RESUMEN
OBJECTIVES: To determine if SAHA, a histone deacetylase inhibitor, decreases ovarian cancer cell viability when combined with paclitaxel in vitro, and to explore molecular alterations of combined paclitaxel+SAHA treatment. METHODS: SKOV3 and Hey ovarian cancer cell lines were treated for 24 h with paclitaxel, then re-treated with SAHA or paclitaxel for an additional 48 h. Protein extracts were prepared at 48 h for western blot analysis. Cell viability was assessed at 72 h using the ApoAlert Annexin V Apoptosis Kit. RESULTS: SAHA causes G1 and G2 cell cycle arrest in ovarian cancer cell lines. Cell viability was significantly reduced by combined paclitaxel+SAHA treatment. In Hey cells, viability was reduced to 67% with paclitaxel, and to 48% with paclitaxel+SAHA (p<0.001). In the SKOV3 cell line, viability was reduced to 70% with continuous paclitaxel treatment, and was further reduced to 57% in the combined treatment group (p<0.05). Increased PARP cleavage was noted in the paclitaxel+SAHA groups. SAHA increased expression of p21cip1/waf1 and p27Kip1, down regulated cyclins A and B, and suppressed CDK1. Paclitaxel induced expression of survivin, an inhibitor of apoptosis protein, was reduced to baseline control levels with the addition of SAHA. The pro-apoptotic protein, Bad, was also increased with SAHA. CONCLUSIONS: Paclitaxel+SAHA reduces cell viability in excess of either agent alone in ovarian cancer cell lines. Cell death is mediated via several mechanisms including G1/G2 arrest from CDK1 downregulation, inhibition of paclitaxel-induced survivin accumulation, and from increased Bad expression.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/biosíntesis , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclina B/antagonistas & inhibidores , Ciclina B/biosíntesis , Sinergismo Farmacológico , Femenino , Humanos , Ácidos Hidroxámicos/administración & dosificación , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , VorinostatRESUMEN
Chemotherapeutic treatment with combinations of drugs is front-line therapy for many types of cancer. Combining drugs which target different signaling pathways often lessens adverse side effects while increasing the efficacy of treatment and reducing patient morbidity. A defined scheduling protocol is described by which histone deacetylase inhibitors (HDIs) facilitate the cytotoxic effectiveness of the topoisomerase I inhibitor camptothecin in the killing of tumor cells. Breast and lung cancer cell lines were treated with camptothecin and sodium butyrate (NaB) or suberoylanilide hydroxamic acid on the day of, the day before, or the day after camptothecin addition. Depending on the time of addition, NaB-treated cells displayed a spectrum of responses from protection to sensitization, indicating the critical nature of timing in the use of HDIs. The IC80 (72-hour assay) dose of 100 nmol/L camptothecin could be lowered to 15 nmol/L camptothecin while maintaining or surpassing cell killing of the single agent if combined with an HDI added 24 to 48 hours after camptothecin. Experiments determined that cells arrested in G2-M by camptothecin were most sensitive to subsequent HDI addition. Western blot analysis indicated that in camptothecin-arrested cells, NaB decreases cyclin B levels, as well as the levels of the antiapoptotic proteins XIAP and survivin. These findings suggest that reducing the levels of these critical antiapoptotic factors may increase the efficacy of topoisomerase I inhibitors in the clinical setting if given in a sequence that does not prevent or inhibit tumor cell progression through the S phase.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de Histona Desacetilasas , Neoplasias Pulmonares/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Butiratos/administración & dosificación , Camptotecina/administración & dosificación , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclina B/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Interacciones Farmacológicas , Inhibidores Enzimáticos/administración & dosificación , Humanos , Ácidos Hidroxámicos/administración & dosificación , Proteínas Inhibidoras de la Apoptosis , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas/metabolismo , Survivin , Inhibidores de Topoisomerasa I , Vorinostat , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Initial chemotherapeutic treatment triggers a stress-related response, which can lead to an increase in the expression of survival proteins. In this study we examine whether paclitaxel (PTX) alters the expression and/or phosphorylation of the translation initiation proteins, eukaryotic initiation factor 4E (eIF-4E) and 4E-binding protein (4E-BP1), a suppressor of eIF-4E in the dephosphorylated state. We found that PTX induced the hyperphosphorylation of 4E-BP1 in the breast cancer cell line, MDA MB 231, which reduced its association with eIF-4E, but did not alter the expression and phosphorylation of eIF-4E. The hyperphosphorylation of 4E-BP1 correlated with G2/M accumulation and with an increase in the phosphorylation of cdk1 substrates. Cotreatment with a histone deacetylase inhibitor (an indirect inhibitor of cdk activity), purvalanol A and roscovitine (direct cdk inhibitors), and the reduction of cyclin B expression using RNA interference decreased the hyperphosphorylation of 4E-BP1 in PTX treated cells. The hyperphosphorylation of 4E-BP1 by PTX increased the association of eIF-4E with eIF-4G, whereas cotreatment with purvalanol A inhibited the association of eIF-4E with eIF-4G in PTX treated cells. Taken together, our data suggest that PTX-increases the functional level of eIF-4E by promoting the hyperphosphorylation and release of 4E-BP1 through a cdk1-dependent mechanism.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas Portadoras/metabolismo , Paclitaxel/farmacología , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , División Celular/efectos de los fármacos , Línea Celular Tumoral , Factor 4E Eucariótico de Iniciación/metabolismo , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Fosforilación/efectos de los fármacos , Unión Proteica , Biosíntesis de ProteínasRESUMEN
To form metastases, tumors must break from the primary tumor site, invade surrounding tissues, enter and survive within the circulation and ultimately colonize a distal tissue. Each of these steps requires the cooperative function of numerous proteins--proteins that facilitate angiogenesis (e.g., VEGF), cell survival (e.g., Bcl-2), invasion (e.g., MMPs), and autocrine growth stimulation (e.g., c-myc, cyclin D1). Although expression of these proteins is regulated at many levels by disparate stimuli, translation of these key malignancy-related proteins is regulated primarily by the activity of the mRNA cap-binding protein eIF-4E, the rate-limiting member of the eIF-4F translation initiation complex. By binding the cap structure at the 5' terminus of cellular mRNAs, eIF-4E recruits mRNAs to the eIF-4F complex, which then scans from the 5' cap through the untranslated region (5'UTR), unwinding secondary structure to reveal the translation initiation codon and to enable ribosome loading. Messenger RNAs with short unstructured 5' UTRs are more easily translated than mRNAs harboring lengthy, highly structured 5' UTRs, as these prohibit efficient scanning and start codon recognition. As such, the translation of these mRNAs, which typically encode proteins involved in angiogenesis (e.g., VEGF), tumor growth (cyclin D1) and survival (Bcl-2), is suppressed except when eIF-4E is engaged with the eIF-4F complex--a common event in many human and experimental cancers. This review focuses on the hypothesis that enhanced eIF-4E function contributes to metastatic progression by selectively upregulating the translation of key malignancy-related proteins that together conspire to drive the metastatic process.
Asunto(s)
Factor 4E Eucariótico de Iniciación/fisiología , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Progresión de la Enfermedad , Humanos , Regulación hacia ArribaRESUMEN
BACKGROUND: Recurrence of drug-resistant disease contributes to the high mortality of ovarian cancer patients, which necessitates the identification of additional chemotherapeutic drugs. Histone deacetylase inhibitors (HDAIs) induce apoptosis in a number of malignant cell types and may represent a new class of drugs clinically relevant in the treatment of ovarian cancer. MATERIALS AND METHODS: Ovarian cancer cells were treated with various combinations of a HDAI and paclitaxel (PTX). Cell death was measured using annexin V/propidium iodide exclusion. RESULTS: The PTX/HDAI drug combination was as efficient in inducing cell death as continuous PTX treatment and superior to continuous HDAI treatment. Reversing the sequence of drug exposure reduced the cytotoxic efficacy of the drug combination. The p53 status of the cell lines did not alter the cytotoxic efficacy of the treatment protocols. CONCLUSION: These results suggest that HDAIs possess possible clinical applications as an adjuvant therapy in the treatment of ovarian cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Butiratos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Proteína p53 Supresora de Tumor/biosíntesis , Butiratos/administración & dosificación , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Inhibidores Enzimáticos/administración & dosificación , Femenino , Humanos , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Proteína p53 Supresora de Tumor/antagonistas & inhibidoresRESUMEN
NME1 is a well-documented metastasis suppressor gene, with suppressor activity demonstrated across a wide spectrum of human cancers including melanoma and carcinomas of the breast, stomach and thyroid. A primary aim of the current study was to identify profiles of genes whose expression is regulated by NME1 in cell lines of melanoma and thyroid carcinoma origin. Impact of NME1 was determined by forcing its expression transiently in cell lines using a novel Ad5-based adenoviral vector (Ad5-NME1), followed 48 h later by analysis of RNA expression profiles using the U133A microarray chip. Robust NME1 expression was achieved following infection with the Ad5-NME1 adenovirus in the human metastasis-derived cell lines WM1158 (melanoma) and WRO82 (follicular thyroid carcinoma), resulting in wide-ranging effects on gene expression in both settings. A substantial proportion of the NME1-regulated genes identified in the analyses were of clear potential relevance to metastasis, such as matrix metalloproteinase-1 (MMP1), angiopoietin-2 (ANGPT2), SERPINB9 and colony stimulating factor receptor-2B (CSFR2B). Nine genes were identified (false discovery rate <0.1) that were regulated by NME1 in both the WM1158 and WRO82 cell lines, each possessing one or more such metastasis-relevant activities as stress fiber formation and focal adhesion (PPM1E, ZYX, PFN1), chemotaxis (CCR1) epithelial-mesenchymal signaling (WNT6), differentiation and morphogenesis (TBX4, ZFP36L2), and G protein modulation (GPR52 and PFN1). In addition, a number of the NME1-regulated genes were shown to be of prognostic value for distant disease-free survival and overall survival in melanoma and breast cancer. The combined expression of three NME1-regulated genes CSFR2B, MSF4A1 and SERPINB9 provided a strongly synergistic correlation with distant disease-free survival in the basal subtype of breast cancer (p<3.5e(-5), hazard ratio=0.33). Our study demonstrates that analysis of NME1-dependent gene expression is a powerful approach for identifying potential modulators of metastatic potential in multiple cancer types, which in turn may represent useful therapeutic targets. The study also highlights NME1-dependent genes as potential prognostic/diagnostic indices, which are profoundly lacking at present in melanoma.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Melanoma/patología , Nucleósido Difosfato Quinasas NM23/genética , Adenoviridae/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Análisis por Conglomerados , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Humanos , Melanoma/mortalidad , Nucleósido Difosfato Quinasas NM23/metabolismo , Metástasis de la Neoplasia , Evaluación del Resultado de la Atención al Paciente , Pronóstico , Neoplasias de la Tiroides/genéticaRESUMEN
OBJECTIVE: To determine effects of suberoylanilide hydroxamic acid (SAHA) with and without paclitaxel in ovarian cancer cells and a nude mouse model. METHODS: Cell viability and apoptosis of ovarian cancer cells (2774) were measured following exposure to control, SAHA, paclitaxel, or SAHA in combination with paclitaxel. Nude mice were injected intraperitoneally (IP) with cancer cells and then groups received variable SAHA doses (25-100 mg/kg/day). In a second experiment, mice were inoculated with cancer and treated IP with vehicle injection, SAHA, paclitaxel, paclitaxel followed by SAHA, or SAHA followed by paclitaxel. Survival, tumor weight, and ascites were evaluated. RESULTS: SAHA decreased viability and increased apoptosis similarly to paclitaxel, but the combination was not statistically significantly different from the single agents. The only significant difference in the SAHA alone mouse study was decreased survival in the 50 mg/kg/daily group. In the combination groups, SAHA followed by paclitaxel, paclitaxel alone, and paclitaxel followed by SAHA improved survival compared with control (p=0.0358, 0.0006, and 0.0001), but SAHA alone did not (p=0.524). The paclitaxel followed by SAHA group had improved survival compared to SAHA followed by paclitaxel (p=0.0002) but not compared to paclitaxel alone (p=0.166). CONCLUSIONS: In vitro, SAHA alone decreased viability and increased apoptosis similarly to paclitaxel. In vivo, paclitaxel followed by SAHA and paclitaxel alone increased survival compared with SAHA alone or SAHA followed by paclitaxel. This suggests adding SAHA to ovarian cancer chemotherapy could increase efficacy and that sequencing of agents is important.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de Histona Desacetilasas , Neoplasias Ováricas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ratones , Ratones Desnudos , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Vorinostat , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hyperoxia is cytotoxic and depresses many cellular metabolic functions including protein synthesis. Translational control is exerted primarily during initiation by two mechanisms: 1) through inhibition of translation initiation complex formation via sequestration of the cap-binding protein, eukaryotic initiation factor (eIF) 4E, with inhibitory 4E-binding proteins (4E-BP); and 2) by prevention of eIF2-GTP-tRNA(i)(Met) formation and eIF2B activity by phosphorylated eIF2alpha. In this report, exposure of human lung fibroblasts to 95% O2 decreased the incorporation of thymidine into DNA at 6 h and the incorporation of leucine into protein beginning at 12 h. The reductions in DNA and protein synthesis were accompanied by increased phosphorylation of eIF4E protein and reduced phosphorylation of 4E-BP1. At 24 h, hyperoxia shifted 4E-BP1 phosphorylation to lesser-phosphorylated isoforms, increased eIF4E expression, and increased the association of eIF4E with 4E-BP1. Although hyperoxia did not change eIF2alpha expression, it increased its phosphorylation at Ser51, but not until 48 h. In addition, the activation of eIF2alpha was not accompanied by the formation of stress granules. These findings suggest that hyperoxia diminishes protein synthesis by increasing eIF4E phosphorylation and enhancing the affinity of 4E-BP1 for eIF4E.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Hiperoxia/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Proliferación Celular , Células Cultivadas , Gránulos Citoplasmáticos/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/ultraestructura , Humanos , Hiperoxia/genética , Hiperoxia/patología , Leucina/metabolismo , Pulmón/metabolismo , Pulmón/patología , Factores de Elongación de Péptidos/metabolismo , Fosforilación , Inhibidores de la Síntesis de la Proteína/metabolismo , Estrés Mecánico , Factores de TiempoRESUMEN
Control of protein synthesis resides at the level of eukaryotic translation initiation (eIF) complex formation. Complex formation is regulated by the mRNA cap-binding protein, eIF4E, whose activity is influenced by phosphorylation and binding to 4E-binding protein 1 (4E-BP1). To provide a link between alterations in protein synthesis and the pathogenesis of oxidant-mediated lung disease, we investigated the effect of hydrogen peroxide (H2O2) on actively growing A549 cells. Cells were exposed to 200 or 400 microM H2O2 for 4 h and then assessed for changes in proliferation, protein synthesis, and eIF4E and 4E-BP1 status over 72 h. We found that both concentrations of H2O2 inhibited [3H]thymidine incorporation and cell division while inducing a G2/M-predominant growth arrest within 24 h. In addition, H2O2 increased cell size, [3H]leucine incorporation/cell, and total cell protein. Although time had little effect on eIF4E and 4E-BP1 expression and phosphorylation state of control cells, H2O2 induced a 2- to 3-fold increase in eIF4E and 4E-BP1 expression, a 5-fold increase in eIF4E phosphorylation, and a shift in the distribution of 4E-BP1 phosphorylation favoring lesser phosphorylated forms. These findings suggest that oxidant-mediated alterations in protein synthesis and cell morphology occur in concert with changes in factors known to regulate translation kinetics.
Asunto(s)
Proteínas Portadoras/metabolismo , Peróxido de Hidrógeno/farmacología , Hipertrofia/metabolismo , Oxidantes/farmacología , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenocarcinoma , Biomarcadores de Tumor/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Tamaño de la Célula/efectos de los fármacos , Factor 4E Eucariótico de Iniciación , Citometría de Flujo , Humanos , Neoplasias Pulmonares , Fosforilación , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Células Tumorales CultivadasRESUMEN
The synthesis of a novel E-ring modified keto ether analogue of camptothecin and homocamptothecin by the cascade radical annulation route is reported. The analogue, Du1441, is an isomer of homocamptothecin, but includes the alpha-hydroxy carbonyl functionality that camptothecin possesses and homocamptothecin lacks. Despite these similarities, the new keto ether analogue is inactive in cell assays, and implications for the structure/activity relationship are discussed.
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Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Camptotecina/síntesis química , Camptotecina/farmacología , Cromatografía Líquida de Alta Presión , Éteres/química , Humanos , Estructura Molecular , Análisis Espectral , Células Tumorales CultivadasRESUMEN
The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5'-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3'-portions of both 5'-SHS strands in either single-stranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5'-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5'-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5'- and 3'-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (-82 to +8). Activity of the negative regulatory region (-1853 to -883), which contains the 5'-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.