title: Inhibition mechanism of the intracellular transporter Ca2+-pump from sarco-endoplasmic reticulum by the antitumor agent dimethyl-celecoxib.
abstract: 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.
authors.affiliation: Departamento de Medicina Clínica, Universidad Miguel Hernández en Campus de San Juan, Alicante, Spain.
authors.forename: Ramón
authors.initials: R
authors.lastname: Coca
authors.affiliation: Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia en Campus de Espinardo, Murcia, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Soler
authors.affiliation: Departamento de Farmacología, Pediatría y Química Orgánica, Universidad Miguel Hernández en Campus de San Juan, Alicante, Spain.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Cortés-Castell
authors.affiliation: Departamento de Medicina Clínica, Universidad Miguel Hernández en Campus de San Juan, Alicante, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Gil-Guillén
authors.affiliation: Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia en Campus de Espinardo, Murcia, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Fernández-Belda
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D020013:Calcium Signaling; D005260:Female; D007700:Kinetics; D062105:Molecular Docking Simulation; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C506698:2,5-dimethylcelecoxib; D000970:Antineoplastic Agents; D011720:Pyrazoles; D013449:Sulfonamides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases