RESUMEN
Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Microtúbulos/efectos de los fármacos , Oligopéptidos/toxicidad , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Sistema Nervioso Periférico/efectos de los fármacos , Moduladores de Tubulina/toxicidad , Tubulina (Proteína)/metabolismo , Transporte Axonal/efectos de los fármacos , Sitios de Unión , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Microtúbulos/metabolismo , Microtúbulos/patología , Mitosis/efectos de los fármacos , Oligopéptidos/metabolismo , Sistema Nervioso Periférico/metabolismo , Sistema Nervioso Periférico/patología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/patología , Unión Proteica , Medición de Riesgo , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Huso Acromático/patología , Moduladores de Tubulina/metabolismoRESUMEN
Eribulin mesylate (Halaven) is a microtubule-targeted anticancer drug used to treat patients with metastatic breast cancer who have previously received a taxane and an anthracycline. It binds at the plus ends of microtubules and has been shown to suppress plus end growth selectively. Because the class III ß tubulin isotype is associated with resistance to microtubule targeting drugs, we sought to determine how ßIII tubulin might mechanistically influence the effects of eribulin on microtubules. We found that while [(3)H]eribulin bound to bovine brain soluble tubulin depleted of ßIII tubulin in a manner similar to that of unfractionated tubulin, it bound to plus ends of microtubules that were depleted of ßIII-depleted tubulin with a maximal stoichiometry (20 ± 3 molecules per microtubule) higher than that of unfractionated microtubules (9 ± 2 molecules per microtubule). In addition, eribulin suppressed the dynamic instability behavior of ßIII-depleted microtubules more strongly than and in a manner different from that of microtubules containing ßIII tubulin. Specifically, with ßIII tubulin present in the microtubules, 100 nM eribulin suppressed the growth rate by 32% and marginally reduced the catastrophe frequency (by 17%) but did not modulate the rescue frequency. However, in the absence of ßIII tubulin, eribulin not only reduced the growth rate but also strongly reduced the shortening rate (by 43%) and the catastrophe and the rescue frequencies (by 49 and 32%, respectively). Thus, when present in microtubules, ßIII tubulin substantially weakens the effects of eribulin.
Asunto(s)
Furanos/farmacología , Cetonas/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Sitios de Unión , Química Encefálica , Bovinos , Furanos/efectos adversos , Furanos/farmacocinética , Humanos , Técnicas In Vitro , Cetonas/efectos adversos , Cetonas/farmacocinética , Microtúbulos/química , Modelos Moleculares , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/químicaRESUMEN
Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.
Asunto(s)
Regulación de la Expresión Génica , Microtúbulos/metabolismo , Proteínas tau/química , Enfermedad de Alzheimer/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Modelos Biológicos , Paclitaxel/farmacología , Fosforilación , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady-state microtubules assembled from MAP-free tubulin in the possible presence of a microtubule-associated drug. As an example of the latter, we both experimentally and theoretically study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. Our model predicts that among the drugs that act locally at the microtubule tip, primary inhibition of the loss of GDP tubulin results in stronger damping of microtubule dynamics than inhibition of GTP tubulin addition. On the other hand, drugs whose action occurs in the interior of the microtubule need to be present in much higher concentrations to have visible effects.
Asunto(s)
Antineoplásicos/farmacología , Maitansina/análogos & derivados , Microtúbulos/efectos de los fármacos , Animales , Antineoplásicos/metabolismo , Sitios de Unión , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Maitansina/metabolismo , Maitansina/farmacología , Microtúbulos/metabolismo , Unión Proteica , Erizos de Mar , Procesos Estocásticos , Tubulina (Proteína)/metabolismoRESUMEN
Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Electroforesis en Gel Bidimensional/métodos , Paclitaxel/farmacología , Estatmina/fisiología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Microtúbulos/efectos de los fármacos , Mitosis , Índice Mitótico , Proteínas de Neoplasias/efectos de los fármacos , Estatmina/genética , Estatmina/metabolismo , Regulación hacia ArribaRESUMEN
Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing dynamic instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086-1095]. Using [(3)H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall K(d) of 46 microM, but also showing a real or apparent very high affinity (K(d) = 0.4 microM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 +/- 1.3 molecules per microtubule (K(d) = 3.5 microM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that the binding of a single eribulin molecule at a microtubule end can potently inhibit its growth. Eribulin does not suppress dynamic instability at microtubule minus ends. Preincubation of microtubules with 2 or 4 microM vinblastine induced additional lower-affinity eribulin binding sites, most likely at splayed microtubule ends. Overall, our results indicate that eribulin binds with high affinity to microtubule plus ends and thereby suppresses dynamic instability.
Asunto(s)
Regulación hacia Abajo , Furanos/metabolismo , Furanos/farmacología , Cetonas/metabolismo , Cetonas/farmacología , Microtúbulos/metabolismo , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Bovinos , Dimerización , Regulación hacia Abajo/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Estabilidad Proteica/efectos de los fármacos , Termodinámica , Vinblastina/farmacologíaRESUMEN
Hesperidin, a flavonoid derived from citrus fruits, has been reported to show various biological effects including anticancer activity. This study investigated whether hesperidin affected the proliferation of MCF-7 human breast cancer cells transfected with green fluorescent protein (GFP)/alpha-tubulin (MCF-7-GFP-Tubulin cells), androgen-independent PC-3 and DU-145 prostate cancer cells, and androgen-dependent LNCaP prostate cancer cells. The results were as follows. (1) Hesperidin inhibited the proliferation of MCF-7-GFP-Tubulin cells, probably not through an antimitotic mechanism. (2) Hesperidin also inhibited both basal and testosterone-induced proliferation of LNCaP cells. (3) However, hesperidin did not significantly affect the cell proliferation of two hormone-independent prostate cancer cells, PC-3 and DU-145. It is concluded that hesperidin can inhibit the proliferation of breast cancer cells through mechanisms other than antimitosis and it is suggested that hesperidin be further investigated for the possible interaction with androgenic receptors and involvement in signaling pathway after receptor binding in prostate cancer cells through future research.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/metabolismo , Proliferación Celular/efectos de los fármacos , Hesperidina/farmacología , Neoplasias de la Próstata/metabolismo , Andrógenos/metabolismo , Línea Celular Tumoral , Femenino , Humanos , MasculinoRESUMEN
Microtubule-targeting agents, such as taxanes and epothilones, block mitosis and cell proliferation by targeting the dynamics of the cytoskeleton. The taxanes are widely used for treatment of various malignancies, but primary and acquired resistance to chemotherapy remains a significant clinical concern. Class I, II, III, IV, and V beta-tubulin isotypes are expressed in human tumors. Overexpression of the betaIII-tubulin isotype is one mechanism that can render tumor cells resistant to taxanes. The relative expression of betaIII-tubulin correlates with clinical outcomes in several tumor types, including breast cancer, non-small cell lung cancer, and ovarian cancer. A novel analogue of epothilone B, ixabepilone, has recently been approved in combination with capecitabine for the treatment of patients with anthracycline- and taxane-resistant locally advanced or metastatic breast cancer and as monotherapy in patients whose tumors are resistant or refractory to an anthracycline, a taxane, and capecitabine. The significant antitumor activity of ixabepilone in taxane-resistant tumors may be related to its preferential suppression of the dynamic instability of alpha/betaIII-microtubules in cells expressing high levels of betaIII-tubulin.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Epotilonas/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Taxoides/uso terapéutico , Tubulina (Proteína)/metabolismo , Animales , Humanos , Resultado del Tratamiento , Tubulina (Proteína)/clasificaciónRESUMEN
Carbendazim (methyl 2-benzimidazolecarbamate) is widely used as a systemic fungicide in human food production and appears to act on fungal tubulin. However, it also inhibits proliferation of human cancer cells, including drug- and multidrug-resistant and p53-deficient cell lines. Because of its promising preclinical anti-tumor activity, it has undergone phase I clinical trials and is under further clinical development. Although it weakly inhibits polymerization of brain microtubules and induces G(2)/M arrest in tumor cells, its mechanism of action in human cells has not been fully elucidated. We examined its mechanism of action in MCF7 human breast cancer cells and found that it inhibits proliferation (IC(50), 10 microM) and half-maximally arrests mitosis at a similar concentration (8 microM), in concert with suppression of microtubule dynamic instability without appreciable microtubule depolymerization. It induces mitotic spindle abnormalities and reduces the metaphase intercentromere distance of sister chromatids, indicating reduction of tension on kinetochores, thus leading to metaphase arrest. With microtubules assembled in vitro from pure tubulin, carbendazim also suppresses dynamic instability, reducing the dynamicity by 50% at 10 microM, with only minimal (21%) reduction of polymer mass. Carbendazim binds to mammalian tubulin (K(d), 42.8 +/- 4.0 microM). Unlike some benzimidazoles that bind to the colchicine site in tubulin, carbendazim neither competes with colchicine nor competes with vinblastine for binding to brain tubulin. Thus, carbendazim binds to an as yet unidentified site in tubulin and inhibits tumor cell proliferation by suppressing the growing and shortening phases of microtubule dynamic instability, thus inducing mitotic arrest.
Asunto(s)
Bencimidazoles/farmacología , Carbamatos/farmacología , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Mitosis/efectos de los fármacos , Neoplasias de la Mama/patología , Colchicina , Relación Dosis-Respuesta a Droga , Humanos , Microtúbulos/fisiología , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/fisiologíaRESUMEN
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.
Asunto(s)
Oligopéptidos/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Ácidos Carboxílicos/farmacocinética , Ácidos Carboxílicos/farmacología , Bovinos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Oligopéptidos/farmacocinética , Profármacos/farmacocinética , Profármacos/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Estilbenos/farmacología , Animales , Inhibidores de la Aromatasa/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Moduladores de los Receptores de Estrógeno/farmacología , Estrógenos/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Tamoxifeno/farmacologíaRESUMEN
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.
Asunto(s)
Centrómero/efectos de los fármacos , Centrómero/metabolismo , Furanos/farmacología , Cetonas/farmacología , Metafase/efectos de los fármacos , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteína B del Centrómero/metabolismo , Furanos/química , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Cetonas/química , Microtúbulos/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Factores de TiempoRESUMEN
Constitutive activation of Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common oncogenic event in certain types of human cancer and is associated with poor patient survival. Small molecule signaling inhibitors have improved the clinical outcomes of patients with various cancer types but attempts to target KRAS have been unsuccessful. Plinabulin represents a novel class of agents that inhibit tubulin polymerization with a favorable safety profile in clinical trials. In the present study, the potency of plinabulin to inhibit tubulin polymerization and growth of KRAS-driven cancer cells was characterized. In vivo efficacy of plinabulin was tested in two different mouse models; one being the RCAS/t-va gene transfer system and the other being a xenograft model. In vitro cell culture tubulin polymerization assays were used to complement the mouse models. There was improved survival in a KRAS-driven mouse gene transfer glioma model, but lack of benefit in a similar model, without constitutively active KRAS, which supports the notion of a KRAS-specific effect. This survival benefit was mediated, at least in part, by the ability of plinabulin to inhibit tubulin polymerization and disrupt endosomal recycling. It was proposed a mechanism of compromised endosomal recycling of displaced KRAS through targeting microtubules that yields inhibition of protein kinase B, but not extracellular signal regulated kinase (ERK) signaling, therefore lending rationale to combination treatments of tubulin- and ERK-targeting agents in KRAS-driven cancer.
RESUMEN
Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5'-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.
Asunto(s)
Microtúbulos/química , Microtúbulos/ultraestructura , Modelos Químicos , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructura , Mezclas Complejas/química , Simulación por Computador , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Conformación ProteicaRESUMEN
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.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/metabolismo , Microtúbulos/efectos de los fármacos , Tiocianatos/farmacología , Acetilación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Procesamiento de Imagen Asistido por Computador , Isotiocianatos , Microscopía Fluorescente , Mitosis/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Sulfóxidos , Moduladores de Tubulina/farmacologíaRESUMEN
Microtubules have been identified as a suitable target for anticancer therapy, primarily based on their biological importance in coordinating chromosomal segregation at mitosis. Two main classes of microtubule-targeted agents, the taxanes and vinca alkaloids, suppress the dynamic behavior of spindle microtubules, inducing mitotic arrest and subsequent apoptotic cell death. Clinical activity of taxanes and first-generation vinca alkaloids in the treatment of solid tumors and hematologic malignancies, respectively, has prompted further research for novel analogs with improved clinical efficacy and safety. Such efforts have led to the development of vinflunine, a bifluorinated vinca alkaloid endowed with unique antitumor properties. Highlighted in this review are the key features of vinflunine that lead to effective suppression of microtubule dynamics and induction of cell death in cancer cells.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Microtúbulos/efectos de los fármacos , Vinblastina/análogos & derivados , Ciclo Celular , Muerte Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Humanos , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Moduladores de Tubulina/farmacología , Vinblastina/farmacologíaAsunto(s)
Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Huso Acromático/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Colchicina/farmacología , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Microtúbulos/fisiología , Neoplasias/fisiopatología , Paclitaxel/farmacología , Huso Acromático/fisiología , Alcaloides de la Vinca/farmacologíaRESUMEN
The neural microtubule-associated protein tau binds to and stabilizes microtubules. Because of alternative mRNA splicing, tau is expressed with either 3 or 4 C-terminal repeats. Two observations indicate that differences between these tau isoforms are functionally important. First, the pattern of tau isoform expression is tightly regulated during development. Second, mutation-induced changes in tau RNA splicing cause neuronal cell death and dementia simply by altering the isoform expression ratio. To investigate whether 3- and 4-repeat tau differentially regulate microtubule behavior in cells, we microinjected physiological levels of these two isoforms into EGFP-tubulin-expressing cultured MCF7 cells and measured the effects on the dynamic instability behavior of individual microtubules by time-lapse microscopy. Both isoforms suppressed microtubule dynamics, though to different extents. Specifically, 4-repeat tau reduced the rate and extent of both growing and shortening events. In contrast, 3-repeat tau stabilized most dynamic parameters about threefold less potently than 4-repeat tau and had only a minimal ability to suppress shortening events. These differences provide a mechanistic rationale for the developmental shift in tau isoform expression and are consistent with a loss-of-function model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal cell death and dementia.
Asunto(s)
Microtúbulos/metabolismo , Proteínas tau/metabolismo , Línea Celular Tumoral , Humanos , Microinyecciones , Microscopía Fluorescente , Microtúbulos/química , Enfermedades Neurodegenerativas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Secuencias Repetitivas de Aminoácido , Factores de Tiempo , Proteínas tau/químicaRESUMEN
Microtubule-targeted drugs such as paclitaxel exhibit potent antiangiogenic activity at very low concentrations, but the mechanism underlying such an effect remains unknown. To understand the involvement of microtubules in angiogenesis, we analyzed the dynamic instability behavior of microtubules in living endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vascular endothelial cells (HUVEC)] following 4 hours of paclitaxel treatment. Unexpectedly, antiangiogenic concentrations of paclitaxel (0.1-5 nmol/L) strongly increased microtubule overall dynamicity in both HMEC-1 (86-193%) and HUVEC (54-83%). This increase was associated with increased microtubule growth and shortening rates and extents and decreased mean duration of pauses. The enhancement of microtubule dynamics by paclitaxel seemed to be specific to antiangiogenic concentrations and to endothelial cells. Indeed, cytotoxic concentration (100 nmol/L) of paclitaxel suppressed microtubule dynamics by 40% and 54% in HMEC-1 and HUVECs, respectively, as observed for all tested concentrations in A549 tumor cells. After 4 hours of drug incubation, antiangiogenic concentrations of paclitaxel that inhibited endothelial cell proliferation without apoptosis (1-5 nmol/L) induced a slight decrease in anaphase/metaphase ratio, which was more pronounced and associated with increased mitotic index after 24 hours of incubation. Interestingly, the in vitro antiangiogenic effect also occurred at 0.1 nmol/L paclitaxel, a concentration that did not alter mitotic progression and endothelial cell proliferation but was sufficient to increase interphase microtubule dynamics. Altogether, our results show that paclitaxel mediates antiangiogenesis by an increase in microtubule dynamics in living endothelial cells and suggest that the impairment of interphase microtubule functions is responsible for the inhibition of angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Células Endoteliales/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Neoplasias/irrigación sanguínea , Paclitaxel/farmacología , Antineoplásicos Fitogénicos/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patologíaRESUMEN
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.