RESUMEN
A bioassay-guided study aiming at identifying inhibitors of the glycation process on the leaves of Ocotea paranapiacabensis afforded four benzylisoquinoline alkaloids (1-4), with 1 and 2 identified as new naturals products, while 3 and 4 were previously described in the literature, with 3 being identified as magnocurarine. Purification was performed by column chromatography and high-performance liquid chromatography. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, and HRMS. The process of skin aging has been recently associated with advanced glycation end products (AGEs), and strategies inhibiting their formation have been addressed by pharmaceutical companies for the development of novel antiaging compounds. Alkaloids 1-4 were evaluated for their potential to inhibit AGE formation and showed inhibition of 62.9%, 83.3%, 26.1%, and 98.2% (150 µM), respectively. The antiaging potential of compounds 1 and 4 were evaluated with a reconstructed human skin model in vitro, and results showed a decrease in dermis contraction (8.7% and 4.2% respectively for 1 and 4) when compared to the glycated control (57.4%). Additionally, absorption, distribution, metabolism, and excretion (ADME) and toxicity properties were predicted using in silico methods, and the results were considered significantly promising for alkaloids 1 and 4 to continue the development of these alkaloids with skincare properties.
Asunto(s)
Alcaloides/farmacología , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Ocotea/química , Envejecimiento de la Piel/efectos de los fármacos , Glicosilación , Humanos , Técnicas In Vitro , Estructura Molecular , Fitoquímicos/farmacología , Hojas de la Planta/químicaRESUMEN
Background: Cutaneous melanoma (CM) is the most aggressive subtype of skin cancer, with increasing incidence over the past several decades. DNA methylation is a key element of several biological processes such as genomic imprinting, cell differentiation and senescence, and deregulation of this mechanism has been implicated in several diseases, including cancer. In order to understand the relationship of DNA methylation in CMs, we searched for an epigenetic signature of cutaneous melanomas by comparing the DNA methylation profiles between tumours and benign melanocytes, the precursor cells of CM. Methods: We used 20 primary CMs and three primary cell cultures of melanocytes as a discovery cohort. The tumours mutational background was collected as previously reported. Methylomes were obtained using the HM450K DNA methylation assay, and differential methylation analysis was performed. DNA methylation data of CMs from TCGA were recovered to validate our findings. Results: A signature of 514 differentially methylated genes (DMGs) was evident in CMs compared to melanocytes, which was independent of the presence of driver mutations. Pathway analysis of this CM signature revealed an enrichment of proteins involved in the binding of DNA regulatory regions (hypermethylated sites), and related to transmembrane signal transducer activities (hypomethylated sites). The methylation signature was validated in an independent dataset of primary CMs, as well as in lymph node and distant metastases (correlation of DNA methylation level: r > 0,95; Pearson's test: p < 2.2e-16). Conclusions: CMs exhibited a DMGs signature, which was independent of the mutational background and possibly established prior to genetic alterations. This signature provides important insights into how epigenetic deregulation contributes to melanomagenesis in general (AU)
Asunto(s)
Humanos , Masculino , Femenino , Neoplasias Cutáneas , Transducción de Señal , Metilación de ADN , Proteínas de Unión al ADN , Transcriptoma/genética , MelanomaRESUMEN
Epigenetic dysregulation is an important emerging hallmark of cutaneous melanoma development. The global loss of DNA methylation in gene-poor regions and transposable DNA elements of cancer cells contributes to increased genomic instability. Long interspersed element-1 (LINE-1) sequences are the most abundant repetitive sequence of the genome and can be evaluated as a surrogate marker of the global level of DNA methylation. In this work, LINE-1 methylation levels were evaluated in cutaneous melanomas and normal melanocyte primary cell cultures to investigate their possible association with both distinct clinicopathological characteristics and tumor mutational profile. A set of driver mutations frequently identified in cutaneous melanoma was assessed by sequencing (actionable mutations in BRAF, NRAS, and KIT genes, and mutations affecting the TER T promoter) or multiplex ligation-dependent probe amplification (MLPA) (CDKN2A deletions). Pyrosequencing was performed to investigate the methylation level of LINE-1 and CDKN2A promoter sequences. The qualitative analysis showed a trend toward an association between LINE-1 hypomethylation and CDKN2A inactivation (p=0.05). In a quantitative approach, primary tumors, mainly the thicker ones (>4 mm), exhibited a trend toward LINE-1 hypomethylation when compared with control melanocytes. To date, this is the first study reporting in cutaneous melanomas a possible link between the dysregulation of LINE-1 methylation and the presence of driver mutations.
Asunto(s)
Metilación de ADN/genética , Elementos de Nucleótido Esparcido Largo/genética , Melanoma/genética , Mutación/genética , Análisis Mutacional de ADN , Humanos , Neoplasias Cutáneas , Melanoma Cutáneo MalignoRESUMEN
BACKGROUND: Matrix metalloproteinases (MMP) are important enzymes in the tumor microenvironment associated with progression of cervical intraepithelial neoplasia (CIN) toward squamous cell carcinoma (SCC) of the cervix. However, the role of MMPs in the inflammatory process associated with Chlamydia trachomatis infection concomitant with the carcinogenic process driven by HPV has not yet been addressed. In the present study, we analyzed the state of the MMP-9-RECK axis in cervical carcinogenesis. METHODS: The levels of MMP-9 and RECK expression were analyzed by immunocytochemistry in liquid-based cytology samples from 136 women with high-grade cervical lesions (CIN2/CIN3) and cervical SCC diagnosed by LLETZ, and in 196 women without cervical neoplasia or CIN1. Real-time qPCR was performed to analyze expression of MMP-9 and RECK in 15 cervical samples. The presence of HPV-DNA and other genital pathogens was evaluated by PCR. RESULTS: We found a higher expression of MMP-9 [OR, 4.2; 95% confidence interval (CI), 2.2-7.8] and lower expression of RECK (OR, 0.4; 95% CI, 0.2-0.7) in women with CIN2/CIN3/SCC when compared with women from the control group (no neoplasia/CIN1). A statistically significant association was also found between MMP-9/RECK imbalance and infection by alpha-9 HPV and C. trachomatis. The prevalence of C. trachomatis infection was significantly higher in women with high-grade cervical disease (OR, 3.7; 95% CI, 1.3-11.3). CONCLUSIONS: MMP-9/RECK imbalance in cervical smears is significantly associated with high-grade cervical diseases and infection by alpha-9 HPV and C. trachomatis. IMPACT: MMP-9/RECK imbalance during cervical inflammation induced by C. trachomatis might play a role in HPV-mediated cervical carcinogenesis.
Asunto(s)
Infecciones por Chlamydia/genética , Proteínas Ligadas a GPI/genética , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 9 de la Matriz/genética , Infecciones por Papillomavirus/genética , Neoplasias del Cuello Uterino/genética , Cervicitis Uterina/genética , Adulto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virología , Cuello del Útero/metabolismo , Cuello del Útero/patología , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/patología , Chlamydia trachomatis/genética , Estudios Transversales , ADN Bacteriano/genética , ADN de Neoplasias/genética , ADN Viral/genética , Femenino , Proteínas Ligadas a GPI/biosíntesis , Regulación Bacteriana de la Expresión Génica , Genotipo , Papillomavirus Humano 16/genética , Humanos , Inmunohistoquímica , Metaloproteinasa 9 de la Matriz/biosíntesis , Prueba de Papanicolaou , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología , Cervicitis Uterina/metabolismo , Cervicitis Uterina/microbiología , Frotis VaginalRESUMEN
In melanoma development, oncogenic process is mediated by genetic and epigenetic mutations, and few studies have so far explored the role of DNA methylation either as predisposition factor or biomarker. We tested patient samples for germline CDKN2A methylation status and found no evidence of inactivation by promoter hypermethylation. We have also investigated the association of clinical characteristics of samples with the DNA methylation pattern of twelve genes relevant for melanomagenesis. Five genes (BAP1, MGMT, MITF, PALB2, and POT1) presented statistical association between blood DNA methylation levels and either CDKN2A-mutation status, number of lesions, or Breslow thickness. In tumors, five genes (KIT, MGMT, MITF, TERT, and TNF) exhibited methylation levels significantly different between tumor groups including acral compared to nonacral melanomas and matched primary lesions and metastases. Our data pinpoint that the methylation level of eight melanoma-associated genes could potentially represent markers for this disease both in peripheral blood and in tumor samples.