Study On The Anti-Tumor Mechanism Of B-Norcholesteryl Benzimidazole Compounds

Steroidal compounds exist widely in nature.The introduction of heteroatoms or heterocyclic ring into the steroid nucleus,using heteroatom to substitute one or more atoms usually leads to the change of its biological characteristics.B-norcholesteryl benzimidazole compounds have a good inhibitory effect on tumor cell proliferation in vitro in our previous studies.In order to further study the mechanism of antitumor activity,three parts were designed in this paper: Firstly,four B-norcholesteryl benzimidazole compounds were used to determine the effects of proliferation activity and cell migration ability of tumor cells in vitro by MTT assay and cell scratch assay.The mechanism of apoptosis induced by compounds were studied by using Annexin V-FITC/PI,cell cycle and mitochondrial membrane potential flow cytometry method.;Secondly,the apoptosis-related protein expression were analysis by western blot;Thirdly,the anti-tumor mechanism target of B-norcholesteryl benzimidazole compounds were explored by UHPLC-QE-MS non-target cell metabolism.These data provided a theoretical basis for drug development of such compounds.The details are as follows:1.The antiproliferative activity of B-norcholesteryl benzimidazole compounds were evaluated by human ovarian cancer cells(SKOV 3),human cervical cancer cells(HeLa),human breast ductal cancer cells(T47D)and human breast cancer cells(MCF-7).The experimental results showed that B-norcholesteryl benzimidazole compounds can effectively inhibit the proliferation of 4 kinds of human gonadal tumor cells,of which SKOV 3 cells have good migration inhibition effect.In addition,B-norcholesteryl benzimidazole compounds can also induce apoptosis of SKOV 3 cells,block its growth S phase,and induce mitochondrial membrane potential collapse.2.The expression levels of related apoptotic factors including PARP,caspase-9,caspase-3,Bax,Bcl-2 and p21 were detected by western blot metheod.The experimental results showed that B-norcholesteryl benzimidazole compounds induced apoptosis of SKOV 3 cells by activating the mitochondrial pathway signaling pathway.3.UHPLC-QE-MS non-target metabolomics analysis method combining positive and negative ion modes,SKOV 3 cell metabolism after drug treatment of B-norcholesteryl benzimidazole compounds 1 and 2 linked with different substituent groups the differences in intracellular metabolites and their metabolic pathways in the negative control group were compared.The results showed that:(1)Compound 1 mainly involved 9 metabolic pathways: Arginine and proline metabolism,Alanine,aspartate and glutamate metabolism,Histidine metabolism,D-Glutamine and D-glutamate metabolism,Cysteine and methionine metabolism,Aminoacyl-tRNA biosynthesis,Purine metabolism,Glutathione metabolism,D-Arginine and D-ornithine metabolism and Nitrogen metabolism.From the perspective of metabolomics,compound 1 inhibited the metabolism of SKOV 3 cells,protein synthesis,and slowed down energy metabolism,thereby inhibiting the proliferation and signal transduction of tumor cells,inhibiting invasion and metastasis,and inducing apoptosis to exert anti-tumor effects.(2)After the compound 2 acts on SKOV 3 cells,the altered metabolic pathway mainly involves Glycerophospholipid metabolism,arginine and proline metabolism,purine metabolism,glycine,serine and Glycine,serine and threonine metabolism,Ether lipid metabolism,five metabolic pathways.The metabolic pathway with the greatest impact and the deepest enrichment is the metabolism of glycerophospholipids.Glycerate plays a major role in cell membrane function and structure,and thus it is known that compound 2 inhibits proliferation mainly by interfering with the cell membrane structure or function of SKOV 3 cells.In addition,it also involves the metabolism of arginine and proline,so compound 2 can also inhibit the invasion and metastasis of SKOV 3 cells and induce apoptosis.