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Cisplatin (cPt) is a commonly used treatment for solid tumors. The main target of its cytotoxicity is the DNA molecule, which makes the DNA damage response (DDR) crucial for cPt-based chemotherapy. Therefore, it is essential to identify biomarkers that can accurately predict the individual clinical response and prognosis. Our goal was to assess the usefulness of alkaline comet assay and immunocytochemical staining of phosphorylated Hsp90α (p-Hsp90α), γH2AX, and 53BP1 as predictive/prognostic markers. Pre-chemotherapy peripheral blood leukocytes were exposed to cPt in vitro and collected at 0, 24 (T24), and 48 (T48) hr post-drug removal. Healthy subjects were also included. Baseline DNA damage was elevated in cancer patients (variability between individuals was observed). After cPt, patients showed increased γH2AX foci/nucleus (T24 and T48). Both in healthy persons and patients, the nuclear p-Hsp90α and N/C (nuclear/cytoplasmic) ratio augmented (T24), decreasing at T48. Favorable clinical response was associated with high DNA damage and p-Hsp90α N/C ratio following cPt. For the first time, p-Hsp90α significance as a predictive marker is highlighted. Post-cPt-DNA damage was associated with longer disease-free survival and overall survival. Our findings indicate that comet assay and p-Hsp90α (a marker of DDR) would be promising prognostic/predictive tools in cP-treated cancer patients.
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Cisplatino , Neoplasias , Humanos , Ensaio Cometa , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Dano ao DNA , LeucócitosRESUMO
PURPOSE: PALB2 variants have been scarcely described in Argentinian and Latin-American reports. In this study, we describe molecular and clinical characteristics of PALB2 mutations found in multi-gene panels (MP) from breast-ovarian cancer (BOC) families in different institutions from Argentina. METHODS: We retrospectively identified PALB2 pathogenic (PV) and likely pathogenic (LPV) variants from a cohort of 1905 MP results, provided by one local lab (Heritas) and SITHER (Hereditary Tumor Information System) public database. All patients met hereditary BOC clinical criteria for testing, according to current guidelines. RESULTS: The frequency of PALB2 mutations is 2.78% (53/1905). Forty-eight (90.5%) are PV and five (9.5%) are LPV. Most of the 18 different mutations (89%) are nonsense and frameshift types and 2 variants are novel. One high-rate recurrent PV (Y551*) is present in 43% (23/53) of the unrelated index cases. From the 53 affected carriers, 94% have BC diagnosis with 14% of bilateral cases. BC phenotype is mainly invasive ductal (78%) with 62% of hormone-receptor positive and 22% of triple negative tumors. Self-reported ethnic background of the cohort is West European (66%) and native Latin-American (20%) which is representative of Buenos Aires and other big urban areas of the country. CONCLUSION: This is the first report describing molecular and clinical characteristics of PALB2 carriers in Argentina. Frequency of PALB2 PV in Argentinian HBOC families is higher than in other reported populations. Y551* is a recurrent mutation that seems to be responsible for almost 50% of PALB2 cases.
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Neoplasias da Mama , Neoplasias Ovarianas , Argentina/epidemiologia , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Neoplasias Ovarianas/epidemiologia , Neoplasias Ovarianas/genética , Estudos RetrospectivosRESUMO
OBJECTIVE: To determine whether circulating heat shock proteins HSP27/HSPB1 and HSP90α/HSPC1 may be useful for early prediction of the occurrence of pre-eclampsia in asymptomatic women. METHODS: We have measured by ELISA the levels of HSPB1, HSPC1, and placental protein 13 (PP13) in serum samples from 44 women in the first trimester (10-12 weeks) and second trimester (17-20 weeks) of pregnancy. Western blot and immunohistochemistry for HSPB1 and HSPC1 were performed. RESULTS: HSPB1 serum levels were higher in women with pre-eclampsia than in normotensive pregnant women at the first and second trimester (P = 0.003), whereas PP13 levels decreased in women with pre-eclampsia only in the first trimester of gestation (P = 0.021). We also observed higher HSPB1 levels in patients with early-onset pre-eclampsia in the first and second trimester (P = 0.014). CONCLUSION: This pilot study points out that circulating HSPB1 levels in first and second trimester might be useful for predicting the occurrence of pre-eclampsia in asymptomatic women. Further validation studies are needed to finally establish this protein as a candidate predictive biomarker of pre-eclampsia.
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Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico , Chaperonas Moleculares , Pré-Eclâmpsia , Biomarcadores , Feminino , Proteínas de Choque Térmico HSP27/sangue , Proteínas de Choque Térmico/sangue , Humanos , Chaperonas Moleculares/sangue , Projetos Piloto , Placenta/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da GravidezRESUMO
Breast cancer can be classified into molecular subtypes. Tumors overexpressing HER2 protein are more aggressive and metastatic; hence, patients have a poor prognosis. Anti-HER2 strategies, such as the monoclonal antibody Trastuzumab (Tz), have therefore been developed. Despite this progress, not all patients respond to the treatment. Retinoic acid (RA) has been proposed as an adjuvant treatment of breast carcinoma because of its ability to inhibit cell growth. We evaluated the effect of Tz in combination with RA on the viability, adhesion, migration, invasion and expression of migration-related proteins in SKBR3 and BT-474 human breast cancer cells. MTT, pharmacological interaction analysis, immunofluorescence, adhesion/migration/invasion and Western blot assays were performed. The coadministration of both drugs synergistically decreased cell survival. Tz+RA significantly decreased adhesion/migration/invasion in both cell types. Tz+RA strongly reduced FAK and HER2 expression and induced nuclear FAK translocation. In addition, a granular distribution of HER2 receptor was observed after the combined treatment. In conclusion, the coadministration of both drugs in patients with this type of cancer could contribute to the improvement of their prognosis and reduce the adverse effects of therapy because the applied Tz doses would be lower due to the adjuvant effect of RA.
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AIM: Accumulated evidence suggests that aberrant methylation of the TP73 gene and increased levels of ΔNp73 in primary tumours correlate with poor prognosis. However, little is known regarding the transcriptional and functional regulation of the TP73 gene in breast cancer. The aim of the present study was to determine the expression of the ΔNp73 isoform, its relationship with DNA methylation of TP73 and their clinical prognostic significance in breast cancer patients. METHODS: TP73 gene methylation was studied in TCGA datasets and in 70 invasive ductal breast carcinomas (IDCs). The expression of p73 isoforms was evaluated by immunohistochemistry (IHC) and Western blot and correlated with clinicopathological variables and clinical outcome. RESULTS: We observed that the methylation of diverse CpG islands of TP73 differed significantly between molecular subtypes. An inverse correlation was found between p73 protein expression and the methylation status of the TP73 gene. The expression of exon 3' of p73 (only expressed in ΔNp73) was significantly higher in patients with wild-type p53. Immunohistochemical analysis revealed that all p73 isoforms were localised in both the nuclear and cytoplasmic compartments. We confirmed a positive association between the expression of ∆Np73 and high histological grade. CONCLUSIONS: Our findings suggest that high expression of ΔNp73 could be used to determine the aggressiveness of IDCs and could be incorporated in the pathologist's report.
Assuntos
Neoplasias da Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Ilhas de CpG/genética , Proteína Tumoral p73/genética , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Metilação de DNA , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Prognóstico , Isoformas de Proteínas , Proteína Tumoral p73/metabolismoRESUMO
Breast cancer is the most common malignancy in women, with metastases being the cause of death in 98%. In previous works we have demonstrated that retinoic acid (RA), the main retinoic acid receptor (RAR) ligand, is involved in the metastatic process by inhibiting migration through a reduced expression of the specific migration-related proteins Moesin, c-Src, and FAK. At present, our hypothesis is that RA also acts for short periods in a non-genomic action to cooperate with motility reduction and morphology of breast cancer cells. Here we identify that the administration of 10(-6) M RA (10-20 min) induces the activation of the migration-related proteins Moesin, FAK, and Paxillin in T-47D breast cancer cells. The phosphorylation exerted by the selective agonists for RARα and RARß, on Moesin, FAK, and Paxillin was comparable to the activation exerted by RA. The RARγ agonist only led to a weak activation, suggesting the involvement of RARα and RARß in this pathway. We then treated the cells with different inhibitors that are involved in cell signaling to regulate the mechanisms of cell motility. RA failed to activate Moesin, FAK, and Paxillin in cells treated with Src inhibitor (PP2) and PI3K inhibitor (WM), suggesting the participation of Src-PI3K in this pathway. Treatment with 10(-6) M RA for 20 min significantly decreased cell adhesion. However, when cells were treated with 10(-6) M RA and FAK inhibitor, the RA did not significantly inhibit adhesion, suggesting a role of FAK in the adhesion inhibited by RA. By immunofluorescence and immunoblotting analysis we demonstrated that RA induced nuclear FAK translocation leading to a reduced cellular adhesion. These findings provide new information on the actions of RA for short periods. RA participates in cell adhesion and subsequent migration, modulating the relocation and activation of proteins involved in cell migration.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Paxilina/metabolismo , Tretinoína/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP72 , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transporte Proteico/efeitos dos fármacos , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico/agonistas , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Quinases da Família src/metabolismoRESUMO
Breast cancer is the most common malignancy in women and the appearance of distant metastases produces the death in 98% of cases. The retinoic acid receptor ß (RARß) is not expressed in 50% of invasive breast carcinoma compared with normal tissue and it has been associated with lymph node metastasis. Our hypothesis is that RARß protein participates in the metastatic process. T47D and MCF7 breast cancer cell lines were used to perform viability assay, immunobloting, migration assays, RNA interference and immunofluorescence. Administration of retinoic acid (RA) in breast cancer cells induced RARß gene expression that was greatest after 72 hrs with a concentration 1 µM. High concentrations of RA increased the expression of RARß causing an inhibition of the 60% in cell migration and significantly decreased the expression of migration-related proteins [moesin, c-Src and focal adhesion kinase (FAK)]. The treatment with RARα and RARγ agonists did not affect the cell migration. On the contrary, the addition of the selective retinoid RARß-agonist (BMS453) significantly reduced cell migration comparable to RA inhibition. When RARß gene silencing was performed, the RA failed to significantly inhibit migration and resulted ineffective to reduce moesin, c-Src and FAK expressions. RARß is necessary to inhibit migration induced by RA in breast cancer cells modulating the expression of proteins involved in cell migration.
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Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Receptores do Ácido Retinoico/metabolismo , Tretinoína/farmacologia , Citoesqueleto de Actina , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/metabolismo , Proteína Tirosina Quinase CSK , Proliferação de Células/efeitos dos fármacos , Feminino , Imunofluorescência , Quinase 1 de Adesão Focal/metabolismo , Humanos , Técnicas Imunoenzimáticas , Proteínas dos Microfilamentos/metabolismo , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética , Retinoides/farmacologia , Células Tumorais Cultivadas , Quinases da Família src/metabolismoRESUMO
Mammary stroma is composed of various cell types, including migratory leukocytes. Although mammary antibody-secreting cells have been extensively studied, reports focusing on mammary T cells are scarce. It is thought that the recruitment mechanism of leukocytes to the mammary gland (MG) is controlled by pregnancy- and lactation-specific stimuli. But whether prolactin (PRL) modulates the T-cell population in MG is still unknown. Our aim was to study the relationship between PRL levels and T and B cells during early lactation (L2, day 2 post partum) and mid-lactation (L12, day 12 of lactation). In order to investigate whether PRL is associated with homing events to MG, female Sprague Dawley (SD) and SD-derived desmoglein 4(-/-) hairless (phenotype with lactation deficit, OFA hr/hr) rats were killed during estrus, pregnancy, and post partum, and blood, MG, and corpora lutea were obtained to perform fluorescent-activated cell sorting (FACS), real-time PCR, and histological and RIA studies. Serum PRL levels were lower in OFA hr/hr rats than in SD rats during early lactation. MG of OFA hr/hr rats showed less secretory material compared with SD rats. FACS analysis showed lower percentage of MG CD3+ cells in OFA hr/hr rats compared with SD rats on L2 and L12. OFA hr/hr rats showed higher absolute numbers of circulating CD3+ cells compared with SD rats on L2 but not on L12. These results show that T-cell population in MG is affected in early lactating OFA hr/hr rats and strongly suggest that serum PRL levels may be involved in the homing events to MG, probably helping antibody-secreting cells and protecting the gland during lactation development.
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Lactação , Glândulas Mamárias Animais/imunologia , Prolactina/fisiologia , Linfócitos T/fisiologia , Animais , Linfócitos B/fisiologia , Feminino , Leucócitos/metabolismo , Contagem de Linfócitos , Masculino , Glândulas Mamárias Animais/crescimento & desenvolvimento , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Quimiocinas/metabolismoRESUMO
Breast carcinogenesis is a multistep process that involves both genetic and epigenetic alterations. Identification of aberrantly methylated genes in breast tumors and their relation to clinical parameters can contribute to improved diagnostic, prognostic, and therapeutic decision making. Our objective in the present study was to identify the methylation status of 34 cancer-involved genes in invasive ductal carcinomas (IDC). Each of the 70 IDC cases analyzed had a unique methylation profile. The highest methylation frequency was detected in the WT1 (95.7%) and RASSF1 (71.4%) genes. Hierarchical cluster analysis revealed three clusters with different distribution of the prognostic factors tumor grade, lymph node metastasis, and proliferation rate. Methylation of TP73 was associated with high histological grade and high proliferation rate; methylation of RARB was associated with lymph node metastasis. Concurrent methylation of TP73 and RARB was associated with high histological grade, high proliferation rate, increased tumor size, and lymph node metastasis. Patients with more than six methylated genes had higher rates of relapse events and cancer deaths. In multivariate analysis, TP73 methylation and the methylation index were associated with disease outcome. Our results indicate that methylation index and methylation of TP73 and/or RARB are related to unfavorable prognostic factors in patients with IDC. These epigenetic markers should be validated in further studies to improve breast cancer management.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Ilhas de CpG , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Adulto , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/patologia , Análise por Conglomerados , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Prognóstico , Receptores do Ácido Retinoico/genética , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , Proteínas WT1/genéticaRESUMO
PURPOSE: The objective of the present study was to examine the consequences of a mild hyperthermia in human tumour cell lines deficient and proficient in the DNA mismatch repair system (MMR) to advance our understanding on the relationship between MMR and heat shock proteins (HSPs). MATERIALS AND METHODS: The human colon carcinoma cell lines HCT116 (parent cells), HCT116 + ch2 (MMR-deficient), and HCT116 + ch3 (MMR-proficient) were used. Cells were incubated at 41°C and 42°C for 1 h and then at 37°C for 4 and 24 h. The expression of Hsp27 and Hsp72 was evaluated by immunocytochemistry. Hsp27, Hsp72, hMLH1 and hMSH2 levels were assessed by western blotting in nuclear and cytoplasmic fractions. The alkaline comet assay was used to evaluate the DNA damage. RESULTS: The mild hyperthermia significantly increased the protein expression levels of Hsp27 and Hsp72 in all cell lines, which was higher in the cytoplasm and nucleus of HCT116 + ch3 cells. We also observed that heat induced translocation of hMLH1 and hMSH2 proteins from the nucleus to the cytoplasm in HCT116 + ch3 cells. The comet assay revealed that HCT116 parent cells were more resistant to heat-induced DNA damage. However, the MMR-proficient and deficient cell lines repaired the DNA damage at the same rate. CONCLUSIONS: The present study demonstrates that hyperthermia induced the nuclear accumulation of Hsp27 and Hsp72 and affected the subcellular localisation of hMLH1 and hMSH2 in HCT116 + ch3 cells. Our findings suggest that the MMR system is not a direct determining factor for the different heat shock response in HCT116 cells.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Reparo de Erro de Pareamento de DNA , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Hipertermia Induzida , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genéticaRESUMO
INTRODUCCIÓN La preeclampsia es una patología del embarazo, etiología desconocida con morbimortalidad materno-perinatal. OBJETIVOS Evaluar los marcadores moleculares Activina A, PAPP-A, PP13 e Inhibina A. Detectar precozmente cuáles son las mujeres con alto riesgo de desarrollar preeclampsia, generar un banco de muestras de sangre, determinar los marcadores en los tres trimestres y establecer si son predictivos y se relacionan con la enfermedad. MÉTODOS Se incorporó a mujeres con embarazo normal y no embarazadas. Se tomaron muestras de sangre entre las semanas 10-12 y 17-20 del embarazo y en el parto. Las muestras fueron alicuotadas y almacenadas a -80 ºC hasta su utilización. Se dosificaron los marcadores por técnica ELISA con kits comerciales. RESULTADOS La concentración de Activina A se incrementó en el primer trimestre en las pacientes con preeclampsia leve. Se observó una disminución en la concentración de PAPP-A en el primer trimestre en las pacientes con desarrollo de preeclampsia, una disminución de PP13 en el primer y el segundo trimestres en las pacientes con preeclampsia leve estadísticamente significativa en el primer trimestre (p=0,038) y una disminución de Inhibina A en el tercer trimestre en las pacientes con preeclampsia severa (p=0,014). DISCUSIÓN La determinación de PP13 en el primer trimestre del embarazo es útil para la predicción de preeclampsia leve. Los resultados hallados son discordantes con los de otros autores que también encuentran diferencias significativas en las concentraciones de los marcadores Activina A, PAPP-A e Inhibina A.
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Pré-Eclâmpsia , Proteína Plasmática A Associada à GravidezRESUMO
INTRODUCTION: Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes) and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. CASE PRESENTATION: A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. CONCLUSION: In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion.
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Genetic and epigenetic events play a critical role in the tumorigenic process of breast cancer. The more genes are studied, the more accurate the epigenetic "signature" can be established. The aim of our work has been to apply the technique Methylation-Specific Multiplex Ligation dependent Probe Amplification (MS-MLPA) to study the methylation profile of 26 cancer related gene regions in breast cancers. Secondly, we aimed to establish if the epigenetic "signature" could serve to detect circulating tumor DNA (ctDNA) in breast cancer patients. The MS-MLPA was successfully setup and allowed to establish which regions were preferentially associated with the tumor process. The analysis permitted also to detect significant concurrent methylation between some genes. The detection of ctDNA could be performed by a "double-round" MS-MLPA (drMS-MLPA) approach and nested-Methyl Specific PCR (Nested-MSP). This development is an important novelty and served to detect a small amount of tumor DNA shaded into the blood stream of breast cancer patients. We conclude that MS-MLPA is an excellent assay to analyze the methylation profile of a tumor. The 82 studied samples presented a specific methylation "mark". These studies serve to enhance the knowledge of the role of epigenetic alterations in breast tumors and can contribute to the development of personalized diagnosis, surveillance and treatment strategies.
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Neoplasias da Mama/genética , Metilação de DNA , Predisposição Genética para Doença/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sondas de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Feminino , Células HeLa , Humanos , Invasividade Neoplásica , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos TestesRESUMO
We have analyzed the predictive/prognostic value of Bcl-2 protein in breast cancer patients treated with neoadjuvant chemotherapy. One hundred and ten patients were submitted to two different chemotherapeutic regimens: a) 5-fluorouracil, adriamycin or epirubicin, and cyclophosphamide (FAC/FEC) during 2-6 cycles before surgery and 3 or 4 additional cycles of FAC/FEC after surgery (n=40) and b) doxorubicin (D) 75 mg/m(2) or epirubicin (E) 120 mg/m(2) during 4 cycles before surgery, and 6 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) after surgery (n=70). Bcl-2 expression, evaluated by immunohistochemistry, did not change significantly after chemotherapy and was not related to clinical/pathological response. In FAC/FEC group, Bcl-2 positive expression after chemotherapy correlated with better disease free survival (DFS) and overall survival (OS) (P=0.008 and P=0.001). In D/E group, Bcl-2 also correlated with better DFS and OS (P=0.03 and P=0.054) in the post-chemotherapy biopsies. An unusual nuclear localization of Bax was observed in some biopsies, but this localization did not correlate with the tumor response or outcome of the patients. We found that a high Bcl-2 expression had no predictive value but had prognostic value in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy.
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Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas c-bcl-2/análise , Antraciclinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Terapia Neoadjuvante/métodos , Prognóstico , Análise de Sobrevida , Resultado do Tratamento , Proteína X Associada a bcl-2/análiseRESUMO
Mismatch repair (MMR) deficiency and higher expression levels of heat shock proteins (Hsps) have been implicated with drug resistance to topoisomerase II poisons (doxorubicin) and to platinum compounds (cisplatin). This study was designed to determine individual influences of doxorubicin and cisplatin treatment on the expression of Hsp27, Hsp70, hMLH1 and hMSH2 proteins and in the DNA damage status in peripheral blood lymphocytes (PBLs). In addition, we studied whether these proteins and the DNA damage correlated with the survival of cancer patients. PBLs from 10 healthy donors and 25 cancer patients (before and after three cycles of chemotherapy) were exposed to in vitro treatments: C (control), HS (heat shock at 42 degrees C), Do or Pt (doxorubicin or cisplatin alone), and HS+Do or HS+Pt (heat shock+doxorubicin or heat shock+cisplatin). PBLs were collected at time 0 (T0: immediately after drug treatment) and after 24h of repair (T24). Hsp27, Hsp70, hMLH1 and hMSH2 were studied by immunocytochemistry and the DNA damage by alkaline comet assay. Immunofluorescence studies and confocal microscopy revealed that hMLH1 and hMSH2 colocalized with Hsp27 and Hsp72 (inducible form of Hsp70). hMLH1 and hMSH2 were significantly induced by Pt and HS+Pt at T24 in cancer patients, but only modestly influenced by Do. Cancer patients presented higher basal expression of total and nuclear Hsp27 and Hsp70 than controls, and these proteins were also increased by HS, Do and HS+Do. The Hsp70 induction by Pt and HS+Pt was noted in cancer patients, especially nuclear Hsp70. In cancer patients, basal DNA damage was slightly higher than in healthy persons; and after Pt and HS+Pt treatments, DNA migration and number of apoptotic cells were higher than controls. Hsps accomplished a cytoprotective function in pre-chemotherapy PBLs (HS before Do or Pt), but not in post-chemotherapy samples. In Pt-treated patients the ratio N/C (nuclear/cytoplasmic) of Hsp27 was related to disease free survival and overall survival, and hMSH2 correlated with overall survival. The results point to the utility of these molecules and of the comet assay as possible predictive markers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Adolescente , Adulto , Antineoplásicos/uso terapêutico , Biomarcadores/análise , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Cisplatino/uso terapêutico , Citoplasma/metabolismo , DNA/análise , Dano ao DNA , Reparo de Erro de Pareamento de DNA , Doxorrubicina/uso terapêutico , Feminino , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico/análise , Humanos , Lactente , Linfócitos/química , Linfócitos/metabolismo , Masculino , Chaperonas Moleculares , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/análise , Proteínas de Neoplasias/análise , Neoplasias/mortalidade , Proteínas Nucleares/análise , PrognósticoRESUMO
Drug resistance is considered the main impediment to successful cancer chemotherapy. The quest for a method useful to predict individual responses to chemotherapy prior to treatment is highly desired. This study was designed to determine the individual influences of doxorubicin and cisplatin on the degree of DNA damage, DNA repair and hMSH2 and the hMLH1 protein expression in peripheral blood lymphocytes (PBL) and their correlations with the clinical response. PBL were obtained from 25 cancer patients (pre- and post-chemotherapy) and from 10 healthy persons, cultured and exposed to doxorubicin or cisplatin. Cells were collected at T0 (immediately after drug treatment) and 24h after damage (T24). The alkaline comet assay was employed to assess the DNA damage and repair function, and immunocytochemistry to study hMLH1 and hMSH2 expression. Clinical response was evaluated after three cycles of chemotherapy. Pre-chemotherapy PBL from cancer patients showed significantly higher levels of basal DNA damage than healthy persons, with appreciable interindividual variations between them. The in vivo administration of antineoplasic drugs was accompanied by significant DNA damage, and an increased in the number of apoptotic cells. Cancer patients with complete response showed a high number of apoptotic cells. The DNA migration increased at T0 and at T24 in cisplatin-treated patients, reflecting a decreased rate of cisplatin adducts repair than that observed in healthy individuals. The ability to repair DNA lesions in doxorubicin-damaged cells was very similar between healthy individuals and cancer patients. Cisplatin-treated patients that died by the disease showed lower DNA migration than the mean value. The expression of hMLH1 and hMSH2 was practically identical between healthy individuals and cancer patients. Nevertheless, chemotherapy induced a depletion mostly of hMLH1. In 83% of cisplatin-treated patients with CR the hMLH1 and hMSH2 expression at T24 was higher than the mean. In this pilot study the alkaline comet assay offered information about the amount of DNA damage and the DNA repair status in PBL from individual patients and this seems useful in predicting the response to chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Antibióticos Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Cisplatino/farmacologia , Ensaio Cometa , Doxorrubicina/farmacologia , Feminino , Humanos , Lactente , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Projetos PilotoRESUMO
This study was designed to assess whether the chemotherapeutic drug paclitaxel can induce DNA damage in peripheral blood lymphocytes of human healthy donors, and to evaluate if such damage could be repaired. Venous blood was collected by routine venipuncture, the lymphocytes were isolated and cultured and then treated with 100nM, 500nM, 10microM, and 30microM of taxol for 4h. The alkaline comet assay technique was used to quantify the level of DNA damage and the DNA repair in lymphocytes. A significant increase in DNA damage was achieved when the cells were incubated with paclitaxel concentrations of 10microM or above. To test the DNA repair capability, the lymphocytes were allowed to recover for 2, 4, 6, and 24h. The DNA damage was almost completely repaired after 24h of incubation demonstrating a time-dependent repair capability. In conclusion, we demonstrate that paclitaxel induces DNA damage in peripheral blood lymphocytes and that this damage can be repaired.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Dano ao DNA , DNA/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Paclitaxel/toxicidade , Adulto , Álcalis , Células Cultivadas , Ensaio Cometa , Feminino , Humanos , Linfócitos/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Doxorubicin is an antineoplastic drug widely used in cancer treatment. However, many tumors are intrinsically resistant to the drug or show drug resistance after an initial period of response. Among the different molecules implicated with doxorubicin resistance are the heat shock proteins (Hsps). At present we do not know with certainty the mechanism(s) involved in such resistance. In the present study, to advance our knowledge on the relationship between Hsps and drug resistance, we have used peripheral blood mononuclear cells obtained from healthy nonsmoker donors to evaluate the capacity of a preliminary heat shock to elicit the Hsp response and to establish the protection against the deoxyribonucleic acid (DNA) damage induced by doxorubicin. DNA damage and repair were determined using the alkaline comet assay. We also measured the expression of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1, hMSH2, and proliferating cell nuclear antigen by immunocytochemistry. The damage induced by doxorubicin was more efficiently repaired when the cells were previously heat shocked followed by a resting period of 24 hours before drug exposure, as shown by (1) the increased number of undamaged cells (P < 0.05), (2) the increased DNA repair capacity (P < 0.05), and (3) the high expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 (P < 0.05). In addition, in the mentioned group of cells, we confirmed by Western blot high expression levels of Hsp27 and Hsp70. We also noted a nuclear translocation of Hsp27 and mainly of Hsp70. Furthermore, inducible Hsp70 was more expressed in the nucleus than Hsc70, showing a possible participation of Hsp70 in the DNA repair process mediated by the MMR system.