Combination of Cl‑IB‑MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR‑dependent autophagy and mitotic catastrophe on human melanoma cells.

Purpose: Metastatic melanoma is the deadliest form of skin cancer. It is highly resistant to conventional therapies,particularly to drugs that cause apoptosis as the main anticancer mechanism. Recently, induction of autophagic cell death is emerging as a novel therapeutic target for apoptotic-resistant cancers. We aimed to investigate the underlying mechanisms elicited by the cytotoxic combination of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5′-N-methyluronamide(Cl-IB-MECA, a selective A(3) adenosine receptor agonist; 10 μM) and paclitaxel (10 ng/mL) on human C32 and A375 melanoma cell lines.

Methods: Cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase leakage assays, after 48-h incubation. Autophagosome and autolysosome formation was detected by fluorescence through monodansylcadaverine-staining and CellLight(®) Lysosomes-RFP-labelling, respectively. Cell nuclei were visualized by Hoechst staining, while levels of p62 were determined by an ELISA kit. Levels of mammalian target of rapamycin (mTOR) and the alterations of microtubule networks were evaluated by immunofluorescence.

Results: We demonstrated, for the first time, that the combination of Cl-IB-MECA with paclitaxel significantly increases cytotoxicity, with apoptosis and autophagy the major mechanisms involved in cell death. Induction of autophagy, using clinically relevant doses,was confirmed by visualization of autophagosome and autolysosome formation, and downregulation of mTOR and p62 levels. Caspase-dependent and caspase-independent mitotic catastrophe evidencing micro- and multinucleation was also observed in cells exposed to our combination.

Conclusions: The combination of Cl-IB-MECA and paclitaxel causes significant cytotoxicity on two melanoma cell lines through multiple mechanisms of cell death. This multifactorial hit makes this therapy very promising as it will help to avoid melanoma multiresistance to chemotherapy and therefore potentially improve its treatment.