RESUMEN
For the treatment and prevention of autoinflammatory diseases, it is essential to develop the drug, regulating the innate immune system. Although differentiation-inducing factor (DIF) derivatives, extracted from the cellular slime mold, Dictyostelium discoideum, exhibit immunomodulatory effects, their effects on the regulation of innate immunity in brain are unknown. In this study, we used the human cerebral microvascular endothelial cell line, hCMEC/D3, to investigate the effects of DIF derivatives on the generation of C-X-C motif chemokine (CXCL) 10 and interferon (IFN)-ß induced by polyinosinic-polycytidylic acid (poly IC). DIF-3 (1-10 µM), but not DIF-1 and DIF-2, dose-dependently inhibited the biosynthesis of not only CXCL10 but also CXCL16 and C-C motif chemokine 2 induced by poly IC. DIF-3 also strongly decreased IFN-ß mRNA expression and protein release from the cells induced by poly IC through the prohibition of p65, a subtype of NF-ĸB, not interferon regulatory transcription factor 3 phosphorylation. In the docking simulation study, we confirmed that DIF-3 had a high affinity to p65. These results suggest that DIF-3 regulates the innate immune system by inhibiting TLR3/IFN-ß signaling axis through the NF-ĸB phosphorylation inhibition.
Asunto(s)
Dictyostelium , Poli I-C , Humanos , Poli I-C/farmacología , Células Endoteliales/metabolismo , FN-kappa B/metabolismo , Inmunidad Innata , Quimiocinas/metabolismo , Quimiocinas/farmacologíaRESUMEN
Differentiation-inducing factor (DIF) defines a group of chlorinated hexaphenones that orchestrate stalk-cell differentiation in the slime mold Dictyostelium discoideum (DD). DIF-1 and 3 have also been reported to have tumor inhibiting properties; however, the mechanisms that underlie the effects of these compounds remain poorly defined. Herein, we show that DIF-3 rapidly triggers Ca2+ release and a loss of mitochondrial membrane potential (MMP) in the absence of cytochrome c and Smac release and without caspase activation. Consistently with these findings, we also detected no evidence of apoptosis in cells treated with DIF-3 but instead found that this compound induced autophagy. In addition, DIF-3 promoted mitochondrial fission in K562 and HeLa cells, as assessed by electron and confocal microscopy analysis. Importantly, DIF-3 mediated the phosphorylation and redistribution of dynamin-related protein 1 (DRP1) from the cytoplasmic to the microsomal fraction of K562 cells. Pharmacological inhibition or siRNA silencing of DRP1 not only inhibited mitochondrial fission but also protected K562 cells from DIF-3-mediated cell death. Furthermore, DIF-3 potently inhibited the growth of imatinib-sensitive and imatinib-resistant K562 cells. It also inhibited tumor formation in athymic mice engrafted with an imatinib-resistant CML cell line. Finally, DIF-3 exhibited a clear selectivity toward CD34+ leukemic cells from CML patients, compared with CD34- cells. In conclusion, we show that the potent anti-leukemic effect of DIF-3 is mediated through the induction of mitochondrial fission and caspase-independent cell death. Our findings may have important therapeutic implications, especially in the treatment of tumors that exhibit defects in apoptosis regulation.
Asunto(s)
Apoptosis/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Hexanonas/farmacología , Leucemia/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dinaminas , Femenino , Humanos , Células K562 , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Fosforilación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Differentiation-inducing factor-1 (DIF-1) produced by Dictyostelium discoideum strongly inhibits the proliferation of various types of cancer cells by suppression of the Wnt/ß-catenin signal transduction pathway. In the present study, we examined the effect of differentiation-inducing factor-3 (DIF-3), a monochlorinated metabolite of DIF-1 that is also produced by D. discoideum, on human colon cancer cell lines HCT-116 and DLD-1. DIF-3 strongly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase. DIF-3 reduced the expression levels of cyclin D1 and c-Myc by facilitating their degradation via activation of GSK-3ß in a time and dose-dependent manner. In addition, DIF-3 suppressed the expression of T-cell factor 7-like 2, a key transcription factor in the Wnt/ß-catenin signaling pathway, thereby reducing the mRNA levels of cyclin D1 and c-Myc. Subsequently, we examined the in vivo effects of DIF-3 in Mutyh(-/-) mice with oxidative stress-induced intestinal cancers. Repeated oral administration of DIF-3 markedly reduced the number and size of cancers at a level comparable to that of DIF-1. These data suggest that DIF-3 inhibits intestinal cancer cell proliferation in vitro and in vivo, probably by mechanisms similar to those identified in DIF-1 actions, and that DIF-3 may be a potential novel anti-cancer agent.