ABSTRACT
BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with Epstein-Barr virus (EBV) infection. Chemoradiotherapy is the mainstream treatment for locally advanced NPC, and chemotherapeutic drugs are an indispensable part of NPC treatment. However, the toxic side-effects of chemotherapy drugs limit their therapeutic value, and new chemotherapy drugs are urgently needed for NPC. Silvestrol, an emerging natural plant anticancer molecule, has shown promising antitumor activity in breast cancer, melanoma, liver cancer, and other tumor types by promoting apoptosis in cancer cells to a greater extent than in normal cells. However, the effects of silvestrol on NPC and its possible molecular mechanisms have yet to be fully explored. METHODS: Cell counting kit-8 (CCK-8), cell scratch, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), and Western blot (WB) assays were used to evaluate the effects of silvestrol on the cell viability, cell cycle, apoptosis, and migration of NPC cells. RNA sequencing (RNA-Seq) was used to study the effect of extracellular signal-regulated kinase (ERK) inhibitors on the cell transcriptome, and immunohistochemistry (IHC) to assess protein expression levels in patient specimens. RESULTS: Silvestrol inhibited cell migration and DNA replication of NPC cells, while promoting the expression of cleaved caspase-3, apoptosis, and cell cycle arrest. Furthermore, silvestrol altered the level of ERK phosphorylation. The ERK-targeted inhibitor LY3214996 attenuated silvestrol-mediated inhibition of NPC cell proliferation but not migration. Analysis of RNA-Seq data and WB were used to identify and validate the downstream regulatory targets of silvestrol. Expression of GADD45A, RAP1A, and hexokinase-II (HK2) proteins was inhibited by silvestrol and LY3214996. Finally, IHC revealed that GADD45A, RAP1A, and HK2 protein expression was more abundant in cancer tissues than in non-tumor tissues. CONCLUSIONS: Silvestrol inhibits the proliferation of NPC cells by targeting ERK phosphorylation. However, the inhibition of NPC cell migration by silvestrol was independent of the Raf-MEK-ERK pathway. RAP1A, HK2, and GADD45A may be potential targets for the action of silvestrol.
Subject(s)
Benzofurans , GADD45 Proteins , Hexokinase , MAP Kinase Signaling System , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms , rap1 GTP-Binding Proteins , Humans , Apoptosis/drug effects , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , MAP Kinase Signaling System/drug effects , Nasopharyngeal Carcinoma/drug therapy , Nasopharyngeal Carcinoma/genetics , Nasopharyngeal Carcinoma/pathology , Nasopharyngeal Neoplasms/drug therapy , Nasopharyngeal Neoplasms/genetics , Nasopharyngeal Neoplasms/pathology , Hexokinase/genetics , Hexokinase/metabolism , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolism , GADD45 Proteins/genetics , GADD45 Proteins/metabolismABSTRACT
OBJECTIVE: To establish a convenient, practical and environmental method for extracting and isolating shikimic acid. METHODS: The content of shikimic acid was measured by RP-ion-pair HPLC, the effects of different pH, temperature, sample concentration and eluate concentration on 717 anion exchange resin were studied. RESULTS: The yield of shikimic acid was 96.52% on the condition of 22 degrees C, pH > 6.5, 7.5 mg /ml sample concentration and 0.03 mol/L HCl eluting. CONCLUSION: This method is feasible and suitable for the extraction and isolation of shikimic acid.