RESUMEN
Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signals that are often constitutively activated in many cancerous or transformed cells and some stromal cells in the tumor microenvironment. Persistent STAT3 activation in malignant cells stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. STAT3 undergoes activation through phosphorylation on tyrosine 705, which facilitates its dimerization. Dimeric STAT3 translocates to the nucleus, where it regulates the transcription of genes involved in cell proliferation, survival, etc. In the present study, a synthetic deguelin analogue SH48, discovered by virtual screening, inhibited the phosphorylation, nuclear translocation, and transcriptional activity of STAT3 in H-ras transformed human mammary epithelial MCF-10A cells (MCF10A-ras). We speculated that SH48 bearing an α,ß-unsaturated carbonyl group could interact with a thiol residue of STAT3, thereby inactivating this transcription factor. Non-electrophilic analogues of SH48 failed to inhibit STAT3 activation, lending support to the above supposition. By utilizing a biotinylated SH48, we were able to demonstrate the complex formation between SH48 and STAT3. SH48 treatment to MCF10A-ras cells induced autophagy, which was verified by staining with a fluorescent acidotropic probe, LysoTracker Red, as well as upregulating the expression of LC3II and p62. In conclusion, the electrophilic analogue of deguelin interacts with STAT3 and inhibits its activation in MCF10A-ras cells, which may account for its induction of autophagic death.
RESUMEN
15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand for PPARγ, has differential effects on cancer cell proliferation and survival depending on the dose and the type of cells. In the present study, we have investigated the effects of 15d-PGJ2 on apoptosis of the Ha-ras transformed human breast epithelial (MCF10A-ras) cells. When MCF10A-ras cells were treated with 15d-PGJ2 (10 µM) for 24 hours, they underwent apoptosis as evidenced by characteristic morphological features, an increased proportion of sub-G0/G1 cell population, a typical pattern of annexin V/propidium iodide staining, perturbation of mitochondrial transmembrane potential (Δψm), and cleavage of caspase-3 and its substrate PARP. A pan-caspase inhibitor, Z-Val-Ala-Asp (OCH3)-fluoromethyl ketone attenuated cytotoxicity and proteolytic cleavage of caspase-3 induced by 15d-PGJ2. The 15d-PGJ2-induced apoptosis was accompanied by enhanced intracellular accumulation of reactive oxygen species (ROS), which was abolished by the antioxidant N-acetyl-L-cysteine (NAC). 15d-PGJ2 inhibited the DNA binding activity of NF-κB which was associated with inhibition of expression and catalytic activity of IκB kinase ß (IKKß). 15d-PGJ2-mediated inhibition of IKKß and nuclear translocation of phospho-p65 was blocked by NAC treatment. 9,10-Dihydro-PGJ2, a non-electrophilic analogue of 15d-PGJ2, failed to produce ROS, to inhibit NF-κB DNA binding, and to induce apoptosis, suggesting that the electrophilic α,ß-unsaturated carbonyl group of 15d-PGJ2 is essential for its pro-apoptotic activity. 15d-PGJ2-induced inactivation of IKKß was also attributable to its covalent thiol modification at the cysteine 179 residue of IKKß. Based on these findings, we propose that 15d-PGJ2 inactivates IKKß-ΝF-κB signaling through oxidative or covalent modification of IKKß, thereby inducing apoptosis in Ha-ras transformed human breast epithelial cells.
RESUMEN
Overproduction of prostaglandin E2 (PGE2) has been linked to enhanced tumor cell proliferation, invasiveness and metastasis as well as resistance to apoptosis. 15-Keto prostaglandin E2 (15-keto PGE2), a product formed from 15-hydroxyprostaglandin dehydrogenase-catalyzed oxidation of PGE2, has recently been shown to have anti-inflammatory and anticarcinogenic activities. In this study, we observed that 15-keto PGE2 suppressed the phosphorylation, dimerization and nuclear translocation of signal transducer and activator of transcription 3 (STAT3) in human mammary epithelial cells transfected with H-ras (MCF10A-ras). 15-Keto PGE2 inhibited the migration and clonogenicity of MCF10A-ras cells. In addition, subcutaneous injection of 15-keto PGE2 attenuated xenograft tumor growth and phosphorylation of STAT3 induced by breast cancer MDA-MB-231â¯cells. However, a non-electrophilic analogue, 13,14-dihydro-15-keto PGE2 failed to inhibit STAT3 signaling and was unable to suppress the growth and transformation of MCF10A-ras cells. These findings suggest that the α,ß-unsaturated carbonyl moiety of 15-keto PGE2 is essential for its suppression of STAT3 signaling. We observed that the thiol reducing agent, dithiothreitol abrogated 15-keto PGE2-induced STAT3 inactivation and disrupted the direct interaction between 15-keto PGE2 and STAT3. Furthermore, a molecular docking analysis suggested that Cys251 and Cys259 residues of STAT3 could be preferential binding sites for this lipid mediator. Mass spectral analysis revealed the covalent modification of recombinant STAT3 by 15-keto PGE2 at Cys259. Taken together, thiol modification of STAT3 by 15-keto PGE2 inactivates STAT3 which may account for its suppression of breast cancer cell proliferation and progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Dinoprostona/análogos & derivados , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Biomarcadores , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Cromatografía Liquida , Dinoprostona/química , Dinoprostona/metabolismo , Dinoprostona/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Fosforilación , Unión Proteica , Proteómica/métodos , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Histone deacetylase inhibitors (HDACIs) cause oncogenetransformed mammalian cell death. Our previous study indicated that HDACIs activate forkhead box O1 (FOXO1) and induce autophagy in liver and colon cancer cells. However, whether FOXO1 is involved in HDACImediated oncogenetransformed mammalian cell death remains unclear. In the present study, Hras transformed MCF10A cells were used to investigate the role of FOXO1 in this pathway. Results showed that trichostatin A (TSA), a HDACI, activated apoptosis in MCF10Aras cells, but not in MCF10A cells. Furthermore, TSA activated FOXO1 via P21 upregulation, whereas the knockdown of FOXO1 reduced TSAinduced cell death. In addition, TSA induced autophagy in MCF10A and MCF10Aras cells by blocking the mammailian target of rapamycin signaling pathway. Furthermore, autophagy inhibition lead to higher MCF10Aras cell death by TSA, thus indicating that autophagy is essential in cell survival. Taken together, the present study demonstrated that TSA causes oncogenetransformed cell apoptosis via activation of FOXO1 and HDACImediated autophagy induction, which served as important cell survival mechanisms. Notably, the present findings imply that a combination of HDACIs and autophagy inhibitors produce a synergistic anticancer effect.