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
All-trans retinoic acid (RA), the primary metabolite of vitamin A, controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, both known as retinoids, are promising agents in treating and chemopreventing different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with tumor metastatic behavior. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that the RA plus FAKi (RA + FAKi) approach could improve the inhibition of tumor progression. By in silico analysis and its subsequent validation by qPCR, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. We also showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. Additionally, we show that both drugs individually, as well as in combination, induce the expression of apoptosis markers such as active-caspase-3 and cleaved-PARP1. We also provided evidence that RA effects are extrapolated to other cancer cells, including T-47D BC and the human cervical carcinoma HeLa cells. In an orthotopic assay of LM3 tumor growth, whereas RA and FAKi administered separately reduced tumor growth, the combined treatment induced a more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduced metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance could reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbate these effects, thus suggesting that the sensitivity to RA therapies could be increased with FAKi coadministration in BC tumors.
Assuntos
Neoplasias da Mama , Tretinoína , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caspase 3 , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HeLa , Humanos , Camundongos , Retinoides/farmacologia , Tretinoína/farmacologia , Tretinoína/uso terapêutico , Vitamina ARESUMO
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
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.
RESUMO
AIM: Perivascular adipose tissue (PVAT) is in intimate contact with the vessel wall and extravascular PVAT-derived inflammatory mediators may adversely influence atherosclerotic plaque formation and stability through outside-to-inside signaling. We sought to investigate the role of PVAT on the atheroma development in an experimental animal model of metabolic syndrome (MS) associated with oxidative stress and low-grade inflammatory state. We also studied the effect of pioglitazone an insulin sensitizer, on the aortic wall and its surrounding PVAT, considering a bi-directional communication between both layers. METHODS: Apolipoprotein E-deficient mice (ApoE-/- ) were fed with standard diet (CD, control diet) or fructose overload (10% w/v) (FD, fructose diet) for 8 weeks and treated with or without pioglitazone the latest 4 weeks. RESULTS: Biochemical variables show that glycemia and lipid peroxidation determined by thiobarbituric acid reactive species (TBARS) significantly increased in FD-fed ApoE-/- mice. FD significantly increased aortic PVAT expression of oxidative stress associated genes: p22phox , Nox1, Nox2, Nox4 and p47phox , and proinflammatory genes: Visfatin, MCP-1, and MMP-9. Pioglitazone diminished PVAT-oxidative damage elicited by fructose treatment and markedly down-regulated proinflammatory markers. Even pioglitazone did not prevent the development of the aortic atheroma plaques stimulated by FD, significantly diminished VCAM-1 expression, MMP-9 expression and activity in aortic media wall and significantly reduced the accumulation of lipids and macrophages in atheroma plaques. CONCLUSION: Our results support the fact that PVAT contributes to the development and progression of cardiovascular disease by underlying mechanisms elicited by "outside-in" signaling. Treatment with pioglitazone may offer a new effect on the whole vessel wall, promoting the stability of advanced atherosclerotic plaques.
Assuntos
Tecido Adiposo/fisiopatologia , Aorta/fisiopatologia , Vasos Sanguíneos/fisiopatologia , Hipoglicemiantes/uso terapêutico , Tiazolidinedionas/uso terapêutico , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/etiologia , Tecido Adiposo/patologia , Animais , Apolipoproteínas E/genética , Glicemia/metabolismo , Vasos Sanguíneos/patologia , Frutose/antagonistas & inibidores , Frutose/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Pioglitazona , Placa Aterosclerótica/patologia , Transdução de Sinais/efeitos dos fármacos , Doenças Vasculares/patologiaRESUMO
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
BACKGROUND: Progesterone (P4) is the main steroid secreted by the corpora lutea (CL) and is required for successful implantation and maintenance of pregnancy. Although adequate circulating levels of thyroid hormone (TH) are needed to support formation and maintenance of CL during pregnancy, TH signaling had not been described in this gland. We determined luteal thyroid hormone receptor isoforms (TR) expression and regulation throughout pregnancy and under the influence of thyroid status, and in vitro effects of triiodothyronine (T3) exposure on luteal P4 synthesis. METHODS: Euthyroid female Wistar rats were sacrificed by decapitation on gestational day (G) 5, G10, G15, G19, or G21 of pregnancy or on day 2 postpartum (L2). Hyperthyroidism and hypothyroidism were induced in female Wistar rats by daily administration of thyroxine (T4; 0.25 mg/kg subcutaneously) or 6-propyl-2-thiouracil (PTU; 0.1 g/L in drinking water), respectively. Luteal TR expression of mRNA was determined using real-time reverse-transcription quantitative polymerase chain reaction, and of protein using Western blot and immunohistochemistry. Primary cultures of luteal cells and of luteinized granulosa cells were used to study in vitro effects of T3 on P4 synthesis. In addition, the effect of T3 on P4 synthesis under basal conditions and under stimulation with luteinizing hormone (LH), prolactin (PRL), and prostaglandin E2 (PGE2) was evaluated. RESULTS: TRα1, TRα2, and TRß1 mRNA were present in CL, increasing during the first half and decreasing during the second half of pregnancy. At the protein level, TRß1 was abundantly expressed during gestation reaching a peak at G19 and decreasing afterwards. TRα1 was barely expressed during early gestation, peaked at G19, and diminished thereafter. Expression of TRß1 and TRα1 at the protein and mRNA level were not influenced by thyroid status. T3 neither modified P4 secretion from CL of pregnancy nor its synthesis in luteinized granulosa cells in culture. CONCLUSIONS: This study confirms for the first time the presence of TR isoforms in the CL during pregnancy and postpartum, identifying this gland as a TH target during gestation. TR expression is modulated in this tissue in accordance with the regulation of P4 metabolism, and the abrupt peripartum changes suggest a role of TH during luteolysis. However, TH actions on the CL do not seem to be related to a direct regulation of P4 synthesis.
Assuntos
Corpo Lúteo/metabolismo , Período Pós-Parto/metabolismo , Receptores dos Hormônios Tireóideos/biossíntese , Animais , Feminino , Hormônio Luteinizante , Gravidez/metabolismo , Progesterona/biossíntese , Prolactina , Propiltiouracila/farmacologia , Isoformas de Proteínas/biossíntese , RNA Mensageiro/metabolismo , Ratos Wistar , Tireotropina/biossíntese , Tri-Iodotironina/farmacologiaRESUMO
Dietary flavonoids, present in different amount in foods, are associated with the prevention of hypertension, but little is known about the interactions between them. The aim of this study was to explore the effect of quercetin (Q), catechin (C) and the mixture, on Angiotensin II (AngII)-induced redox-dependent signalling pathways and cell behaviour. Mesenteric smooth muscle cells (MesSMC) from spontaneously hypertensive rats (SHR) were incubated with AngII (0.1 µmol/L) alone, or with the mixture of low concentrations of Q and C. AngII-increased ROS production was reduced by the mixture of separately ineffective low concentration of Q (15 µmol/L) plus C (20 µmol/L). This mixture reduced AngII-stimulated NAD(P)H oxidase activation and p47phox translocation to the cell membrane, without affecting Nox2 expression. Co-incubation of Q + C significantly inhibited AngII-induced migration and proliferation, and these effects were independent of p-ERK1/2 and related with reduced p38MAPK phosphorylation. These findings demonstrated that low concentrations of singly non-effective flavonoids when are combined exert a synergistic effect in inhibiting AngII-induced redox-sensitive signalling pathways.