Anti - Tumor Activity And Mechanism Of New Compounds In

Nowadays,cancer is one of the major problems.tumor incidence The incidence of cancer deaths is increasing, and more effective drugs are still urgent needs for its treatment.Microtubule inhibitor is a class of anticancer drugs which applied to destroy the homeostasis of tubulin, thereby preventing tumor cell proliferation,and has been used as key components of chemotherapeutic regiments for various solid tumors.Microtubule-binding agents target specific molecules, binding to tubulin,promote or inhibit the normal structure and function of microtubule assembly, interfere with the normal structure and function of the mitotic spindle.Besides interfering with both mitosis, cell proliferation and tumor therapeutic effect, the microtubule inhibitor has a potent inhibition of antiangiogenic and vascular-disrupting effects. WX-123-27, WX-127-07, WX-132-18B were three novel molecules which were screened by our institution and had a strong anti-tumor activity indicate that These compounds may act on tubulin in line with the data of preliminary experiments.This research project is built on high content analysis (HCA) antitumor technology platform. The platform is built for the study of pharmacodynamic effects and mechanisms. HCA has systematic and efficient features which can detect the antitumor activity and molecular mechanism of the three new compounds.This will help to develop a comprehensive understanding of pharmacological effect of these active molecules,which will lay the foundation for further development.In the study, the well known microtubule-targeting drugs were used as main positive drugs. The anti-proliferation activity was observed by SRB assay. The cells cycle arrest was examined by flow cytometry. The multiplexed changes and different pathways about tumor, inflammation etc. were detected and analyzed on IN Cell Analyzer1000HCS platform.These tumor-associated signaling pathways includ2GPCRs,6nuclear receptors,2growth factor receptors and kinases,7pathways about DNA damage and cell cycle,6pathways about stress and inflammatory.8pathways unrelated to tumor-associated signaling pathways were also been analyzed in the experiment. Tryptic digestion assay and competition inhibition of colchicine or FL-vinblastine were used to confirm the binding site on microtubule in vitro.RESULTS All the tested componds could obviously inhibit the growth of A549, HepG2, HeLa, HLF and HUVEC cell, the IC50values of WX127-07were4.47±0.05nM,5.18±0.08nM,4.90±0.19nM,4.10±0.16nM,5.04±0.08nM; the IC50values of WX123-27were6.83±0.15nM,6.72±0.19nM,6.78±0.31nM,5.5±0.11nM,5.37±0.20nM; the IC50values of WX132-18B were1.03±0.02nM,1.01±0.01nM,1.03±0.06nM,0.86±0.02nM,0.76±0.04nM.Their anti-proliferation activity were stronger than colchicine,the IC50values were21.17±1.22nM, 14.19±0.53nM,43.80±1.64nM,145.89±10.97nM,27.67±1.79nM respectively,and also stronger than vincristine, the IC50values were16.51±0.36nM,16.76±0.33nM,27.80±2.75nM,43.80±1.48nM,9.15±0.78nM respectively.and stronger or similar to taxol, the IC50values were10.68±0.61nM,12.86±0.25nM,4.81±0.61nM,102.07±15.17nM,3.04±0.12nM respectively.High content multi-parameter analysis revealed that the tested compounds induced a dose dependent microtube depolymerization with the same pattern as colchicine and vincristine, but with superior activity.The EC50values of taxol,vincristine,colchicine, WX-123-27, WX-127-07and WX-132-18B were75.84±6.73nM,293.2nM,105.5nM,25.91nM,17.31nM,9.43nM respectively. All these microtubule targeting agents can induce the cell cycle arrest in A549cells.The distribution of Go/G1, S, G2/M phase of cells in the normal control group were71.74%,21.95%,6.31%.After treated wiht taxol, vincristine, colchicine, WX-123-27, WX-127-07and WX-132-18B,the cells distribution of G0/G1was significantly reduced to18.17%,11.63%,19.15%,7.41%,5.62%,3.74%respectively, while the G2/M phase was significantly increased to62.90%,71.33%,63.61%,80.35%,72.06%respectively.The same as positive compounds,the tested compounds increased nuclear membrane permeability and early signs of apoptosis in HepG2cells. In the tests,both cancer related pathway and inflammatory related pathway were not affected by the tested drugs, either.We just found that the area of Rad51increased slightly,but the increasing of was just about a quarter of the area induced by camptothecin.Microtubule competition inhibition assay showed that the tested compounds inhibited the binding of colchicine with tubulin, the IC50values of WX-123-27, WX-127-07, WX-132-18B were1.75±0.54μM,1.28±0.08μM,0.47±0.10μM. There were also some non-specific inhibition of the vincristine binding site under high concentration. Tryptic digestion of tubulin-compound premixture shows similar electrophoretic band with that of tubulin-colchicine premixture.To be conclusive, we found that our agents WX-123-27, WX-127-07and WX-132-18B show prominent anti-proliferative activity for both tumor cell and vascular endothelial cell in vitro. Mechanism evaluation revealed that they exert antitumor activity by disturbing microtubule normal function through interaction with the generally acknowledged colchicine binding site in microtubule, and they possess characters shared by other microtubule-targeted agents. All these characters suggest their further development as new-generation microtubule depolymerizing agent in antitumor drug development.