RESUMO
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.
Assuntos
Cisplatino , Neoplasias , Humanos , Ensaio Cometa , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Dano ao DNA , LeucócitosRESUMO
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.
Assuntos
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
Heat shock proteins (HSPs), also known as molecular chaperones, participate in important cellular processes, such as protein aggregation, disaggregation, folding, and unfolding. HSPs have cytoprotective functions that are commonly explained by their antiapoptotic role. Their involvement in anticancer drug resistance has been the focus of intense research efforts, and the relationship between HSP induction and DNA repair mechanisms has been in the spotlight during the past decades. Because DNA is permanently subject to damage, many DNA repair pathways are involved in the recognition and removal of a diverse array of DNA lesions. Hence, DNA repair mechanisms are key to maintain genome stability. In addition, the interactome network of HSPs with DNA repair proteins has become an exciting research field and so their use as emerging targets for cancer therapy. This article provides a historical overview of the participation of HSPs in DNA repair mechanisms as part of their molecular chaperone capabilities.
Assuntos
Reparo do DNA , Proteínas de Choque Térmico/metabolismo , Animais , Dano ao DNA , Humanos , Modelos Biológicos , Transdução de SinaisRESUMO
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
We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O6-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.
Assuntos
Apoptose/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dacarbazina/farmacologia , Glioma/patologia , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imuno-Histoquímica , Chaperonas Moleculares , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , TemozolomidaRESUMO
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.
Assuntos
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
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.