RESUMEN
Parthenolide (PTL) is a sesquiterpene lactone isolated from feverfew and exhibits potent antitumor activity against various cancers. Many studies indicate that PTL treatment leads to apoptosis, however, the mechanism has not been defined. Here, we observed that cells underwent autophagy shortly after PTL treatment. Inhibition of autophagy by knocking out autophagy associated gene atg5 blocked PTL-induced apoptosis. Surprisingly, PTL decreased the level of translation initiation factor eIF4E binding protein 1 (4E-BP1) in correlation with autophagy. Ectopic expression or shRNA knockdown of 4E-BP1 further verified the effect of 4E-BP1 on PTL-induced autophagy. Meanwhile, PTL elevated the cellular reactive oxygen species (ROS) which located upstream of the depletion of 4E-BP1, and contributed to the consequent autophagy. This study revealed 4E-BP1 as a trigger for PTL-induced autophagy and may lead to therapeutic strategy to enhance the efficacy of anticancer drugs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Autofagia/efectos de los fármacos , Proteínas Portadoras/metabolismo , Fosfoproteínas/metabolismo , Sesquiterpenos/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Proteínas de Ciclo Celular , Factores Eucarióticos de Iniciación , Fibroblastos/metabolismo , Células HEK293 , Células HL-60 , Células HeLa , Humanos , Ratones , Fagosomas/metabolismo , Fosforilación/efectos de los fármacos , Plásmidos , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Vav proteins are guanine nucleotide exchange factors (GEFs) that activate a group of small G proteins (GTPases). Vav1 is predominantly expressed in hematopoietic cells, whereas Vav2 and Vav3 are ubiquitously distributed in almost all human tissues. All three Vav proteins contain conserved structural motifs and associate with a variety of cellular activities including proliferation, migration, and survival. Previous observation with Jurkat leukemia T cells showed that Vav1 possessed anti-apoptotic activity by enhancing Bcl-2 transcription. However the mechanism has not been unveiled. Here, we explored the effectors of Vav1 in promoting Bcl-2 expression in Jurkat cells and revealed that Rac2-Akt was specifically evoked by the expression of Vav1, but not Vav2 or Vav3. Although all three Vav isoforms existed in Jurkat cells, Rac2 was distinguishably activated by Vav1 and that led to enhanced Bcl-2 expression and cell survival. Akt was modulated downstream of Vav1-Rac2, and the activation of Akt was indispensable in the enhanced transcription of Bcl-2. Intriguingly, neither Vav2 nor Vav3 was able to activate Rac2-Akt pathway as determined by gene silencing approach. Our data illustrated a unique role of Vav1 in T leukemia survival by selectively triggering Rac2-Akt axis and elevating the expression of anti-apoptotic Bcl-2.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Apoptosis , Células HEK293 , Humanos , Células Jurkat , Leucemia/metabolismo , Leucemia/patología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-vav/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-vav/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/genética , Proteína RCA2 de Unión a GTPRESUMEN
Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17ß-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not ß. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-vav/genética , Activación Transcripcional , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular , Línea Celular Tumoral , Femenino , Humanos , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
Vav1 is a guanine nucleotide exchange factor (GEF) specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor (TCR)-mediated signal transduction, whereas Vav2 and Vav3 function as GEFs that overlap with Vav1 on TCR-induced cytoskeleton reorganization. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal 20 amino acids of Vav1 in the calponin homology (CH) domain to be essential for its interaction with calmodulin (CaM) that leads to TCR-induced calcium mobilization. Substitution of the 1-20 amino acids of Vav1 with those of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells (NFAT) activation and IL-2 production. This study highlights the importance of the N-terminal 20 aa of Vav1 for CaM binding, and provides new insights into the distinguished and irreplaceable role of Vav1 in T cell activation and signal transduction.
Asunto(s)
Señalización del Calcio/fisiología , Activación de Linfocitos/fisiología , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Células HeLa , Humanos , Interleucina-2/biosíntesis , Interleucina-2/genética , Mutación , Unión Proteica/fisiología , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-vav/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citologíaRESUMEN
AIM: To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells. METHODS: Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (ΔΨ(m)) was measured using DiOC(6)(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays. RESULTS: Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (ΔΨ(m)) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression. CONCLUSION: Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity.