Apoptotic Activity Of Harmine And Its Derivative ZC-14 Against Spodoptera Frugiperda Cultured Sf9 Cell Line

Harmine extracted from the roots of Peganum harmala L.belongs to one of the beta-carboline alkaloids.It's reported that harmine has significant pharmacological activities,including anti-tumor activity,anti-depressive activity,anti-bacterial and insecticidal activity.However the spectrum of insecticidal effects is narrow.Sf9 cells are ovary cells of Spodoptera frugiperda,which are ideal materials for cytotoxicity study.In this paper,base on the interaction between harmine and DNA topoisomerase I analyzed by molecular docking technology,the derivative ZC-14 was directional structure modified and synthesized.In addition,the cytotoxicity of ZC-14 was evaluated in Sf9 cells.It was confirmed that the the apoptosis induced by derivative ZC-14 could be the mechanism of proliferation inhibition.Further studies showed that mitochondria participate in ZC-14-induced apoptosis of Sf9 cells.The research results were summarized as follows.1.According to the result of molecular docking,it was demonstrated that harmine could interacted with DNA topoisomerase I.Furthermore,the derivative ZC-14 was synthesized through skeleton growth design,and the interaction of ZC-14 and topoisomerase I was stronger than harmine.2.Enzyme activity assay in vitro showed that harmine and derivative ZC-14 could inhibit DNA topoisomerase I,and the inhibitory effect of ZC-14 was stronger than harmine.3.The cytotoxicity of harmine and ZC-14 was determined by MTT assay.The results showed that the IC50 values of ZC-14 at 24 and 48 h were 17.33 ?g/m L and 8.54 ?g/m L,respectively;the IC50 values of harmine were 59.32 ?g/m L and 50.59 ?g/m L.4.The mechanism of cell proliferation inhibition showed that ZC-14 inhibited cell proliferation by inducing Sf9 cells apoptosis.The main research results were summarized as follows.Apoptotic bodies appeared under morphological observation with 7.5 ?g/m L of ZC-14 treatment.Nuclear condensation was detected by Hoechst staining.DNA ladder was detected by Agarose gel electrophoresis.PI staining results showed that cell cycle arrest inG2/M phase.FITC/PI double staining showed that apoptosis rate was different with different treatment times,and the apoptosis rate of Sf9 cells was 32.1% after treatment for 24 h.Caspase activity assay showed that caspase-3 activity was significantly up-regulated.At the same time,q RT-PCR confirmed that the m RNA expression level of caspase-1 increased significantly.Furthermore,the protein expression of Cleaved-Caspase-3 protein expression was significantly upregulated.5.Mitochondria involved in ZC-14-induced Sf9 cells apoptosis.Mainly research results were showed as follows.Rhodamine 123 staining showed that the green fluorescence decreased with the extension of treatment time,indicating the decrease of mitochondrial membrane potential.Western Blot results confirmed that cytochrome c released from mitochondria to cytoplasm in Sf9 cells with ZC-14 treatment.This paper discussed the target and the function of apoptosis induction of ZC-14,and proposed further research content in the future.The results of this paper provided important references for developing new pesticides with harmine as the lead compound.