RESUMO
BACKGROUND: The detection of homologous recombination deficiency (HRD) can identify patients who are more responsive to platinum and poly ADP ribose polymerase inhibitors (PARPi). MyChoice CDx (Myriad) is the most used HRD test in ovarian cancer (OC). However, some limitations of commercial tests exist, because of the high rate of inconclusive results, costs, and the impossibility of evaluating functional resistance mechanisms. PATIENTS AND METHODS: Two academic genomic tests and a functional assay, the RAD51 foci, were evaluated to detect HRD. One hundred patients with high-grade OC enrolled in the MITO16A/MaNGO-OV2 trial and treated with first-line therapy with carboplatin, paclitaxel, and bevacizumab were analyzed. RESULTS: The failure rate of the two genomic assays was 2%. The sensitivity in detecting HRD when compared with Myriad was 98.1% and 90.6%, respectively. The agreement rate with Myriad was 0.92 and 0.87, with a Cohen's κ coefficient corresponding to 0.84 and 0.74, respectively. For the RAD51 foci assay, the failure rate was 30%. When the test was successful, discordant results for deficient and proficient tumors were observed, and additional HRD patients were identified compared to Myriad; sensitivity was 82.9%, agreement rate was 0.65, and Cohen's κ coefficient was 0.18. The HRD detected by genomic assays and residual tumor at primary surgery and stage was correlated with progression-free survival at multivariate analysis. CONCLUSIONS: Results suggest the feasibility of academic tests for assessing HRD status that show robust concordance with Myriad and correlation with clinical outcome. The contribution of the functional information related to the RAD51 foci test to the genomic data needs further investigation.
Assuntos
Mangifera , Neoplasias Ovarianas , Feminino , Humanos , Bevacizumab/uso terapêutico , Carboplatina/uso terapêutico , Recombinação Homóloga , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Paclitaxel/uso terapêutico , Platina/uso terapêutico , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
In cancer, myeloid cells have tumor-supporting roles. We reported that the protein GPNMB (glycoprotein nonmetastatic B) was profoundly upregulated in macrophages interacting with tumor cells. Here, using mouse tumor models, we show that macrophage-derived soluble GPNMB increases tumor growth and metastasis in Gpnmb-mutant mice (DBA/2J). GPNMB triggers in the cancer cells the formation of self-renewing spheroids, which are characterized by the expression of cancer stem cell markers, prolonged cell survival and increased tumor-forming ability. Through the CD44 receptor, GPNMB mechanistically activates tumor cells to express the cytokine IL-33 and its receptor IL-1R1L. We also determined that recombinant IL-33 binding to IL-1R1L is sufficient to induce tumor spheroid formation with features of cancer stem cells. Overall, our results reveal a new paracrine axis, GPNMB and IL-33, which is activated during the cross talk of macrophages with tumor cells and eventually promotes cancer cell survival, the expansion of cancer stem cells and the acquisition of a metastatic phenotype.
Assuntos
Fibrossarcoma/patologia , Receptores de Hialuronatos/metabolismo , Interleucina-33/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Apoptose , Proliferação de Células , Fibrossarcoma/etiologia , Fibrossarcoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Interleucina-33/genética , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos DBA , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Sarcoma Experimental/etiologia , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Trabectedina/farmacologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Biologia de Sistemas/métodos , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive disease, that can be described as a member of the family of small round blue cell tumors. The molecular diagnostic marker is the t(11;22)(p13;q12) translocation, which creates an aberrant transcription factor, EWS-WT1, that underlies the oncogenesis of DSRCT. Current treatments are not very effective so new active drugs are needed. Trabectedin, now used as a single agent for the treatment of soft tissue sarcoma, was reported to be active in some pre-treated DSRCT patients. Using JN-DSRCT-1, a cell line derived from DSRCT expressing the EWS-WT1 fusion protein, we investigated the ability of trabectedin to modify the function of the chimeric protein, as in other sarcomas expressing fusion proteins. After detailed characterization of the EWS-WT1 transcripts structure, we investigated the mode of action of trabectedin, looking at the expression and function of the oncogenic chimera. METHODS: We characterized JN-DSRCT-1 cells using cellular approaches (FISH, Clonogenicity assay) and molecular approaches (Sanger sequencing, ChIP, GEP). RESULTS: JN-DSRCT-1 cells were sensitive to trabectedin at nanomolar concentrations. The cell line expresses different variants of EWS-WT1, some already identified in patients. EWS-WT1 mRNA expression was affected by trabectedin and chimeric protein binding on its target gene promoters was reduced. Expression profiling indicated that trabectedin affects the expression of genes involved in cell proliferation and apoptosis. CONCLUSIONS: The JN-DSRCT-1 cell line, in vitro, is sensitive to trabectedin: after drug exposure, EWS-WT1 chimera expression decreases as well as binding on its target promoters. Probably the heterogeneity of chimera transcripts is an obstacle to precisely defining the molecular mode of action of drugs, calling for further cellular models of DSRCT, possibly growing in vivo too, to mimic the biological complexity of this disease.
Assuntos
Tumor Desmoplásico de Pequenas Células Redondas/tratamento farmacológico , Dioxóis/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/efeitos dos fármacos , Tetra-Hidroisoquinolinas/farmacologia , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Tumor Desmoplásico de Pequenas Células Redondas/metabolismo , Tumor Desmoplásico de Pequenas Células Redondas/fisiopatologia , Dioxóis/uso terapêutico , Humanos , Proteínas de Fusão Oncogênica/genética , Proteína EWS de Ligação a RNA , Tetra-Hidroisoquinolinas/uso terapêutico , Trabectedina , Proteínas WT1RESUMO
BACKGROUND: Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. PATIENTS AND METHODS: We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. RESULTS AND CONCLUSIONS: Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, 'Activins/Inhibins' and 'Hedgehog' signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.
Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Prognóstico , Adulto , Idoso , Carcinoma Epitelial do Ovário , Intervalo Livre de Doença , Feminino , Humanos , MicroRNAs/genética , Análise em Microsséries , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologiaRESUMO
BACKGROUND: Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen. PATIENTS AND METHODS: One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis. RESULTS: Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P < 0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P < 0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-ß activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P < 0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P < 0.001). CONCLUSIONS: This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.
Assuntos
Cistadenocarcinoma Seroso/tratamento farmacológico , MicroRNAs/biossíntese , Terapia Neoadjuvante , Neoplasias Ovarianas/tratamento farmacológico , Proteína Smad2/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Biópsia , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Prognóstico , Proteína Smad2/genéticaRESUMO
BACKGROUND: The majority of patients with stage III-IV epithelial ovarian cancer (EOC) relapse after initially responding to platinum-based chemotherapy, and develop resistance. The genomic features involved in drug resistance are unknown. To unravel some of these features, we investigated the mutational profile of genes involved in pathways related to drug sensitivity in a cohort of matched tumors obtained at first surgery (Ft-S) and second surgery (Sd-S). PATIENTS AND METHODS: Matched biopsies (33) taken at Ft-S and Sd-S were selected from the 'Pandora' tumor tissue collection. DNA libraries for 65 genes were generated using the TruSeq Custom Amplicon kit and sequenced on MiSeq (Illumina). Data were analyzed using a high-performance cluster computing platform (Cloud4CARE project) and independently validated. RESULTS: A total of 2270 somatic mutations were identified (89.85% base substitutions 8.19% indels, and 1.92% unknown). Homologous recombination (HR) genes and TP53 were mutated in the majority of Ft-S, while ATM, ATR, TOP2A and TOP2B were mutated in the entire dataset. Only 2% of mutations were conserved between matched Ft-S and Sd-S. Mutations detected at second surgery clustered patients in two groups characterized by different mutational profiles in genes associated with HR, PI3K, miRNA biogenesis and signal transduction. CONCLUSIONS: There was a low level of concordance between Ft-S and Sd-S in terms of mutations in genes involved in key processes of tumor growth and drug resistance. This result suggests the importance of future longitudinal analyses to improve the clinical management of relapsed EOC.
Assuntos
Adenocarcinoma de Células Claras/genética , Adenocarcinoma Mucinoso/genética , Cistadenocarcinoma Seroso/genética , Neoplasias do Endométrio/genética , Genes Neoplásicos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação/genética , Neoplasias Ovarianas/genética , Adenocarcinoma de Células Claras/mortalidade , Adenocarcinoma de Células Claras/secundário , Adenocarcinoma de Células Claras/terapia , Adenocarcinoma Mucinoso/mortalidade , Adenocarcinoma Mucinoso/secundário , Adenocarcinoma Mucinoso/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Terapia Combinada , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/secundário , Cistadenocarcinoma Seroso/terapia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias do Endométrio/mortalidade , Neoplasias do Endométrio/secundário , Neoplasias do Endométrio/terapia , Feminino , Seguimentos , Recombinação Homóloga , Humanos , Estudos Longitudinais , Metástase Linfática , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Estadiamento de Neoplasias , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Dioxóis/farmacologia , Lipossarcoma Mixoide/tratamento farmacológico , Proteínas de Fusão Oncogênica/genética , Proteína FUS de Ligação a RNA/genética , Tetra-Hidroisoquinolinas/farmacologia , Fator de Transcrição CHOP/genética , Adulto , Animais , Biópsia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Doxorrubicina/farmacologia , Feminino , Humanos , Lipossarcoma Mixoide/genética , Camundongos Nus , Proteínas de Fusão Oncogênica/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Trabectedina , Fator de Transcrição CHOP/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Dioxóis/farmacologia , Lipossarcoma Mixoide/genética , Lipossarcoma Mixoide/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Apoptose , Proteína C-Reativa/genética , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Metilação de DNA , Metilases de Modificação do DNA/deficiência , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fibronectinas/genética , Humanos , Lipossarcoma Mixoide/tratamento farmacológico , Lipossarcoma Mixoide/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Componente Amiloide P Sérico/genética , Transdução de Sinais , Temozolomida , Trabectedina , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Raios UltravioletaRESUMO
The p73 locus gene has a complex structure encoding a plethora of isoforms. The different DeltaN truncated isoforms of p73 may exert different activities depending on the cellular context. The beta isoform of DeltaNp73 seems to have a particular pattern of action even if its role in cell cycle and mitosis is still under investigation. To gain further knowledge of DeltaNp73beta's function, we investigated the effects of its over-expression in tumour cellular models, using the tetracycline-inducible expression system. In the human lung carcinoma cell line H1299, DeltaNp73beta over-expression resulted in suppression of cell growth and in cell death. Surprisingly stable over-expression of DeltaNp73beta impaired the genomic stability of tumour cells, leading to the formation of tetraploid cells. The cells become enlarged and multinucleate, with incorrect mitotic figures, and died by apoptotic-independent pathways. Our data suggest that DeltaNp73beta-induced aberrant mitosis evades the control of the mitotic spindle assay checkpoint, leading to tetraploidy and cell death through mitotic catastrophe rather than apoptosis. The various C-terminal regions of DeltaNp73 may influence the final cellular phenotype and we assume that the beta one in particular could be important in both cell growth control and regulation of mitosis.
Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Proteínas Nucleares/genética , Poliploidia , Carcinoma de Pequenas Células do Pulmão/genética , Proteínas Supressoras de Tumor/genética , Apoptose/fisiologia , Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: P63 belongs to the 'p53 family' whose role in cancer progression has been recently revisited in light of the plethora of splicing variants that are generated. We analyzed the expression of the full-length TAp63 gene and its dominant-negative form deltaNp63 in ovarian cancer biopsies to correlate their expression with clinical outcome. MATERIALS AND METHODS: Real-time RT-PCR analysis was used to determine the levels of TAp63 and deltaNp63 in 83 stage I and in 86 stage III ovarian cancer biopsies and in seven human ovarian cancer cell. RESULTS: TAp63 levels were comparable in stage I and stage III, but deltaNp63 levels increased 77-fold in stage III, independently of the p53 status. Patients with high deltaNp63 expression had the worst overall survival (OS); patients with a deltaNp63/TAp63 ratio >2 had a poor OS. Patients with a high deltaNp63/TAp63 ratio were those with a poor response to platinum-based therapy. CONCLUSIONS: Data indicate a role for deltaNp63 as a potential biomarker to predict patient's outcome and tumor progression in ovarian cancer. This would have particularly clinical relevance in ovarian cancer where the high rate of mortality reflects our lack of knowledge of molecular mechanisms underlying cell progression toward malignancy.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/análise , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Transativadores/análise , Proteínas Supressoras de Tumor/análise , Biópsia , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Neoplasias Ovarianas/patologia , Análise de Sobrevida , Fatores de TranscriçãoRESUMO
The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73alpha expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73alpha. In vitro the two DNp73alpha overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73alpha both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73alpha does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxiciclina/farmacologia , Expressão Gênica , Genes Supressores de Tumor , Células HCT116 , Humanos , Camundongos , Neoplasias/genética , Fenótipo , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
ET-743 (Yondelis(TM), Trabectedin) isolated from the tunicate Ecteinascidia turbinata, is being tested in phase II clinical trials in Europe and the United States of America (USA). Studies with different solid tumours have shown antitumour activity in advanced, pre-treated sarcomas as well as in drug-resistant breast and ovarian cancer. The primary mechanism of action for ET-743 has not been fully elucidated and different models have been suggested to explain its molecular mechanism of action. ET-743 binds tightly to the minor groove of DNA and previous data have suggested that ET-743 acts by interfering with RNA transcription. To further investigate the mechanism of in vitro drug resistance, we evaluated the gene expression profile in ovarian and chondrosarcoma cell lines selected for resistance to ET-743. We found 70 genes whose expression was modulated in both drug-resistant cell lines when compared with their respective parental drug-sensitive cell lines. This pattern of gene expression seems to be selective for ET-743-resistant cells, since ovarian cancer cells resistant to paclitaxel did not share the same gene expression changes. Data presented in this study reveal different molecular pathways that could be involved in the cellular mechanism of ET-743 resistance.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Condrossarcoma/tratamento farmacológico , Dioxóis/uso terapêutico , Isoquinolinas/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Antineoplásicos , Antineoplásicos Alquilantes/farmacocinética , Linhagem Celular Tumoral , Condrossarcoma/genética , Dioxóis/farmacocinética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Isoquinolinas/farmacocinética , Neoplasias Ovarianas/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tetra-Hidroisoquinolinas , TrabectedinaRESUMO
The conventional chemotherapy is mostly based on the evidence that proliferating cells are more sensitive to anticancer agents than non-dividing cells. This is the main reason why these compounds are not tumour specific and their selectivity is generally in favour of rapidly growing cells (haematopoietic or intestine. i.e.) rather than discriminating against any fundamental biological difference between normal and tumour cells. The critical issue is at present to identify how tumour cells differ from normal cells and how those differences can be exploited therapeutically for designing and synthesising new drugs with a selective mechanism of action and thus with an improved therapeutic index. This topic and the strategies to identify these new targets will be discussed in details in the review. The expanding knowledge on molecular biology of cancer cells has allowed in the last years the identification of different molecular pathways altered in cancer that could be exploited as potential therapeutic targets. For most of the pathways previously disclosed it has been a problem to develop selective molecules with a relevant clinic impact. To target those specific genetics defects, different kind of molecules (antibodies, "antisense oligonucleotides", short peptides and small molecules) have been made and some of them are currently under investigation. This review will be focused mainly on three different classes of compounds: I. Compounds designed to hit or inhibit crucial molecular targets. II. Novel DNA minor groove binders. III. Products of marine origin that exhibit novel mode of action.
Assuntos
Antineoplásicos , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Defects in DNA mismatch repair (MMR) are associated with a predisposition to tumorigenesis and with drug resistance owing to high mutation rates and failure to engage DNA-damage-induced apoptosis. DNA minor groove binders (MGBs) are a class of anticancer agents highly effective in a variety of human cancers. Owing to their mode of action, DNA MGB-induced DNA damage may be a substrate for DNA MMR. This study was aimed at investigating the effect of loss of MMR on the sensitivity to brostallicin (PNU-166196), a novel synthetic alpha-bromoacrylic, second-generation DNA MGB currently in Phase II clinical trials and structurally related to distamycin A. Brostallicin activity was compared to a benzoyl mustard derivative of distamycin A (tallimustine). We report that the sensitivities of MLH1-deficient and -proficient HCT116 human colon carcinoma cells were comparable after treatment with brostallicin, while tallimustine resulted in a three times lower cytotoxicity in MLH1-deficient than in -proficient cells. MSH2-deficient HEC59 parental endometrial adenocarcinoma cells were as sensitive as the proficient HEC59+ch2 cells after brostallicin treatment, but were 1.8-fold resistant after tallimustine treatment as compared to the MSH2-proficient HEC59+ch2 counterpart. In addition, p53-deficient mouse fibroblasts lacking PMS2 were as sensitive to brostallicin as PMS2-proficient cells, but were 1.6-fold resistant to tallimustine. Loss of neither ATM nor DNA-PK affected sensitivity to brostallicin in p53-deficient mouse embryonic fibroblasts, indicating that brostallicin-induced cytotoxicity in a p53-deficient genetic background does not seem to require these kinases. These data show that, unlike other DNA MGBs, MMR-deficient cells retain their sensitivity to this new alpha-bromoacrylic derivative, indicating that brostallicin-induced cytotoxicity does not depend on functional DNA MMR. Since DNA MMR deficiency is common in numerous types of tumours, brostallicin potentially offers the advantage of being effective against MMR-defective tumours that are refractory to several anticancer agents.
Assuntos
Pareamento Incorreto de Bases , Neoplasias do Colo/patologia , Reparo do DNA , Proteínas de Ligação a DNA , Guanidinas/farmacologia , Proteínas Proto-Oncogênicas , Pirróis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Proteínas de Transporte , Morte Celular , Distamicinas/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fibroblastos , Humanos , Camundongos , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/genética , Compostos de Mostarda Nitrogenada/farmacologia , Proteínas Nucleares , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53RESUMO
The cytotoxic effect of Aplidin was investigated on fresh leukaemia cells derived from children with B-cell-precursor (BCP) acute lymphoblastic leukaemia (ALL) by using stromal-layer culture system and on four cell lines, ALL-PO, Reh, ALL/MIK and TOM-1, derived from patients with ALL with different molecular genetic abnormalities. In ALL cell lines Aplidin was cytotoxic at nanomolar concentrations. In the ALL cell lines the drug-induced cell death was clearly related to the induction of apoptosis and appeared to be p53-independent. Only in ALL-PO 20 nM Aplidin treatment caused a block of vascular endothelial growth factor (VEGF) secretion and downregulation of VEGF-mRNA, but Aplidin cytotoxicity does not seem to be related to VEGF inhibition since the sensitivity of ALL-PO cells to Aplidin is comparable to that observed for the other cells used. Aplidin induced a G(1) and a G(2) M block in ALL cell lines. In patient-derived leukaemia cells, Aplidin induced a strong cytotoxicity evidenced in a stroma-supported immunocytometric assay. Cells from children with genetic abnormalities such as t(9;22) and t(4;11) translocations, associated with an inferior treatment outcome, were sensitive to Aplidin to the same extent as that observed in other BCP-ALL cases. Aplidin exerted a strong cell killing effect (>88%) against primary culture cells from five relapsed ALL cases, at concentrations much lower than those reported to be achieved in plasma of patients receiving Aplidin at recommended doses. Taken together these data suggest that Aplidin could be a new anticancer drug to be investigated in ALL patients resistant to available therapy.
Assuntos
Antineoplásicos/farmacologia , Depsipeptídeos , Resistencia a Medicamentos Antineoplásicos , Peptídeos Cíclicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Adolescente , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cariotipagem , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Espectrometria de Massas , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The mechanism by which aplidine, a marine natural product in early clinical development as an anticancer agent, induces cell growth inhibition and apoptosis has been investigated in the human leukemia cell line MOLT-4. This cell line is characterized not only by the ability to secrete VEGF, but also for the presence on its surface of the VEGF receptor-1 (VEGFR-1). Previous studies from our laboratory concerned with evaluating early changes in gene expression induced by aplidine in MOLT-4 cells have shown that the drug decreases the expression of VEGFR-1 (Marchini et al. Proc Am Assoc Cancer Res 2000; 41: 833). Here, we report the ability of aplidine to block the VEGF/VEGFR-1 loop. We found that aplidine blocked VEGF secretion that was temporally followed by a decrease in both VEGF and VEGFR-1 production. Aplidine did not directly affect either VEGF transcription or stabilization of its mRNA. Transfection of MOLT-4 cells with an antisense VEGF cDNA construct, resulted in inhibition of colony formations. One clone, transfected with sense VEGF cDNA, secreting 8-10 times more VEGF than parental cells, was less sensitive to aplidine-induced cytotoxicity and apoptosis than control cells. Moreover, addition of VEGF in the medium decreased the activity of aplidine in MOLT-4 cells. These data demonstrate that aplidine inhibits the growth and induces apoptosis in MOLT-4 cells through the inhibition of VEGF secretion which blocks the VEGF/VEGFR-1 autocrine loop necessary for the growth of these cells.
Assuntos
Antineoplásicos/farmacologia , Depsipeptídeos , Fatores de Crescimento Endotelial/antagonistas & inibidores , Leucemia de Células T/tratamento farmacológico , Linfocinas/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Comunicação Autócrina , Divisão Celular/efeitos dos fármacos , Primers do DNA/química , Dactinomicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Meia-Vida , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Luciferases/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio VascularRESUMO
Microarray technique was employed to study differences in gene expression profile induced by Aplidine treatment in the Molt-4 human leukemic T cell line. Aplidine is a novel marine compound purified from caribbean tunicate (sea squirt) Aplidium Albicans. Despite promising anti-tumor activity, few data are available on its mechanism of action. Exponentially growing cells were treated with Aplidine concentrations close to its 5IC50 for 1 hour and RNA samples collected after 0.5, 1, 6 and 24 hours of recovery in drug free medium. 32P labelled cDNAs were hybridized against Atlas Human Cancer arrays onto which 588 cDNAs were spotted. Genes involved in different cellular pathways, (such as growth factors, signal transduction or transcription factors) were found modulated by the drug. Even if the data obtained in the present study cannot be conclusive, several hypothesis on Aplidine's mechanism of action are indicated that will be the subject of future studies.
Assuntos
Anticarcinógenos/uso terapêutico , Depsipeptídeos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos Cíclicos/uso terapêutico , Northern Blotting , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Background: It is known that antioxidant liposoluble vitamins and carotenoids are reduced in liver cirrhosis, but little is known about chronic viral hepatitis, where oxidative damage has to be taken into account. Methods: Fifty-five patients with chronic hepatitis, mainly C virus-related, were matched with 16 patients with biliary stones and 20 healthy controls. Plasma and liver analyses were carried out using a well-tried HPLC technique that affords an accurate quantification of retinol, tocopherol, alpha- and beta-carotene, cryptoxanthin, and lycopene. Results: Plasma concentration of retinol, tocopherol, beta-carotene, and lycopene was significantly decreased in both patient groups, particularly in those with chronic hepatitis. In contrast, liver concentration of both esterified and free retinol, tocopherol, and some carotenoids was better preserved in the hepatitis group than in the cholelithiasis group. A strict correspondence between aminotransferases and the amount of liver-stored retinol was documented. Conclusions: Plasma vitamin and carotenoid depletion co-existing with preserved liver storage may indicate a functional defect in liver pool mobilization or even a real depletion of the antioxidant defenses, which play a key role in averting cellular damage. The implications for nutrition and therapy need to be taken into account.
RESUMO
4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548) belongs to a novel class of antitumor compounds (termed alkycyclines) and is currently undergoing Phase II clinical trial. In the present study, we investigated the in vitro and in vivo antitumor activity, the pharmacokinetics, and the toxicological profile of this compound. PNU-159548 showed good cytotoxic activity in murine and human cancer cells growing in vitro, with an average concentration for 50% growth inhibition of 15.8 ng/ml. The drug showed strong antitumor efficacy in vivo after i.v. and p.o. administration against rapidly proliferating murine leukemias and slowly growing transplantable human xenografts. At non-toxic doses, PNU-159548 produced complete regression and cures in ovarian, breast, and human small cell lung carcinomas. Fourteen of 16 models studied, including colon, pancreatic, gastric, and renal carcinomas, astrocytoma and melanoma, were found to be sensitive to PNU-159548. In addition, PNU-159548 was effective against intracranially implanted tumors. Toxicological studies revealed myelosuppression as the main toxicity in both mice and dogs. The maximum tolerated doses, after a single administration, were 2.5 mg/kg of body weight in mice, 1.6 mg/kg in rats, and 0.3 mg/kg in dogs. In the cyclic studies, the maximum tolerated doses were 0.18 mg/kg/day (cumulative dose/cycle: 0.54 mg/kg) in rats and 0.05 mg/kg/day (cumulative dose/cycle: 0.15 mg/kg) in dogs. PNU-159548 showed minimal cardiotoxicity, when compared with doxorubicin in the chronic rat model at a dose level inducing similar myelotoxicity. Animal pharmacokinetics, carried out in mice, rats, and dogs, was characterized by high volumes of distribution, plasma clearance of the same order of the hepatic blood flow, and short terminal half-life. These findings support the conclusion that PNU-159548 is an excellent candidate for clinical trials in the treatment of cancer.