RESUMEN
Purpose The combretastatins (CAs) are known to exhibit anti-tumour activity but the underlying mechanism remains to be fully elucidated. Inflammation plays a critical role in altering the function of cancer cells and evasion of cell death and increased proliferation are characteristics of transformed malignancies. Many of the proteins involved in these pathways are regulated by the transcription factor NF-κB which can be activated by tumour necrosis factor (TNF-α), a pro-inflammatory cytokine released by both malignant and immune cells within the tumour microenvironment. In this study, we examined the ability of combretastatin A-4 (CA-4) and its novel, cis-restricted analogue CA-432 to target the NF-κB signalling pathway in T cells. Methods Effects of the CAs on the viability of DND-41 leukaemia and Jurkat lymphoma T-cell lines was assessed by the alamar blue assay. Induction of apoptosis and effects on expression levels of key apoptotic proteins was established though flow cytometry and western blotting. Modulation of the NF-κB signalling pathway was determined through western blotting and through assessment of NF-κB reporter gene activity. Results CA-4 and CA-432 reduced cell viability and induced apoptosis in DND-41 and Jurkat T cells and sensitised the cells to TNF-α-induced apoptosis through inhibition of the NF-κB signalling pathway. Suppression of the NF-κB pathway downregulated NF-κB-dependent gene products involved in cell survival (IAPs, Bcl-2 and Mcl-1), proliferation (cyclin D1) and inflammation (COX-2). Furthermore, both CA-4 and CA-432 inhibited TNF-α-induced NF-κB activation through the inhibition of IκBα degradation and p65 nuclear translocation and decreased NF-κB reporter gene activity. Conclusions Our data indicate that the anti-cancer properties of comebretastatins may be mediated in part through targeting the NF-κB pathway. This study provides new insights into the molecular mechanisms of CA compounds and a potential application of combretastatins for inflammatory diseases such as cancers, which are associated with abnormal NF-κB activation.
Asunto(s)
Antineoplásicos/farmacología , Bibencilos/farmacología , FN-kappa B/metabolismo , Linfocitos T/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Células Jurkat , Transducción de Señal/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The C-KIT receptor tyrosine kinase is constitutively activated in the majority of gastrointestinal stromal tumours (GIST). Imatinib (IM) a selective inhibitor of C-KIT, is indicated for the treatment of KIT-positive unresectable and/or metastatic GIST, and has tripled the survival time of patients with metastatic GIST. However, the majority of patients develop IM-resistance and progress. Although IM elicits strong antiproliferative effects, it fails to induce sufficient levels of apoptosis; acquired IM-resistance and disease recurrence remain an issue, a more effective drug treatment is greatly needed. We examined the effect of a novel microtubule-targeting agent (MTA), pyrrolo-1,5-benzoxazepine (PBOX)-15 in combination with IM on GIST cells. PBOX-15 decreased viability and in combination with IM synergistically enhanced apoptosis in both IM-sensitive and IM-resistant GIST cells, decreased the anti-apoptotic protein Mcl-1, and enhanced activation of pro-caspase-3 and PARP cleavage. The combination treatment also led to an enhanced inhibition of C-KIT-phosphorylation and inactivation of C-KIT-dependent signalling in comparison to either drug alone; CDC37, a key regulator of C-KIT in GIST was also dramatically decreased. Furthermore, PBOX-15 reduced CKII expression, an enzyme which regulates the expression of CDC37. In conclusion, our findings indicate the potential of PBOX-15 to improve the apoptotic response of IM in GIST cells and provide a more effective treatment option for GIST patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Mesilato de Imatinib/uso terapéutico , Oxazepinas/uso terapéutico , Pirroles/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chaperoninas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Citometría de Flujo , Tumores del Estroma Gastrointestinal/patología , Humanos , Mesilato de Imatinib/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Oxazepinas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirroles/farmacología , Transducción de Señal/efectos de los fármacos , Resultado del TratamientoRESUMEN
Several prodrugs of the naturally occurring combretastatins have undergone extensive clinical evaluation as vascular targeting agents (VTAs). Their increased selectivity toward endothelial cells together with their innate ability to rapidly induce vascular shutdown and inhibit tumor growth at doses up to 10-fold less than the maximum tolerated dose led to the clinical evaluation of combretastatins as VTAs. Tubulin is well established as the molecular target of the combretastatins and the vast majority of its synthetic derivatives. Furthermore, tubulin is a highly validated molecular target of many direct anticancer agents routinely used as front-line chemotherapeutics. The unique vascular targeting properties of the combretastatins have somewhat overshadowed their development as direct anticancer agents and the delineation of the various cell death pathways and anticancer properties associated with such chemotherapeutics. Moreover, the ongoing clinical trial of OXi4503 (combretastatin-A1 diphosphate) together with preliminary preclinical evaluation for the treatment of refractory acute myelogenous leukemia has successfully highlighted both the indirect and direct anticancer properties of combretastatins. In this review, we discuss the development of the combretastatins from nature to the clinic. The various mechanisms underlying combretastatin-induced cell cycle arrest, mitotic catastrophe, cell death, and survival are also reviewed in an attempt to further enhance the clinical prospects of this unique class of VTAs.
Asunto(s)
Antineoplásicos/farmacología , Estilbenos/farmacología , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Huso Acromático/efectos de los fármacos , Estilbenos/química , Estilbenos/uso terapéutico , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacología , Moduladores de Tubulina/uso terapéuticoRESUMEN
A series of azetidin-2-ones substituted at positions 1, 3 and 4 of the azetidinone ring scaffold were synthesised and evaluated for antiproliferative, cytotoxic and tubulin-binding activity. In these compounds, the cis double bond of the vascular targeting agent combretastatin A-4 is replaced with the azetidinone ring in order to enhance the antiproliferative effects displayed by combretastatin A-4 and prevent the cis/trans isomerisation that is associated with inactivation of combretastatin A-4. The series of azetidinones was synthetically accessible via the Staudinger and Reformatsky reactions. Of a diverse range of heterocyclic derivatives, 3-(2-thienyl) analogue 28 and 3-(3-thienyl) analogue 29 displayed the highest potency in human MCF-7 breast cancer cells with IC(50) values of 7 nM and 10nM, respectively, comparable to combretastatin A-4. Compounds from this series also exhibited potent activity in MDA-MB-231 breast cancer cells and in the NCI60 cell line panel. No significant toxicity was observed in normal murine breast epithelial cells. The presence of larger, bulkier groups at the 3-position, for example, 3-naphthyl derivative 21 and 3-benzothienyl derivative 26, resulted in relatively lower antiproliferative activity in the micromolar range. Tubulin-binding studies of 28 (IC(50)=1.37 µM) confirmed that the molecular target of this series of compounds is tubulin. These novel 3-(thienyl) ß-lactam antiproliferative agents are useful scaffolds for the development of tubulin-targeting drugs.
Asunto(s)
Azetidinas/química , Azetidinas/farmacología , Tubulina (Proteína)/metabolismo , Animales , Azetidinas/síntesis química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Cristalografía por Rayos X , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Ratones , Modelos Moleculares , Embarazo , Relación Estructura-Actividad , beta-Lactamas/síntesis química , beta-Lactamas/química , beta-Lactamas/farmacologíaRESUMEN
Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (ß-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected ß-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead ß-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and ß-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Azetidinas/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Guayacol/análogos & derivados , Estilbenos/farmacología , beta-Lactamas/química , Antineoplásicos Fitogénicos/química , Azetidinas/química , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Guayacol/química , Guayacol/farmacología , Células HL-60 , Humanos , Células K562 , Microscopía Confocal , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Estructura Molecular , Estereoisomerismo , Estilbenos/química , Tubulina (Proteína)/metabolismoRESUMEN
We have synthesized a series of polymethoxylated rigid analogs of combretastatin A-4 which contain a benzoxepin ring in place of the usual ethylene bridge present in the natural combretastatin products. The compounds display antiproliferative activity when evaluated against the MCF-7 and MDA human breast carcinoma cell lines. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepine (11g) was found to be the most potent product when evaluated against the MCF-7 breast cancer cell line. A brief computational study of the structure-activity relationship for the synthesized compounds is presented. These 4,5-diarylbenzoxepins are identified as potentially useful scaffolds for the further development of antitumor agents which target tubulin.
Asunto(s)
Antineoplásicos Fitogénicos , Benzoxepinas , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Estilbenos/química , Tubulina (Proteína)/metabolismo , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Benzoxepinas/síntesis química , Benzoxepinas/química , Benzoxepinas/farmacología , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Intrinsic or acquired resistance to chemotherapy is a major clinical problem that has evoked the need to develop innovative approaches to predict and ultimately reverse drug resistance. A prolonged G(2)M arrest has been associated with apoptotic resistance to various microtubule-targeting agents (MTAs). In this study, we describe the functional significance of the mitotic spindle checkpoint proteins, BubR1 and Bub3, in maintaining a mitotic arrest after microtubule disruption by nocodazole and a novel series of MTAs, the pyrrolo-1,5-benzoxazepines (PBOXs), in human cancer cells. Cells expressing high levels of BubR1 and Bub3 (K562, MDA-MB-231, and HeLa) display a prolonged G(2)M arrest after exposure to MTAs. On the other hand, cells with low endogenous levels of mitotic spindle checkpoint proteins (SK-BR-3 and HL-60) transiently arrest in mitosis and undergo increased apoptosis. The phosphorylation of BubR1 correlated with PBOX-induced G(2)M arrest in four cell lines tested, indicating an active mitotic spindle checkpoint. Gene silencing of BubR1 by small interfering RNA interference reduced PBOX-induced G(2)M arrest without enhancing apoptotic efficacy. Further analysis demonstrated that PBOX-treated BubR1-depleted cells were both mononucleated and multinucleated with a polyploid DNA content, suggesting a requirement for BubR1 in cytokinesis. Taken together, these results suggest that BubR1 contributes to the mitotic checkpoint induced by the PBOXs.
Asunto(s)
Mitosis/fisiología , Oxazepinas/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Pirroles/farmacología , Huso Acromático/metabolismo , Tubulina (Proteína)/biosíntesis , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Células HL-60 , Humanos , Células K562 , Mitosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Oxazepinas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Pirroles/uso terapéutico , Huso Acromático/efectos de los fármacos , Huso Acromático/genética , Tubulina (Proteína)/genéticaRESUMEN
Estrogen receptor α (ERα) is an important target for the design of drugs such as tamoxifen (2a) and fulvestrant (5). Three series of ER-ligands based on the benzoxepin scaffold structure were synthesized: series I containing an acrylic acid, series II with an acrylamide, and series III with a saturated carboxylic acid substituent. These compounds were shown to be high affinity ligands for the ER with nanomolar IC50 binding values. Series I acrylic acid ligands were generally ERα selective. In particular, compound 13e featuring a phenylpenta-2,4-dienoic acid substituent was shown to be antiproliferative and downregulated ERα and ERß expression in MCF-7 breast cancer cells. Interestingly, from series III, the phenoxybutyric acid derivative compound 22 was not antiproliferative and selectively downregulated ERß. A docking study of the benzoxepin ligands was undertaken. Compound 13e is a promising lead for development as a clinically relevant SERD, while compound 22 will be a useful experimental probe for helping to elucidate the role of ERß in cancer cells.
Asunto(s)
Benzoxepinas/química , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Cristalografía por Rayos X , Receptor alfa de Estrógeno/química , Receptor beta de Estrógeno/química , Humanos , Ligandos , Células MCF-7 , Modelos Moleculares , Simulación del Acoplamiento Molecular , Proteolisis/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/síntesis química , Relación Estructura-ActividadRESUMEN
Glucuronidation by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) is a cause of intrinsic drug resistance in cancer cells. Glucuronidation of combretastatin A-4 (CA-4) was previously identified as a mechanism of resistance in hepatocellular cancer cells. Herein, we propose chemical manipulation of ß-lactam bridged analogues of Combretastatin A-4 as a novel means of overcoming drug resistance associated with glucuronidation due to the expression of UGTs in the CA-4 resistant human colon cancer HT-29 cells. The alkene bridge of CA-4 is replaced with a ß-lactam ring to circumvent potential isomerisation while the potential sites of glucuronate conjugation are deleted in the novel 3-substituted-1,4-diaryl-2-azetidinone analogues of CA-4. We hypothesise that glucuronidation of CA-4 is the mechanism of drug resistance in HT-29 cells. Ring B thioether containing 2-azetidinone analogues of CA-4 such as 4-(4-(methylthio)phenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27) and 3-hydroxy-4-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (45) were identified as the most potent inhibitors of tumour cell growth, independent of UGT status, displaying antiproliferative activity in the low nanomolar range. These compounds also disrupted the microtubular structure in MCF-7 and HT-29 cells, and caused G2/M arrest and apoptosis. Taken together, these findings highlight the potential of chemical manipulation as a means of overcoming glucuronidation attributed drug resistance in CA-4 resistant human colon cancer HT-29 cells, allowing the development of therapeutically superior analogues.
Asunto(s)
Estilbenos/química , beta-Lactamas/farmacología , Antineoplásicos Fitogénicos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Resistencia a Antineoplásicos , Glucuronatos/metabolismo , Células HT29 , Humanos , Inactivación Metabólica , Estilbenos/metabolismo , Estilbenos/farmacocinética , Relación Estructura-Actividad , beta-Lactamas/químicaRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive hematopoietic malignancy prone to relapse and drug resistance. Half of all T-ALL patients exhibit mutations in Notch1, which leads to aberrant Notch1 associated signaling cascades. Notch1 activation is mediated by the γ-secretase cleavage of the Notch1 receptor into the active intracellular domain of Notch1 (NCID). Clinical trials of γ-secretase small molecule inhibitors (GSIs) as single agents for the treatment of T-ALL have been unsuccessful. The present study demonstrated, using immunofluorescence and western blotting, that blocking γ-secretase activity in T-ALL cells with N-[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester (DAPT) downregulated NCID and upregulated the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5). Upregulation of DR5 restored the sensitivity of T-ALL cells to TRAIL. Combination index revealed that the combined treatment of DAPT and TRAIL synergistically enhanced apoptosis compared with treatment with either drug alone. TRAIL combined with the clinically evaluated γ-secretase inhibitor 3-[(1r, 4s)-4-(4-chlorophenylsulfonyl)-4-(2, 5-difluorophenyl) cyclohexyl] propanoic acid (MK-0752) also significantly enhanced TRAIL-induced cell death compared with either drug alone. DAPT/TRAIL apoptotic synergy was dependent on the extrinsic apoptotic pathway and was associated with a decrease in BH3 interacting-domain death agonist and x-linked inhibitor of apoptosis. In conclusion, γ-secretase inhibition represents a potential therapeutic strategy to overcome TRAIL resistance for the treatment of T-ALL.
RESUMEN
Apoptotic defects are frequently associated with poor outcome in pediatric acute lymphoblastic leukaemia (ALL) hence there is an ongoing demand for novel strategies that counteract apoptotic resistance. The death ligand TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) and its selective tumour receptor system has attracted exceptional clinical interest. However, many malignancies including ALL are resistant to TRAIL monotherapy. Tumour resistance can be overcome by drug combination therapy. TRAIL and its agonist antibodies are currently undergoing phase II clinical trials with established chemotherapeutics. Herein, we present promising therapeutic benefits in combining TRAIL with the selective anti-leukaemic agents, the pyrrolo-1,5-benzoxazepines (PBOXs) for the treatment of ALL. PBOX-15 synergistically enhanced apoptosis induced by TRAIL and a DR5-selective TRAIL variant in ALL-derived cells. PBOX-15 enhanced TRAIL-induced apoptosis by dual activation of extrinsic and intrinsic apoptotic pathways. The specific caspase-8 inhibitor, Z-IETD-FMK, identified the extrinsic pathway as the principal mode of apoptosis. We demonstrate that PBOX-15 can enhance TRAIL-induced apoptosis by upregulation of DR5, reduction of cellular mitochondrial potential, activation of the caspase cascade and downregulation of PI3K/Akt, c-FLIP, Mcl-1 and IAP survival pathways. Of note, the PI3K pathway inhibitor LY-294002 significantly enhanced the apoptotic potential of TRAIL and PBOX-15 validating the importance of Akt downregulation in the TRAIL/PBOX-15 synergistic combination. Considering the lack of cytotoxicity to normal cells and ability to downregulate several survival pathways, PBOX-15 may represent an effective agent for use in combination with TRAIL for the treatment of ALL.
Asunto(s)
Sinergismo Farmacológico , Oxazepinas/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pirroles/administración & dosificación , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/biosíntesis , Caspasa 8/biosíntesis , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Fosfatidilinositol 3-Quinasas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated, leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl "A" ring and 4-phenyl "B" ring for potent antiproliferative activity and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC50 values of 38 and 19 nM, respectively, in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular structure in MCF-7 cells as visualized by confocal microscopy, and caused G2/M arrest and apoptosis. Compound 90b possessed a mean GI50 value of 22 nM in the NCI60 cell line screen, displayed minimal cytotoxicity, and was shown to interact at the colchicine-binding site on ß-tubulin. Phosphate and amino acid prodrugs of both 78b and 90b were synthesized, of which the alanine amide 102b retained potency and is a promising candidate for further clinical development.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Azetidinas/síntesis química , Azetidinas/farmacología , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/efectos de los fármacos , Apoptosis/efectos de los fármacos , Sitios de Unión/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Fase G2/efectos de los fármacos , Humanos , Microtúbulos/efectos de los fármacos , Modelos Moleculares , Relación Estructura-Actividad , beta-Lactamas/farmacologíaRESUMEN
Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Oxazepinas/farmacología , Pirroles/farmacología , Receptores de Estrógenos/análisis , Animales , Ciclo Celular/efectos de los fármacos , Supervivencia Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/fisiología , Receptores de Estrógenos/fisiología , Células Tumorales CultivadasRESUMEN
The synthesis and biochemical activities of novel water-soluble ß-lactam analogues of combretastatin A-4 are described. The first series of compounds investigated, ß-lactam phosphate esters 7a, 8a and 9a, exhibited potent antiproliferative activity and caused microtubule disruption in human breast carcinoma-derived MCF-7 cells. They did not inhibit tubulin polymerisation in vitro, indicating that biotransformation was necessary for their antiproliferative and tubulin binding effects in MCF-7 cells. The second series of compounds, ß-lactam amino acid amides (including 10k and 11l) displayed potent antiproliferative activity in MCF-7 cells, disrupted microtubules in MCF-7 cells and also inhibited the polymerisation of tubulin in vitro. This indicates that the ß-lactam amides did not require metabolic activation to have antiproliferative effects, in contrast to the phosphate series. Both series of compounds caused mitotic catastrophe and apoptosis in MCF-7 cells. Molecular modelling studies indicated potential binding conformations for the ß-lactam amino acid amides 10k and 11l in the colchicine-binding site of tubulin. Due to their aqueous solubility and potent biochemical effects, these compounds are promising candidates for further development as microtubule-disrupting agents.
Asunto(s)
Aminoácidos/farmacología , Antineoplásicos/farmacología , Microtúbulos/efectos de los fármacos , Fosfatos/farmacología , Estilbenos/farmacología , Aminoácidos/síntesis química , Aminoácidos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Células MCF-7 , Modelos Moleculares , Estructura Molecular , Fosfatos/síntesis química , Fosfatos/química , Solubilidad , Estilbenos/síntesis química , Estilbenos/química , Relación Estructura-ActividadRESUMEN
The pyrrolo-1,5-benzoxazepines (PBOXs) are a novel group of selective apoptotic agents displaying promising therapeutic potential in both ex vivo chemotherapy-refractory patient samples and in vivo murine carcinoma models. In this report, we present novel data concerning the induction of autophagy by the PBOXs in adenocarcinoma-derived colon cancer cells. Autophagy is a lysosome-dependent degradative pathway recently associated with chemotherapy. However, whether autophagy facilitates cell survival in response to chemotherapy or contributes to chemotherapy-induced cell death is highly controversial. Autophagy was identified by enhanced expression of LC3B-II, an autophagosome marker, an increase in the formation of acridine orange-stained cells, indicative of increased vesicle formation and electron microscopic confirmation of autophagic structures. The vacuolar H+ ATPase inhibitor bafilomycin-A1 (BAF-A1) inhibited vesicle formation and enhanced the apoptotic potential of PBOX-6. These findings suggest a cytoprotective role of autophagy in these cells following prolonged exposure to PBOX-6. Furthermore, BAF-A1 and PBOX-6 interactions were determined to be synergistic and caspase-dependent. Potentiation of PBOX-6-induced apoptosis by BAF-A1 was associated with a decrease in the levels of the anti-apoptotic protein, Mcl-1. The data provide evidence that autophagy functions as a survival mechanism in colon cancer cells to PBOX-6-induced apoptosis and a rationale for the use of autophagy inhibitors to further enhance PBOX6induced apoptosis in colon cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Autofagia/genética , Neoplasias del Colon/tratamiento farmacológico , Macrólidos/administración & dosificación , Oxazepinas/administración & dosificación , Pirroles/administración & dosificación , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/patología , Sinergismo Farmacológico , Humanos , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
In recent years an extensive series of synthetic combretastatin A-4 (CA-4)-azetidinone (ß-lactam) hybrids were designed and synthesised with a view to improve the stability, therapeutic efficacy and aqueous solubility of CA-4. Lead compounds containing a 3,4,5-trimethoxy aromatic ring at position 1 and a variety of substitution patterns at positions 3 and 4 of the ß-lactam ring were screened in three adenocarcinoma-derived colon cancer cell lines (CT-26, Caco-2 and the CA-4 resistant cell line, HT-29). In both CT-26 and Caco-2 cells all ß-lactam analogues analysed displayed potent therapeutic efficacy within the nanomolar range. Substitution of the ethylene bridge of CA-4 with the ß-lactam ring together with the aforementioned aryl substitutions improved the therapeutic efficacy of CA-4 up to 300fold in the combretastatin refractory HT-29 cells. The lead compound combretazet-3 (CAZ-3); chemical name [4-(3-hydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one] demonstrated improved chemical stability together with enhanced therapeutic efficacy as compared with CA-4 whilst maintaining the natural biological properties of CA-4. Furthermore, CAZ-3 demonstrated significant tumour inhibition in a murine model of colon cancer. Our results suggest that combretastatin-azetidinone hybrids represent an effective novel therapy for the treatment of combretastatin resistant carcinomas.
Asunto(s)
Antineoplásicos/farmacología , Azetidinas/farmacología , Neoplasias del Colon/tratamiento farmacológico , Guayacol/análogos & derivados , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/metabolismo , Autofagia/efectos de los fármacos , Azetidinas/administración & dosificación , Azetidinas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/patología , Estabilidad de Medicamentos , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Guayacol/administración & dosificación , Guayacol/metabolismo , Guayacol/farmacología , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Combretastatin A-4 (CA-4) is a naturally occurring microtubular-destabilising agent that possesses potent anti-tumour and anti-vascular properties both in vitro and in vivo. Clinical trials to date indicate that its water-soluble prodrug, combretastatin A-4 phosphate (CA-4P), is well tolerated at therapeutically useful doses. However, the stilbenoid structure of CA-4, consisting of two phenyl rings linked by an ethylene bridge, renders the compound readily susceptible to isomerisation from its biologically active cis-conformation to its more thermodynamically stable but inactive trans-isomer. To circumvent this problem, we synthesised a series of cis-restricted CA-4 analogues. Replacement of the ethylene bridge with a 1,4-diaryl-2-azetidinone (ß-lactam) ring provided a rigid scaffold thus preventing cis-trans isomerisation. We previously documented that these tubulin-depolymerising ß-lactam compounds potently induced cell cycle arrest and apoptosis in a variety of cancerous cell lines (including those displaying multidrug resistance) and ex vivo patient samples, whilst exerting only minimal toxicity to normal cells. The purpose of this study was to elucidate the effect of the ß-lactam compounds on both tumour vascularisation and tumour cell migration, two critical elements that occur during the growth and metastatic progression of tumours. We established that two representative ß-lactam compounds, CA-104 and CA-432, exerted both anti-endothelial effects [G2/M arrest and apoptosis of primary human umbilical vein endothelial cells (HUVECs)] and anti-angiogenic effects [inhibition of HUVEC migration and differentiation and reduced vascular endothelial growth factor (VEGF) release from MDA-MB-231 breast adenocarcinoma cells]. In addition, we established that lead analogue, CA-432, abrogated the migration of MDA-MB-231 cells indicating an anti-metastatic function for these compounds. In summary, our results to date collectively indicate that these cis-restricted ß-lactam CA-4 analogues may prove to be useful alternatives to CA-4 in the treatment of cancer but with the added advantage of improved stability of the cis-isomer.
Asunto(s)
Adenocarcinoma/metabolismo , Antineoplásicos/farmacología , Azetidinas/farmacología , Neoplasias de la Mama/metabolismo , Movimiento Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Guayacol/análogos & derivados , beta-Lactamas/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Guayacol/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Microtúbulos/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Recent clinical data demonstrated that the vascular targeting agent Combretastatin-A4 phosphate (CA-4P) prolonged survival of patients with advanced anaplastic thyroid cancer without any adverse side effects. However, as a single agent CA-4 failed to reduce tumour growth in the murine CT-26 adenocarcinoma colon cancer model. Furthermore, the molecular mechanism of the innate resistance of HT-29 human adenocarcinoma cells to CA-4 is largely unknown. In this report, we demonstrate for the first time that prolonged exposure to CA-4 and an azetidinone cis-restricted analogue, CA-432 (chemical name; 4-(3-Hydroxy-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one) induced autophagy in adenocarcinoma-derived CT-26, Caco-2 and HT-29 cells but not in fibrosarcoma-derived HT-1080 cells. Autophagy is a fundamental self-catabolic process which can facilitate a prolonged cell survival in spite of adverse stress by generating energy via lysosomal degradation of cytoplasmic constituents. Autophagy was confirmed by acridine orange staining of vesicle formation, electron microscopy and increased expression of LC3-II. Combretastatin-induced autophagy was associated with a loss of mitochondrial membrane potential and elongation of the mitochondria. Furthermore, inhibition of autophagy by the vacuolar H(+)ATPase inhibitor Bafilomycin-A1 (BAF-A1) significantly enhanced CA-432 induced HT-29 cell death. Both CA-4 and its synthetic derivative, CA-432 induced the formation of large hyperdiploid cells in Caco-2 and CT-26 cells. The formation of these polyploid cells was significantly inhibited by autophagy inhibitor, BAF-A1. Results presented within demonstrate that autophagy is a novel response to combretastatin exposure and may be manipulated to enhance the therapeutic efficacy of this class of vascular targeting agents.
Asunto(s)
Adenocarcinoma/inmunología , Autofagia/efectos de los fármacos , Neoplasias del Colon/inmunología , Estilbenos/farmacología , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Citometría de Flujo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiologíaRESUMEN
BubR1 is a well-defined guardian of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome alignment across the mitotic plate. However, the role of BubR1 in combretastatin-induced cell death remains unknown. In this study, we describe the effects of combretastatin A-4 (CA-4) and a synthetic cis-restricted 3,4-diaryl-2-azetidinone (ß-lactam) analogue (CA-432) on the modulation and phosphorylation of BubR1 in human cervical cancer-derived cells. We demonstrate that CA-4 and CA-432 depolymerise the microtubular network of human cervical carcinoma-derived cells. Both compounds induced the disassembly of the microtubules and the loss of microtubule tension led to the early phosphorylation of BubR1 and the late cleavage of BubR1. The phosphorylation of BubR1 correlated with the onset of G2M cell cycle arrest whilst the cleavage of BubR1 coincided with apoptosis induced by the combretastatins. The combretastatin-induced apoptosis and the BubR1 cleavage were caspase-dependent. In vitro enzyme digests demonstrated that combretastatin-activated BubR1 is a substrate for caspase-3. Gene silencing of BubR1 with small interfering RNA severely compromised combretastatin-induced G2M cell cycle arrest with a corresponding increase in the formation of polyploid cells in both cervical and breast cancer-derived cells. In summary, BubR1 is required to maintain the G2M arrest and limit the formation of polyploid cells in response to continued combretastatin exposure. Moreover, substitution of the ethylene bridge with 3,4-diaryl-2-azetidinone did not alter the tubulin depolymerising properties or the subsequent mitotic spindle checkpoint response to CA-4 in human cancer cells.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Azetidinas/farmacología , Guayacol/análogos & derivados , Mitosis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Estilbenos/farmacología , Antineoplásicos Fitogénicos/síntesis química , Apoptosis/efectos de los fármacos , Azetidinas/síntesis química , Caspasa 3/química , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Guayacol/síntesis química , Guayacol/farmacología , Humanos , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Estilbenos/síntesis química , Tubulina (Proteína)/metabolismoRESUMEN
The structure-activity relationships of antiproliferative ß-lactams, focusing on modifications at the 4-position of the ß-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7 cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an IC(50) value of 0.22 µM. The mechanism of action was demonstrated to be by inhibition of tubulin polymerisation. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of the cell cycle and induction of apoptosis. Additionally, mitotic catastrophe for combretastatin A-4 and for 26 was demonstrated in breast cancer cells for the first time, as evidenced by the formation of giant, multinucleated cells.