RESUMO
Prostate cancer (PCa) is the second most prevalent cancer in men worldwide. The majority of PCa incidences eventually progress to castration-resistant PCa (CRPC), thereby establishing an urgent need for new effective therapeutic strategies. This study aims to examine the effects of morusin, a prenylated flavonoid isolated from Morus alba L., on PCa progression and identify the regulatory mechanism of morusin. Cell growth, cell migration and invasion, and the expression of EMT markers were examined. Cycle progression and cell apoptosis were examined using flow cytometry and a TUNEL assay, while transcriptome analysis was performed using RNA-seq with results being further validated using real-time PCR and western blot. A xenograft PCa model was used to examine tumor growth. Our experimental results indicated that morusin significantly attenuated the growth of PC-3 and 22Rv1 human PCa cells; moreover, morusin significantly suppressed TGF-[Formula: see text]-induced cell migration and invasion and inhibited EMT in PC-3 and 22Rv1 cells. Significantly, morusin treatment caused cell cycle arrest at the G2/M phase and induced cell apoptosis in PC-3 and 22Rv1 cells. Morusin also attenuated tumor growth in a xenograft murine model. The results of RNA-seq indicated that morusin regulated PCa cells through the Akt/mTOR signaling pathway, while our western blot results confirmed that morusin suppressed phosphorylation of AKT, mTOR, p70S6K, and downregulation of the expression of Raptor and Rictor in vitro and in vivo. These results suggest that morusin has antitumor activities on regulating PCa progression, including migration, invasion, and formation of metastasis, and might be a potential drug for CRPC treatment.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Linhagem Celular Tumoral , Transdução de Sinais/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células/genética , Apoptose/genética , Movimento CelularRESUMO
BACKGROUND: Bavachin is a natural product isolated from Psoralea corylifolia L. that has been applied as a traditional medicine in Asian countries. However, the anti-inflammatory effects of bavachin on LPS-induced inflammation and NLRP3 inflammasome activation by macrophages remain unclear. PURPOSE: We investigated the anti-inflammatory effects of bavachin on LPS-activated murine macrophage cell line J774A.1 cells and murine peritoneal macrophages. METHODS: J774A.1 cells and murine peritoneal macrophages were pre-treated with bavachin following LPS treatment. The concentrations of NO, PGE2, IL-6 and IL-12p40 in cell culture supernatant were analyzed. The expressions of iNOS, COX-2, mPGES-1 and MAPKs were analyzed using Western blotting, while NF-κB activity was detected using promoter reporter assay. To examine the activation of NLRP3 inflammasome, J774A.1 cells were incubated with LPS, and then treated with bavachin following treatment with ATP. The concentration of IL-1ß in the cell culture supernatant was measured. The expressions of NLRP3, ASC, caspase-1 and IL-1ß were analyzed using Western blotting. The formation of inflammasome complex was observed by immunofluorescence microscopy. RESULTS: Bavachin suppressed LPS-induced NO and PGE2 production, and decreased iNOS and mPGES-1 expression. Bavachin also reduced LPS-induced IL-6 and IL-12p40 production and decreased the activation of MAPKs and NF-κB. Additionally, bavachin suppressed NLRP3 inflammasome-derived IL-1ß secretion, decreased caspase-1 activation, repressed mature IL-1ß expression, and inhibited inflammasome complex formation. Furthermore, bavachin also suppressed the production of NO, IL-6 and IL-12p40 by LPS-stimulated murine peritoneal macrophages. CONCLUSION: Our experimental results indicated anti-inflammatory effects of bavachin exhibit attenuation of LPS-induced inflammation and inhibit activation of NLRP3 inflammasome in macrophages. These results suggest that bavachin might have potential in treating inflammatory and autoimmune diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Flavonoides/farmacologia , Inflamassomos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Dinoprostona/metabolismo , Inflamassomos/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/metabolismo , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Prostaglandina-E Sintases/metabolismoRESUMO
The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1-DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1-DNA complex is estimated to be 0.55 microM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.