RESUMEN
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 , Ensayo Cometa , Cisplatino/farmacología , Cisplatino/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Daño del ADN , LeucocitosRESUMEN
Platinum analogs are commonly used for cancer treatment. There is increasing interest in finding biomarkers which could predict and overcome resistance, because to date there is no reliable predictive/prognostic marker for these compounds. Here we studied the immunohistochemical expression of proteins involved in DNA damage response and repair (γH2AX, 53BP1, ERCC1, MLH1, and MSH2) in primary tumor tissues from patients treated with platinum-based chemotherapy. Levels and localization of Heat Shock Protein (HSP)27 and phospho-(Thr5/7)-HSP90α (p-HSP90α) were also determined. The implications in clinical response, disease-free survival and overall survival were analyzed. High γH2AX and 53BP1 expressions were associated with poor clinical response. Nuclear p-HSP90α, as well as nuclear absence and low cytoplasmic expression of HSP27 correlated with good response. Patients with high γH2AX and high cytoplasmic HSP27 expressions had shorter overall survival and disease-free survival. MLH1, MSH2, or ERCC1 were not associated with clinical response or survival. We report the potential utility of p-HSP90α, HSP27, γH2AX, and 53BP1 as predictive/prognostic markers for platinum-based chemotherapy. We present the first study that evaluates the predictive and prognostic value of p-HSP90α in primary tumors. Our research opens new possibilities for clinical oncology and shows the usefulness of immunohistochemistry for predicting chemotherapy response and prognosis in cancer.
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Proteínas de Choque Térmico HSP27 , Neoplasias , Antineoplásicos/uso terapéutico , Biomarcadores , Biomarcadores de Tumor/metabolismo , Daño del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/metabolismo , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico/genética , Humanos , Proteína 2 Homóloga a MutS/genética , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Compuestos de Platino/uso terapéutico , PronósticoRESUMEN
Double heterozygosity pathogenic variants in BRCA1 and BRCA2 genes are a very rare finding, particularly in non-Ashkenazi individuals. We described the first case of double heterozygosity variants in a non-Ashkenazi Argentinean woman with metachronous bilateral breast cancer. The proband is a 65-year-old female diagnosed with invasive ductal carcinoma in the left breast at 45 years old and invasive carcinoma in the right breast at 65 years old. She underwent a multi-gene panel testing indicating the presence of two concurrent heterozygous germline deleterious variants NM_007300.4(BRCA1):c.4201C>T (p.Gln1401Ter), and NM_000059.3(BRCA2):c.5146_5149del (p.Tyr1716fs). . The patient's son (40 years-old) was found to have the inherited pathogenic variant in BRCA2 gene. There are few reports of double heterozygosity variants in BRCA1 and BRCA2 genes in Latin America. The two pathogenic variants identified in our patient have not been described together so far.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Neoplasias Primarias Secundarias/genética , Mutación Puntual , Eliminación de Secuencia , Adulto , Anciano , Femenino , Asesoramiento Genético , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Heterocigoto , Humanos , Masculino , Linaje , Análisis de Secuencia de ADNRESUMEN
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 , Preeclampsia , Biomarcadores , Femenino , Proteínas de Choque Térmico HSP27/sangre , Proteínas de Choque Térmico/sangre , Humanos , Chaperonas Moleculares/sangre , Proyectos Piloto , Placenta/metabolismo , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del EmbarazoRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Islas de CpG/genética , Proteína Tumoral p73/genética , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Metilación de ADN , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Pronóstico , Isoformas de Proteínas , Proteína Tumoral p73/metabolismoRESUMEN
PURPOSE: Hyperthermia is used in combination with conventional anticancer agents to potentiate their cytotoxicity. One of its key events is the synthesis of heat shock proteins (HSPs), which are able to associate with components from DNA repair mechanisms. However, little is known about their relationship with the mismatch repair system (MMR). Our aim was to study the effects of hyperthermia on cisplatin (cPt) sensitivity and to determine whether MLH1 and MSH2 associate with Hsp27 and Hsp72 in MMR-deficient(-)/-proficient(+) cells. MATERIALS AND METHODS: HCT116+ch2 (MMR-) and HCT116+ch3 (MMR+) cell lines were exposed to cPt with or without previous hyperthermia (42 °C, 1 h). Clonogenic survival assays, MTT, confocal immunofluorescence, immunoprecipitation, immunoblotting and flow cytometry were performed. RESULTS: Hyperthermia increased the cPt resistance in MMR- cells 1.42-fold. Immunofluorescence revealed that after cPt, Hsp27 and Hsp72 translocated to the nucleus and colocalisation coefficients between these proteins with MLH1 and MSH2 increased in MMR+ cells. Immunoprecipitation confirmed the interactions between HSPs and MMR proteins in control and treated cells. Hyperthermia pretreatment induced cell cycle arrest, increased p73 expression and potentiated cPt sensitivity in MMR+ cells. CONCLUSIONS: This is the first report showing in a MMR-/+ cellular model that MLH1 and MSH2 are client proteins of Hsp27 and Hsp72. Our study suggests that p73 might participate in the cellular response to hyperthermia and cPt in a MMR-dependent manner. Further functional studies will confirm whether HSPs cooperate with the MMR system in cPt-induced DNA damage response or whether these protein interactions are only the result of their chaperone functions.
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Antineoplásicos/uso terapéutico , Cisplatino/efectos adversos , Neoplasias del Colon/tratamiento farmacológico , Daño del ADN/genética , Reparación de la Incompatibilidad de ADN/genética , Fiebre/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas del Choque Térmico HSP72/metabolismo , Línea Celular Tumoral , Cisplatino/toxicidad , HumanosRESUMEN
BACKGROUND: The brain is a common target of metastases for melanoma patients. Little is known about the genetic and epigenetic alterations in melanoma brain metastases (MBMs). Unraveling these molecular alterations is a key step in understanding their aggressive nature and identifying novel therapeutic targets. METHODS: Genome-wide DNA methylation analyses of MBMs (n = 15) and normal brain tissues (n = 91) and simultaneous multigene DNA methylation and gene deletion analyses of metastatic melanoma tissues (99 MBMs and 43 extracranial metastases) were performed. BRAF and NRAS mutations were evaluated in MBMs by targeted sequencing. RESULTS: MBMs showed significant epigenetic heterogeneity. RARB, RASSF1, ESR1, APC, PTEN, and CDH13 genes were frequently hypermethylated. Deletions were frequently detected in the CDKN2A/B locus. Of MBMs, 46.1% and 28.8% had BRAF and NRAS missense mutations, respectively. Compared with lung and liver metastases, MBMs exhibited higher frequency of CDH13 hypermethylation and CDKN2A/B locus deletion. Mutual exclusivity between hypermethylated genes and CDKN2A/B locus deletion identified 2 clinically relevant molecular subtypes of MBMs. CDKN2A/B deletions were associated with multiple MBMs and frequently hypermethylated genes with shorter time to brain metastasis. CONCLUSIONS: Melanoma cells that colonize the brain harbor numerous genetically and epigenetically altered genes. This study presents an integrated genomic and epigenomic analysis that reveals MBM-specific molecular alterations and mutually exclusive molecular subtypes.
Asunto(s)
Neoplasias Encefálicas/genética , Metilación de ADN , Eliminación de Gen , Melanoma/genética , Mutación/genética , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/secundario , Estudios de Cohortes , Epigénesis Genética , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Masculino , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Tasa de Supervivencia , Adulto JovenRESUMEN
Neoadjuvant chemotherapy is used in patients with locally advanced breast cancer to reduce tumor size before surgery. Unfortunately, resistance to chemotherapy may arise from a variety of mechanisms. Heat shock proteins (HSPs), which are highly expressed in mammary tumor cells, have been implicated in anticancer drug resistance. In spite of the widely described value of HSPs as molecular markers in cancer, their implications in breast tumors treated with anthracycline-based neoadjuvant chemotherapy has been poorly explored. In this study, we have evaluated, by immunohistochemistry, the expression of HSP27 (HSPB1) and HSP70 (HSPA) in serial biopsies from locally advanced breast cancer patients (n = 60) treated with doxorubicin (DOX)- or epirubicin (EPI)-based monochemotherapy. Serial biopsies were taken at days 1, 3, 7, and 21, and compared with prechemotherapy and surgical biopsies. After surgery, the patients received additional chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil. High nuclear HSPB1 and HSPA expressions were found in invasive cells after DOX/EPI administration (P < 0.001), but the drug did not affect the cytoplasmic expression of the HSPs. Infiltrating lymphocytes showed high nuclear HSPA (P < 0.01) levels at postchemotherapy. No correlations were found between HSPs expression and the clinical and pathological response to neoadjuvant therapy. However, in postchemotherapy biopsies, high nuclear (>31 % of the cells) and cytoplasmic HSPA expressions (>11 % of the tumor cells) were associated with better DFS (P = 0.0348 and P = 0.0118, respectively). We conclude that HSPA expression may be a useful prognostic marker in breast cancer patients treated with neoadjuvant DOX/EPI chemotherapy indicating the need to change the administered drugs after surgery for overcoming drug resistance.
Asunto(s)
Antraciclinas/uso terapéutico , Antibióticos Antineoplásicos/uso terapéutico , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Mama/patología , Proteínas HSP70 de Choque Térmico/análisis , Terapia Neoadyuvante , Adulto , Anciano , Mama/efectos de los fármacos , Neoplasias de la Mama/patología , Doxorrubicina/uso terapéutico , Epirrubicina/uso terapéutico , Femenino , Estudios de Seguimiento , Proteínas de Choque Térmico HSP27/análisis , Humanos , Inmunohistoquímica , Persona de Mediana Edad , PronósticoRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Islas de CpG , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Adulto , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/diagnóstico , Carcinoma Ductal de Mama/patología , Análisis por Conglomerados , Proteínas de Unión al ADN/genética , Femenino , Humanos , Persona de Mediana Edad , Proteínas Nucleares/genética , Pronóstico , Receptores de Ácido Retinoico/genética , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genética , Proteínas WT1/genéticaRESUMEN
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 Transductoras de Señales/metabolismo , Reparación de la Incompatibilidad de ADN , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Hipertermia Inducida , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genéticaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Metilación de ADN , Predisposición Genética a la Enfermedad/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Línea Celular Tumoral , Sondas de ADN , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Femenino , Células HeLa , Humanos , Invasividad Neoplásica , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/mortalidad , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Antraciclinas/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Terapia Neoadyuvante/métodos , Pronóstico , Análisis de Supervivencia , Resultado del Tratamiento , Proteína X Asociada a bcl-2/análisisRESUMEN
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.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Resistencia a Antineoplásicos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Adolescente , Adulto , Antineoplásicos/uso terapéutico , Biomarcadores/análisis , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Niño , Preescolar , Cisplatino/uso terapéutico , Citoplasma/metabolismo , ADN/análisis , Daño del ADN , Reparación de la Incompatibilidad de ADN , Doxorrubicina/uso terapéutico , Femenino , Proteínas de Choque Térmico HSP27 , Proteínas HSP70 de Choque Térmico/análisis , Proteínas de Choque Térmico/análisis , Humanos , Lactante , Linfocitos/química , Linfocitos/metabolismo , Masculino , Chaperonas Moleculares , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/análisis , Proteínas de Neoplasias/análisis , Neoplasias/mortalidad , Proteínas Nucleares/análisis , PronósticoRESUMEN
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.
Asunto(s)
Antineoplásicos/uso terapéutico , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Linfocitos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Antibióticos Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Niño , Preescolar , Cisplatino/farmacología , Ensayo Cometa , Doxorrubicina/farmacología , Femenino , Humanos , Lactante , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proyectos PilotoRESUMEN
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.