title: Clotrimazole decreases human breast cancer cells viability through alterations in cytoskeleton-associated glycolytic enzymes.
abstract: Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.
authors.affiliation: Laboratório de Enzimologia e Controle do Metabolismo (LabECoM), Dept. Fármacos, Fac. Farmácia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
authors.forename: Débora Dummer
authors.initials: DD
authors.lastname: Meira
authors.forename: Mônica M
authors.initials: MM
authors.lastname: Marinho-Carvalho
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Teixeira
authors.forename: Venício F
authors.initials: VF
authors.lastname: Veiga
authors.forename: Andrea T
authors.initials: AT
authors.lastname: Da Poian
authors.forename: Carla
authors.initials: C
authors.lastname: Holandino
authors.forename: Marta S
authors.initials: MS
authors.lastname: de Freitas
authors.forename: Mauro
authors.initials: M
authors.lastname: Sola-Penna
mesh_terms: D000199:Actins / Q000187:drug effects / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003022:Clotrimazole / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D010590:Phalloidine / Q000737:chemistry / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000187:drug effects / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D006131:Growth Inhibitors; D012235:Rhodamines; D010590:Phalloidine; D019344:Lactic Acid; C009434:tetramethylrhodamine isothiocyanate; D019809:Streptavidin; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase; D005634:Fructose-Bisphosphate Aldolase; D003022:Clotrimazole; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose