Antimitotic effect of the retinoid 4-oxo-fenretinide through inhibition of tubulin polymerization: a novel mechanism of retinoid growth-inhibitory activity.
The retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR), a metabolite of fenretinide (4-HPR) present in plasma of 4-HPR-treated patients, is very effective in inducing growth inhibition and apoptosis in several cancer cell lines. 4-Oxo-4-HPR and 4-HPR have different mechanisms of action because 4-oxo-4-HPR, unlike 4-HPR, causes marked cell accumulation in G2-M phase. Here, we investigated the molecular events involving 4-oxo-4-HPR-induced cell cycle perturbation in ovarian (A2780 and IGROV-1) and breast (T47D, estrogen receptor+ and BT-20, estrogen receptor-) cancer cells. 4-Oxo-4-HPR induced a delay of mitosis (with mitotic index increasing 5- to 6-fold in all cell lines) without progression beyond the anaphase, as shown by cyclin B1 expression. 4-Oxo-4-HPR induced multipolar spindle formation and phosphorylation of BUBR1, resulting in activation of the spindle checkpoint. Multipolar spindles were not due to impairment of pole-focusing process, loss of centrosome integrity, or modulation of the expression levels of molecules associated with spindle aberrations (Kif 1C, Kif 2A, Eg5, Tara, tankyrase-1, centractin, and TOGp). We show here that 4-oxo-4-HPR targets microtubules because, in treated cells, it interfered with the reassembly of cold-depolymerized spindle microtubules and decreased the polymerized tubulin fraction. In cell-free assays, 4-oxo-4-HPR inhibited tubulin polymerization (50% inhibition of microtubule assembly at 5.9 micromol/L), suggesting a direct molecular interaction with tubulin. In conclusion, by showing that 4-oxo-4-HPR causes mitotic arrest through antimicrotubule activities, we delineate a new molecular mechanism for a retinoid.
Appierto
Valentina
V
Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
Tiberio
Paola
P
Cavadini
Elena
E
Casalini
Patrizia
P
Cappelletti
Graziella
G
Formelli
Franca
F
D000199:Actins / Q000235:genetics; D050256:Antimitotic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D005260:Female; D017313:Fenretinide / Q000031:analogs & derivatives* / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D016547:Kinesins / Q000235:genetics; D008840:Microfilament Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008940:Mitotic Index; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011108:Polymers / Q000378:metabolism; D012176:Retinoids / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D008941:Spindle Apparatus / Q000187:drug effects; D038501:Tankyrases / Q000235:genetics; D014404:Tubulin / Q000378:metabolism*
D000199:Actins; D050256:Antimitotic Agents; D017313:Fenretinide; D006131:Growth Inhibitors; D016547:Kinesins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D011108:Polymers; D012176:Retinoids; D038501:Tankyrases; D014404:Tubulin; C494898:4-oxofenretinide; C454180:CKAP5 protein, human; C470243:KIF11 protein, human; C114063:KIF1C protein, human; C509805:KIF2A protein, human; C425471:TRIOBP protein, human; C076834:centractin; C418109:TNKS protein, human