthcare, Chandler, AZ) containing approximately 55,000 30-mer probes. Six (Mia-PaCa-2) or five (SK-Mel-28) hybridizations were performed for each condition (Amblyomin-X treated or untreated control cells) using cRNA targets obtained from independent preparations. Cy5-labeled hybridized targets were detected with an arrayWoRx scanner (Applied Precision Inc., WA, USA).