RESUMEN
There are seven ß-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of ß-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct ß-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for ß-tubulin from different sources. To determine the exact amount of drug that binds to different ß-tubulin isotypes, bovine brain tubulin was photolabeled and the isotypes resolved by high-resolution isoelectrofocusing. All bands were analyzed by mass spectrometry following cyanogen bromide digestion, and the identity and relative quantity of each ß-tubulin isotype determined. It was found that compared with other ß-tubulin isotypes, ßIII-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of ß-tubulin near the Taxol binding site indicated that, in addition to the M-loop that is known to be involved in drug binding, the leucine cluster region of ßIII-tubulin contains a unique residue, alanine, at 218, compared with other isotypes that contain threonine. Molecular dynamic simulations indicated that the frequency of Taxol-accommodating conformations decreased dramatically in the T218A variant, compared with other ß-tubulins. Our results indicate that the difference in residue 218 in ßIII-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.
Asunto(s)
Taxoides/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión , Células HeLa , Humanos , Mutación/genética , Polimerizacion , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Alineación de Secuencia , Tubulina (Proteína)/químicaRESUMEN
Several next-generation taxanes have been reported to possess high potency against Taxol-resistant cancer cell lines overexpressing ßIII-tubulin and/or P-glycoprotein (P-gp), both of which are involved in drug resistance. Using a photoaffinity Taxol analogue, 2-( m-azidobenzoyl)taxol, two potent next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibited distinct inhibitory effects on photolabeling of ß-tubulin from different eukaryotic sources that differ in ß-tubulin isotype composition. They also specifically inhibited photolabeling of P-gp, and the inhibitory effect correlated well with the steady-state accumulation of [3H]vinblastine in a multidrug resistant (MDR) cell line, SKVLB1. Several microtubule-stabilizing agents (MSAs)-resistant cell lines from the human ovarian cancer cell line Hey were isolated, and their MDR1 and ßIII-tubulin levels determined. Distinct potencies of the two taxanes against different MSA-resistant cells expressing unique levels of MDR1 and ßIII-tubulin were found. Cytotoxicity assays, done in the presence of verapamil, indicated that SB-T-1214 is a substrate, although not as good as Taxol, for P-gp. The mechanisms involved in drug resistance are multifactorial, and the effectiveness of new Taxol analogues depends on the interaction between the drugs and all possible targets; in this case the two major cellular targets are ß-tubulin and P-gp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Paclitaxel/farmacología , Tubulina (Proteína)/metabolismo , Femenino , Humanos , Microtúbulos/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Taxoides/farmacología , Células Tumorales Cultivadas , Verapamilo/farmacología , Vinblastina/farmacologíaRESUMEN
(+)-Discodermolide is a microtubule-stabilizing agent with potential for the treatment of taxol-refractory malignancies. (+)-Discodermolide congeners containing the C-3'-phenyl side chain of taxol (paclitaxel) were synthesized based on computational docking models predicting this moiety would fill an aromatic pocket of ß-tubulin insufficiently occupied by (+)-discodermolide, thereby conferring improved ligand-target interaction. It was recently demonstrated, however, that the C-3'-phenyl side chain occupied a different space, instead extending toward the M-loop of ß-tubulin, where it induced a helical conformation, hypothesized to improve lateral contacts between adjacent microtubule protofilaments. This insight led us to evaluate the biological activity of hybrid congeners using a panel of genetically diverse cancer cell lines. Hybrid molecules retained the same tubulin-polymerizing profile as (+)-discodermolide. Since (+)-discodermolide is a potent inducer of accelerated senescence, a fate that contributes to drug resistance, congeners were also screened for senescence induction. Flow cytometric and transcriptional analysis revealed that the hybrids largely retained the senescence-inducing properties of (+)-discodermolide. In taxol-sensitive cell models, the congeners had improved dose-response parameters relative to (+)-discodermolide and, in some cases, were superior to taxol. However, in cells susceptible to senescence, EMax increased without concomitant improvements in EC50 such that overall dose-response profiles resembled that of (+)-discodermolide.
Asunto(s)
Alcanos/administración & dosificación , Carbamatos/administración & dosificación , Lactonas/administración & dosificación , Paclitaxel/administración & dosificación , Pironas/administración & dosificación , Antineoplásicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Humanos , Microtúbulos/metabolismo , Transcripción Genética/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
Taxol®, an antitumor drug with significant activity, is the first microtubule stabilizing agent described in the literature. This short review of the mechanism of action of Taxol® emphasizes the research done in the Horwitz' laboratory. It discusses the contribution of photoaffinity labeled analogues of Taxol® toward our understanding of the binding site of the drug on the microtubule. The importance of hydrogen/deuterium exchange experiments to further our insights into the stabilization of microtubules by Taxol® is addressed. The development of drug resistance, a major problem that arises in the clinic, is discussed. Studies describing differential drug binding to distinct ß-tubulin isotypes are presented. Looking forward, it is suggested that the ß-tubulin isotype content of a tumor may influence its responses to Taxol®.
Asunto(s)
Paclitaxel/farmacología , Moduladores de Tubulina/farmacología , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Humanos , Microtúbulos/química , Microtúbulos/metabolismo , Paclitaxel/análogos & derivados , Paclitaxel/química , Unión Proteica , Isoformas de Proteínas , Subunidades de Proteína , Relación Estructura-Actividad , Moduladores de Tubulina/químicaRESUMEN
OBJECTIVE: To test if TP53 hot spot mutations (HSMs) confer differential chemotherapy resistance or survival outcomes, the effects of microtubule stabilizers on human ovarian carcinoma cells (OCCs) expressing TP53 HSMs were studied in vitro. Survival outcomes of patients with high grade serous epithelial ovarian carcinoma (HGS EOC) expressing matched HSMs were compared using The Cancer Genome Atlas (TCGA) data. METHODS: Growth inhibition of OCCs transfected with a HSM (m175, m248 or m273) was measured during treatment with paclitaxel, epothilone B (epoB), or ixabepilone. Effects of epoB on p53 expression, phosphorylation, and acetylation, as well as p53-regulated expression of p21 and mdm2 proteins, were determined by Western blot analysis. Expression of p53 target genes P21, GADD45, BAX, PIDD, NF-kB2, PAI-1, and MDR1 was measured by RT-PCR. cBioPortal.org identified patients with codon R175, R248 or R273 HSMs from TCGA data. Survival outcomes were characterized. RESULTS: p53-m248 confers chemoresistance and is not acetylated during epoB treatment. m273 demonstrated high MDR1 expression and resistance to paclitaxel. P21, GADD45 and PAI-1 expression were down-regulated in mutant OCCs. Optimally cytoreduced patients with codon R273 (n=17), R248 (n=13), R175 (n=7) HSMs, or any other TP53 mutation demonstrated median 14.9, 17.6, 17.8 and 16.9months (p=0.806) progression free survival and 84.1, 33.6, 62.1 and 44.5months (p=0.040) overall survival, respectively. CONCLUSIONS: Human OCCs harboring different TP53 HSMs were selectively resistant to microtubule stabilizers. Patients with different HSMs had significantly different overall survival. Both in vitro data and clinical experience support further studying the outcomes of particular TP53 HSMs.
Asunto(s)
Genes p53 , Mutación , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Moduladores de Tubulina/farmacología , Carcinoma Epitelial de Ovario , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Epotilonas/farmacología , Femenino , Humanos , Persona de Mediana Edad , Paclitaxel/farmacología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Ovarian cancer constitutes nearly 4% of all cancers among women and is the leading cause of death from gynecologic malignancies in the Western world. Standard first line adjuvant chemotherapy treatments include Paclitaxel (Taxol) and platinum-based agents. Taxol, epothilone B (EpoB) and discodermolide belong to a family of anti-neoplastic agents that specifically interferes with microtubules and arrests cells in the G2/M phase of the cell cycle. Despite initial success with chemotherapy treatment, many patients relapse due to chemotherapy resistance. In vitro establishment of primary ovarian cancer cells provides a powerful tool for better understanding the mechanisms of ovarian cancer resistance. We describe the generation and characterization of primary ovarian cancer cells derived from ascites fluids of patients with epithelial ovarian cancer. METHODS: Chemosensitivity of these cell lines to Taxol, EpoB and discodermolide was tested, and cell cycle analysis was compared to that of immortalized ovarian cancer cell lines SKOV3 and Hey. The relationship between drug resistance and αß-tubulin and p53 status was also investigated. RESULTS: All newly generated primary cancer cells were highly sensitive to the drugs. αß-tubulin mutation was not found in any primary cell lines tested. However, one cell line that harbors p53 mutation at residue 72 (Arg to Pro) exhibits altered cell cycle profile in response to all drug treatments. Immortalized ovarian cancer cells respond differently to EpoB treatment when compared to primary ovarian cancer cells, and p53 polymorphism suggests clinical significance in the anti-tumor response in patients. CONCLUSIONS: The isolation and characterization of primary ovarian cancer cells from ovarian cancer patients' specimens contribute to further understanding the nature of drug resistance to microtubule interacting agents (MIAs) currently used in clinical settings.
RESUMEN
Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRA(TM)), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αß-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.
Asunto(s)
Química Encefálica , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Epotilonas/química , Lactonas/química , Macrólidos/química , Microtúbulos/química , Proteínas del Tejido Nervioso/química , Moduladores de Tubulina/química , Tubulina (Proteína)/química , Animales , Sitios de Unión , Encéfalo , Bovinos , Espectrometría de Masas , Estructura Secundaria de ProteínaRESUMEN
OBJECTIVE: Ovarian cancer is the leading cause of death from gynecologic cancers in the United States. Epothilone B (EpoB), Taxol and vinblastine are anti-neoplastic agents that interfere with microtubules and arrest the cell cycle in the G2/M phase. EpoB is being evaluated in phase III clinical trials, and its analogs are currently being used in the treatment of taxane-resistant metastatic breast cancer. Little is known about the effect of these drugs on the immune response to tumors. Cancer cells evade immune recognition by down-regulating HLA Class I expression, allowing escape from immune surveillance and destruction. Our data illustrates the effect of microtubule-interacting agents on HLA Class I and HLA-A2 expression as well as the modulation of cytokine expression in ovarian cancer cells. METHODS: Ovarian cancer cells were treated with different concentrations of drugs. Cell surface expression and mRNA transcription of HLA Class I molecules and HLA-A2 was examined. IFNα, IL1ß, IL12 and IL6 mRNA expression was also evaluated upon EpoB treatment. RESULTS: Low-dose EpoB, Taxol and vinblastine greatly increased expression of HLA Class I and HLA-A2 molecules in Hey ovarian cancer cells. EpoB does not modulate HLA expression in drug-resistant ovarian cancer cells. The expression of IFNα, IL1ß, IL12 and IL6 is also markedly increased upon EpoB treatment. CONCLUSIONS: Nanomolar concentrations of microtubule-interacting agents enhance immune-visibility of ovarian cancer cells by increasing HLA Class I and pro-inflammatory cytokine expression. Immune recognition of tumor cells may be improved.
Asunto(s)
Epotilonas/farmacología , Antígenos HLA/biosíntesis , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Línea Celular Tumoral , Citocinas/biosíntesis , Citocinas/genética , Citocinas/inmunología , Relación Dosis-Respuesta a Droga , Femenino , Antígenos HLA/genética , Antígenos HLA/inmunología , Antígeno HLA-A2/biosíntesis , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Neoplasias Ováricas/genética , Paclitaxel/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Moduladores de Tubulina/farmacología , Vinblastina/farmacologíaRESUMEN
OBJECTIVES: Epothilone B (EpoB), like Taxol, stabilizes microtubules resulting in an inhibition of microtubule dynamic instability. The drug is being evaluated in phase III clinical trials. An EpoB analog, Ixabepilone, was approved by the FDA for the treatment of taxane-resistant metastatic breast cancer. Epithelial cell adhesion antigen (EpCAM) expression is significantly higher in epithelial ovarian cancer cells compared to normal cells. The effects of EpoB and other microtubule-interacting agents on surface EpCAM expression were studied. METHODS: Biochemical methods, immunofluorescence and flow cytometry were used to identify EpCAM expression on the surface of the ovarian cancer cell line, Hey, after exposure to EpoB. The relationship between EpoB-mediated surface EpCAM expression and EpoB-induced α-tubulin acetylation, a surrogate marker for stable microtubules, in Hey cells also was investigated. RESULTS: Nanomolar concentrations of EpoB, Taxol, discodermolide or vinblastine caused a marked increase in surface EpCAM expression in Hey cells. Alpha-tubulin acetylation was increased following treatment with Taxol, EpoB and discodermolide, but not with vinblastine, indicating that drug-enhanced surface EpCAM expression does not correlate with tubulin acetylation or stabilization. Unexpectedly, EpoB did not have a significant effect on EpCAM mRNA expression, nor did it alter the level of total cellular EpCAM in Hey cells. CONCLUSIONS: The results indicate that disruption of the microtubule cytoskeleton is associated with the redistribution of cell surface antigens in ovarian cancer cells. The increase in cell surface EpCAM antigen density may facilitate the antibody targeting of EpCAM-positive ovarian cancer cells.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Epotilonas/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Acetilación/efectos de los fármacos , Alcanos/farmacología , Carbamatos/farmacología , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial , Femenino , Humanos , Ácidos Hidroxámicos/farmacología , Lactonas/farmacología , Paclitaxel/farmacología , Pironas/farmacología , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacología , Vinblastina/farmacologíaRESUMEN
A Taxol-resistant cell line, K20T, which does not express P-glycoprotein, was selected with Taxol from human MDA-MB-231 breast adenocarcinoma cells and maintained in the presence of 20nM Taxol. K20T cells were approximately 18-fold resistant to Taxol, displayed cross-resistance to Taxotere and the epothilones, but little cross-resistance to discodermolide. Sequence analysis of the class I beta-tubulin indicated that it harbored an A593G mutation resulting in a change from glutamate to glycine at amino acid 198, which is near the intradimer interface within the alpha/beta-tubulin heterodimer. An HA-tagged wild-type class I beta-tubulin expression vector was transfected into the K20T cells. Immunofluorescence studies demonstrated that this exogenous tubulin was incorporated into cellular microtubules and Western blot analysis indicated that the K20T transfectants predominantly expressed the exogenous wild-type class I beta-tubulin. The transfected cells were only approximately 5-fold resistant to Taxol. Our results, plus the knowledge that Glu198 is the target for other anti-tubulin agents, suggest that glutamate198 in beta-tubulin is a critical determinant for microtubule stability and Taxol resistance.
Asunto(s)
Resistencia a Antineoplásicos/genética , Mutación Missense , Paclitaxel/farmacología , Tubulina (Proteína)/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Alcanos/farmacología , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carbamatos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Dimerización , Docetaxel , Epotilonas/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Ácido Glutámico/genética , Glicina/genética , Humanos , Lactonas/farmacología , Modelos Moleculares , Estructura Cuaternaria de Proteína , Pironas/farmacología , Taxoides/farmacología , Transfección , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
To explore the molecular mechanisms underlying the actions of Taxol and the functionally related molecule epothilone B (EpoB), we have analyzed the gene expression profiles in A549 cells in response to increasing concentrations of these microtubule-stabilizing drugs. An almost identical expression pattern was observed in cells treated with either Taxol or EpoB. Low concentrations of the drugs induced aberrant mitosis including asymmetric and multipolar cell divisions. At drug concentrations that triggered G(2)-M arrest, cells escaped from a prolonged mitotic arrest without cell division, resulting in tetraploid G(1) cells. This mitotic slippage is correlated with diminished expression of cdc2 kinase, topoisomerase IIalpha, BUB3, and BUB2-like protein 1, as well as with an increased expression of 14-3-3-sigma. Poly(ADP-ribose) polymerase cleavage, an early indicator of apoptosis, occurred in cells undergoing mitotic slippage and in aneuploid cells resulting from aberrant mitosis. In contrast, cells arrested in mitosis demonstrated no signal for apoptosis but had an increased expression of survivin, an inhibitor of apoptosis. Induction of aneuploid or tetraploid G(1) cells was accompanied by increased expression of CD95, p21, and BTG2 that may contribute to cell death because their expression was diminished in an EpoB-resistant cell line. In contrast, expression of GADD45 and PTGF-beta could promote cell survival. We conclude that abnormal mitotic exit is required for apoptotic cell death induced by microtubule-stabilizing drugs.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Epotilonas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Paclitaxel/farmacología , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Microtúbulos/efectos de los fármacos , Mitosis/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Poliploidía , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
A 95-fold epothilone B (EpoB)-resistant, but not dependent, A549 human lung carcinoma cell line, A549.EpoB40 (EpoB40), has a Gln to Glu mutation at residue 292 that is situated near the M-loop of betaI-tubulin. Further selection of this cell line with higher concentrations of EpoB produced A549.EpoB480 (EpoB480), which is approximately 900-fold resistant to EpoB. This cell line, like EpoB40, exhibits cross-resistance to Taxol and extreme sensitivity to vinblastine, but in contrast to EpoB40 it is unusually dependent on EpoB, requiring a minimum of 125 nmol/L EpoB to maintain normal growth. Sequence analysis of the beta-tubulin and Kalpha1-tubulin genes in EpoB480 showed that, in addition to the beta292 mutation, beta60 was mutated from Val to Phe and alpha195 was mutated from Leu to Met. Mass spectrometry indicated that both the Val(60)Phe and Leu(195)Met mutations in betaI- and Kalpha1-tubulin, respectively, were expressed at the protein level. Molecular modeling indicated that beta60 is located at the end of the H1-S2 loop that has been implicated as a principal partner of the M-loop for contacts between protofilaments. A mutation at beta60 could inhibit the lateral contacts between protofilaments, thereby destabilizing microtubules. alpha195 is located at the external surface of the microtubule that has been proposed as the domain that interacts with a variety of endogenous proteins, such as stathmin and microtubule-associated protein 4. A mutation at alpha195 could modulate the interactions between tubulin and regulatory proteins. We propose that the betaVal(60)Phe mutation plays a critical role in the drug-dependent phenotype of EpoB480 cells.
Asunto(s)
Resistencia a Antineoplásicos/genética , Epotilonas/farmacología , Mutación/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Proteínas de Microtúbulos/metabolismo , Modelos Moleculares , Paclitaxel/farmacología , Fosfoproteínas/metabolismo , Estructura Cuaternaria de Proteína , Estatmina , Tubulina (Proteína)/químicaRESUMEN
Nanomolar concentrations of Taxol, and other antimitotic agents that interact with microtubules, mediate serine phosphorylation of the 66-kDa Shc isoform (p66shc) in A549 human lung carcinoma cells, 9-18 h after drug treatment. This event coincides with the release of PARP cleavage fragments that are early indicators of apoptosis. Taxol-induced serine phosphorylation of p66shc results from a MEK-independent signaling pathway that is activated in A549 cells that have a prolonged or abnormal mitotic phase of the cell cycle [Cancer Res. 60 (2000) 5171]. In contrast, in murine macrophage RAW 264.7 cells, micromolar concentrations of Taxol but not other microtubule-interacting agents induced serine phosphorylation of p66shc that correlated with the phosphorylation of Raf-1 and extracellular signal-regulated kinase (ERK1/2), within 15-30 min after Taxol treatment. This event also was induced by lipopolysaccharide (LPS). The MEK-inhibitor, U0126, that specifically inhibits the activation of ERK also blocked the phosphorylation of p66shc and Raf-1, suggesting that these processes were MEK-dependent, quite different from that which was observed in A549 cells. Taxol also induced phosphorylation of p38 and JNK MAP kinases within 8-15 min after drug treatment. It is known that Taxol, but not other microtubule-interacting agents, induces the production of cytokines, such as tumor necrosis factor alpha (TNF-alpha) in mouse macrophages. The time course of Taxol-induced TNF-alpha expression coincides with that of Taxol-induced p66shc phosphorylation, and U0126 inhibits significantly Taxol-induced TNF-alpha expression in RAW 264.7 cells. Our data indicate that the Taxol-induced serine phosphorylation of p66shc in RAW 264.7 cells is microtubule-independent and may be related to increased TNF-alpha expression after Taxol and LPS treatment. It is concluded that the mechanisms involved in Taxol-induced p66shc phosphorylation are distinct in A549 and RAW 264.7 cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Antineoplásicos Fitogénicos/farmacología , Paclitaxel/farmacología , Proteínas/efectos de los fármacos , Proteínas/metabolismo , Animales , Butadienos/farmacología , Línea Celular , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Fosforilación , Proteínas/química , Piridinas/farmacología , Serina/química , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Treonina/química , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Despite the clinical success of microtubule-interacting agents (MIA), a significant challenge for oncologists is the inability to predict the response of individual patients with cancer to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellular resistance to the class of MIAs known as microtubule-stabilizing agents (MSA). The human lung cancer cell line A549 was compared with two drug-resistant daughter cell lines, a taxol-resistant cell line (AT12) and an epothilone B (EpoB)-resistant cell line (EpoB40). The ovarian cancer cell line Hey was compared with two drug-resistant daughter cell lines, an EpoB-resistant cell line (EpoB8) and an ixabepilone-resistant cell line (Ixab80). All 2D DIGE results were validated by Western blot analyses. A variety of cytoskeletal and cytoskeleton-associated proteins were differentially expressed in drug-resistant cells. Differential abundance of 14-3-3σ, galectin-1 and phosphorylation of stathmin are worthy of further studies as candidate predictive biomarkers for MSAs. This is especially true for galectin-1, a ß-galactose-binding lectin that mediates tumor invasion and metastasis. Galectin-1 was greatly increased in EpoB- and ixabepilone-resistant cells and its suppression caused an increase in drug sensitivity in both drug-sensitive and -resistant Hey cells. Furthermore, the growth medium from resistant Hey cells contained higher levels of galectin-1, suggesting that galectin-1 could play a role in resistance to MSAs.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Resistencia a Antineoplásicos/genética , Microtúbulos/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Electroforesis en Gel Bidimensional , Epotilonas/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de Neoplasias/genética , Paclitaxel/administración & dosificación , ProteómicaRESUMEN
Abstract Eribulin mesylate is a synthetic analog of halichondrin B known to bind tubulin and microtubules, specifically at their protein rich plus-ends, thereby dampening microtubule (MT) dynamics, arresting cells in mitosis, and inducing apoptosis. The proteins which bind to the MT plus-end are known as microtubule plus-end tracking proteins (+TIPs) and have been shown to promote MT growth and stabilization. Eribulin's plus-end binding suggests it may compete for binding sites with known +TIP proteins such as End-binding 1 (EB1). To better understand the impact of eribulin plus-end binding in regard to the proteins which normally bind there, cells expressing GFP-EB1 were treated with various concentrations of eribulin. In a concentration dependent manner, GFP-EB1 became dissociated from the MT plus-ends following drug addition. Similar results were found with immuno-stained fixed cells. Cells treated with low concentrations of eribulin also showed decreased ability to migrate, suggesting the decrease in MT dynamics may have a downstream effect. Extended exposure of eribulin to cells leads to total depolymerization of the MT array. Taken together, these data show eribulin effectively disrupts EB1 +TIP complex formation, providing mechanistic insights into the impact of eribulin on MT dynamics.
Asunto(s)
Furanos/metabolismo , Cetonas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Línea Celular Tumoral , Humanos , Tubulina (Proteína)/metabolismoRESUMEN
Potential binding modes of (+)-discodermolide at the paclitaxel binding site of tubulin have been identified by computational studies based on earlier structural and SAR data. Examination of the prospective binding modes reveal that the aromatic pocket occupied by the paclitaxel side chain is unoccupied by (+)-discodermolide. Based on these findings, a small library of (+)-discodermolide-paclitaxel hybrids have been designed and synthesized. Biological evaluation reveals a two- to eight-fold increase in antiproliferative activity compared to the parent molecule using the A549 and MCF-7 cancer cell lines.
Asunto(s)
Alcanos/síntesis química , Antineoplásicos/síntesis química , Carbamatos/síntesis química , Lactonas/síntesis química , Paclitaxel/análogos & derivados , Paclitaxel/síntesis química , Pironas/síntesis química , Alcanos/farmacología , Antineoplásicos/farmacología , Sitios de Unión , Carbamatos/farmacología , Línea Celular Tumoral , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lactonas/farmacología , Microtúbulos/química , Modelos Moleculares , Conformación Molecular , Paclitaxel/farmacología , Unión Proteica , Pironas/farmacología , Soluciones , Estereoisomerismo , Relación Estructura-Actividad , Tubulina (Proteína)/químicaRESUMEN
Centromere associated protein-E (CENP-E), a mitotic checkpoint protein, is required for efficient, stable microtubule capture at kinetochores during mitosis. Absence of CENP-E results in misaligned chromosomes leading to metaphase arrest. Microtubule-interacting agents such as Taxol and epothilone B (EpoB), at concentrations that induce mitotic arrest, transiently increase expression of CENP-E in a variety of cancer cell lines. The CENP-E level in an EpoB-resistant A549 cell line, EpoB40, is ~ 2-fold higher than in A549 cells. CENP-E overexpression, after transfection with CENP-E cDNA into drug sensitive cells, does not alter Taxol or EpoB sensitivity. However, suppression of CENP-E expression by CENP-E siRNA results in a moderate increase in drug sensitivity, suggesting that a minimal quantity of CENP-E is required for maintaining its function. It is known that CENP-E binds to BubR1 and enhances its recruitment to each unattached kinetochore. Suppression of CENP-E results in a decrease in BubR1 levels in EpoB40 cells. During metaphase, both targeting of CENP-E and BubR1 to the kinetochores and the interaction between CENP-E and BubR1 are significantly reduced in EpoB40 cells, compared to A549 cells. In addition, the distance between the two centrosomes during metaphase is shorter in EpoB40 than in A549 cells, suggesting that defects in the spindle-assembly checkpoint have occurred in EpoB40 cells during the development of drug resistance. These results indicate that defects in the mitotic checkpoint may have a role in, or be the result of, the development of EpoB resistance.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Epotilonas/farmacología , Mitosis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular Tumoral , Centrosoma/efectos de los fármacos , Centrosoma/metabolismo , Humanos , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Metafase/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacosRESUMEN
Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death. Concurrent suppression of MEK and mTOR inhibited ribosomal biogenesis by 40% within 24 h and was associated with a decreased polysome/monosome ratio that is indicative of reduced protein translation efficiency. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone or PD0325901 at the maximum tolerated dose in nude mice bearing human lung tumor xenografts or heterotransplants. Except for a PTEN mutant, all tumor models had sustained tumor regressions and minimal toxicity. These data (a) provide evidence that both pathways converge on factors that regulate translation initiation and (b) support therapeutic strategies in lung cancer that simultaneously suppress the RAS and AKT signaling network.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Proteínas Quinasas/química , Animales , Benzamidas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Retroalimentación Fisiológica , Humanos , Immunoblotting , Inmunosupresores/farmacología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosfohidrolasa PTEN , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TORRESUMEN
Taxol is widely used for the treatment of human cancer. Its mechanism of action in cells is dependent on drug concentration. At low concentrations of Taxol (5-10 nM), cells exhibit aberrant mitosis, including aneuploidy, in the absence of mitotic arrest. At higher concentrations of Taxol (>20 nM), the cell cycle is blocked at metaphase by spindle checkpoint activation. Here we demonstrate that low concentrations of Taxol cause mitotic delay, and result in an aneuploid population of cells after exit from mitosis. Low concentrations of Taxol dissociated p55CDC-Mad2 or p55CDC-BubR1 complexes after mitosis, whereas high concentrations of Taxol sustained the protein complex formation leading to mitotic block. The induction of apoptosis and aneuploidy by low concentrations of Taxol may result from chromosome missegregation caused by spindle checkpoint defects.