Study On Antitumor Mechanism Of Novel Microtubule Inhibitor A3

Background:Tumor is a common and multiple disease that still threatens human life and health today.There are many factors that cause tumors,such as physical,chemical,and biological carcinogenic factors in external environmental factors,intrinsic autoimmune functions and genetic factors.The reason why tumors are difficult to overcome is mainly due to the occurrence of tumors,which are a collection of related diseases induced by the abnormal growth of tumor cells and invading other normal parts of the body.Microtubules play an important role in cell mitosis.This important role makes microtubules an important target for antitumor therapy,and tubulin is an essential basic unit that constitutes a microtubule system.If tubulin is destroyed,the microtubule system loses its stability,and ultimately the effect of cell mitosis is self-evident.Based on the important behavior of tubulin in cells,drugs that interact with tubulin have also been increasingly developed and studied,and some have been clinically approved for the treatment of tumors.Objective:This experiment evaluated the activity of novel microtubule inhibitor A3 against human cervical cancer(HeLa)cells and studied its anti-tumor mechanism.Methods:The effect of paclitaxel,CA-4 and compound A3 on inhibition of tubulin polymerization was examined using a tubulin polymerization screening kit.The effect of different concentrations of A3 on tubulin in HeLa cells by Immunofluorescence.The effect of A3 on the growth and proliferation of HeLa cells was detected by MTT assay.The effect of A3 on the morphology and intracellular tubulin of HeLa cells was observed by inverted fluorescence microscope.The effect of A3 on mitochondrial membrane potential and reactive oxygen species in HeLa cells was examined using a high-throughput live cell imaging system.The effect of A3 on the cycle(PI),apoptosis(FITC Annexin V/PI),mitochondrial membrane potential(JC-1)and reactive oxygen species(DHE)of HeLa cells were detected by flow cytometry.The effect of A3 on the migration of HeLa cells was detected by cell scratch and Transwell chamber assay.The effect of A3 on the changes of HeLa cells cycle-associated proteins(cyclinB1,cyclinD1),apoptosis-related proteins(Caspase-3,Caspase-8,Caspase-9,Bax,XIAP)and Cytochrome C was detected by Western blotting.HUVEC was used as a tubule formation assay to test the anti-angiogenic ability of A3.Results:1.The tubulin polymerization screening kit detected that A3 inhibited the polymerization of tubulin and was dose-dependent.2.Immunofluorescence assay revealed that A3 could affect the changes of the microtubule protein in HeLa cells,and make its distribution diffuse.3.The MTT assay showed that A3 had a significant inhibitory effect on the growth of HeLa cells in a dose-and time-dependent manner.4.Through the observation of cell morphology,A3 induced HeLa cell apoptosis.5.Flow cytometry showed that A3 could induce significant G2/M phase arrest and apoptosis of HeLa cells,and also caused a decrease in intracellular mitochondrial membrane potential and changes in reactive oxygen species.6.Cell scratches and Transwell experiments showed that A3 could inhibit the migration of HeLa and HUVEC cells.7.Western blotting showed that A3 induced the expression of apoptosis-associated proteins and apoptosis-related proteins in a time and dose-dependent manner.8.Tubule formation experiments showed that A3 was able to inhibit the formation of tubules in human umbilical vein endothelial cells(HUVECs)and demonstrated anti-angiogenic effects.Conclusions:The novel microtubule inhibitor A3 showed significant cytotoxicity to HeLa cells.It mainly uses tubulin as a target to block the polymerization of intracellular tubulin and cause cell cycle arrest.The mitochondrial apoptotic pathway induces the occurrence of apoptosis,at the same time,it inhibits the migration of tumor cells and their peripheral angiogenesis,and ultimately exerts an anti-tumor effect.