RESUMEN
Some of the more than 200 known HPV types are essential for cervical cancer development, the third type of cancer most incident in the female population. However, for the malignant transformation occur, some cofactors are needed, as the reactive oxygen species (ROS), which can be neutralized by the antioxidant system. The SOD2 enzyme, encoded by the same name gene, is found in mitochondria and is part of the first line of defense against oxidative stress damage. Genetic polymorphisms can act by altering the efficiency of the enzyme, among which the most studied is the rs4880. Thus, the purpose of the present study was to evaluate the association of this polymorphism with HPV infection and the development of low and high grade squamous intraepithelial lesions (LSIL and HSIL) and cervical cancer, in 407 women attended by the public health system in Brazil. HPV detection in cervical secretion samples was carried out by polymerase chain reaction (PCR) and blood samples were used for polymorphism genotyping through PCR followed by restriction fragment length polymorphism (RFLP). PCR and restriction products were subjected to 10% polyacrylamide gel electrophoresis. HPV negative group (control) included 158 women and the HPV positive group (case) 249 women. The infected group was divided into No Lesion (n = 90), LSIL (n = 20), HSIL (n = 67) and cervical cancer (n = 72). The data found on socio-epidemiological characteristics and habits corroborated with data found in the literature. The distribution of genotypes in the control group was 51.9% women TC, 29.8% TT and 18.3% CC. In the case group, the distribution was 55.0% women TC, 26.1% TT and 18.9% CC. This is the first study evaluating the influence of SOD2 rs4880 polymorphism on HPV infection, the development of cervical intraepithelial lesions and cervical cancer in a Brazilian population, although additional studies are needed to corroborate the results.
Asunto(s)
Biomarcadores de Tumor/genética , Polimorfismo de Nucleótido Simple , Lesiones Intraepiteliales Escamosas/genética , Superóxido Dismutasa/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Adulto , Brasil , Estudios Transversales , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Fenotipo , Medición de Riesgo , Factores de Riesgo , Lesiones Intraepiteliales Escamosas/enzimología , Lesiones Intraepiteliales Escamosas/patología , Lesiones Intraepiteliales Escamosas/virología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Adulto Joven , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
Secundiflorol G (SG) is an isoflavan isolated from the root bark of Aeschynomene fascicularis, a Mayan medicinal plant used to treat cancer-like symptoms. SG has been shown to have cytotoxic effects on cervical cancer cells (HeLa). Assays were done to identify the mechanisms of SG's cytotoxic effect.HeLa cells treated with SG exhibited early and late apoptosis, and caspase-9, -8 and -3 activities. It also induces generation of reactive oxygen species and disrupted mitochondrial membrane potential.SG isolated from A. fascicularis induces apoptosis through extrinsic and intrinsic pathways on HeLa cells. SG could be a candidate for in vivo studies and a promising natural compound in cervical cancer treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzopiranos/aislamiento & purificación , Benzopiranos/farmacología , Fabaceae/química , Isoflavonas/aislamiento & purificación , Isoflavonas/farmacología , Plantas Medicinales/química , Neoplasias del Cuello Uterino/patología , Antineoplásicos/farmacología , Benzopiranos/química , Caspasas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Células HeLa , Humanos , Isoflavonas/química , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/enzimología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
ST3GAL4 gene expression is altered in different cancer types, including cervical cancer. Several mRNA transcripts have been reported for this gene; however, their expression levels in cervical cancer have not been analyzed. ST3GAL4 encodes for ßgalactosidase α2,3sialyltransferase 4, involved in the biosynthesis of the tumour antigens sLe(x) and sulfosLe(x). The present study evaluated the presence of three mRNA variants (V1, V2 and V3) in cervical cancer cell lines, detecting the three variants. Additionally, the expression level of the V1 transcript of the ST3GAL4 gene was determined by reverse transcriptionquantitative polymerase chain reaction in cervical cell lines and in normal, premalignant and cervical cancer tissue. The V1 transcript of the ST3GAL4 demonstrated significant decreased expression in premalignant and malignant cervical tissues. The results suggested that deregulation of this gene could occur prior to the presence of cancer and demonstrated the importance of evaluating the expression level of V1, and its association with disease progression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Isoformas de ARN/genética , Sialiltransferasas/genética , Neoplasias del Cuello Uterino/enzimología , Adolescente , Adulto , Anciano , Progresión de la Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Neoplasias del Cuello Uterino/genética , Adulto JovenRESUMEN
PURPOSE To analyze the relation between the cytological findings and telomerase activity (TA). METHODS Cervical samples were evaluated and classified according to the Bethesda System. Telomerase activity was measured total product generated values (TPG) using the TRAP assay (telomeric repeat amplification protocol); data were analyzed statistically using the χ2 test, with the level of significance set at p<0.05. RESULTS The study was conducted on 102 patients. Of these, 3.9% showed normal cytological findings, 8.8% showed cervicitis; 2% showed Atypical Squamous Cells of Undetermined Significance (ASCUS); 67.6% showed Low Grade Squamous Intraepithelial Lesion (LSIL); 11.8% showed High Grade Squamous Intraepithelial Lesion (H-SIL) and 5.9% showed Squamous Carcinoma. Among telomerase-positive samples, the TPG values were cervicitisAsunto(s)
Carcinoma de Células Escamosas/enzimología
, Telomerasa/metabolismo
, Neoplasias del Cuello Uterino/enzimología
, Adulto
, Femenino
, Humanos
, Frotis Vaginal
, Displasia del Cuello del Útero
RESUMEN
Abstract PURPOSE To analyze the relation between the cytological findings and telomerase activity (TA). METHODS Cervical samples were evaluated and classified according to the Bethesda System. Telomerase activity was measured total product generated values (TPG) using the TRAP assay (telomeric repeat amplification protocol); data were analyzed statistically using the χ2 test, with the level of significance set at p<0.05. RESULTS The study was conducted on 102 patients. Of these, 3.9% showed normal cytological findings, 8.8% showed cervicitis; 2% showed Atypical Squamous Cells of Undetermined Significance (ASCUS); 67.6% showed Low Grade Squamous Intraepithelial Lesion (LSIL); 11.8% showed High Grade Squamous Intraepithelial Lesion (H-SIL) and 5.9% showed Squamous Carcinoma. Among telomerase-positive samples, the TPG values were cervicitis<normal<ASCUS<L-SIL<H-SIL<Carcinoma. CONCLUSION Results show increased telomerase activity with increasing severity of lesion, supporting the association between TA and type of lesion.
Resumo OBJETIVO Analisar a relação entre os achados citológicos e atividade da telomerase (AT). MÉTODOS Amostras cervicais foram avaliadas e classificadas pelo sistema Bethesda. A AT foi medida como valores de produto total gerado (PTG), utilizando o protocolo de amplificação repetida da telomerase (TRAP); os dados foram analisados estatisticamente usando o teste do χ2, com nível de significância de p<0,05. RESULTADOS Cento e dois pacientes foram analisados: 3,9% com achados citológicos normais, 8,8% com cervicite, 2% com células escamosas atípicas de significado indeterminado (ASCUS), 67,6% com lesão escamosa intraepitelial baixo grau (LEI-BG), 11,8 % com lesão intraepitelial escamosa alto grau (LEI-AG) e 5,9% com carcinoma escamoso. Valores PTG para amostras positivas AT foram: cervicite<normal<ASCUS<LEI-BG<LEI-AG<Carcinoma. CONCLUSÃO Os resultados mostram um aumento na AT com o aumento da lesão, sustentando a associação entre a AT e o tipo de lesão.
Asunto(s)
Humanos , Femenino , Adulto , Carcinoma de Células Escamosas/enzimología , Telomerasa/metabolismo , Neoplasias del Cuello Uterino/enzimología , Displasia del Cuello del Útero , Frotis VaginalRESUMEN
BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.
Asunto(s)
Bacteriófago P22/química , Cápside/química , Sistema Enzimático del Citocromo P-450/administración & dosificación , Portadores de Fármacos/química , Proteínas de la Cápside/química , Línea Celular Tumoral , Sistema Enzimático del Citocromo P-450/uso terapéutico , Terapia Enzimática , Femenino , Células HeLa , Humanos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/enzimologíaRESUMEN
The DEAD box RNA helicase DDX5 is a multifunctional protein involved in the regulatory events of gene expression. Herein, we presented evidence indicating that DDX5 is transcriptionally upregulated by calcitriol, the hormonal form of vitamin D3. In silico analysis revealed the presence of two putative vitamin D response elements (VDREs) in the DDX5 promoter region. Using luciferase reporter assays, we demonstrated that the DDX5 promoter containing these putative VDREs significantly increased the luciferase activity in vitamin D receptor (VDR)-positive SiHa cells upon calcitriol treatment. Electrophoretic mobility shift assays showed the ability of VDR and retinoid X receptor to interact only with the most proximal VDRE, while chromatin immunoprecipitation analysis confirmed the occupancy of this VDRE by the VDR. Finally, we demonstrated that calcitriol significantly increased both DDX5 mRNA and protein in SiHa cells. In summary, this study shows that DDX5 gene is transcriptionally upregulated by calcitriol through a VDRE located in its proximal promoter. Given the importance of DDX5 as a master regulator of differentiation programs, our study suggests that the pro-differentiating properties of calcitriol may be related with the induction of DDX5.
Asunto(s)
Calcitriol/farmacología , ARN Helicasas DEAD-box/metabolismo , Receptores de Calcitriol/agonistas , Transcripción Genética/efectos de los fármacos , Neoplasias del Cuello Uterino/enzimología , Elemento de Respuesta a la Vitamina D/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , ARN Helicasas DEAD-box/genética , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores X Retinoide/metabolismo , Transfección , Regulación hacia Arriba , Neoplasias del Cuello Uterino/genéticaRESUMEN
BACKGROUND: Human papillomavirus (HPV) inactivates the retinoblastoma 1 (RB1) gene by promoter methylation and reduces cellular E-cadherin expression by overexpression of DNA methyltransferase 1 (DNMT1). The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions. METHODS: Sixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP. RESULTS: HPV frequency was associated with cervical cancer cases (p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%). CONCLUSIONS: The methylation status of both genes increased with disease progression. With EBV, RB1 methylation was a tumor-associated event because only the cancer group presented methylated RB1 with HPV infection. HPV infection was shown to be significantly correlated with cancer conditions. The global methylation frequency was higher when HPV was present, showing its epigenetic role in cervical carcinogenesis. Nevertheless, EBV seems to be a cofactor and needs to be further investigated. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1159157579149317 .
Asunto(s)
Cadherinas/genética , Metilación de ADN , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/genética , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/genética , Lesiones Intraepiteliales Escamosas de Cuello Uterino/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Antígenos CD , Cadherinas/metabolismo , Estudios de Casos y Controles , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Progresión de la Enfermedad , Epigénesis Genética , Infecciones por Virus de Epstein-Barr/virología , Femenino , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 4/patogenicidad , Interacciones Huésped-Patógeno , Pruebas de ADN del Papillomavirus Humano , Humanos , Clasificación del Tumor , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa , Proteína de Retinoblastoma/metabolismo , Lesiones Intraepiteliales Escamosas de Cuello Uterino/enzimología , Lesiones Intraepiteliales Escamosas de Cuello Uterino/patología , Lesiones Intraepiteliales Escamosas de Cuello Uterino/virología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
INTRODUCTION: Cervical cancer is characterized by an immunosuppressive microenvironment and a Th2-type cytokine profile. Expression of arginase (ASE), the enzyme that converts L-arginine into L-ornithine and urea, is stimulated by Th2-type cytokines. OBJECTIVE: To assess the association of ASE activity and L-Arg metabolism products with cervical cancer. METHODS: Sera of 87 and 41 women with histologically confirmed by colposcopy-directed biopsy SCC and CIN3 respectively and 79 with normal cytology or Low-Grade Squamous Intraepithelial Lesion (LSIL), were evaluated. Cytokines were measured using Milliplex Human cytokine/chemokine kit. Arginase (ASE) activity was determined using an enzymatic assay. Levels of L-arginine, L-ornithine, putrescine and spermine were determined by HPLC. RESULTS: Significantly higher levels of ASE activity were observed in women with CIN3 (age-adjusted OR: 24.3; 95%CI: 3.82-155) and SCC (AOR: 9.8; 95%CI: 2.34-40.8). As expected, possibly due to high levels of ASE activity, higher levels of l-Arg were negatively associated with CIN3 (AOR: 0.03; 95%CI: 0.004-0.19) and SSC (AOR: 0.06; 95%CI: 0.02-0.24). Consistent with the role of ASE in the conversion of L-arginine to L-ornithine and polyamine production therefrom, women with cervical cancer had higher levels of spermine and putrescine. A correlation analysis revealed a significant albeit weak relationship between high levels of IL-10 and high levels of ASE (Pearson r=0.32, p-value=0.003) in women with cervical cancer. CONCLUSION: This study indicates that ASE activity and L-Arg degradation mechanisms of immunosuppression are present in cervical cancer. The results foster research in the design of possible strategies to inhibit ASE activity for therapy of cervical cancer.
Asunto(s)
Arginasa/metabolismo , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/inmunología , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/inmunología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/inmunología , Adulto , Anciano , Arginina/metabolismo , Carcinoma de Células Escamosas/sangre , Femenino , Humanos , Tolerancia Inmunológica , Persona de Mediana Edad , Neoplasias del Cuello Uterino/sangre , Adulto Joven , Displasia del Cuello del Útero/sangreRESUMEN
We performed a case-control association study to evaluate the association between common polymorphisms in MTHFR (C677T and A1298C) and the Arg72Pro polymorphism in the p53 gene and the risk for cervical intraepithelial neoplasia (CIN) or invasive cervical cancer (ICC) in Mexican HPV-infected women. We included 131 women with diagnosis of CIN grade I-II and 78 with CIN III or ICC; as controls we also included 274 women with normal Pap smear and negative HPV test. Genotyping for MTHFR and p53 polymorphisms was performed by PCR-RFPLs. HPV was tested by Hybrid Capture II. Odds ratios and 95% confidence intervals were estimated. Genotype frequencies for the 3 studied polymorphisms were distributed according to the Hardy-Weinberg equilibrium. The A1298C-MTHFR polymorphism showed significant differences for the heterozygous AC genotype and the C allele, whereas the AA genotype and A allele resulted to be genetic risk factors for CIN or ICC (p<0.03). The Arg72Pro-p53 polymorphism showed for the genotypes Arg/Pro and Pro/Pro, and for the Pro allele, a significant association only to the risk for CIN (p<0.03). The MTHFR/p53 interaction showed that the genotype combinations AA/ArgArg and AA/ArgPro were associated, respectively, to the risk of ICC and CIN (p<0.05). This study suggests that the A1298C-MTHFR polymorphism contributes to the genetic risk for both CIN and ICC, whereas the Arg72Pro-p53 polymorphism only contributes to the risk for CIN. The MTHFR/p53 genetic combinations AA/ArgArg and AA/ArgPro are associated genetic risk factors for ICC and CIN in Mexican HPV-infected women.
Asunto(s)
Genes p53 , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Infecciones por Papillomavirus/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , México , Persona de Mediana Edad , Infecciones por Papillomavirus/enzimología , Polimorfismo Genético , Factores de Riesgo , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/virologíaRESUMEN
The level of beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) mRNA, encoded by the gene siat1, is increased in malignant tissues. Expression is regulated by different promoters - P1, P2 and P3 - generating three mRNA isoforms H, X and YZ. In cervical cancer tissue the mRNA isoform H, which results from P1 promoter activity, is increased. To study the regulation of P1 promoter, different constructs from P1 promoter were evaluated by luciferase assays in cervical and hepatic cell lines. Deletion of a fragment of 1048 bp (-89 to +24 bp) increased 5- and 3-fold the promoter activity in C33A and HepG2 cell lines, respectively. The minimal region with promoter activity was a 37 bp fragment in C33A cells. The activity of this region does not require the presence of an initiator sequence. In HepG2 cells the minimal promoter activity was detected in the 66 bp fragment. Sp1 (-32) mutation increased the promoter activity only in HepG2 cells. HNF1 mutation decreased promoter activity in HepG2 cell line but not in C33A cells. We identified a large region that plays a negative regulation role. The regulation of promoter activity is cell type specific. Our study provides new insights into the complex transcriptional regulation of siat1 gene.
Asunto(s)
Antígenos CD/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mutación , Sialiltransferasas/genética , Neoplasias del Cuello Uterino/genética , Antígenos CD/metabolismo , Secuencia de Bases , Sitios de Unión , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Femenino , Expresión Génica , Células Hep G2 , Factor Nuclear 1 del Hepatocito/genética , Humanos , Neoplasias Hepáticas/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Isoformas de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sialiltransferasas/metabolismo , Factor de Transcripción Sp1/inmunología , Neoplasias del Cuello Uterino/enzimologíaRESUMEN
Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.
Asunto(s)
Neoplasias/enzimología , Neoplasias/genética , Fosfofructoquinasa-1/metabolismo , Animales , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , ADN Complementario/genética , Activación Enzimática , Femenino , Células HeLa , Humanos , Cinética , Hígado/enzimología , Neoplasias Hepáticas Experimentales/enzimología , Neoplasias Hepáticas Experimentales/genética , Miocardio/enzimología , Fosfofructoquinasa-1/genética , Fosfofructoquinasa-1 Tipo Hepático/genética , Fosfofructoquinasa-1 Tipo Hepático/metabolismo , Fosfofructoquinasa-1 Tipo Muscular/genética , Fosfofructoquinasa-1 Tipo Muscular/metabolismo , Fosfofructoquinasa-1 Tipo C/genética , Fosfofructoquinasa-1 Tipo C/metabolismo , Polimorfismo Genético , Ratas , Ratas Wistar , Especificidad por Sustrato , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/genéticaRESUMEN
INTRODUCTION: Cervical cancer remains the second leading cause of death among women. Intraepithelial neoplasias and uterine invasive cancer are frequently associated with disturbances in coagulation and changes in the concentrations of adenine nucleotides. This work intended to analyze changes in extracellular adenine nucleotide hydrolysis and blood platelet aggregation in patients diagnosed for cervical intraepithelial neoplasia in different stages as well as uterine invasive cancer. PATIENTS AND METHODS: NTPDase, E-NPP, 5'-nucleotidase, total ADA and its isoforms (ADA1 and ADA2), as well as the platelet aggregation from patients with different stages of cervical intraepithelial neoplasia (NICs I, NIC II, NIC III) and uterine invasive cancer were verified. RESULTS: Neither ATP hydrolysis nor E-NPP activity was changed by the neoplasia stage. On the other hand, ADP and AMP hydrolysis as well as ADA activity were enhanced in NIC I group. AMP hydrolysis was also increased in the cancer group. ADA 1 was the ADA isoform found in platelets from both control and patient groups. CONCLUSION: Our results showed for the first time that NTPDase, 5'-nucleotidase, E-NPP and ADA are not sensible regarding the grade of neoplasia development, since no significant difference was found between the groups studied. Only ADP hydrolysis and ADA activity showed a significant enhancement in NIC I group related to the other stages possibly as a result of the beginning of the neoplasic transformation. This increase could be reflecting a body's reaction against the probable high adenosine levels. We propose for the first time that the ADA isoform present in platelets is ADA 1.
Asunto(s)
Adenosina Desaminasa/metabolismo , Agregación Plaquetaria , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/patología , Estudios de Casos y Controles , Femenino , Humanos , Hidrólisis , Isoenzimas , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias del Cuello Uterino/enzimología , Displasia del Cuello del Útero/enzimologíaRESUMEN
The metalloproteinases (MMP) 11 and 12 have been shown to be expressed in cervical cancer (CC). In order to extend our previous results, these MMPs were evaluated in cervical precursor lesions. One hundred seventeen cervical scrapes: thirty-six normal, thirty-six low grade squamous lesions (LSIL), thirty-six high grade (HSIL), nine CC; and, also ninety-nine paraffin-embedded cervical lesions: fifteen normal cervices, thirty eight LSIL, sixteen HSIL, and five CC were collected. The samples were analyzed for relative expression by real time RT-PCR or immunohistochemistry assay. We were able to identify a relative increased expression of MMP11 in 75% and 78% from LSIL and HSIL samples, respectively. While MMP12 expression was 64% and 75% in LSIL and HSIL, respectively. Positive samples for MMP11 expression were also positive for MMP12 expression and also increased according to illness progression. In the tissues, MMP11 or MMP12 expression was observed in the cytoplasm of the neoplastic cells, while in the normal epithelium was absent. The reaction was always stronger for MMP12 than MMP11. MMP11 expression was present in 77% and 66% of LSIL and HSIL, while MMP12 expression was 73% and 68%. There was a relationship between MMP11 or MMP12 expression and HPV infection. Our data are showing a relationship between diagnostic of precursor lesions and the MMP11 and 12 expressions, suggesting that their expression could be an early event in the neoplastic lesions of the cervix and could have clinical significance.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Metaloproteinasa 11 de la Matriz/análisis , Metaloproteinasa 12 de la Matriz/análisis , Lesiones Precancerosas/enzimología , Neoplasias del Cuello Uterino/enzimología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Distribución de Chi-Cuadrado , ADN Viral/análisis , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Papillomavirus Humano 16/genética , Humanos , Inmunohistoquímica , Metaloproteinasa 11 de la Matriz/genética , Metaloproteinasa 12 de la Matriz/genética , México , Clasificación del Tumor , Adhesión en Parafina , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Lesiones Precancerosas/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Matrices Tisulares , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Frotis VaginalRESUMEN
BACKGROUND: Uterine cervical neoplasia is an important worldwide malignancy sometimes associated with thrombosis. Ectonucleotidases are membrane-bound enzymes which participate in thromboregulation by hydrolyzing adenine nucleotides in the extracellular medium. In this sense, we aimed to investigate their activity in patients with uterine cervical neoplasia. METHODS: We evaluated NTPDase and 5'-nucleotidase activities from patients previously treated for uterine cervical neoplasia with either conization or radiotherapy (RTX). These patients were divided into four groups: two conization groups (I and II) and two RTX groups (III and IV), which were further divided based on the amount of time that had passed since the conclusion of their treatment, where groups I and III were extended-remission-period groups (patients with 1 to 5 years elapsed after the conclusion of treatment), and groups II and IV were recently treated patients (treated up to three months before). RESULTS: For both conization and RTX groups, ATP and ADP hydrolysis decreased in the extended-remission groups when compared to the control and recently treated groups. On the other hand, AMP hydrolysis was decreased in all the treated groups (both conization and RTX) compared to the control. CD39 expression was decreased in extended-remission groups (I and III) when compared to the other groups. CONCLUSIONS: NTPDase protects against platelet aggregation and 5'-nucleotidase is more involved in the control of adenosine formation.
Asunto(s)
Antígenos CD/sangre , Apirasa/sangre , Plaquetas/enzimología , Conización , Neoplasias del Cuello Uterino/radioterapia , Neoplasias del Cuello Uterino/cirugía , 5'-Nucleotidasa/sangre , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Pruebas de Coagulación Sanguínea , Femenino , Humanos , Persona de Mediana Edad , Agregación Plaquetaria , Recuento de Plaquetas , Plasma Rico en Plaquetas , Neoplasias del Cuello Uterino/sangre , Neoplasias del Cuello Uterino/enzimología , Frotis VaginalRESUMEN
Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose- and time-dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase-8 and -9; BID cleavage, cytochrome C release and PARP cleavage. Statin-sensitive cancers expressed high levels of HMG-CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG-CoA reductase since mevalonate pre-incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG-CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies.
Asunto(s)
Neoplasias de los Genitales Femeninos/enzimología , Neoplasias de los Genitales Femeninos/patología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lípidos/química , Agua/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de los Genitales Femeninos/genética , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Lovastatina/farmacología , Ácido Mevalónico/farmacología , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Fosfatos de Poliisoprenilo/farmacología , Pravastatina/farmacología , Sesquiterpenos/farmacología , Transducción de Señal/efectos de los fármacos , Simvastatina/farmacología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
Altered sialylation observed during oncogenic transformation, tumor metastases and invasion, has been associated with enhanced sialyltransferases (STs) transcription. Increased mRNA expression of STs (ST6Gal I, ST3Gal III) has been detected in invasive cervical squamous cell carcinoma. A study of the sialic acid concentration in local tissue of cervix and in serum showed a slight elevation in benign inflammatory lesions and a moderate elevation in severe neoplasia, but to date, altered expression of STs in cervical intraepithelial neoplasia has not yet been evaluated. This study investigates the changes in mRNA expression of three STs (ST6Gal I, ST3Gal III, and ST3Gal IV) in cervical intraepithelial lesions (CIN). Alterations of these STs mRNA expression were examined in 35 cervix specimens classified as normal, CIN 1, CIN 2 and CIN 3, by semiquantitative reverse transcription-polymerase chain reaction, mRNA expression of the three STs was enhanced in CIN 1, CIN 2 and CIN 3 with respect to normal tissue, with a significant difference of p < 0.001 (Mann-Whitney U test) for all the enzymes. Our results suggest that altered expression of ST3Gal III, ST3Gal IV and ST6Gal I in CIN could play an important role during malignant transformation and could be related with the enhanced sialic acid expression detected in neoplasic tissues.
Asunto(s)
Proteínas de Neoplasias/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Sialiltransferasas/genética , Displasia del Cuello del Útero/enzimología , Neoplasias del Cuello Uterino/enzimología , Transformación Celular Neoplásica , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Neoplasias/biosíntesis , Procesamiento Proteico-Postraduccional , Estudios Retrospectivos , Sialiltransferasas/biosíntesis , Transcripción Genética , Neoplasias del Cuello Uterino/genética , Displasia del Cuello del Útero/genéticaRESUMEN
PURPOSE: The present study evaluated mRNA expression of interferon-alpha (IFN-alpha), IFN-alpha receptor subunits (IFNAR-1 and IFNAR-2) and an IFN-stimulated gene encoding the enzyme 2',5'-oligoadenylate synthetase (2'5'OAS) in biopsies on patients with varying grades of cervical intraepithelial neoplasia (CIN I, II and III). METHODS: Uterine cervix biopsies were collected from women with CIN I, II and III (n = 28) and controls without CIN lesions or human papilloma virus (HPV) infection (n = 17). The presence of high and low-risk HPV DNA was determined using hybrid capture. The mRNA levels of IFNAR-1, IFNAR-2, IFN-alpha and 2'5'OAS were determined by RT-PCR with specific primers. RESULTS: The control group exhibited a greater frequency of IFNAR-1 expression (10/17; 58.3%) than the CIN samples (4/28; 14.2%) (P = 0.0018), while, the expression of IFNAR-2 was also greater in the control samples (11/17; 64.7%) than in the patients with lesions (2/28; 7.1%) (P = 0.0018). Importantly, simultaneous expression of both receptors was observed only in the control group (8/17; 47.0%) (P = 0.0001). Among the CIN samples, there was one case of low expression of mRNA of IFNAR-1 and IFNAR-2. IFN-alpha was present in 14.2% (4/28) of the CIN samples but was not expressed in the control group. mRNA 2'5'OAS were expressed in 28.5% (8/28) of the CIN samples and 11.7% (2/17) of the control samples (not statistically significant). Fifty percent (14/28) of the CIN samples were positive for HPV DNA. CONCLUSIONS: Cervical biopsy samples from control women or those without neoplasia or HPV infection displayed higher IFN-alpha receptor expression than those with CIN, while simultaneous expression of both IFN-alpha receptor subunits was found only in the control group. There was no significant difference in mRNA expression of IFN-alpha and 2'5'OAS between the control and CIN groups. Then we concluded that the samples obtained from patients with CIN present low levels of the IFN-alpha receptor mRNA.
Asunto(s)
Interferón-alfa/biosíntesis , Receptor de Interferón alfa y beta/biosíntesis , Displasia del Cuello del Útero/inmunología , Neoplasias del Cuello Uterino/inmunología , 2',5'-Oligoadenilato Sintetasa/biosíntesis , 2',5'-Oligoadenilato Sintetasa/genética , Adolescente , Estudios de Casos y Controles , ADN Viral/análisis , Femenino , Humanos , Interferón-alfa/genética , Persona de Mediana Edad , Papillomaviridae/genética , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor de Interferón alfa y beta/genética , Transducción de Señal , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Adulto Joven , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/virologíaRESUMEN
The relation between the detection of clue cells in cervical smears of women with CIN and the expression of COX-2 in these lesions were determined. Samples from 228 women, treated due to CIN and who underwent cervical conization, were obtained. Hybrid Capture II and Pap smear samples were collected immediately before performing conization. Pathological diagnoses were 11 (5%) normal cervix, 35 (15%) CIN1, 31 (14%) CIN2, and 151 (66%) CIN3. COX-2 immunoreactivity grading on the pathological specimens was based on the German ImmunoReactive score. In cervical smears, 20 fields (40x) were examined, each of them with a minimum count of 10 epithelial cells. When 20% or more of clue cells were detected the sample was considered positive for clue cells. The prevalence of clue cells was similar across histological strata (P = 0.42). Although the expression of COX-2 did not differ in lesions with varying severities (P = 0.24), there was a negative association between the expression of COX-2 and the presence of clue cells in Pap smear (OR = 0.4; 95% CI = 0.2-0.9): only 12% of women with moderate and strong expression of COX-2 had clue cells in their smears, contrasted to 22% of those with negative and weak expression of COX-2. HPV infection was associated in a borderline manner to the expression of COX-2 (P = 0.04; OR = 2.3 95% CI = 1.0-5.4). The reduced expression of COX-2 in CIN specimens may suggest that clue cells interfere with the inflammatory component of the carcinogenic process that lead to CIN.
Asunto(s)
Carcinoma in Situ/enzimología , Carcinoma in Situ/patología , Ciclooxigenasa 2/metabolismo , Células Epiteliales/enzimología , Células Epiteliales/patología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/patología , Carcinoma in Situ/diagnóstico , Cuello del Útero/virología , Ciclooxigenasa 2/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Prueba de Papanicolaou , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/patología , Estudios Retrospectivos , Neoplasias del Cuello Uterino/diagnóstico , Frotis Vaginal/métodosRESUMEN
Human papillomavirus (HPV) is the main risk factor for cervical cancer; however, some carcinomas occur in the absence of the virus. IGF-IR and an isoform of the insulin receptor, IR-A, play important roles in cancer. In this study we assessed the role of the IGF/insulin receptors in cervical cancer cell lines with different HPV status, SiHa (HPV positive), and C33a (HPV negative). Different patterns of receptor expression were found; while SiHa expressed IGF-IR, IR-A and IR-B, and IR/IGF-IR hybrid receptors, C33a cells expressed the IR-A only. Tyrosine phosphorylation of these receptors in response to their corresponding ligands correlated with the expression level of these receptors in the cell lines. Activation of PI3-K and MAPK pathways was revealed in both cell lines, however, no effects on proliferation, migration, or invasion were observed. Here we show that cervical cancer cell lines--positive and negative for HPV--differ in the type of insulin and IGF-1 receptors expressed. Additional studies are needed for characterization of the role of IR-A in cervical carcinogenesis.