RESUMO
The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.
Assuntos
Apoptose , Transformação Celular Neoplásica/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Citoproteção , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteína Fosfatase 2CRESUMO
Elevated serum levels of hepatocyte growth factor (HGF) and high tumor expression of c-Met are both indicators of poor overall survival from ovarian cancer (OVCA). In the present study, we evaluated the role of the HGF signaling pathway in OVCA cell line chemoresistance and OVCA patient overall survival as well as the influence of HGF/c-Met signaling inhibition on the sensitivity of OVCA cells to combinational carboplatin plus paclitaxel therapy. The prevalence of the HGF receptor, c-Met, was determined by immunohistochemistry in primary OVCA samples (n=79) and OVCA cell lines (n=41). The influence of the c-Met-specific inhibitor MK8033 on OVCA cell sensitivity to combinations of carboplatin plus paclitaxel was examined in a subset of OVCA cells (n=8) by CellTiter-Blue cell viability assays. Correlation tests were used to identify genes associated with response to MK8033 and carboplatin plus paclitaxel. Identified genes were evaluated for influence on overall survival from OVCA using principal component analysis (PCA) modeling in an independent clinical OVCA dataset (n=218). Immunohistochemistry analysis indicated that 83% of OVCA cells and 92% of primary OVCA expressed the HGF receptor, c-Met. MK8033 exhibited significant anti-proliferative effects against a panel of human OVCA cell lines. Combination index values determined by the Chou-Talalay isobologram equation indicated synergistic activity in combinations of MK8033 and carboplatin plus paclitaxel. Pearson's correlation identified a 47-gene signature to be associated with MK8033-carboplatin plus paclitaxel response. PCA modeling indicated an association of this 47-gene response signature with overall survival from OVCA (P=0.013). These data indicate that HGF/c-Met pathway signaling may influence OVCA chemosensitivity and overall patient survival. Furthermore, HGF/c-Met inhibition by MK8033 represents a promising new therapeutic avenue to increase OVCA sensitivity to carboplatin plus paclitaxel.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzocicloeptenos/farmacologia , Carboplatina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Sulfonamidas/farmacologia , Benzocicloeptenos/administração & dosagem , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sinergismo Farmacológico , Feminino , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Paclitaxel/administração & dosagem , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/administração & dosagemRESUMO
OBJECTIVE: Most women with advanced-stage epithelial ovarian cancer (OVCA) ultimately develop chemoresistant recurrent disease. Therefore, a great need to develop new, more active, and less toxic agents and/or to optimize the efficacy of existing agents exists. METHODS: In this study, we investigated the activity of Avemar, a natural, nontoxic, fermented wheat germ extract (FWGE), against a range of OVCA cell lines, both alone and in combination with cisplatin chemotherapy and delineated the molecular signaling pathways that underlie FWGE activity at a genome-wide level. RESULTS: We found that FWGE exhibited significant antiproliferative effects against 12 human OVCA cell lines and potentiated cisplatin-induced apoptosis. Pearson correlation of FWGE sensitivity and gene expression data identified 2142 genes (false discovery rate < 0.2) representing 27 biologic pathways (P < 0.05) to be significantly associated with FWGE sensitivity. A parallel analysis of genomic data for 59 human cancer cell lines matched to chemosensitivity data for 2,6-dimethoxy-p-benzoquinone, a proposed active component of FWGE, identified representation of 13 pathways common to both FWGE and 2,6-dimethoxy-p-benzoquinone sensitivity. CONCLUSIONS: Our findings confirm the value of FWGE as a natural product with anticancer properties that may also enhance the activity of existing therapeutic agents. Furthermore, our findings provide substantial insights into the molecular basis of FWGE's effect on human cancer cells.
Assuntos
Antineoplásicos/uso terapêutico , Benzoquinonas/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Ovarianas/genética , Extratos Vegetais/farmacologia , Transdução de SinaisRESUMO
Ovarian cancer (OVCA) is the most lethal gynecological malignancy. The high mortality rate associated with this disease is due in large part to the development of resistance to chemotherapy; however, the biological basis of this remains unclear. Gemcitabine is frequently used for the treatment of patients with platinum-resistant OVCA. We report molecular signaling pathways associated with OVCA response to gemcitabine. Forty-one OVCA cell lines were subjected to gene expression analysis; in parallel, IC50 values for gemcitabine were quantified using CellTiter-Blue viability assays. Pearson's correlation coefficients were calculated for gene expression and gemcitabine IC50 values. The genes associated with gemcitabine sensitivity were subjected to pathway analysis. For the identified pathways, principal component analysis was used to derive pathway signatures and corresponding scores, which represent overall measures of pathway expression. Expression levels of the identified pathways were then evaluated in a series of clinico-genomic datasets from 142 patients with stage III/IV serous OVCA. We found that in vitro gemcitabine sensitivity was associated with expression of 131 genes (p<0.001). These genes include significant representation of three molecular signaling pathways (p<0.02): O-glycan biosynthesis, Role of Nek in cell cycle regulation and Antiviral actions of Interferons. In an external clinico-genomic OVCA dataset (n=142), expression of the O-glycan pathway was associated with overall survival, independent of surgical cytoreductive status, grade and age (p<0.001). Expression levels of Role of Nek in cell cycle regulation and Antiviral actions of Interferons were not associated with survival (p=0.31 and p=0.54, respectively). Collectively, expression of the O-glycan biosynthesis pathway, which modifies protein function via post-translational carbohydrate binding, is independently associated with overall survival from OVCA. Our findings shed light on the molecular basis of OVCA responsiveness to gemcitabine and also identify a signaling pathway that may influence patient survival.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Ovarianas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Vias Biossintéticas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Glicosilação , Humanos , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Polissacarídeos/biossíntese , Análise de Componente Principal , Transdução de Sinais , Transcrição Gênica , GencitabinaRESUMO
OBJECTIVE: The BCL2 family proteins are critical mediators of cellular apoptosis and, as such, have been implicated as determinants of cancer cell chemo-sensitivity. Recently, it has been demonstrated that the phosphorylation status of the BCL2 antagonist of cell death (BAD) protein may influence ovarian cancer (OVCA) cell sensitivity to cisplatin. Here, we sought to evaluate how kinase and phosphatase components of the BAD apoptosis pathway influence OVCA chemo-sensitivity. METHODS: Protein levels of cyclin-dependent kinase 1 (CDK1) and protein phosphatase 2C (PP2C) were measured by immunofluorescence in a series of 64 primary advanced-stage serous OVCA patient samples. In parallel, levels of cAMP-dependent protein kinase (PKA), AKT, and PP2C were quantified by Western blot analysis in paired mother/daughter platinum-sensitive/resistant OVCA cell lines (A2008/C13, A2780S/A2780CP, Chi/ChiR). BAD pathway kinase CDK1 was depleted using siRNA transfection, and the influence on BAD phosphorylation and cisplatin-induced apoptosis was evaluated. RESULTS: OVCA patient samples that demonstrated complete responses to primary platinum-based therapy demonstrated 4-fold higher CDK1 (p<0.0001) and 2-fold lower PP2C (p=0.14) protein levels than samples that demonstrated incomplete responses. Protein levels of PP2C were lower in the platinum-resistant versus that shown in the platinum-sensitive OVCA cell line sub-clones. Levels of PKA were higher in all platinum-resistant than in platinum-sensitive OVCA cell line sub-clones. Selective siRNA depletion of CDK1 increased sensitivity to cisplatin-induced apoptosis (p<0.002). CONCLUSION: BAD pathway kinases and phosphatases, including CDK1 and PP2C, are associated with OVCA sensitivity to platinum and may represent therapeutic opportunities to enhance cytotoxic efficacy.
RESUMO
OBJECTIVE: To identify pathways that influence endometrial cancer (EC) cell sensitivity to cisplatin and to characterize the BCL2 antagonist of cell death (BAD) pathway as a therapeutic target to increase cisplatin sensitivity. METHODS: Eight EC cell lines (Ishikawa, MFE296, RL 95-2, AN3CA, KLE, MFE280, MFE319, HEC-1-A) were subjected to Affymetrix Human U133A GeneChip expression analysis of approximately 22,000 probe sets. In parallel, endometrial cell line sensitivity to cisplatin was quantified by MTS assay, and IC(50) values were calculated. Pearson's correlation test was used to identify genes associated with response to cisplatin. Genes associated with cisplatin responsiveness were subjected to pathway analysis. The BAD pathway was identified and subjected to targeted modulation, and the effect on cisplatin sensitivity was evaluated. RESULTS: Pearson's correlation analysis identified 1443 genes associated with cisplatin resistance (P<0.05), which included representation of the BAD-apoptosis pathway. Small interfering RNA (siRNA) knockdown of BAD pathway protein phosphatase PP2C expression was associated with increased phosphorylated BAD (serine-155) levels and a parallel increase in cisplatin resistance in Ishikawa (P=0.004) and HEC-1-A (P=0.02) cell lines. In contrast, siRNA knockdown of protein kinase A expression increased cisplatin sensitivity in the Ishikawa (P=0.02) cell line. CONCLUSION: The BAD pathway influences EC cell sensitivity to cisplatin, likely via modulation of the phosphorylation status of the BAD protein. The BAD pathway represents an appealing therapeutic target to increase EC cell sensitivity to cisplatin.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Eletroporação , Neoplasias do Endométrio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fosforilação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
PURPOSE: Despite initial sensitivity to chemotherapy, ovarian cancers (OVCA) often develop drug resistance, which limits patient survival. Using specimens and/or genomic data from 289 patients and a panel of cancer cell lines, we explored genome-wide expression changes that underlie the evolution of OVCA chemoresistance and characterized the BCL2 antagonist of cell death (BAD) apoptosis pathway as a determinant of chemosensitivity and patient survival. EXPERIMENTAL DESIGN: Serial OVCA cell cisplatin treatments were performed in parallel with measurements of genome-wide expression changes. Pathway analysis was carried out on genes associated with increasing cisplatin resistance (EC(50)). BAD-pathway expression and BAD protein phosphorylation were evaluated in patient samples and cell lines as determinants of chemosensitivity and/or clinical outcome and as therapeutic targets. RESULTS: Induced in vitro OVCA cisplatin resistance was associated with BAD-pathway expression (P < 0.001). In OVCA cell lines and primary specimens, BAD protein phosphorylation was associated with platinum resistance (n = 147, P < 0.0001) and also with overall patient survival (n = 134, P = 0.0007). Targeted modulation of BAD-phosphorylation levels influenced cisplatin sensitivity. A 47-gene BAD-pathway score was associated with in vitro phosphorylated BAD levels and with survival in 142 patients with advanced-stage (III/IV) serous OVCA. Integration of BAD-phosphorylation or BAD-pathway score with OVCA surgical cytoreductive status was significantly associated with overall survival by log-rank test (P = 0.004 and P < 0.0001, respectively). CONCLUSION: The BAD apoptosis pathway influences OVCA chemosensitivity and overall survival, likely via modulation of BAD phosphorylation. The pathway has clinical relevance as a biomarker of therapeutic response, patient survival, and as a promising therapeutic target.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Feminino , Humanos , Neoplasias Ovarianas/mortalidade , Fosforilação , Transdução de Sinais , Células Tumorais Cultivadas , Proteína de Morte Celular Associada a bcl/genéticaRESUMO
Modulation of estrogen signaling is one of the most successful modalities for the treatment of estrogen receptor (ER)-positive breast cancer, yet de novo and acquired resistance are frequent. Recent data suggests that the induction of autophagy may play a considerable role in promoting tumor cell survival and resistance to anti-estrogen therapy. Hence, bypassing autophagy may offer a novel strategy to enhance the anti-tumor efficacy of anti-estrogens. Histone deacetylases (HDAC) are involved in the regulation of steroid hormone receptor mediated cell signaling and their inhibition potentiates the anti-tumor effects of anti-estrogens. However, the mechanism underlying this anti-tumor activity is poorly understood. In this report, we show that the addition of an HDAC inhibitor redirects the response of ER-positive breast cancer cells when treated with tamoxifen from growth arrest to apoptotic cell death. This redirection requires functional ER signaling and is mediated by a depletion of Bcl-2 and an induction of Bax and Bak, manifesting in cytochrome C release and PARP cleavage. With combined treatment, a subpopulation of cells is refractory to apoptosis and exhibit a strong induction of autophagy. Inhibition of autophagy in these cells, using siRNA directed against Beclin-1 or treatment with chloroquine, further promotes the induction of apoptosis. Thus, supporting prior reports that autophagy acts as a survival mechanism, our findings demonstrate that HDAC and autophagy inhibition directs autophagy-protected cells into apoptotic cell death, which may impair development of tamoxifen resistance.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Tamoxifeno/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Neoplasias da Mama , Linhagem Celular Tumoral , Sinergismo Farmacológico , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Indóis , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Panobinostat , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais , Ácido Valproico/farmacologia , VorinostatRESUMO
Histone deacetylases (HDAC) may have a prominent role in the development of cancer and the response to anticancer therapy. However, the therapeutic relevance and tissue specificity of individual HDAC enzymes remain largely unknown. HDAC inhibitors may function as sensitizing agents to chemotherapies that target DNA through their effects on chromatin structure and plasticity. Here, we report a new role for HDAC2 as a regulator of chromatin compaction status and the mediator of HDAC inhibitor-induced sensitization to chemotherapy. The selective depletion of HDAC2 by small interfering RNA led to reduced expression of heterochromatin maintenance proteins and morphologic changes indicative of chromatin decondensation. Furthermore, depletion of HDAC2 but not HDAC1 or HDAC6 was sufficient to sensitize breast cancer cells to topoisomerase inhibitor-induced apoptosis. The levels of HDAC2 expression appear to correlate with the degree of HDAC inhibitor-induced histone acetylation in a surrogate tissue in patients. These data suggest that HDAC2 may be a relevant pharmacologic and biological target for combination therapy involving drugs that target DNA.
Assuntos
Neoplasias da Mama/genética , Cromatina/metabolismo , DNA de Neoplasias/metabolismo , DNA/genética , Histona Desacetilases/fisiologia , Proteínas Repressoras/fisiologia , Acetilação , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Epirubicina/farmacologia , Perfilação da Expressão Gênica , Histona Desacetilase 1 , Histona Desacetilase 2 , Desacetilase 6 de Histona , Histonas , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia , Células Tumorais CultivadasRESUMO
PURPOSE: The novel topoisomerase I inhibitor karenitecin (KTN) shows activity against melanoma. We examined whether histone deacetylase inhibition could potentiate the DNA strand cleavage, cytotoxicity as well as the clinical toxicity, and efficacy of KTN in melanoma. EXPERIMENTAL DESIGN: Apoptosis, COMET, and xenograft experiments were carried out as described previously. A phase I/II trial of valproic acid (VPA) and KTN was conducted in patients with stage IV melanoma, with any number of prior therapies, Eastern Cooperative Oncology Group performance status 0-2, and adequate organ function. RESULTS: VPA pretreatment potentiated KTN-induced apoptosis in multiple melanoma cell lines and in mouse A375 xenografts. VPA increased KTN-induced DNA strand breaks. In the phase I/II trial, 39 patients were entered, with 37 evaluable for toxicity and 33 evaluable for response. Somnolence was the dose-limiting toxicity. The maximum tolerated dose for VPA was 75 mg/kg/d; at maximum tolerated dose, serum VPA was approximately 200 microg/mL (1.28 mmol/L). At the dose expansion cohort, 47% (7 of 15) of patients had stable disease; median overall survival and time to progression were 32.8 and 10.2 weeks, respectively. Histone hyperacetylation was observed in peripheral blood mononuclear cells at maximum tolerated dose. CONCLUSION: VPA potentiates KTN-induced DNA strand breaks and cytotoxicity. VPA can be combined at 75 mg/kg/d for 5 days with full-dose KTN without overlapping toxicities. In metastatic poor prognosis melanoma, this combination is associated with disease stabilization in 47% of patients. Further testing of this combination appears warranted.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/análogos & derivados , Inibidores Enzimáticos/uso terapêutico , Inibidores de Histona Desacetilases , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Inibidores da Topoisomerase I , Ácido Valproico/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Feminino , Histonas/metabolismo , Humanos , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Cutâneas/metabolismo , Ácido Valproico/administração & dosagem , Ácido Valproico/efeitos adversosRESUMO
PURPOSE: The aim was to study the biological and molecular effects of the histone deacetylase (HDAC) inhibitor, valproic acid, in patients with solid tumor malignancies. EXPERIMENTAL DESIGN: A phase I dose escalation of valproic acid given on days 1 to 3 followed by epirubicin (day 3) was followed by a dose expansion of valproic acid combined with 5-fluorouracil, epirubicin, and cyclophosphamide (FEC100). Pharmacodynamic and pharmacokinetic studies entailed valproic acid and epirubicin plasma levels and their interaction, the effects of valproic acid on histone acetylation in peripheral blood mononuclear cells (PBMC) and tumor cells at baseline and day 3, and baseline expression of HDAC2 and HDAC6 as therapeutic targets. RESULTS: Forty-four patients were enrolled in the phase I part, with a disease-specific cohort expansion of 15 breast cancer patients (median age, 55 years; range, 28-66 years) receiving 120 mg/kg/day valproic acid followed by FEC100. Partial responses were seen in 9 of 41 (22%) patients during the phase I part. Objective responses were seen in 9 of 14 (64%) evaluable patients at the dose expansion with a median number of 6 administered cycles. Predominant toxicities were valproic acid-associated somnolence and epirubicin-induced myelosuppression. Valproic acid plasma levels were associated with short-term, reversible depletion of WBC and neutrophils within 48 hours. Histone acetylation in tumor samples and in PBMCs correlated with valproic acid levels and was further linked to baseline HDAC2 but not to HDAC6 expression. CONCLUSION: Valproic acid is a clinically relevant HDAC inhibitor, and PBMCs may serve as a surrogate for tumor histone acetylation in solid tumor malignancies. HDAC2 should be further considered as a relevant therapeutic target.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epirubicina/uso terapêutico , Inibidores de Histona Desacetilases , Neoplasias/tratamento farmacológico , Ácido Valproico/uso terapêutico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Plaquetas/efeitos dos fármacos , Ciclofosfamida/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Epirubicina/administração & dosagem , Epirubicina/farmacocinética , Feminino , Fluoruracila/administração & dosagem , Histonas/metabolismo , Humanos , Leucócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Neoplasias/metabolismo , Ácido Valproico/administração & dosagem , Ácido Valproico/farmacocinéticaRESUMO
Several histone deacetylases (HDAC) are involved in estrogen receptor (ER)-mediated gene transactivation, and HDAC inhibitors have been reported to restore sensitivity to antihormonal therapy. The modulation of ER is the most promising approach to ER-expressing breast cancers. Recent studies further suggest a critical role of the progesterone receptor (PR) on ER signaling. Although HDAC inhibitors modulate ER, little is known about their effects on PR. We evaluated the roles of specific HDAC isoenzymes and their inhibition on both ER and PR signaling and their importance in response to endocrine therapy. The roles of individual HDAC isoenzymes on ER and PR expression and their functions were evaluated by depletion of select HDAC enzymes using siRNA or pharmacologic inhibition. Cotreatment of breast cancer cell lines with HDAC inhibitors and the antiestrogen, tamoxifen, resulted in synergistic antitumor activity with simultaneous depletion of both ER and PR. Selective inhibition of HDAC2, but not HDAC1 or HDAC6, was sufficient to potentiate tamoxifen-induced apoptosis in ER/PR-positive cells. Depletion of HDAC1 and HDAC6 was associated with down-regulation of ER but not PR. Only the selective depletion of HDAC2 siRNA down-regulated both ER and PR expression, and was sufficient to potentiate tamoxifen. Selective depletion of HDAC2 resulted in simultaneous depletion of ER and PR, and potentiated the effects of antihormonal therapy in ER-positive cells. A more effective pharmacologic inhibition of HDAC2 and evaluation of HDAC2 and PR as therapeutic targets or as predictive markers in hormonal therapy may be considered when combining HDAC inhibitors and hormonal therapy.
Assuntos
Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Apoptose , Western Blotting , Linhagem Celular Tumoral , Inativação Gênica , Histona Desacetilase 2 , Humanos , Isoenzimas , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Tamoxifeno/farmacologiaRESUMO
PURPOSE: To determine the safety, toxicity, and maximum-tolerated dose of a sequence-specific combination of the histone deacetylase inhibitor (HDACi), valproic acid (VPA), and epirubicin in solid tumor malignancies and to define the clinical feasibility of VPA as an HDACi. PATIENTS AND METHODS: Patients were treated with increasing doses of VPA (days 1 through 3) followed by epirubicin (day 3) in 3-week cycles. The study evaluated pharmacokinetic and pharmacodynamic end points, toxicities, and tumor response. RESULTS: Forty-eight patients were enrolled, and 44 received at least one cycle of therapy. Patients (median age, 54 years; range, 39 to 78 years) received the following doses of VPA: 15, 30, 45, 60, 75, 90, 100, 120, 140, and 160 mg/kg/d. Dose-limiting toxicities were somnolence (n = 1), confusion (n = 3), and febrile neutropenia (n = 1). No exacerbation of epirubicin-related toxicities was observed. Partial responses were seen across different tumor types in nine patients (22%), and stable disease/minor responses were seen in 16 patients (39%), despite a median number of three prior regimens (range, zero to 10 prior regimens). Patients received a median number of four treatment cycles (range, one to 10 cycles), and treatment was stopped after reaching maximal epirubicin doses rather than progression in 13 (32%) of 41 patients patients. Total and free VPA plasma concentrations increased linearly with dose and correlated with histone acetylation in peripheral-blood mononuclear cells. CONCLUSION: The maximum-tolerated dose and recommended phase II dose was VPA 140 mg/kg/d for 48 hours followed by epirubicin 100 mg/m2. Sustained plasma concentrations of VPA exceeding those required for in vitro synergy were achieved with acceptable toxicity. Noteworthy antitumor activity was observed in heavily pretreated patients and historically anthracycline-resistant tumors.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Epirubicina/uso terapêutico , Inibidores de Histona Desacetilases , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase II , Ácido Valproico/uso terapêutico , Adulto , Idoso , Antibióticos Antineoplásicos/farmacocinética , Quimioterapia Combinada , Epirubicina/farmacocinética , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/patologia , Taxa de Sobrevida , Resultado do Tratamento , Ácido Valproico/farmacocinéticaRESUMO
Histone deacetylase inhibitors (HDACi) are a promising class of anticancer agents, yet the specific biological effects resulting in cell death are still poorly understood and clinically relevant markers of response are not adequately defined. The anticonvulsant valproic acid has recently emerged as an HDACi, and in vitro studies suggested that valproic acid may potentiate cytotoxic agents. We evaluated the pharmacologic and biological effects of valproic acid on histone acetylation, chromatin structure, and DNA damage induced by topoisomerase II inhibitors in mice bearing breast cancer tumors and developed an ex vivo methodology for response prediction using comet assays. The exposure of mice to valproic acid before exposure to epirubicin led to tumor regression when valproic acid was given for 48 hours at concentrations sufficient for histone hyperacetylation, down-regulation of heterochromatin maintenance proteins, and chromatin decondensation. Tumor response was accurately predicted by ex vivo comet moments. Valproic acid did not exacerbate epirubicin-related toxicity. Antitumor effects were not observed with valproic acid alone despite biologically active valproic acid concentrations. These findings suggest that exposure of tumor-bearing mice to valproic acid potentiated the antitumor effects of topoisomerase II inhibitors without enhancing toxicity. The HDACi-induced histone acetylation and modulation of heterochromatin correlated with potentiation of epirubicin-mediated DNA damage. However, these effects did not result in antitumor activity when using a HDACi alone and hence should not be considered a surrogate marker. Ex vivo comet assays may be useful as a predictive tool when tumor cells are limited and serial biopsies are difficult to obtain.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Inibidores da Topoisomerase II , Acetilação , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Ensaio Cometa , Epirubicina/farmacologia , Feminino , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Microscopia Eletrônica , Ácido Valproico/farmacologiaRESUMO
BACKGROUND: DNA topoisomerase II inhibitors and poisons are among the most efficacious drugs for the treatment of cancer. Sensitivity of cancer cells to the cytotoxic effects of topoisomerase II targeting agents is thought to depend on the expression of the topoisomerase IIalpha isoform, and drug resistance is often associated with loss or mutation of topoisomerase IIalpha. Histone deacetylase inhibitors (HDACi) are a novel class of compounds that potentiate the antitumor effects of topoisomerase II-targeting agents. METHODS: The interaction between HDACi and topoisomerase II-targeting agents in cancer cells was evaluated as a function of topoisomerase IIalpha and topoisomerase IIbeta expression. Topoisomerase II isoforms were selectively depleted using small interfering RNA and antisense. Drug-induced formation of cleavable complexes involving topoisomerase IIalpha and topoisomerase IIbeta was evaluated by trapped-in-agarose DNA immunostaining and band depletion assays in the presence and absence of HDACi. RESULTS: Preexposure to HDACi increased the cytotoxicity of topoisomerase II poisons. This was associated with a down-regulation of topoisomerase IIalpha expression but had no effects on topoisomerase IIbeta. In the setting of HDACi-induced chromatin decondensation and topoisomerase IIalpha depletion, topoisomerase II poison cytotoxicity was mediated through topoisomerase IIbeta cleavable complex formation. The HDACi-induced sensitization was also observed in cells with target-specific resistance to topoisomerase II poisons. CONCLUSIONS: The recruitment of topoisomerase IIbeta as a target may overcome primary or emergent drug resistance to topoisomerase II-targeting agents and hence may broaden the applicability of this important class of anticancer agents.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase II , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Butiratos/farmacologia , Cromatina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA Antissenso/farmacologia , Interações Medicamentosas , Sinergismo Farmacológico , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Mitoxantrona/farmacologia , Neoplasias/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia , Teniposídeo/intoxicação , Células Tumorais Cultivadas , Ácido Valproico/farmacologiaRESUMO
Histone acetylation and deacetylation are crucial in the regulation of gene expression. Dynamic changes in gene expression may affect chromatin structure and, consequently, the interaction of chromatin with regulatory factors. In this study, the effects of the antiseizure drug valproic acid (VPA) on the expression of genes that regulate the structure of chromatin and the access of macromolecules to the DNA were investigated. Exposure of breast cancer cells to VPA resulted in rapid dose-dependent hyperacetylation of the histones H3 and H4. VPA further induced a depletion of several members of the structural maintenance of chromatin (SMC) proteins, SMC-associated proteins, DNA methyltransferase, and heterochromatin proteins. Down-regulation of these proteins was associated with chromatin decondensation. The observed alterations of chromatin structure correlated with enhanced sensitivity of DNA to nucleases and increased interaction of DNA with intercalating agents. VPA-induced chromatin decondensation led to a sequence-specific potentiation of DNA-damaging agents in cell culture and xenograft models. Modulation of heterochromatin maintenance proteins was not a direct, but a downstream, effect of histone acetylation. The effects on the chromatin structure were reversible upon drug withdrawal, but obligatory for the potentiation of DNA-damaging agents. In addition to their antitumor activity as single agents, the chromatin decondensation induced by histone deacetylase inhibitors may enhance the efficacy of cytotoxic agents that act by targeting DNA. The proposed mechanism of action suggests an effect of drug sequencing on the antitumor activity of these drugs.
Assuntos
Neoplasias da Mama/genética , Cromatina/efeitos dos fármacos , Ácido Valproico/farmacologia , Acetilação/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Histonas/metabolismo , Humanos , Substâncias Intercalantes/administração & dosagem , Substâncias Intercalantes/farmacologia , Camundongos , Camundongos Nus , Ácido Valproico/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cyclin-dependent kinase activating kinase (CAK) is a trimeric complex composed of cdk7, cyclin H and MAT1. CAK/cdk7 functions as a master cell cycle regulator by phosphorylating cyclin-dependent kinases for cell cycle progression. We have previously reported that protein kinase C-iota (PKC-iota) associates with CAK/cdk7. In this investigation, immunofluorescence confocal microscopy was used to provide further evidence for the co-localization of PKC-iota with CAK/cdk7. PKC-iota was labeled with Alexa Fluor 488 (green fluorescent dye) and CAK/cdk7 was labeled with Alexa Fluor 555 (red fluorescent dye). The fusion of the red and green fluorescent colors produced a yellow color, which was used to quantify co-localization of PKC-iota and CAK/cdk7. Confocal microscopy revealed the co-localization of PKC-iota with CAK/cdk7 in both the cytoplasm and nucleus of U-373 MG cells.
Assuntos
Núcleo Celular/enzimologia , Quinases Ciclina-Dependentes/metabolismo , Citoplasma/enzimologia , Glioma/enzimologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Linhagem Celular Tumoral , Imunofluorescência , Humanos , Microscopia Confocal , Transdução de Sinais , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Acetylation of histones leads to conformational changes of DNA. We have previously shown that the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induced cell cycle arrest, differentiation, and apoptosis. In addition to their antitumor effects as single agents, HDAC inhibitors may cause conformational changes in the chromatin, rendering the DNA more vulnerable to DNA damaging agents. We examined the effects of SAHA on cell death induced by topo II inhibitors in breast cancer cell lines. Topo II inhibitors stabilize the topo II-DNA complex, resulting in DNA damage. Treatment of cells with SAHA promoted chromatin decondensation associated with increased nuclear concentration and DNA binding of the topo II inhibitor and subsequent potentiation of DNA damage. While SAHA-induced histone hyperacetylation occurred as early as 4 h, chromatin decondensation was most profound at 48 h. SAHA-induced potentiation of topo II inhibitors was sequence-specific. Pre-exposure of cells to SAHA for 48 h was synergistic, whereas shorter pre-exposure periods abrogated synergy and exposure of cells to SAHA after the topo II inhibitor resulted in antagonistic effects. Synergy was not observed in cells with depleted topo II levels. These effects were not limited to specific types of topo II inhibitors. We propose that SAHA significantly potentiates the DNA damage induced by topo II inhibitors; however, synergy is dependent on the sequence of drug administration and the expression of the target. These findings may impact the clinical development of combining HDAC inhibitors with DNA damaging agents.