ABSTRACT
This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.
Subject(s)
Humans , Triple Negative Breast Neoplasms/metabolism , Matrix Metalloproteinase 2/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Movement , Ubiquitination , RNA, Messenger/metabolism , Epithelial-Mesenchymal Transition , Transcription Factors/metabolismABSTRACT
AIM To establish an HPLC method for the simultaneous content determination of four constituents in Hydnocarpus anthelmintica Pierre.METHODS The analysis of 60％ ethanol extract from H.anthelmintica was carried out on a 35 ℃ thermostatic Diamonsil C18column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of 80％ acetonitrile-0.1％ phosphoric acid flowing at 1.0 mL/min in a gradient manner,and the detection wavelength was set at 347 nm.RESULTS Luteolin,hydnocarpusol,chrysoeriol and hydnocarpin showed good linear relationships within the ranges of 0.0523 6-1.047 μg (r =0.999 9),0.011 24-0.224 8 μg (r =0.999 9),0.029 46-0.589 2 μg (r =0.999 9) and 0.130 5-2.609 μg (r =0.999 9),whose average recoveries were 102.45％ (RSD=1.9％),98.66％ (RSD=1.8％),97.60％ (RSD=1.6％) and 97.88％ (RSD=1.4％),respectively.CONCLUSION This simple,accurate and sensitive method can be used for the quality control of H.anthelmintica.
ABSTRACT
OBJECTIVE: To study the chemical constituents whole plant of Bidens frondosa L.