RESUMO
Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is mainly formed as a byproduct of glycolysis. Elevated MGO level is known to induce apoptosis of vascular endothelial cells, which is implicated with progression of atherosclerosis and diabetic complications. However, the underlying mechanisms have not been exhaustively investigated yet. Here, we further characterized the mechanisms how MGO induced apoptosis in human umbilical vein endothelial cells (HUVECs). Our data revealed that cytosolic phospholipase A2 (cPLA2) played an important role in MGO-induced cell apoptosis. It was found that MGO could increase both the activity and expression of cPLA2. Inhibition of cPLA2 by Pyrrophenone (PYR) or siRNA significantly attenuated the MGO-induced apoptosis. Additionally, MGO time-dependently decreased the phosphorylation of nuclear factor κB (NF-κB). Pretreatment of the cells with NF-κB inhibitor, BAY11-7082, further increased MGO-induced apoptosis of HUVECs, indicating that NF-κB played a survival role in this MGO-induced apoptosis. Furthermore, in the presence of si-cPLA2 or PYR, MGO no longer decreased NF-κB phosphorylation. Beyond that, the antioxidant N-acetyl cysteine (NAC) could reverse the changes of both cPLA2 and NF-κB caused by MGO. p38, the upstream of cPLA2, was also significantly phosphorylated by MGO. However, p38 inhibitor failed to reverse the apoptosis induced by MGO. This study gives an important insight into the downstream signaling mechanisms of MGO, cPLA2-NF-κB, in endothelial apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Fosfolipases A2 Citosólicas/metabolismo , Aldeído Pirúvico/toxicidade , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Fosfolipases A2 Citosólicas/antagonistas & inibidores , Fosfolipases A2 Citosólicas/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Peripheral corticotropin-releasing hormone receptors (CRHRs) are G protein-coupled receptors that play different roles depending on tissue types. Previously, we discovered the mechanism of CRHR-mediated apoptosis of mouse prostate cancer cell line (RM-1) to be a change of Bcl-2:Bax ratio, and CRH was found to inhibit transforming growth factor ß migration of breast cancer cells via CRHRs. In the present study, we investigated cytosolic calcium-dependent phospholipase A2 (cPLA2) bridging CRHR activations and Bcl-2:Bax ratio and the effect of CRHR activation on cell migration. Silencing of cPLA2 attenuated a CRHR1 agonist, CRH-induced apoptosis, and the decrease of the Bcl-2:Bax ratio, whereas silencing of cPLA2 aggravated CRHR2 agonist, Urocortin 2 (Ucn2)-inhibited apoptosis, and the increase of the Bcl-2:Bax ratio. CRH in a time- and concentration-dependent manner increased cPLA2 expression mainly through interleukin 1ß (IL1ß) upregulation. Ucn2 decreased cPLA2 expression through neither tumor necrosis factor α nor IL1ß. CRH-suppressed decay of cPLA2 mRNA and Ucn2 merely suppressed its production. Overexpression of CRHR1 or CRHR2 in HEK293 cells correspondingly upregulated or downregulated cPLA2 expression after CRH or Ucn2 stimulation respectively. In addition, both CRH and Ucn2 induced migration of RM-1 cells. Our observation not only established a relationship between CRHRs and cell migration but also for the first time, to our knowledge, demonstrated that cPLA2 participates in CRHR1-induced apoptosis and CRHR2-inhibited apoptosis.
Assuntos
Apoptose/fisiologia , Movimento Celular/fisiologia , Fosfolipases A2/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Animais , Linhagem Celular Tumoral , Hormônio Liberador da Corticotropina/antagonistas & inibidores , Hormônio Liberador da Corticotropina/metabolismo , Dactinomicina/farmacologia , Regulação para Baixo , Células HEK293 , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Masculino , Camundongos , Mitomicina/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfolipases A2/biossíntese , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Pirimidinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Tapsigargina/farmacologia , Fator de Necrose Tumoral alfa/genética , Regulação para Cima , Urocortinas/antagonistas & inibidores , Urocortinas/metabolismo , Proteína X Associada a bcl-2/biossínteseRESUMO
Urocortin (UCN) is a member of corticotrophin-releasing factor (CRF) family, which has been reported to play a role in many biological processes, including inflammation and cancer development. Growing evidence shows that PLA2 (phospholipase A2) enzymes also participate in inflammation and tumor development. The primary aim of the present study was to identify a novel signaling pathway of CRF receptor activation leading to migration of two kinds of hepatoma carcinoma cell lines, HepG2 and SMMC-7721, linking the stimulation of PLA2 expression by UCN to UCN-induced tumor cell migration. Pharmacological inhibitors and genetic approaches (such as stable transfection and siRNAs) were used in this study. Unlike HepG2 cells which express both CRF receptors themselves, SMMC-7721 cells which hardly express these two CRF receptors needed stable transfection with CRFR1 or CRFR2 to observe the effect of UCN. Two types of PLA2 enzymes, cPLA2 and iPLA2, were found to be regulated by UCN. Our data showed that UCN raised cPLA2 expression but lowered iPLA2 expression. Moreover, UCN was found to act on the certain region of iPLA2 promoter to reduce its transcription. UCN promoted tumor cell migration by up-regulating cPLA2 expression via CRFR1 whereas it suppressed tumor cell migration by down-regulating iPLA2 expression via CRFR2. These results indicate the dual roles for UCN in the hepatoma carcinoma cell migration, which involve the regulation of both cPLA2and iPLA2.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fosfolipases A2 Independentes de Cálcio/genética , Fosfolipases A2 Citosólicas/genética , Urocortinas/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , NF-kappa B/metabolismo , Naftalenos/farmacologia , Fosfolipases A2 Independentes de Cálcio/antagonistas & inibidores , Fosfolipases A2 Independentes de Cálcio/metabolismo , Fosfolipases A2 Citosólicas/antagonistas & inibidores , Fosfolipases A2 Citosólicas/metabolismo , Regiões Promotoras Genéticas , Pironas/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Urocortinas/farmacologiaRESUMO
Patients with ulcerative colitis are at a very high risk of developing colorectal cancer. Corticotrophin-releasing hormone (CRH) family peptides and their receptors (CRHRs) are found to modulate inflammation and tumor cell growth. However, the role of CRH family peptides and their receptors in the inflammation-related colon cancer is still unknown. The aim of this study was to investigate the functions of CRHR1 signaling on the development of colitis-associated cancer (CAC). Crhr1-deficient (Crhr1(-/-)) mice were used to explore the role of CRHR1 in the development of azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced CAC. WT (Crhr1(+/+)) littermates were set as control. We found that the expression of CRHR1 and its endogenous ligands: urocortin and CRH were enhanced in the colon of Crhr1(+/+) mice during treatment with AOM and DSS. Tumorigenesis was significantly reduced in Crhr1(-/-) mice, determined by analysis of survival rate (increased by 20%), weight loss (decreased by 10%), tumor formation (decreased by 60% in tumor number), histological scores (decreased by 58%), and cytokine production. During early CAC tumorigenesis, Crhr1(-/-) mice exhibited much less tumorigenesis, accompanied by lower inflammatory response, including decreased IL1ß, IL6 and TNFα expression and macrophage infiltration and increased IL10 expression. Moreover, Crhr1(-/-) mice displayed a reduced activation of NFκB and STAT3 phosphorylation with decreased proliferating and enhanced apoptotic cells in the colon. In conclusion, CRHR1 has a proinflammatory and therefore a protumorigenesis effect in terms of CAC, which may be helpful to develop new therapeutic approaches for inflammation and cancer prevention and treatment.