ABSTRACT
Objective: To research the mechanism of ebracteolatain A against breast cancer cells, screening the genes with significant changes using second-generation sequencing, and explore the anti-breast cancer mechanism of action of ebracteolatain A at the transcriptomics level. Methods: The acetyl phloroglucinol compound ebracteolatain A was extracted from Euphorbia ebracteolata, interferencing with MCF7 cells (luminal A type of breast cancer cells) to observe differential gene expression between the interfered cells and normal cells. High-throughput transcriptome sequencing and data analysis were performed on three groups of control groups and three experimental groups using Illumina Hi-Seq sequencing technology. Results: A total of 123 656 848, 123 974 262 available reads were obtained in the control group and experimental group, respectively, the reads on the reference genome were 119 762 214, 119 881 622, respectively, accounting for 96.85% and 96.69% of the total; Two groups of transcriptome controls were available: the total number of differential genes was 1 695, of which 770 were up-regulated, 925 were down-regulated, and 3 874 genes were clearly annotated. Bio-enrichment analysis was carried out by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). GO analysis found that these 3 874 genes mainly involved in biological processes (1 270), cell composition (1 322) and molecular function (1 282), 45 subcategories of three major categories, including cell growth and development, signaling protein activity, membrane and regulation of gene expression. KEGG analysis revealed that the differentially expressed genes involved 263 signaling pathways; The main metabolic pathways were: PI3K-Akt signaling pathway, MAPK signaling pathway, carbohydrate metabolism, myocardial system and cellular reproductive system and etc. Conclusion: The results showed that 1 695 differential genes were screened and identified by Illumina Hi-Seq sequencing technology, and the relationship between the genes of ebracteolatain A and MCF7 cells was further understood, which provided some theoretical cornerstones for breast cancer treatment.
ABSTRACT
Objective Breast cancer is one of the deadliest malignancies in the world. ebracteolatain A (EA) is a kind of acetylphloroglucinol extracted from ebracteolatain. To explore the specific mechanism of EA inhibiting the proliferation of breast cancer cell MCF-7, so as to provide a new approach for the clinical treatment of breast cancer. Methods EA with different concentrations were added to breast cancer cell MCF-7 to detect changes in PKD1 protein expression. The plasmid with overexpressed PKD1 was constructed and transfected into cells, and the mRNA and protein expression levels of PKD1 were detected by real-time fluorescence quantitative PCR and Western Blot assay. CCK-8 assay was used to detect changes in cell proliferation capacity. Western Blot assay was used to detect the expression level of PKD1 and its related signaling pathways. Results EA inhibited the expression of PKD1 protein in breast cancer cells with a dose-dependent manner (P< 0.05). When transfected with the overexpressed plasmid, PKD1 was significantly increased in mRNA and protein levels (P<0.001). At the same time, PKD1 overexpression significantly reversed inhibition of EA on MCF-7 proliferation (P<0.001). It was confirmed by signaling pathway analysis that EA might affect the proliferation ability of breast cancer cells by inhibiting PKD1-mediated MEK/ERK and PI3K/AKT signaling activity (P<0.05). Conclusion EA could inhibit the proliferation of breast cancer cells by regulating PKD1-mediated MEK/ERK and PI3K/AKT signaling pathways.
ABSTRACT
Objective To isolate and purify ebracteolatain A from the roots of Euphorbia ebracteolata Hayata, and to explore its anti-breast cancer activity. Methods Ebracteolatain A was isolated and purified from the roots of Euphorbia ebracteolata Hayata using reflux extraction, solvent extraction and absorption chromatography techniques. The chemical structure was identified by mass spectrometry and nuclear magnetic resonance spectrometry. The antibreast cancer activity of ebracteolatain A was determined by MTT assay in the breast cancer cell lines including SUM149 (triple-negative), MCF-7 (luminal A), ZR-75-1 (luminal B) and SK-BR-3 (HER2-positive). The effect of ebracteolatain A on cell cycle was determined by flow cytometry. A tumor model was established in nude mice by transplanting SUM149 cells, and the inhibitory effect of ebracteolatain A on breast cancer was evaluated. Results The ebracteolatain A was 3, 3’-diacetyl-2, 4’-dimethoxy-2’, 4, 6, 6’-tetrahydroxy-5’-methyl diphenylmethane. The half maximal inhibitory concentrations of ebracteolatain A on SUM149, MCF-7, ZR-75-1 and SK-BR-3 cells were 5.50, 6.16, 7.08 and 8.64 μmol/L, respectively. With the increase of drug concentration (2.5, 5, 10 μmol/L), the percentage of the cells at G0/G1 phase was decreased (P<0.05, P<0.01) and the percentage of the cells at S phase was increased (P<0.05, P<0.01) after treatment with ebracteolatain A for 12, 24 and 48 h. After intraperitoneal injection of ebracteolatain A 35 mg/kg, the inhibition rates of the tumor volume and mass in nude mice were 37.94% and 41.38%, respectively. Conclusion Ebracteolatain A can inhibit the proliferation of the four types of breast cancer cells and the growth of transplanted-SUM149 cell tumor on nude mice, which may be related to suppressing the transition of cell cycle from S phase to G2/M phase.
ABSTRACT
Objective To establish a method for determination of ebracteolatain A content in the root of traditional Chinese medicine white Langdu, including Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata. Methods An HPLC-DAD method was created for determination at the following condition:the column was Agilent Zorbax SB-C18 (4.6 mm×150 mm, 5 μm);mobile phase was acetonitrile:0.1% formic acid in water=55:45(V:V), isocratic elution, the flow rate was 1.0 mL·min-1, the temperature of column was 25°C, the detection wavelength was set at 290 nm, the injection volume was 20 μL, and the running time was 20 min. Results Ebracteolatain A was separated from the interference in the baseline, and the linear range was 4.545-227.3 μg·mL-1, with the linear correlation being 0.9999 for ebracteolatain A. The result of intra-day and inter-day precisions were both within 2%(n=3), and the average recovery was (99.14±3.4)%(n=6). The content of ebracteolatain A in two batches of Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata were (56.73±1.09) μg/g, (18.98±2.11) μg/g and (235.2±2.4) μg/g (n=6), respectively. Conclusion The present method is simple, rapid, accurate and convenient for determination of ebracteolatain A in the root of traditional Chinese medicine white Langdu.