Inhibition mechanism of the intracellular transporter Ca2+-pump from sarco-endoplasmic reticulum by the antitumor agent dimethyl-celecoxib

• Published in: PloS one 2014-07, Vol.9 (7), p.e102083-e102083
• Main Authors: Coca, Ramón, Soler, Fernando, Cortés-Castell, Ernesto, Gil-Guillén, Vicente, Fernández-Belda, Francisco
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatase; Animals; Anticancer properties; Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacology; Apoptosis; Binding; Binding Sites; Biology and Life Sciences; Ca2+-transporting ATPase; Calcium (intracellular); Calcium (reticular); Calcium ions; Calcium Signaling; Calcium transport; Cancer; Celecoxib; Conformation; COX-2 inhibitors; Cyclooxygenase-2; Cytotoxicity; Docking; Drug dosages; Endoplasmic reticulum; Female; Inhibition; Intracellular; Kinetics; Molecular Docking Simulation; Muscles; Oxygenase; Phosphorylation; Prostaglandin endoperoxide synthase; Pyrazoles - chemistry; Pyrazoles - pharmacology; Rabbits; Sarcoplasmic reticulum; Sarcoplasmic Reticulum - drug effects; Sarcoplasmic Reticulum - metabolism; Sarcoplasmic Reticulum Calcium-Transporting ATPases - antagonists & inhibitors; Sarcoplasmic Reticulum Calcium-Transporting ATPases - chemistry; Sarcoplasmic Reticulum Calcium-Transporting ATPases - metabolism; Skeletal muscle; Sulfonamides - chemistry; Sulfonamides - pharmacology; Transport; Transporter
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0102083

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 Ca2+-ATPase activity and ATP-dependent Ca2+ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-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 Ca2+-pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-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 Ca2+ transporter at the inhibition site of hydroquinones.