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1.
Carcinogenesis ; 29(12): 2360-8, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18952594

ABSTRACT

Sulforaphane (SFN), a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. SFN arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g. taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-green fluorescent protein-alpha-tubulin breast tumor cells by approximately 50% (~15 microM), SFN significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic concentration required to inhibit mitosis by 50%, SFN suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. SFN suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by SFN in cells is due to a direct effect on the microtubules. The results indicate that SFN arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for SFN's chemopreventive activity.


Subject(s)
Anticarcinogenic Agents/pharmacology , Breast Neoplasms/metabolism , Microtubules/drug effects , Thiocyanates/pharmacology , Acetylation , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Flow Cytometry , Humans , Image Processing, Computer-Assisted , Isothiocyanates , Microscopy, Fluorescence , Mitosis/drug effects , Spindle Apparatus/drug effects , Sulfoxides , Tubulin Modulators/pharmacology
2.
Mol Cancer Ther ; 7(7): 2003-11, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18645010

ABSTRACT

Eribulin (E7389), a synthetic analogue of halichondrin B in phase III clinical trials for breast cancer, binds to tubulin and microtubules. At low concentrations, it suppresses the growth phase of microtubule dynamic instability in interphase cells, arrests mitosis, and induces apoptosis, suggesting that suppression of spindle microtubule dynamics induces mitotic arrest. To further test this hypothesis, we measured the effects of eribulin on dynamics of centromeres and their attached kinetochore microtubules by time-lapse confocal microscopy in living mitotic U-2 OS human osteosarcoma cells. Green fluorescent protein-labeled centromere-binding protein B marked centromeres and kinetochore-microtubule plus-ends. In control cells, sister chromatid centromere pairs alternated under tension between increasing and decreasing separation (stretching and relaxing). Eribulin suppressed centromere dynamics at concentrations that arrest mitosis. At 60 nmol/L eribulin (2 x mitotic IC(50)), the relaxation rate was suppressed 21%, the time spent paused increased 67%, and dynamicity decreased 35% (but without reduction in mean centromere separation), indicating that eribulin decreased normal microtubule-dependent spindle tension at the kinetochores, preventing the signal for mitotic checkpoint passage. We also examined a more potent, but in tumors less efficacious antiproliferative halichondrin derivative, ER-076349. At 2 x IC(50) (4 nmol/L), mitotic arrest also occurred in concert with suppressed centromere dynamics. Although media IC(50) values differed 15-fold between the two compounds, the intracellular concentrations were similar, indicating more extensive relative uptake of ER-076349 into cells compared with eribulin. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic arrest indicates that the primary mechanism by which eribulin blocks mitosis is suppression of spindle microtubule dynamics.


Subject(s)
Centromere/drug effects , Centromere/metabolism , Furans/pharmacology , Ketones/pharmacology , Metaphase/drug effects , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Centromere Protein B/metabolism , Furans/chemistry , Green Fluorescent Proteins/metabolism , Heterocyclic Compounds, 4 or More Rings/chemistry , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Ketones/chemistry , Microtubules/drug effects , Protein Transport/drug effects , Spindle Apparatus/drug effects , Time Factors
3.
Cancer Res ; 67(12): 5717-26, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17575138

ABSTRACT

Secondary resistance to hormonal therapy for breast cancer commonly develops after an initial response to tamoxifen or aromatase inhibitors. Agents to abrogate these adaptive changes would substantially enhance the long-term benefits of hormonal therapy. Our studies with a stilbene derivative called TMS (2,3',4,5'-tetramethoxystilbene) identified unexpected effects with potential utility for treatment of breast tumors secondarily resistant to hormonal therapy. TMS was originally developed as an inhibitor of cytochrome P450 1B1 to block the conversion of estradiol to 4-OH-estradiol. While studying this agent in three models of hormone resistance, we detected direct antitumor effects not related to its role as an inhibitor of catecholestrogens. During examination of the mechanisms involved, we showed that treatment with 3 micromol/L TMS for 24 h inhibited tubulin polymerization and microtubule formation, caused a cell cycle block at the G2-M phase, and induced apoptosis. TMS also inhibited activated focal adhesion kinase (FAK), Akt, and mammalian target of rapamycin (mTOR) and stimulated c-jun-NH2-kinase and p38 mitogen-activated protein kinase activity. With respect to antitumor effects, TMS at a concentrations of 0.2 to 0.3 micromol/L inhibited the growth of long-term tamoxifen-treated MCF-7 cells by 80% and fulvestrant-treated MCF-7 cells by 70%. In vivo studies, involving 8 weeks of treatment with TMS via a 30-mg s.c. implant, reduced tumor volume of tamoxifen-resistant MCF-7 breast cancer xenografts by 53%. Our data suggest that TMS is a promising therapeutic agent because of its unique ability to block several pathways involved in the development of hormone resistance.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/physiology , Mammary Neoplasms, Experimental/drug therapy , Stilbenes/pharmacology , Animals , Aromatase Inhibitors/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Estrogen Receptor Modulators/pharmacology , Estrogens/metabolism , Female , Fluorescent Antibody Technique , Humans , Immunoblotting , In Situ Nick-End Labeling , Mammary Neoplasms, Experimental/metabolism , Mice , Tamoxifen/pharmacology
4.
Cancer Res ; 67(8): 3767-76, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17440090

ABSTRACT

Tasidotin (ILX-651), an orally active synthetic microtubule-targeted derivative of the marine depsipeptide dolastatin-15, is currently undergoing clinical evaluation for cancer treatment. Tasidotin inhibited proliferation of MCF7/GFP breast cancer cells with an IC(50) of 63 nmol/L and inhibited mitosis with an IC(50) of 72 nmol/L in the absence of detectable effects on spindle microtubule polymer mass. Tasidotin inhibited the polymerization of purified tubulin into microtubules weakly (IC(50) approximately 30 micromol/L). However, it strongly suppressed the dynamic instability behavior of the microtubules at their plus ends at concentrations approximately 5 to 10 times below those required to inhibit polymerization. Its major actions were to reduce the shortening rate, the switching frequency from growth to shortening (catastrophe frequency), and the fraction of time the microtubules grew. In contrast with all other microtubule-targeted drugs thus far examined that can inhibit polymerization, tasidotin did not inhibit the growth rate. In contrast to stabilizing plus ends, tasidotin enhanced microtubule dynamic instability at minus ends, increasing the shortening length, the fraction of time the microtubules shortened, and the catastrophe frequency and reducing the rescue frequency. Tasidotin C-carboxylate, the major intracellular metabolite of tasidotin, altered dynamic instability of purified microtubules in a qualitatively similar manner to tasidotin but was 10 to 30 times more potent. The results suggest that the principal mechanism by which tasidotin inhibits cell proliferation is by suppressing spindle microtubule dynamics. Tasidotin may be a relatively weak prodrug for the functionally active tasidotin C-carboxylate.


Subject(s)
Oligopeptides/pharmacology , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Carboxylic Acids/pharmacokinetics , Carboxylic Acids/pharmacology , Cattle , Cell Growth Processes/drug effects , Cell Line, Tumor , Humans , Microtubules/drug effects , Microtubules/metabolism , Mitosis/drug effects , Oligopeptides/pharmacokinetics , Prodrugs/pharmacokinetics , Prodrugs/pharmacology
5.
Mol Cancer Ther ; 5(9): 2225-33, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16985056

ABSTRACT

2-Methoxyestradiol (2ME2), a metabolite of estradiol-17beta, is a novel antimitotic and antiangiogenic drug candidate in phase I and II clinical trials for the treatment of a broad range of tumor types. 2ME2 binds to tubulin at or near the colchicine site and inhibits the polymerization of tubulin in vitro, suggesting that it may work by interfering with normal microtubule function. However, the role of microtubule depolymerization in its antitumor mechanism of action has been controversial. To determine the mechanism by which 2ME2 induces mitotic arrest, we analyzed its effects on microtubule polymerization in vitro and its effects on dynamic instability both in vitro and in living MCF7 cells. In vitro, 2ME2 (5-100 micromol/L) inhibited assembly of purified tubulin in a concentration-dependent manner, with maximal inhibition (60%) at 200 micromol/L 2ME2. However, with microtubule-associated protein-containing microtubules, significantly higher 2ME2 concentrations were required to depolymerize microtubules, and polymer mass was reduced by only 13% at 500 micromol/L 2ME2. In vitro, dynamic instability was inhibited at lower concentrations. Specifically, 4 micromol/L 2ME2 reduced the mean growth rate by 17% and dynamicity by 27%. In living interphase MCF7 cells at the IC50 for mitotic arrest (1.2 micromol/L), 2ME2 significantly suppressed the mean microtubule growth rate, duration and length, and the overall dynamicity, consistent with its effects in vitro, and without any observable depolymerization of microtubules. Taken together, the results suggest that the major mechanism of mitotic arrest at the lowest effective concentrations of 2ME2 is suppression of microtubule dynamics rather than microtubule depolymerization per se.


Subject(s)
Antimitotic Agents/pharmacology , Estradiol/analogs & derivatives , Microtubules/drug effects , Tubulin Modulators/pharmacology , 2-Methoxyestradiol , Angiogenesis Inhibitors/pharmacology , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cattle , Cell Line, Tumor , Estradiol/pharmacokinetics , Estradiol/pharmacology , Humans , Interphase/drug effects , Interphase/physiology , Mice , Microtubules/metabolism , Microtubules/physiology , Mitosis/drug effects , Mitosis/physiology , Spindle Apparatus/drug effects , Spindle Apparatus/physiology , Strongylocentrotus purpuratus , Tubulin/metabolism , Tubulin Modulators/pharmacokinetics
6.
Mol Cancer Ther ; 4(7): 1086-95, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16020666

ABSTRACT

E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G(2)-M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified microtubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predominantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disassembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/anaphase checkpoint.


Subject(s)
Antimitotic Agents/pharmacology , Furans/pharmacology , Ketones/pharmacology , Microtubules/drug effects , Microtubules/metabolism , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/ultrastructure , Cattle , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Female , Furans/administration & dosage , Humans , Interphase/drug effects , Ketones/administration & dosage , Mitosis/drug effects , Spindle Apparatus/drug effects , Tubulin/drug effects , Tubulin/metabolism , Tumor Cells, Cultured
7.
Mol Cancer Ther ; 2(5): 427-36, 2003 May.
Article in English | MEDLINE | ID: mdl-12748304

ABSTRACT

Vinflunine is a novel fluorinated Vinca alkaloid currently in Phase II clinical trials, which in preclinical studies exhibited superior antitumor activity to that of two clinically useful Vinca alkaloids, vinorelbine and vinblastine. All three of the drugs block mitosis at the metaphase/anaphase transition, leading to apoptosis. The mechanism of the mitotic block is not known. On the basis of results with purified microtubules and in living interphase cells, we hypothesized that it involves suppression of spindle microtubule dynamics. Here we measured the effects of the three Vinca alkaloids on dynamics of centromeres and spindle kinetochore-microtubules by a novel approach involving quantitative time-lapse confocal microscopy in living mitotic human U2OS cells. Green fluorescent protein-labeled centromere-binding protein B was used to mark centromeres and kinetochore-microtubule plus ends. In controls, pairs of centromeres on sister chromatids alternated under tension between increasing and decreasing separation (stretching and relaxing). All three of the Vinca alkaloids suppressed centromere dynamics similarly at concentrations that block mitosis. At concentrations approximating the IC(50)s for mitotic accumulation (18.8 nM vinflunine, 7.3 nM vinorelbine, and 6.1 nM vinblastine), centromere dynamicity decreased by 44%, 25%, and 26%, respectively, and the time centromeres spent in a paused state increased by 63%, 52%, and 36%, respectively. Centromere relaxation rates, stretching durations, and transition frequencies all decreased. Thus all three of the drugs decreased the normal microtubule-dependent spindle tension at the centromeres/kinetochores, thereby preventing the signal for mitotic checkpoint passage. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic block indicates that the primary mechanism by which the Vinca alkaloids block mitosis is suppression of spindle microtubule dynamics.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Centromere/drug effects , Spindle Apparatus/drug effects , Vinblastine/analogs & derivatives , Vinblastine/pharmacology , Bone Neoplasms/pathology , Cell Division/drug effects , Cell Movement/drug effects , Centromere/metabolism , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Microtubules/drug effects , Mitosis/drug effects , Osteosarcoma/pathology , Subcellular Fractions , Tumor Cells, Cultured , Vinorelbine
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