Afatinib triggers a Ni²⁺ -resistant Ca²⁺ influx pathway in A549 non-small cell lung cancer cells

Afatinib is used to treat non-small cell lung cancer cells (NSCLC), and its mechanism involves irreversible inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. In this study, we examined if afatinib had cytotoxic action against NSCLC other than inhibition of tyrosine kinase. Afatinib (1-30 μM) caused apoptotic death in A549 NSCLC in a concentration-dependent manner. Afatinib triggered Ca²⁺ influx without causing Ca²⁺ release, and the Ca²⁺ influx was unaffected by sodium orthovanadate (SOV, an inhibitor of tyrosine phosphatase), suggesting that afatinib-triggered Ca²⁺ response was unrelated to its inhibition of tyrosine kinase. Addition of afatinib also promoted Mn²⁺ influx. Ca²⁺ influx triggered by afatinib was resistant to SKF96365 and ruthenium red (two general blockers of TRP channels) and, unexpectedly, Ni²⁺ (a non-specific Ca²⁺ channel blocker). Afatinib caused an increase in mitochondrial Ca²⁺ level, an initial mitochondrial hyperpolarization (4 h) and followed by mitochondrial potential collapse (24-48 h). Afatinib-induced cell death was slightly but significantly alleviated in low extracellular Ca²⁺ condition or under pharmacological block of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A. Therefore, in addition to tyrosine kinase inhibition as a major anti-cancer mechanism of afatinib, stimulation of an atypical Ca²⁺ influx pathway, mitochondrial Ca²⁺ overload, and potential collapse in part contribute to afatinib-induced cell death.