Preliminary Study On Anti-tumor Activity And Mechanism Of Brasilamide E Derivative C1050

Natural products(NPs)are characterized by rich chemical structure and variety biological activities,and are vital treasures for modern drug development.However,NPs-based drug discovery is facing with numerous challenges.In addition to the challenge of finding out diverse monomeric structures,the lack of proper high-throughput screening(HTS)methods and limited understanding the molecular mechanisms of NPs and their targets had severely slowed down the discovery and clinical transformation of NP drugs.In recent years,the rapid development of various omics and bioinformatics technologies,as well as the increasing perfection of drug screening technology represented by high content analysis,have greatly accelerated the recognition speed of NPs' characteristics,and provided a new opportunity for efficient drug discovery of NPs.Compound C1050 is a novel structural compound modified by Professor Che Yongsheng according to the chemical structure of sesquiterpenoid(XY172)found in the fermentation product of the endophytic fungus Paraconiothyrium brasiliense.Preliminary cellular experiments had found that this compound not only retains the unique antitumor cell selectivity of XY172,but also presents stronger antitumor activity.In order to further evaluate the anti-tumor activity and selectivity of compound C1050,and provide a basis for further research and development,the anti-tumor activities of compounds C1050 and XY172 were first assayed by MTT and CCK8 on 24 solid tumor cell lines(including two sources of HeLa and HepG2 cells,three sources of MCF7 cells and HO8910),2 blood tumors cells(K562 and HL-60),as well as normal cells HUVEC and NIH3T3.Furthermore,the SRB method was used to confirm the anti-tumor activity of the compounds on positive cells and 7 other related cells,including the self-preserved HeLa,HeLa-ATCC,self-preserved HepG2,HepG2-ATCC,C33 A,BEL7402,and PLC/PRF/5.The anti-tumor activity of C1050 was further evaluated by tumor cell colony formation and 3D tumor ball assay.Subsequently,we systematically screened and studied the anti-tumor mechanism of compound C1050 at the levels of the cell phenotype profile and the molecular mechanism through high-connotation analysis(HCA),flow cytometry(FCM),transcriptome sequencing,qRT-PCR and Western blot.According to these studies,the results are presented as follows:1.Compound C1050 inhibited the activity and proliferation of four tumor cells,including HeLa-ATCC,HepG2 preserved in the lab,HO8910 and MCF7 preserved in the lab 1,with IC50 values of 0.64,0.93,3.26 and 4.33 ?M respectively.The inhibitory activities on the other 22 tumor cells(including the same background different source solid tumors and blood tumor cells)were less than 40% at 50 ?M(less than 20% on 7 strains of cells),and less than 10% on normal cells were.C1050 had an anti-tumor activity that was 7-15 times stronger than that of its lead XY172,but remained similar selectivity on tumor cell types.2.Compound C1050 inhibited the colony formation of HeLa-ATCC and HepG2-preserved in the lab cells in a concentration-dependent manner and inhibited the growth of HeLa-ATCC tumor cell spheres.3.Compound C1050 concentration-dependently induced apoptosis in Caspase-dependent HeLa-ATCC cells.C1050 induced tumor cell death,which was not affected by the programmed necrosis inhibitor Necrostatin-1 or the cell autophagic blocker Hydroxychloroquine.4.Cytotoxic phenotype,cytoskeleton phenotype and cell cycle analysis showed that compound C1050 increased nuclear membrane permeability(NMP)of HeLa-ATCC cell in a concentration-dependent(0.1-30 ?M)manner,and decreased cell survival,altered nuclear morphology,decreased mitochondrial membrane potential(MMP)at 0.3-30 ?M,leading to the S-phase arrest at 1-3 ?M.Meanwhile,it led to the increased content of microfilaments and microtubules,the tension microfilament thickening,the growth of cell volume and the microtubule aggregation round the nuclear at 3-30 ?M.Additionally,the screening of 14 tumor-associated signaling pathways and five cellular stress response pathways showed that C1050 only inhibited EGFR activation at 3 ?M,with a maximum inhibition rate of approximately 50%,but had no effect on other pathways.5.The GO analysis of transcriptional sequencing showed that the unfolded protein reaction and the endoplasmic reticulum stress-related genes were significantly up-regulated in a time-dependent manner at 6-12 hours after treatment with C1050.Ribosomal function genes were significantly up-regulated,and mitosis and microtubule cytoskeleton-related genes were significantly down-regulated at 24 hours.The results on HepG2-preserved in the lab were similar to those of HeLa-ATCC cells.qRT-PCR validation experiments confirmed that the expression levels of endoplasmic reticulum stress-related genes DDIT3,CTH,ATF3,FGF21,and CHAC1 were elevated.6.Western blot analysis showed that C1050 increased the expression of p-PERK and CHOP protein in a time-dependent manner(0.5,1,2,6,12 h),while the expression levels of IRE1 alpha,p-IRE1 alpha,PERK and ATF4 protein increased initially and then decreased with time,indicating that C1050 activated endoplasmic reticulum stress-mediated apoptosis-related pathways.Thus,according to the above results,we concluded that the novel structural compound C1050 selectivity inhibits HeLa tumor at the in-vitro level.The anti-tumor effect of C1050 may be achieved by causing endoplasmic reticulum stress through inducing activation of the p-PERK-CHOP pathway,and inducing caspase-dependent apoptosis.An increase in NMP and a decrease in MMP may also be involved in C1050-induced tumor cell apoptosis.This study is expected to contribute to the further development of compound C1050.