Preliminary Study On Anti-tumor Activity And Mechanism Of Eupenifeldin And Its Analogue C1057

Cancer is growing to be the leading cause of human death.Effective drugs have been urgently needed all over the world.Due to the diversity in structure and biological activity,natural products have been the important resource for discovering novel anticancer drug.Tropolone,the secondary metabolites derived mainly from fungi,is characterized by anti-bacterial,anti-fungal,anti-viral,anti-inflammatory,insecticidal and anti-tumor bio-activities,et al.The therapeutic potential of tropolone in tumors has become a focus in recent years.However,till now,no breakthrough had been made in this area and the mechanism of action is still unclear due to the complexity of structures and availability of these natural products.Eupenifeldin is a derivative of tropolone that was first discovered from the Eupenicillium brefeldianum ATCC74184 in 1993.Then it was isolated from Phoma sp.XZ068 with high yield and purity and numbered with C1055 by Professor Che Yongsheng.Previous studies indicated that eupenifeldin has apparent inhibition on multiple tumor cells,and prolonged the survival time in P388 leukemia mice.However,as a representative molecule of tropolone,its anti-tumor efficacy and mechanism still needs systematic evaluation and investigation.This study was to validate and evaluate the anti-tumor activity of eupenifeldin?C1055?and C1057?C1055 derivative?at in-vitro level,and to screen and study their mechanism of action.The paper includes the following four parts.1.The anti-tumor activity of C1055 and C1057 was evaluated by MTT,SRB and tumor cell colony test.The results showed that these two compounds had sound inhibition on the viability and proliferation of tumor cells.The IC₅₀ value of C1055 on BxPC3,H4,U251,HepG2,SKOV3,PANC-1,SH-SY5Y and HeLa cells in SRB tests was:?6.95±2.19?,?8.40±3.59?,?8.93±0.95?,?10.33±0.60?,?10.97±0.44?,?24.60±10.10?,?29.71±2.70?and?40.30±7.53?nM,respectively,and the IC₅₀0 of C1057 was?0.56±0.14?,?0.73±0.09?,?0.63±0.12?,?0.57±0.05?,?0.65±0.03?,?0.48±0.13?,?1.14±0.038?,?2.07±0.62??M,respectively.Additionally,C1055?1,3,10nM?and C1057?10,30,100nM?significantly inhibited the colony formation of BxPC3 and H4 cells.2.The mechanisms of action of C1055 and C1057 were systematically screened based on the dynamic changes of 14 parameters of cytotoxicity,16 reporter cells with stable expression of EGFP-tumor related signaling pathway marker proteins and 5 reporter cells with stable expression EGFP-cell stress response pathway marker proteins using high content analysis?HCA?based on cell fluorescence imaging.The results showed 24-h treatment with C1055 concentration-dependently?1-100nM?reduced the number of cells and the content of GSH.ROS,MnSOD and nuclear membrane permeability?NMP?were significantly increased by 30-100nM.Differently,the change of p H2AX?DNA damage?,LC3B?autophagy marker?,lysosome number,and mitochondrial membrane potential?MMP?were detected only at the maximum concentration?100nM?,which was accompanied by 30%cell death.In addition,C1055?30nM?and C1057?0.3?M?time-dependently?6-24h?decreased GSH content in HepG2 cells,yet with fewer cell loss.C1055?300nM?and C1057?3?M?induced formation of Rad 51 foci?85%and 48%,respectively?in Rad51-EGFP-SW480 cells,which was accompanied by 30%cell death;and inhibited EGFR redistribution?22%and 66%,respectively?in EGFR-EGFP-U2OS cells.3.In BxPC3 and H4 cells,C1055 and C1057 lowered GSH content in concentration-and time-dependent manner.On this basis,the gene expression of the enzymes related to GSH synthesis and catabolism,glutamate-cysteine ligase?GCL?including the catalytic subunit GCLC and the regulatory subunit GCLM,glutathione synthetase?GSS?,glutathione reductase?GR?and glutathione S-transferase?GST?were assayed.Then we assayed GST activity after 1,3,6,9,12 h treatment with C1055 and C1057.The results showed that C1055 and C1057 concentration-dependently up-regulated mRNA of the enzymes related to GSH synthesis and catabolism and enhanced GST activity.These results suggested that C1055 and C1057 reduced intracellular GSH content most probably by increasing the activity of GST,since neither of these two compounds could induce the nuclear translocation of Nrf2.4.The C1055 and C1057 induced cell death manner was screened by detecting the cell lactate dehydrogenase?LDH?release,and combined with specific inhibitors on cell death signal pathways.The results showed that Z-VAD-FMK,3-MA,necrostatin-1and ferrostatin-1 reversed C1055 or C1057 induced LDH release in BxPC3 cells;Z-VAD-FMK reversed both of C1055-and C1057-induced LDH release,whereas ferrostatin-1only rescued C1055-induced LDH release in H4 cells.Apoptosis Flow Cytometry and LC3B and LPO?lipid peroxidation?HCA further revealed that these two compounds induced cell apoptosis,autophagy and ferroptosis in Bx PC3 cells,and mainly induced cell apoptosis in H4 cells,meanwhile,C1055 also induced ferroptosis in H4.Based on the above results,we can conclude that:1.C1055 and its derivative C1057 have definite antitumor effect at in-vitro.The IC₅₀value of SRB testing is 8-29nM and 0.5-2?M for C1055 and C1057 respectively.The IC₅₀ values of both compounds are no more than 10 times,which tells a broad spectrum of inhibitory effects on tumor cells.2.C1055 and C1057 could decrease the GSH content of tumor cells in a time-and concentration-dependent manner.Promoted GST activity may be associated with the GSH depletion of C1055 and C1057.3.The cell death manner induced by C1055 and C1057 depends on the cell type.Both C1055 and C1057 can induce apoptosis and autophagy in BxPC3 cells,while,they only induce apoptosis in H4 cells.C1055 show more apparent effect on ferroptosis in BxPC3and H4 cells.4.Given that GSH depletion has been a strategy for cancer therapy,the comprehensive anti-tumor effect of C1055 and C1057 deserve further study.