RESUMO
BACKGROUND: DNA damage caused by exposure to metal mixtures and the potential modulating role of genes involved in DNA repair and the antioxidant response have not been evaluated in newborns. AIM: The aim was to evaluate the association between prenatal exposure to metal mixtures and DNA repair capacity (DRC) in newborns from the Metropolitan Area of Mexico City (MAMC), a heavily polluted area, and the impact of variants in genes involved in DNA repair and the antioxidant response on this association. METHODS: We analyzed cord blood samples obtained at delivery from 125 healthy newborns from the MAMC. Twenty-four elements were determined by inductively coupled plasma mass spectrometry (ICPâMS), but only 12 (Cu, I, Se, Zn, As, Ba, Cs, Mn, Sb, Sr, Pb, and Ti) were quantified in most samples. DRC was assessed by the challenge-comet assay, and OGG1, PARP1, and NFE2L2 genotyping was performed with TaqMan probes. Metal mixtures were identified and analyzed using principal component analysis (PCA) and weighted quantile sum (WQS) regression. Independent adjusted linear regression models were used to evaluate the associations. RESULTS: A null DRC was observed in 46% of newborns. The metals with the highest concentrations were Mn, Sr, Ti, and Pb. Essential elements showed normal levels. Only the mixture characterized by increased As, Cs, Cu, Se, and Zn levels was inversely associated with DRC. As was the principal contributor (37.8%) in the negative direction in the DRC followed by Ba and Sb, according to the WQS regression. Newborns carrying of the derived (G) allele of the PARP1 rs1136410 variant showed decreased DRC by exposure to some potentially toxic metals (PTMs) (As, Cs, and Ba). CONCLUSION: Prenatal exposure to metal mixtures negatively affected DRC in newborns, and the PARP1 rs1136410 variant had a modulating role in this association.
Assuntos
Antioxidantes , Efeitos Tardios da Exposição Pré-Natal , Gravidez , Feminino , Recém-Nascido , Humanos , Chumbo , Dano ao DNA , Reparo do DNA , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switches between distinct cell death pathways, and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite's nucleolus. We also describe that TcPARP is present in a thread-like structure that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this thread during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Trypanosoma cruzi/genética , Inibidores de Poli(ADP-Ribose) Polimerases , Ribose/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Núcleo Celular/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Dano ao DNA , Adenosina Difosfato Ribose/metabolismo , Mitose , Doença de Chagas/metabolismoRESUMO
Malignant melanoma is the main cause of death in patients with skin cancer. Overexpression of Proteolipid protein 2 (PLP2) increased tumor metastasis and the knockdown of PLP2 inhibited the growth and metastasis of melanoma cells. In the present work, we studied the antitumor activity of peptide Rb4 derived from protein PLP2. In vitro, Rb4 induced F-actin polymerization, prevented F-actin depolymerization and increased the ER-derived cytosolic calcium. Such effects were associated with necrosis of murine melanoma B16F10-Nex2 cells and with inhibition of the viability of human cancer cell lines. Loss of plasma membrane integrity, dilation of mitochondria, cytoplasm vacuolation and absence of chromatin condensation characterized tumor cell necrosis. Cleavage of PARP-1 and inhibition of RIP1 expression were also observed. In vivo, peptide Rb4 reduced the lung metastasis of tumor cells and delayed the subcutaneous melanoma growth in a syngeneic model. Rb4 induced the expression of two DAMPs molecules, HMGB1 and calreticulin, in B16F10-Nex2. Our results suggest that peptide Rb4 acts directly on tumor cells inducing the expression of DAMPs, which trigger the immunoprotective effect in vivo against melanoma cells. We suggest that peptide Rb4 is a promising compound to be developed as an anticancer drug.
Assuntos
Morte Celular/genética , Expressão Gênica/genética , Expressão Gênica/fisiologia , Proteínas com Domínio MARVEL/genética , Proteínas com Domínio MARVEL/farmacologia , Melanoma/genética , Melanoma/patologia , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteolipídeos/genética , Proteolipídeos/farmacologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Animais , Antineoplásicos , Calreticulina/genética , Calreticulina/metabolismo , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Proteínas com Domínio MARVEL/metabolismo , Proteínas com Domínio MARVEL/fisiologia , Camundongos , Necrose , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Peptídeos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteolipídeos/metabolismo , Proteolipídeos/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND/AIM: Experimental oncology commonly uses cells as oncological models, providing a framework for the testing of drugs, and investigation of cytotoxicity, mutagenesis and carcinogenesis. Investigations into poly-ADP-ribose polymerase 1 (PARP1) inhibition have become ever more relevant due to its approval as a therapeutic option for tumors with BRCA1/2 DNA repair-associated mutation and the seemingly high PARP expression levels in some tumor subtypes. In this study, we aimed to determine PARP1 gene expression of different hematological cancer-derived cell lineages and compare them to that of normal cell lines. MATERIALS AND METHODS: PARP1 gene expression in seven different neoplastic lineages, representing three different hematological disorders (chronic myeloid leukemia, Burkitt lymphoma and acute lymphoblastic leukemia), was quantified by quantitative real-time polymerase chain reaction. RESULTS: All hematological malignant lineages in this study overexpressed PARP1 when compared to the normal cell line MRC-5, with Burkitt's lymphoma cells having the highest expression values (fold change: 93). CONCLUSION: Overexpression of PARP1 in hematological malignant lineages is a finding of crucial importance to future studies exploring possible cellular oncogenic pathways and supports investigations into the effectiveness of PARP1 inhibitors against hematological disorders.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/genética , Oncologia/métodos , Poli(ADP-Ribose) Polimerase-1/genética , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Hematológicas/patologia , Humanos , Células Jurkat , Células K562 , Leucemia/genética , Leucemia/patologiaRESUMO
Trypanosoma cruzi is the etiologic agent of Chagas' disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2-related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.
Assuntos
Doença de Chagas/parasitologia , Dano ao DNA , Interações Hospedeiro-Parasita , Estresse Oxidativo , Trypanosoma cruzi/fisiologia , Antioxidantes/metabolismo , Morte Celular , Linhagem Celular , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , Regulação para Baixo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
OBJECTIVES: To evaluate the association of allelic and genotypic frequencies of PSCA (rs2976392), TNF-α (rs1800629), PARP1 (rs1136410) and TP53 (rs368771578) SNPs with GC susceptibility in a Brazilian population. MATERIALS AND METHODS: This is a retrospective study, which included 102 paraffin-embedded adenocarcinoma tissue samples > 5 years of obtention, with 204 alleles for each studied SNP. Other 102 healthy tissue samples were included as controls. For analysis, the genotyping method Dideoxy Single Allele-Specific - PCR was used. Statistical analysis was performed with the Bioestat software 5.3, determining Hardy-Weinberg's equilibrium for the genotypic frequencies p-values < 0.05 were considered significant. RESULTS: PSCA (rs2976392) and TNF-α (rs1800629) SNPs were associated with GC in the analyzed samples (X2=10.3/102 and p<0.001/0.00001, respectively). TNF-α (rs1800629) SNP presented also a statistically significant relationship between its genotypes and the morphological pattern (intestinal/diffuse) (p<0.032). However, PARP1 (rs1136410) and TP53 (rs368771578) SNPs were in Hardy-Weinberg's equilibrium and, therefore, were not significantly associated with GC in these samples (X2=0.73/2.89 and p<0.39/0.08). CONCLUSIONS: PSCA (rs2976392) and TNF-α (rs1800629) SNPs are potential molecular markers of susceptibility to GC development. PARP1 (rs1136410) and TP53 (rs368771578) SNPs were not associated with the risk of GC development.
Assuntos
Antígenos de Neoplasias/genética , Predisposição Genética para Doença/genética , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias Gástricas/genética , Fator de Necrose Tumoral alfa/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Alelos , Biomarcadores Tumorais/genética , Brasil , Estudos de Casos e Controles , Feminino , Proteínas Ligadas por GPI/genética , Frequência do Gene/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
The developmental period in utero is a critical window for environmental exposure. Epigenetic fetal programming via DNA methylation is a pathway through which metal exposure influences the risk of developing diseases later in life. Genetic damage repair can be modified by alterations in DNA methylation, which, in turn, may modulate gene expression due to metal exposure. We investigated the impact of prenatal metal exposure on global and gene-specific DNA methylation and mRNA expression in 181 umbilical cord blood samples from newborns in Mexico City. Global (LINE1) and promoter methylation of DNA-repair (OGG1 and PARP1) and antioxidant (Nrf2) genes was evaluated by pyrosequencing. Prenatal metal exposure (As, Cu, Hg, Mn, Mo, Pb, Se, and Zn) was determined by ICP-MS analysis of maternal urine samples. Multiple regression analyses revealed that DNA methylation of LINE1, Nrf2, OGG1, and PARP1 was associated with potentially toxic (As, Hg, Mn, Mo, and Pb) and essential (Cu, Se, and Zn) elements, and with their interactions. We also evaluated the association between gene expression (mRNA levels quantified by p-PCR) and DNA methylation. An increase in OGG1 methylation at all sites and at CpG2, CpG3, and CpG4 sites was associated with reduced mRNA levels; likewise, methylation at the CpG5, CpG8, and CpG11 sites of PARP1 was associated with reduced mRNA expression. In contrast, methylation at the PARP1 CpG7 site was positively associated with its mRNA levels. No associations between Nrf2 expression and CpG site methylation were observed. Our data suggest that DNA methylation can be inï¬uenced by prenatal metal exposure, which may contribute to alterations in the expression of repair genes, and therefore, result in a lower capacity for DNA damage repair in newborns.
Assuntos
Antioxidantes/metabolismo , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/genética , Metais Pesados/farmacologia , População Urbana , Adolescente , Adulto , Dano ao DNA , DNA Glicosilases/genética , Feminino , Humanos , Recém-Nascido , Metais Pesados/administração & dosagem , México , Fator 2 Relacionado a NF-E2/genética , Poli(ADP-Ribose) Polimerase-1/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Adulto JovemRESUMO
Poly- adenosine diphosphate (ADP)-ribose (PAR) is a polymer synthesized as a posttranslational modification by some poly (ADP-ribose) polymerases (PARPs), namely PARP-1, PARP-2, tankyrase-1, and tankyrase-2 (TNKS-1/2). PARP-1 is nuclear and has also been detected in extracellular vesicles. PARP-2 and TNKS-1/2 are distributed in nuclei and cytoplasm. PARP or PAR alterations have been described in tumors, and in particular by influencing the Epithelial- Mesenchymal Transition (EMT), which influences cell migration and drug resistance in cancer cells. Pro-EMT and anti-EMT effects of PARP-1 have been reported while whether PAR changes occur specifically during EMT is currently unknown. The PARP-1/2 inhibitor Olaparib (OLA) is approved by FDA to treat certain patients harboring cancers with impaired homologous recombination. Here, we studied PAR changes and OLA effects on EMT. Total and nuclear PAR increased in EMT while PAR belts were disassembled. OLA prevented EMT, according to: (i) molecular markers evaluated by immuno-cytofluorescence/image quantification, Western blots, and RNA quantitation, (ii) morphological changes expressed as anisotropy, and (iii) migration capacity in the scratch assay. OLA also partially reversed EMT. OLA might work through unconventional mechanisms of action (different from synthetic lethality), even in non-BRCA (breast cancer 1 gene) mutated cancers.
Assuntos
Glândulas Mamárias Animais/citologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Fator de Crescimento Transformador beta/efeitos adversos , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
This study investigated the effects of exercise training in regulating inflammatory processes, endoplasmic reticulum stress, and apoptosis in hypothalamic neurons of obese mice. Swiss mice were distributed into three groups: Lean mice (Lean), sedentary animals fed a standard diet; obese mice (Obese), sedentary animals fed a high-fat diet (HFD); trained obese mice (T. Obese), animals fed with HFD and concurrently subjected to an endurance training protocol for 8 weeks. In the endurance training protocol, mice ran on a treadmill at 60% of peak workload for 1 hr, 5 days/week for 8 weeks. Twenty-four hours after the last exercise session, the euthanasia was performed. Western blot, quantitative real-time polymerase chain reaction, and terminal deoxynucleotide transferase biotin-dUTP nick end-labeling (TUNEL) techniques were used for the analysis of interest. The results show exercise training increased phosphorylation of leptin signaling pathway proteins (pJAK2/pSTAT3) and reduced the content of tumor necrosis factor α, toll-like receptor 4, suppressor of cytokine signaling 3, protein-tyrosine phosphatase 1B as well as the phosphorylation of IkB kinase in the hypothalamus of T. Obese animals. A reduction of macrophage activation and phosphorylation of eukaryotic initiation factor 2α, and protein kinase RNA-like endoplasmic reticulum kinase (PERK) were also observed in exercised animals. Furthermore, exercise decreased the expression of the proapoptotic protein (PARP1) and increased anti-inflammatory (IL-10) and antiapoptotic (Bcl2) proteins. Using the TUNEL technique, we observed that the exercised animals had lower DNA fragmentation. Finally, physical exercise preserved pro-opiomelanocortin messenger RNA content. In conclusion, exercise training was able to reorganize the control of the energy balance through anti-inflammatory and antiapoptotic responses in hypothalamic tissue of obese mice.
Assuntos
Treino Aeróbico , Inflamação/fisiopatologia , Obesidade/terapia , Condicionamento Físico Animal , Animais , Apoptose/genética , Dieta Hiperlipídica , Metabolismo Energético/genética , Regulação da Expressão Gênica , Humanos , Hipotálamo/metabolismo , Hipotálamo/patologia , Inflamação/terapia , Interleucina-10/genética , Camundongos , Camundongos Obesos , Neurônios/metabolismo , Neurônios/patologia , Obesidade/fisiopatologia , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Breast cancer is one of the major health issues confronting women; however, treatment with conventional chemotherapeutic drugs is limited. Currently, paclitaxel is used as a therapeutic clinical agent to treat breast cancer that exerts antitumor activity in numerous types of cancer cell. Curcumin (diferuloylmethane), a polyphenol derived from turmeric (Curcuma longa), possesses several properties that could enable it to exert an anticancer effect. Previous reports have demonstrated the synergistic effects of several chemotherapeutic drugs in combination with curcumin. Therefore, the aim of the current study was to evaluate cell death induced by curcumin and paclitaxel alone and in combination in human breast cancer cell lines: MCF7, an epithelial and luminal-like adenocarcinoma cell line triple positive for estrogen and progesterone receptor, and MDA-MB-234, a metastatic human breast cancer cell line triple negative for such receptors, as well as MCF-10F as a normal breast cell line. The results indicated that curcumin and paclitaxel induced apoptosis and necrosis, which was demonstrated through multiple methods, including assays of caspase-3/7 activity, Annexin V, poly(ADP-ribose) polymerase-1 activation and protein expression of caspase-3, nuclear factor (NF)-κB transcription factor and proliferating cell nuclear antigen. The results identified that the combination of curcumin and paclitaxel had a decreased effect on apoptosis in the malignant MDA-MB-231 cell line compared with in MCF7 or MCF-10F. It was demonstrated that the combined treatment with curcumin and paclitaxel resulted in a higher level of apoptosis compared with either substance alone in breast cancer cell lines. Therefore, breast cancer treatment may benefit from the use of a combination of drugs in chemotherapy.