Inhibition Mechanism of the Intracellular Transporter Ca(2+)-Pump from Sarco-Endoplasmic Reticulum by the Antitumor Agent Dimethyl-Celecoxib

Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca(2+)-ATPase activity and ATP-dependent Ca(2+) transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca(2+)-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca(2+) transport was evaluated. Since Ca(2+) transport was more sensitive to inhibition than Ca(2+)-ATPase activity the drugs under study caused Ca(2+)/P(i) uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca(2+)-pump functioning. Dimethyl-celecoxib prevented Ca(2+) binding by stabilizing the inactive Ca(2+)-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca(2+)–pump was exposed to the drug in the presence of Ca(2+) before phosphorylation. This provided evidence of non-preferential interaction with the Ca(2+)-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca(2+) was consistent with the mentioned effect on Ca(2+) binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca(2+)-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and dimethyl-celecoxib with the intracellular Ca(2+) transporter at the inhibition site of hydroquinones.