Direct Binding of Cisplatin to p22phox, an Endoplasmic Reticulum (ER) Membrane Protein, Contributes to Cisplatin Resistance in Oral Squamous Cell Carcinoma (OSCC) Cells

Prolonged treatment with cisplatin (CDDP) frequently develops chemoresistance. We have previously shown that p22phox, an endoplasmic reticulum (ER) membrane protein, confers CDDP resistance by blocking CDDP nuclear entry in oral squamous cell carcinoma (OSCC) cells; however, the underlying mechanism remains unresolved. Using a fluorescent dye-labeled CDDP, here we show that CDDP can bind to p22phox in both cell-based and cell-free contexts. Subsequent detection of CDDP-peptide interaction by the Tris-Tricine-based electrophoresis revealed that GA-30, a synthetic peptide matching a region of the cytosolic domain of p22phox, could interact with CDDP. These results were further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis, from which MA-11, an 11-amino acid subdomain of the GA-30 domain, could largely account for the interaction. Amino acid substitutions at Cys50, Met65 and Met73, but not His72, significantly impaired the binding between CDDP and the GA-30 domain, thereby suggesting the potential CDDP-binding residues in p22phox protein. Consistently, the p22phox point mutations at Cys50, Met65 and Met73, but not His72, resensitized OSCC cells to CDDP-induced cytotoxicity and apoptosis. Finally, p22phox might have binding specificity for the platinum drugs, including CDDP, carboplatin and oxaliplatin. Together, we have not only identified p22phox as a novel CDDP-binding protein, but further highlighted the importance of such a drug-protein interaction in drug resistance.