Identification of tubulin as the molecular target of proapoptotic pyrrolo-1,5-benzoxazepines.
We have demonstrated previously that certain members of a series of novel pyrrolo-1,5-benzoxazepine (PBOX) compounds potently induce apoptosis in a variety of human chemotherapy-resistant cancer cell lines and in primary ex vivo material derived from cancer patients. A better understanding of the molecular mechanisms underlying the apoptotic effects of these PBOX compounds is essential to their development as antineoplastic therapeutic agents. This study sought to test the hypothesis that proapoptotic PBOX compounds target the microtubules. We show that a representative proapoptotic PBOX compound, PBOX-6, induces apoptosis in both the MCF-7 and K562 cell lines. An accumulation of cells in G2/M precedes apoptosis in response to PBOX-6. PBOX-6 induces prometaphase arrest and causes an accumulation of cyclin B1 levels and activation of cyclin B1/CDK1 kinase in a manner similar to that of two representative antimicrotubule agents, nocodazole and paclitaxel. Indirect immunofluorescence demonstrates that both PBOX-6 and another pro-apoptotic PBOX compound, PBOX-15, cause microtubule depolymerization in MCF-7 cells. They also inhibit the assembly of purified tubulin in vitro, whereas a nonapoptotic PBOX compound (PBOX-21) has no effect on either the cellular microtubule network or on the assembly of purified tubulin. This suggests that the molecular target of the pro-apoptotic PBOX compounds is tubulin. PBOX-6 does not bind to either the vinblastine or the colchicine binding site on tubulin, suggesting that it binds to an as-yet-uncharacterised novel site on tubulin. The ability of PBOX-6 to bind tubulin and cause microtubule depolymerization confirms it as a novel candidate for antineoplastic therapy.
School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland.
Jude M
JM
Mulligan
Lisa M
LM
Greene
Suzanne
S
Cloonan
Margaret M
MM
Mc Gee
Valeria
V
Onnis
Giuseppe
G
Campiani
Caterina
C
Fattorusso
Mark
M
Lawler
D Clive
DC
Williams
Daniela M
DM
Zisterer
D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002219:Carbamates / Q000494:pharmacology; D002455:Cell Division; D045744:Cell Line, Tumor; D003078:Colchicine / Q000378:metabolism / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase; D006801:Humans; D020014:K562 Cells; D008677:Metaphase / Q000187:drug effects; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D010077:Oxazepines / Q000378:metabolism / Q000494:pharmacology*; D011758:Pyrroles / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism*; D014747:Vinblastine / Q000378:metabolism / Q000494:pharmacology
C514160:4-acetoxy-5-(1-(naphthyl))naphtho(2,3-b)pyrrolo(2,1-d)(1,4)oxazepine; C514161:7-((diethylcarbamoyl)oxy)-6-p-tolylpyrrolo(2,1-d)(1,5)benzoxazepine; D000972:Antineoplastic Agents, Phytogenic; C534699:CCNB1 protein, human; D002219:Carbamates; D019926:Cyclin B; D056744:Cyclin B1; D010077:Oxazepines; D011758:Pyrroles; D014404:Tubulin; D014747:Vinblastine; D016203:CDC2 Protein Kinase; C418868:PBOX-6; D003078:Colchicine