Anticancer Activity Of Emodin Derivatives Of Quaternary Ammonium Salt In Vitro And Their Apoptosis Induction Effect Study

Objective:By investigating the anticancer activity of emodin derivatives of quaternary ammonium salt in vitro, high efficiency and low toxicity drugs were selected. And their anticancer mechanism was studied, aiming to provide theory reference for developing new drugs.Methods:Human malignant melanoma cell A375, human gastric cancer cell BGC-823, human hepatoma cell HepG2 and human embryonic lung fibroblast cell HELF were treated by series of emodin of quaternary ammonium salt derivatives for 24 h, and their cell toxicity was evaluated by MTT assay. High efficiency and low toxicity drugs were selected for intensive research by structure-activity relationship analysis. Drug-induced cell morphological changes were assessed by light microscope and fluorescence microscope after AO/EB and DAPI staining. After mitochondria fluorescent probe staining, the subcellular localization of drugs in cell was analysed by LSCM. FCM analysis after DiOC6 staining was used to research the drug effect on cell mitochondrial membrane potentia(△ψ m). ROS level in cell was tested by reactive oxygen detection kit. At last, Western blot technology was to investigate the impact on the expression of Bcl-2 after cell treated with drugs,Results:MTT data demonstrated the anticancer activity of all derivatives was improved in different levels, when compared to emodin. Anticancer activity of different compounds that 1,3,8-positions replaced with methyl or ethyl, carbon chain connected in 3-position or in N atom of quaternary ammonium salt and 3-position substituted by short chain or long chain was similar. But the place of substituent group in 1,8-position had effect on anticancer activity. The length and amount of carbon chain connected on quaternary ammonium salt impacted anticancer effect great, and carbon chain of moderate length compounds were best. IC50 to cancer cell of compounds 11c and 13a was less than 5 μM while to normal cell more than 10 μM. Meanwhile, the cell sensibility to emodin derivatives of quaternary ammonium salt was different and the order was A375>BGC-823>HepG2>HELF. Light microscope observation and AO/EB and DAPI staining methods proved compound 11c and 13a induced A375 cell apoptosis morphological changes, such as incomplete cell morphological and chromatin condensation. Some of drugs located in mitochondria observed by LSCM. FCM analysis indicated mitochondrial membrane potential(ATm) was down and ROS level was up, and both compounds down-regulated the expression of Bcl-2.Conclusion:Emodin derivatives of quaternary ammonium salt exhibited good anticancer activity. The possible anticancer mechanism to A375 of Compound 11c and 13a can be concluded as down-regulating the expression of Bcl-2, drugs accumulated in mitochondria, improving ROS level, as well as declining mitochondrial membrane potential(△ψm), and finally inducing apoptosis and cell death.