RESUMO
The objective of this study was to explore the role of the SULF2-mediated ERK/AKT signaling pathway in cervical cancer. SULF2 expression was detected in tumor tissues and tumor-adjacent normal tissues from cervical cancer patients. HeLa cells were divided into six groups: control group, NC group, SULF2 siRNA group, SULF2 group, SULF2 + LY294002 group, and SULF2 + U0125 group. In each group, HeLa cells received the corresponding treatment, followed by measurement of the cellular biological characteristics and expression of the ERK/AKT signaling pathway. We also confirmed the effect of SULF2 in vivo using a xenograft model in nude mice. SULF2 was upregulated in cervical cancer tissues, which was specifically associated with the clinical stage, histological differentiation, and lymphatic metastasis. Compared to the control group, the SULF2 siRNA group displayed decreased expression of SULF2, concomitant with reduced proliferation, migration, and invasion, but there was an increase in the apoptosis rate of HeLa cells, as well as downregulation of the p-Akt/Akt, p-ERK/ERK, and Bax/Bcl-2 ratios and cyclin D1. Additionally, tumor growth was significantly inhibited in the xenograft model of nude mice. The results in the SULF2 group were quite the opposite in which SULF2 facilitated the growth of cervical cancer cells, which was reversed by LY294002 or U0126. SULF2 is highly expressed in cervical cancer, and thus, downregulation of SULF2 can inhibit the ERK1/2 and AKT signaling pathways to suppress the proliferation, invasion, and migration of cervical cancer cells while facilitating apoptosis.
Assuntos
Apoptose , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sulfatases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Adulto , Idoso , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-akt/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Sulfatases/genéticaRESUMO
The objective of this study was to explore the role of the SULF2-mediated ERK/AKT signaling pathway in cervical cancer. SULF2 expression was detected in tumor tissues and tumor-adjacent normal tissues from cervical cancer patients. HeLa cells were divided into six groups: control group, NC group, SULF2 siRNA group, SULF2 group, SULF2 + LY294002 group, and SULF2 + U0125 group. In each group, HeLa cells received the corresponding treatment, followed by measurement of the cellular biological characteristics and expression of the ERK/AKT signaling pathway. We also confirmed the effect of SULF2 in vivo using a xenograft model in nude mice. SULF2 was upregulated in cervical cancer tissues, which was specifically associated with the clinical stage, histological differentiation, and lymphatic metastasis. Compared to the control group, the SULF2 siRNA group displayed decreased expression of SULF2, concomitant with reduced proliferation, migration, and invasion, but there was an increase in the apoptosis rate of HeLa cells, as well as downregulation of the p-Akt/Akt, p-ERK/ERK, and Bax/Bcl-2 ratios and cyclin D1. Additionally, tumor growth was significantly inhibited in the xenograft model of nude mice. The results in the SULF2 group were quite the opposite in which SULF2 facilitated the growth of cervical cancer cells, which was reversed by LY294002 or U0126. SULF2 is highly expressed in cervical cancer, and thus, downregulation of SULF2 can inhibit the ERK1/2 and AKT signaling pathways to suppress the proliferation, invasion, and migration of cervical cancer cells while facilitating apoptosis.
Assuntos
Humanos , Animais , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Coelhos , Sulfatases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Apoptose , Sistema de Sinalização das MAP Quinases/fisiologia , Sulfatases/genética , Imuno-Histoquímica , Células HeLa , Transdução de Sinais , Estudos de Casos e Controles , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Camundongos Nus , Estadiamento de NeoplasiasRESUMO
Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.
Assuntos
Condroitina Sulfatases/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Células Cultivadas , Condroitina Sulfatases/química , Condroitina Sulfatases/genética , Condroitina Sulfatases/isolamento & purificação , Endocitose , Estabilidade Enzimática , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Pichia/genética , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sulfatases/genética , Sulfatases/metabolismo , TemperaturaRESUMO
It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.
Assuntos
Animais , Masculino , Catepsina B/metabolismo , Diabetes Mellitus Experimental/enzimologia , Fígado/enzimologia , Lisossomos/enzimologia , Albuminas/análise , Western Blotting , Glicemia/efeitos dos fármacos , Catepsina L/metabolismo , Creatinina/urina , Cisteína Proteases/metabolismo , Sulfato de Dextrana/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Glucuronidase/metabolismo , Hexosaminidases/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , RNA , Sulfatases/metabolismoRESUMO
It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.
Assuntos
Catepsina B/metabolismo , Diabetes Mellitus Experimental/enzimologia , Fígado/enzimologia , Lisossomos/enzimologia , Albuminas/análise , Animais , Glicemia/efeitos dos fármacos , Western Blotting , Catepsina L/metabolismo , Creatinina/urina , Cisteína Proteases/metabolismo , Sulfato de Dextrana/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Glucuronidase/metabolismo , Hexosaminidases/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Masculino , RNA/isolamento & purificação , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Sulfatases/metabolismoAssuntos
Humanos , Feminino , Neoplasias da Mama/etiologia , Neoplasias da Mama/terapia , Progestinas/biossíntese , Progestinas/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Estrogênios/biossíntese , Moduladores de Receptor Estrogênico/uso terapêutico , Pós-Menopausa , Sulfatases/metabolismoRESUMO
Interleukin (IL)-13 is a central mediator in allergic asthma. Our previous results have indicated that sulfatase-modifying factor 2 (SUMF2) interacts with IL-13 and inhibits its secretion. In this study, we investigated the interactions between SUMF2 subtypes and 2 types of IL-13. Wild type IL-13 (wh-IL-13) and its mutated counterpart (mh-IL-13) were analyzed and cloned into pSos yeast expression vectors. Protein was expressed in host cdc25H yeast strains. A quartet of agar growth plates was prepared for the yeast two-hybrid system, which was used to detect IL-13 and SUMF2 subtype interactions. Both yeast expression vectors, pSos/whIL-13 and pSos/whIL-13, and recombinant expression vectors for the 5 subtypes of SUMF2 (pMyr/SUMF2-Vx) were constructed. Our data showed that all of the SUMF2 subtypes bound to whIL-13 and mhIL-13 in the CytoTrap system. Five SUMF2 subtypes - SUMF2-V2, SUMF2-V3, SUMF2-V4, SUMF2-V5, and SUMF2-V7--interacted with whIL-13 and mhIL-13. These subtypes may contribute to allergic asthma by mediating IL-13 release.
Assuntos
Interleucina-13/metabolismo , Sulfatases/metabolismo , Humanos , Interleucina-13/genética , Mutação , Ligação Proteica , Sulfatases/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). The activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of ß-glucuronidase, N-acetyl-ß-d-glucosaminidase, and N-acetyl-ß-d-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. The main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Rim/enzimologia , Lisossomos/enzimologia , Animais , Catepsinas/genética , Catepsinas/metabolismo , Diabetes Mellitus Experimental/genética , Regulação para Baixo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Masculino , Ratos , Ratos Wistar , Sulfatases/genética , Sulfatases/metabolismoRESUMO
Mucopolysaccharidosis (MPS) IVA is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate sulfatase (GALNS), which leads to the accumulation of keratan sulfate and chondroitin 6-sulfate, mainly in bone. To explore the possibility of gene therapy for Morquio A disease, we transduced the GALNS gene into HEK293 cells, human MPS IVA fibroblasts and murine MPS IVA chondrocytes by using adeno-associated virus (AAV)-based vectors, which carry human GALNS cDNA. The effects of the promoter and the cotransduction with the sulfatase-modifying factor 1 gene (SUMF1) on GALNS activity levels was evaluated. Downregulation of the cytomegalovirus (CMV) immediate early enhancer/promoter was not observed for 10 days post-transduction. The eukaryotic promoters induced equal or higher levels of GALNS activity than those induced by the CMV promoter in HEK293 cells. Transduction of human MPS IVA fibroblasts induced GALNS activity levels that were 15-54% of those of normal human fibroblasts, whereas in transduced murine MPS IVA chondrocytes, the enzyme activities increased up to 70% of normal levels. Cotransduction with SUMF1 vector yielded an additional four-fold increase in enzyme activity, although the level of elevation depended on the transduced cell type. These findings suggest the potential application of AAV vectors for the treatment of Morquio A disease, depending on the combined choice of transduced cell type, selection of promoter, and cotransduction of SUMF1.
Assuntos
Condroitina Sulfatases/genética , Condroitina Sulfatases/metabolismo , Dependovirus/genética , Mucopolissacaridose IV/genética , Regiões Promotoras Genéticas/genética , Sulfatases/genética , Sulfatases/metabolismo , Animais , Linhagem Celular , Vetores Genéticos/genética , Humanos , Camundongos , Mucopolissacaridose IV/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , TransfecçãoRESUMO
Morquio A is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to the lysosomal accumulation of keratan-sulfate and chondroitin-6-sulfate. We evaluated in HEK293 cells the effect of the cytomegalovirus immediate early enhancer/promoter (CMV) or the elongation factor 1alpha (EF1alpha) promoters, and the coexpression with the sulfatase modifying factor 1 (SUMF1) on GALNS activity. Four days postransfection GALNS activity in transfected cells with CMV-pIRES-GALNS reached a plateau, whereas in cells transfected with EF1alpha-pIRES-GALNS continued to increase until day 8. Co-transfection with pCXN-SUMF1 showed an increment up to 2.6-fold in GALNS activity. Finally, computational analysis of transcription factor binding-sites and CpG islands showed that EF1alpha promoter has long CpG islands and high-density binding-sites for Sp1 compared to CMV. These results show the advantage of the SUMF1 coexpression on GALNS activity and indicate a considerable effect on the expression stability using EF1alpha promoter compared to CMV.