Inhibition Mechanism of the Intracellular Transporter Ca2+-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.