RESUMO
OBJECTIVES: BCL2L12 is a recently discovered and cloned gene from members of our research team. It is a novel member of the BCL2 gene family, members of which are implicated in different hematological malignancies. In the present study, we investigated and studied the expression profile of BCL2L12 in acute myeloid leukemia (AML). DESIGN AND METHODS: Total RNA was isolated from peripheral blood of 67 AML patients and healthy donors. The expression profile of BCL2L12 was studied using real-time PCR methodology (SYBR Green chemistry). We also evaluated the association of the BCL2L12 mRNA expression level with clinical and pathological disease parameters, as well with disease-free survival (DFS) and overall survival (OS), using the chi-square (χ(2)) test or the Fisher's exact test, where appropriate. RESULTS: Leukemia patients expressing high level of BCL2L12 were 3 times more likely to relapse (p=0.004) or die (p=0.007) than patients with low level of BCL2L12 expression. Additionally, statistically significant relationships were revealed between BCL2L12 expression level and CD117 expression, the presence of splenomegaly and chemotherapy response. CONCLUSIONS: Our results suggest that BCL2L12 can be considered as a new independent prognostic and chemotherapy response marker in AML.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mieloide Aguda/sangue , Proteínas Musculares/sangue , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas c-bcl-2/sangue , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Etoposídeo/administração & dosagem , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatomegalia/sangue , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Pessoa de Meia-Idade , Análise Multivariada , Proteínas Musculares/genética , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-bcl-2/genética , Esplenomegalia/sangue , Resultado do Tratamento , Regulação para Cima , Adulto JovemRESUMO
BCL2L12 is a recently identified gene belonging to the BCL2 family, members of which are implicated in hematologic malignancies, including chronic lymphocytic leukemia (CLL). The aim of this study was to analyze the mRNA expression of the novel apoptosis-related gene BCL2L12 in patients with CLL and to examine its prognostic and predictive value and potential clinical application as a novel molecular biomarker for CLL. For this purpose, total RNA was isolated from peripheral blood of 65 CLL patients and 23 healthy donors. An ultrasensitive quantitative real-time polymerase chain reaction methodology for BCL2L12 and BCL2 mRNA quantification was developed using SYBR Green chemistry. After preparing cDNA by reverse transcription, relative quantification analysis was performed using the comparative C(T) (2(-ΔΔCT)) method. Furthermore, analysis of IGHV mutational status, CD38 expression, and detection of early apoptosis by double staining with Annexin V-FITC and propidium iodide were performed. According to our findings, BCL2L12 mRNA expression is significantly higher in CLL patients than in healthy donors. Receiver operating characteristic analysis demonstrated that BCL2L12 expression had significant discriminatory value, distinguishing very efficiently CLL patients from the non-leukemic population. Moreover, BCL2L12 expression predicts the presence of CLL, as demonstrated by both univariate and multivariate logistic regression analyses. Finally, high BCL2L12 mRNA levels are associated with advanced clinical stage and predict shorter overall survival in CLL patients.
Assuntos
Biomarcadores Tumorais/biossíntese , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Musculares/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Análise de SequênciaRESUMO
We synthesized, characterized and studied the anticancer properties of a new water-soluble peroxo niobium complex (K(3)[Nb(Asc)(O(2))(3)]·4H(2)O (Asc = ascorbate anion C(6)H(6)O(6)(2-))), as well as that of ascorbic acid, in human leukemic cells. The complex was synthesized and characterized by elemental, IR, Raman, thermogravimetric analysis, detailed NMR and mass spectra analysis. The cytotoxic activity of the complex on HL-60 and K562 human leukemia cell lines has been investigated by assessing vital cellular mechanisms, such as the metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT) and the proliferation capacity (growth curves) of leukemia cells, as well as the structural integrity of cell membrane (trypan blue assay). The complex exerts an increased antiproliferative effect primarily on HL60 human leukemia cells, compared to ascorbic acid alone, as well as an inhibitory effect on necrosis caused by ascorbic acid. Its effect on K562 cells concerns mainly its inhibitory effect upon cell necrosis induced by ascorbic acid alone. Our results support a concentration- and time-dependent enhanced antileukemic effect of the complex, suggesting its significance as a promising tool in the confrontation of leukemia.
Assuntos
Antineoplásicos/síntese química , Complexos de Coordenação/síntese química , Nióbio , Antineoplásicos/química , Antineoplásicos/farmacologia , Ácido Ascórbico/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Células HL-60 , Humanos , Concentração Inibidora 50 , Células K562 , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Espectrofotometria Infravermelho , Análise Espectral Raman , TermogravimetriaRESUMO
Taxol and vincristine belong to a group of anticancer drugs that target microtubules, subsequently arresting cells at the mitotic phase of the cell cycle and inducing programmed cell death. The BCL2 (bcl-2) family of genes is of known implication in apoptosis induced by various stimuli, among which BCL2L12, a new member of the family, cloned by our group. For further insights into the mechanisms and molecular targets implicated and modified as a result of apoptosis induced by these two mitosis-arresting drugs, we studied the possible alterations, at the mRNA level, of various apoptosis-related genes (BCL2, BAX, BCL2L12, CASPASE-3, FAS) after leukemia cell (HL-60) treatment with these drugs. The kinetics of cell toxicity were evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method, trypan blue staining, and cell proliferation efficiency; apoptosis induction was assayed by endonucleosomal cleavage of DNA (DNA laddering); and the expression levels of the genes were analysed by RT-PCR, using gene-specific primers. The percentage of nonviable cells was upregulated with increasing cell exposure time and drug concentrations to both taxol and vincristine. Distinct modulations of apoptosis-related genes at the mRNA level were also observed, mainly concerning BCL2 and BCL2L12 along apoptosis induction. Our results indicate and support the hypothesis that the apoptosis-related genes BCL2 and BCL2L12 respond similarly to treatment of the human, acute, myelocytic leukemia HL60 cells with the anticancer drugs vincristine and taxol though in a drug-specific and time-dependent manner.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Musculares/genética , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Vincristina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/genética , Caspase 3/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2/genética , Receptor fas/genéticaRESUMO
Several of the novel kallikrein-related peptidases (tissue kallikreins; KLKs) are emerging new serum and/or tissue biomarkers for prostate cancer (CaP) diagnosis, prognosis and monitoring. In the present research approach, our objective was to investigate the possible alterations in the mRNA expression levels of KLK5 and KLK11 genes in prostate cancer cells PC3 as a response to treatment with mitoxantrone, etoposide, doxorubicin and carboplatin. Viability was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after cell treatment with either mitoxantrone (2 microM), etoposide (20 microM), doxorubicin (1 microM), or carboplatin (15 microM), for 24, 48 and 72 hours. Additionally, trypan blue staining revealed that in PC3 cells all drugs displayed almost the same limited necrotic effects which appeared mainly at 72 hours of treatment. PC3 prostate cancer cells showed a concentration- and time-dependent increased cytotoxicity to the drugs under study which was mainly due to reduction of cell proliferation efficiency. Distinct modulations of KLK5 and KLK11 genes, at the mRNA level, were observed, supporting a drug-dependent cell response. Our experimental data demonstrate that the molecular profile mainly of KLK5 gene may serve as a new potential molecular biomarker predicting treatment response in CaP cells.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Carboplatina/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Calicreínas/genética , Mitoxantrona/farmacologia , Neoplasias da Próstata/genética , Serina Endopeptidases/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Calicreínas/metabolismo , Masculino , Necrose , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , RNA Mensageiro/metabolismo , Serina Endopeptidases/metabolismo , Fatores de TempoRESUMO
We assessed changes in the apoptosis-related genes BCL2, BAX, BCL2L12, FAS and CASPASE-3 in OVCAR-3 human ovarian cancer cells and BT-20 human breast cancer cells to provide an insight into the molecular mechanisms involved in the response of these cells to treatment with anticancer drugs and to assess their value as potential biomarkers of chemotherapy response in breast and ovarian cancer. Cells were treated with different chemotherapeutic drugs (cisplatin, carboplatin, doxorubicin, etoposide and taxol) and assessed for changes in the expression of apoptosis-related genes at the mRNA level. Total RNA was extracted, reverse-transcribed into cDNA and amplified by PCR using gene-specific primers. GAPDH was used as a housekeeping gene. Cytotoxicity was assessed by MTT assay. Both cancer cell lines responded differentially at the molecular level to the drug treatments. OVCAR-3 cells showed more pronounced sensitivity and changes compared to BT-20 cells at the mRNA level for different apoptosis-related genes, leading to cell and cancer type dependence in conjunction with drug dependence.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Neoplasias Ovarianas/patologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Carboplatina/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BCL2 (bcl-2) gene family members are important regulators of apoptosis. Increasing evidence supports their modulated expression in breast cancer cells and in many cases their relation to chemotherapy response, outcome, and overall prognosis, as well as their value as important potent therapeutic targets. Investigation and increased understanding of their transcriptional regulation and their specific roles in cancer progression and therapy response will be useful for focusing research on the development of novel therapies targeted against this gene family members' expression status. In the present review, we describe current knowledge of the molecular profile of the classical and novel members of the BCL2 family of genes as a response of breast cancer cells to cytotoxic/cytostatic drugs.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Citostáticos/uso terapêutico , Genes bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias da Mama/genética , Regulação para Baixo , Humanos , Regulação para CimaRESUMO
We studied the antitumor properties of the dinuclear copper(II) complex of l-carnitine [Cu 2( l-carnitine) 2Cl 2(H 2O) 2]Cl 2, as well as those of l-carnitine and copper chloride dihydrate, in human leukemic cells. The complex was synthesized and characterized using EPR, (1)H NMR, (13)C NMR, IR, and UV-vis analyses. Its cytotoxic effect on the human leukemia cell lines HL-60 and K562 was studied by assessing the metabolic activity of cells (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT method), the structural integrity of cell membrane using Trypan blue assay, and the proliferation capacity of cells studying growth curves. Both leukemia cell lines showed a concentration-specific increased cytotoxicity of the complex, compared to l-carnitine or copper chloride dihydrate, with distinct underlying mechanisms, which were decreased proliferation efficiency for HL-60 cells and increased necrotic phenomena for K562 cells. Our results are indicative of a concentration-specific enhanced antileukemic effect of the complex, implying its value as a tool in the implementation of leukemia.
Assuntos
Carnitina/química , Carnitina/toxicidade , Cloretos/química , Cobre/química , Água/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Estrutura MolecularRESUMO
PURPOSE: Most anticancer drugs show their antiproliferative and cytotoxic activity via induction of apoptosis. In the present study we assessed the implication and role of cordycepin, a polyadenylation-specific inhibitor and a well-known chemotherapeutic drug, in apoptosis, induced by the anticancer drug etoposide. METHODS: For this purpose, a variety of leukemia and lymphoma cell lines (U937, K562, HL-60, Daudi, Molt-4) were treated with the anticancer drugs etoposide and/or cordycepin and assessed for poly(A) polymerase (PAP) activity and isoforms by the highly sensitive PAP activity assay and western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, DAPI staining, caspase-6 activity assay and DeltaPsi m reduction, whereas cytotoxicity and cell cycle status were assessed by Trypan blue staining, MTT assay and flow cytometry. RESULTS AND CONCLUSIONS: The results showed that PAP changes in all cell lines, in response to apoptosis induced by etoposide, in many cases even prior to hallmarks of apoptosis (endonucleosomal cleavage of DNA, DeltaPsi(m) reduction). A further elucidation to this apoptosis-polyadenylation correlation was added, by cell treatment with cordycepin, resulting in either suppression (U937, K562) or induction (HL-60) of the apoptotic process, according to the cell type. However, inhibition of polyadenylation did not influence the cell lines Daudi and Molt-4 used, where alternative apoptotic pathways are induced through cleavage of DNA into high molecular weight fragments.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Desoxiadenosinas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Poli A/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Caspase 6/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Etoposídeo/farmacologia , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Processamento de Imagem Assistida por Computador , Indóis , Leucemia/tratamento farmacológico , Leucemia/patologia , Potenciais da Membrana/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Neoplasias/patologiaRESUMO
PURPOSE: Posttranscriptional modifications, such as polyadenylation, are very often implicated in the regulation and dysregulation of cell death, through regulation of the expression of specific genes. Based on the fact that an increasing number of adenosine analogues show their antiproliferative and cytotoxic activity via induction of apoptosis, we assessed the effect of cordycepin, a polyadenylation specific inhibitor, an adenosine analogue and a well-known chemotherapeutic drug, on two human leukemia and lymphoma cell lines. METHODS: Cells were treated with the anticancer drug cordycepin and assessed for poly(A) polymerase (PAP) activity and isoforms by the highly sensitive PAP activity assay and western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, DAPI staining and Deltapsi(m) reduction, whereas cytotoxicity and cell cycle status were assessed by Trypan blue staining, MTT assay and flow cytometry. RESULTS AND CONCLUSIONS: The results showed that the differentiated modulations of PAP in the two cell lines may be a result of the additive effect of the changes in cell cycle and apoptotic pathway induced.
Assuntos
Antineoplásicos/farmacologia , Desoxiadenosinas/farmacologia , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Processamento de Imagem Assistida por Computador , Indóis , Leucemia/tratamento farmacológico , Leucemia/patologia , Linfoma/tratamento farmacológico , Linfoma/patologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Necrose , Polinucleotídeo Adenililtransferase/metabolismo , Sais de Tetrazólio , TiazóisRESUMO
Recently, a new member of the human SR (Ser/Arg-rich) superfamily of pre-mRNA splicing factors, SRA1 (SR-A1), has been discovered and cloned by members of our group, the gene for which was found to be overexpessed in a series of human tumors. In the present study, we investigated the significance of alterations at the mRNA expression levels of the SRA1 gene after treatment of HL-60 human promyelocytic leukemia cells with the anticancer drugs cisplatin and bleomycin. The kinetics of apoptosis and cell toxicity were investigated by DNA laddering and the MTT and trypan blue assays, respectively. Total RNA was extracted and cDNA was prepared by reverse transcription. The splicing-related genes SRA1 and SC35, as well as the apoptosis-related gene BCL2 (Bcl-2), were amplified by PCR using gene-specific primers. The results showed that mRNA levels of SRA1 were up-regulated upon treatment with the antibiotic bleomycin, whereas they were down-regulated by treatment of HL-60 human promyelocytic leukemia cells with cisplatin. Our results support the hypothesis that mRNA expression analysis of SRA1 may serve as a new prospective molecular marker, playing an important role in chemotherapy outcome in human leukemia.
Assuntos
Bleomicina/farmacologia , Cisplatino/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Promielocítica Aguda/genética , Proteínas Nucleares/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Células HL-60 , Humanos , Fatores de Processamento de Serina-Arginina , Fatores de TempoRESUMO
Most apoptosis-related genes regulate cellular fate as a response to anticancer drugs. Modulations at the mRNA levels of such genes often correlate with the sensitivity of various types of cancer cells to chemotherapeutic reagents. The drugs cisplatin, carboplatin, and doxorubicin exhibit anticancer activity, the mechanism of which is not yet completely clarified, although they are known to modulate the expression of several genes including apoptosis-related genes, such as members of the BCL2 (Bcl-2) family. In order to define the significance of the expression patterns of such genes as a response to anticancer drug cytotoxic activity, we studied the possible alterations in the mRNA expression levels of various apoptosis-related genes, including the new member, BCL2L12, after cell treatment with distinct anticancer drugs (cisplatin, carboplatin, and doxorubicin), in the breast cancer cell line, MCF-7. The kinetics of cell toxicity was evaluated by the MTT method, whereas the expression levels of distinct apoptosis-related genes were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), using gene-specific primers. The percentage of nonviable cells was upregulated with increasing concentrations and cell exposure time to the different anticancer drugs. Distinct modulations of apoptosis-related genes, at the mRNA level, were also observed. However, further work is required in order to ascertain whether the mRNA expression profile of such genes may provide evidence for their contribution to more specific and sensitive prediction of breast cancer response to treatment and therefore the rationale for individualized, more appropriate, and successful treatment.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/metabolismo , Carboplatina/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , HumanosRESUMO
We synthesized and studied the antitumor properties of the novel complex compound 2,5-dihydroxybenzoate molybdenum(VI) with tetraphenylphosphonium as counterion, which also acts as cancer cell growth inhibitor. A novel complex compound, the 2,5-dihydroxybenzoate molybdenum(VI) complex, (PPh4)2[Mo3O6(mu-O)2(2,5-DHBA)2] was synthesized. 1H NMR, 13C NMR, IR, and UV-Vis analyses were used for its molecular characterization. The human leukemia cell lines HL-60 and K562 were tested for their viability by assessing the metabolic activity of cells (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT), the structural integrity of the cell membrane (Trypan blue assay), and cell proliferation ability (growth curves). We showed that both leukemia cell lines are induced to decreased proliferation efficiency after treatment with the novel complex, compared to 2,5-dihydroxybenzoate, tetraphenyl-phosphonium polyoxomolybdate, or tetraphenylphosphonium chloride as individual entities, in a time- and concentration-dependent manner. Our results suggest that the new 2,5-dihydroxybenzoate molybdenum(VI) complex may provide a valuable tool in cancer chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Gentisatos/farmacologia , Oniocompostos/farmacologia , Compostos Organometálicos/farmacologia , Compostos Organofosforados/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Gentisatos/química , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide , Espectroscopia de Ressonância Magnética , Necrose , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Espectrofotometria Infravermelho , Espectrofotometria UltravioletaRESUMO
The neutral mononuclear copper complex with the quinolone antibacterial drug N-propyl-protected norfloxacin, Hpr-norfloxacin, in the presence of the nitrogen donor heterocyclic ligand 2,2'-bipyridine has been prepared and characterized. The crystal structure of (chloro)(2,2'-bipyridine)(pr-norfloxacinato)copper(II), 1, has been determined and refined with X-ray crystallography. X-band electron paramagnetic resonance (=EPR) spectroscopy at liquid helium temperatures from powdered samples indicates the presence of dimeric units in consistency with the crystal structure. In aqueous solutions of 1 the EPR behavior indicates mixture of dimeric and monomeric species. The antimicrobial activity of the complex has been tested on three different microorganisms and the best inhibition (MIC=0.25mugmL(-1)) has been exhibited against Escherichia coli. The study of the interaction of the complex with calf-thymus DNA has been performed with diverse spectroscopic techniques and has shown that complex 1 is bound to calf-thymus DNA by the intercalative mode. Potential anticancer cytostatic and cytotoxic effects of complex 1 on human promyelocytic leukemia HL-60 and human chronic myelogenous leukemia K562 cell lines have been investigated. Complex 1 shows an increased antiproliferative and necrotic effect on both HL-60 and K562 human leukemia cells in comparison to the free pr-norfloxacin.
Assuntos
2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Cobre/química , Norfloxacino/análogos & derivados , Animais , Bovinos , Linhagem Celular Tumoral , Dicroísmo Circular , DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Norfloxacino/química , Norfloxacino/farmacologia , Espectrofotometria Infravermelho , Espectrofotometria UltravioletaRESUMO
Many members of BCL2 (Bcl-2) apoptosis-related genes were found to be differentially expressed in various malignancies and were proposed as prognostic cancer biomarkers. Recently, a new member of the BCL2 gene family, BCL2L12, was cloned and was found to be expressed in mammary gland. In the present study, 55 specimens from patients with, histologically confirmed, epithelial breast carcinoma were analyzed for BCL2 and BCL2L12 gene expression by RT-PCR. Increased expression of BCL2 gene was found in patients belonging to the age groups <45 or >55 years, as well as in estrogen receptors (ER)-positive patients and in BCL2L12-positive tumors. In addition, BCL2 or BCL2L12-positive patients were found to be almost four times less likely to relapse or die in comparison to BCL2 or BCL2L12-negative patients, respectively. Multivariate analysis revealed that BCL2 and BCL2L12 might be used as independent prognostic biomarkers in breast cancer.
Assuntos
Neoplasias da Mama/genética , Genes bcl-2 , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Adulto , Idoso , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/análise , Análise de Sobrevida , Fatores de TempoRESUMO
We studied alterations in the mRNA expression levels of BCL2 (Bcl-2), BCL2L12, BAX, FAS and CASPASE-9 genes in the MCF-7 breast cancer cell line in response to treatment with two anticancer drugs. Cell toxicity was evaluated by the MTT method, trypan blue staining and DNA laddering, whereas the expression levels of the apoptosis-related genes were analysed by RT-PCR using gene-specific primers. In the case of etoposide, down-regulation of the BCL2L12-A gene variant and of CASPASE-9, as well as upregulation of BAX, was observed, whereas treatment of MCF-7 cells with taxol led to down-regulation of the mRNA levels of all genes examined. Our results support the idea that after long-term clinical studies, mRNA expression analysis of BCL2L12 and other members of the BCL2 gene family may serve as useful molecular markers predicting chemotherapy response in breast cancer.
Assuntos
Caspase 9/metabolismo , Etoposídeo/farmacologia , Proteínas Musculares/metabolismo , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Receptor fas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 9/efeitos dos fármacos , Caspase 9/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Fatores de Tempo , Proteína X Associada a bcl-2/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Receptor fas/efeitos dos fármacos , Receptor fas/genéticaRESUMO
One of the most effective ways to combat different types of cancer is through early diagnosis and administration of effective treatment, followed by efficient monitoring that will allow physicians to detect relapsing disease and treat it at the earliest possible time. Apoptosis, a normal physiological form of cell death, is critically involved in the regulation of cellular homeostasis. Dysregulation of programmed cell death mechanisms plays an important role in the pathogenesis and progression of cancer as well as in the responses of tumours to therapeutic interventions. Many members of the BCL2 (B-cell CLL/lymphoma 2; Bcl-2) family of apoptosis-related genes have been found to be differentially expressed in various malignancies, and some are useful prognostic cancer biomarkers. We have recently cloned a new member of this family, BCL2L12, which was found to be differentially expressed in many tumours. Most of the BCL2 family genes have been found to play a central regulatory role in apoptosis induction. Results have made it clear that a number of coordinating alterations in the BCL2 family of genes must occur to inhibit apoptosis and provoke carcinogenesis in a wide variety of cancers. However, more research is required to increase our understanding of the extent to which and the mechanisms by which they are involved in cancer development, providing the basis for earlier and more accurate cancer diagnosis, prognosis and therapeutic intervention that targets the apoptosis pathways. In the present review, we describe current knowledge of the function and molecular characteristics of a series of classic but also newly discovered genes of the BCL2 family as well as their implications in cancer development, prognosis and treatment.
Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Genes bcl-2/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Marcação de Genes/métodos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/terapiaRESUMO
Apoptotic cell death is a highly regulated process, which plays a crucial role in many biological events. Etoposide is an antineoplastic drug, which targets the DNA unwinding enzyme, topoisomerase II. The aim of the present research approach to investigate the expression of the apoptosis-related genes BCL2 (Bcl-2), FAS, Caspase-3, BAX and the new member BCL2L12, cloned by our group, along with treatment of HL-60 leukemia cells with etoposide. The kinetics of apoptosis induction and cell toxicity was evaluated by DNA laddering and MTT method, respectively. The mRNA expression levels of the genes were analyzed by RT-PCR using gene-specific primers. Beta-actin was used as a control gene. An important downregulation of BCL2L12 was observed at 4 h of drug treatment, whereas BAX was upregulated at the same time point. No alteration in the expression pattern of the other apoptosis-related genes was detected. Since, the main anticarcinogenic effect of etoposide is due to the induction of apoptosis, these changes observed in the mRNA expression levels of the genes may be an underlying mechanism.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/genética , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Sequência de Bases , Caspase 3/genética , Primers do DNA , Células HL-60 , Humanos , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2/genética , Receptor fas/genéticaRESUMO
Cancer results from an imbalance between cell cycle progression and apoptosis. Therefore, most anticancer drugs exert their antiproliferative and cytotoxic activity via cell cycle arrest and induction of apoptosis, a controlled form of cell death that is dysregulated in cancer. Many polyadenylation trans-acting factors, including polyadenylate polymerase (PAP), are increasingly found to be involved in cell cycle, apoptosis and cancer prognosis. The objective of the present study was to identify PAP modulations in the response of two epithelial cancer cell lines (HeLa and MCF-7) to apoptosis induction by the anticancer drugs etoposide and cordycepin. Cells were assessed for PAP activity and isoforms by the highly sensitive PAP activity assay and Western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, 4'6-diamidino-2-phenylindol (DAPI) staining and caspase-6 activity assay, whereas cytotoxicity and cell cycle status were assessed by trypan blue staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Our results indicate that PAP changes very early in response to either etoposide or cordycepin treatment, even prior to the hallmarks of apoptosis (chromatin condensation and cleavage), in both cell lines tested, but in a different mode. Our results suggest, for the first time, that in the epithelial cancer cell lines used, PAP modulations follow cell cycle progression rather than the course of apoptosis.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Ciclo Celular , Desoxiadenosinas/farmacologia , Etoposídeo/farmacologia , Polinucleotídeo Adenililtransferase/metabolismo , Neoplasias da Mama , Caspase 6 , Caspases/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA , Feminino , Humanos , Isoenzimas/metabolismo , Neoplasias Epiteliais e Glandulares , Fatores de Tempo , Neoplasias do Colo do ÚteroRESUMO
Apoptosis is a type of programmed cell death involved in many crucial biological processes. It represents the basic mechanism for the action of chemotherapeutic agents, such as doxorubicin and carboplatin. Both are able to cause cell death through the induction of apoptosis in the human leukemic cell line HL-60. We investigated the possible alterations in the expression of apoptosis-related genes, including the novel BCL2L12 gene, which was recently cloned in our group. The kinetics of apoptosis induction and cell toxicity was investigated by DNA laddering and by the MTT method, respectively. Total RNA was extracted and cDNA was prepared by reverse transcription. BCL2 , BAX , FAS , caspase-9, caspase-3 and BCL2L12 were amplified by PCR. Overexpression of FAS , BCL2L12 and caspase-3 was observed after treatment of HL-60 cells for 3 or 6 h with carboplatin, while their expression was decreased after a 12-h treatment, demonstrating that these genes may take part in the early stages of apoptosis. Overexpression of the same genes was also observed after 6 h of treatment with doxorubicin (concomitantly with DNA laddering). In the case of carboplatin-induced apoptosis we detected down-regulation of BAX , BCL2 and caspase-9, whereas in the case of doxorubicin, BAX and BCL2 remained at control levels and caspase-9 was increased.