Unraveling The Mechanism That Two Small Molecules Inhibit Cell Proliferation Via Targeting Microtubule

Cell division is perhaps the most fundamental progression of cell proliferation and essential to human growth and development.Mitosis is the most dramatic progression of the cell division,involving a major reorganization of virtually all cell components.The goal of mitosis is to equally distribute the genetic material into the two daughter cells.Erroneous mitosis or more generally mutations in genes encoding components of the mitotic machinery are linked to further pathologies,such as cancer,malformation of the central nervous system,aging and infertility.Striking rearrangement of microtubule is responsible for faithful chromosome segregation between daughter cells during mitosis.For this reason,most microtubule-targeting agents have been developed with different aims,including as antifungals,antiparasitics,antineoplastic and powerful probes that dissect the temporal and spatial mechanisms of mototic processes:Although treatments for these mitosis-related diseases have resulted in improved long-term outcomes,the majority of patients are suffering from pain now.Therefore,there is an urgent demand for the development of candidates with improved treatment outcomes.Besides,despite a vast knowledge about the molecular processes governing mitosis,the exact molecular mechanism of mitosis and physiological contribution of the mitotic machinery in pathogenesis are still under investigation,in some cases,controversial.Hence,there is an urgent need to develop more tools and methods to investigate complicated mitotic progression.This paper is divided into four chapter.In the first chapter,we presented the overview of recent progress in the regulation of mitosis and microtubule,as well as the development of microtubule-targeting agents.In the second chapter,we provided the detail information of experimental material and method.In the third chapter,we did an in depth exploration into the mechanism that a small molecule CS1 inhited HeLa cell proliferation and made a try to probe mitotic progression using CS1.Due to the short duration and dynamicity of mitosis,it is a great challenge to dissect temporal and spatial mechanisms in this processes.Small molecules achieve greater prominence for fast,temporal and reversible regulation of mitotic progression that take place in second to minute time scales.An ocean of antimitotic agents have been developed to dissect the mechanism of the drastic reorganization in cellular architecture during mitosis.Despite that great success of these agents,our understanding of mitotic progress is still limiting.Therefore,there is an urgent demand for more antimitotic compounds to control over complicated mitotic progression.In this study,we characterized the small molecule CS1 as a special antimitotic agent.CS1 potently inhibited microtubule polymerization via interaction with the colchicine-binding pocket of tubulin in vitro and inhibited the formation of the spindle apparatus by reducing the bulk of growing microtubules in HeLa cells,which led to activation of the spindle assembly checkpoint(SAC)and mitotic arrest of HeLa cells.Compared with colchicine,CS1 impaired the progression of sister chromatid resolution independent of cohesin dissociation,and this was reversed by the removal of CS1.Additionally,CS1 induced unique histone phosphorylation patterns distinct from those induced by colchicine.In conclusions,CS1 is a unique antimitotic small molecule and a powerful tool with complementary value to colchicine that makes it possible to specifically and conditionally perturb mitotic progression.In the fourth chapter,we focused on the effects of the small molecule PMN on CRC cells.Colorectal cancer(CRC)is the third most prevalent malignancy worldwide,leading to 1,361,000 new cases and over 690,000 deaths in 2012.Despite extraordinary progress in the treatment of CRC,new understandings about this disease are urgently required to guide clinical therapies.In this study,we investigated the anti-mitotic mechanism of a small molecule PMN and its application in colorectal cancer treatment.PMN dose-dependently inhibited cell proliferation through inducing mitotic arrest and apoptosis in three CRC cell lines:RKO,HCT116 and SW480.PMN induced mitotic arrest via the disruption of spindle apparatus by inhibiting microtubule polymerization.PMN-induced mitotic arrest led to apoptosis and p53 upregulation.Furthermore,PMN sensitized p53-positive cells to apoptosis,as evidenced by the fact that p53 deficiency suppressed apoptosis in PMN-treated cells.In conclusion,PMN exerted antitumor activity through inducing mitotic arrest and p53-dependent apoptosis via the inhibition of microtubule polymerization.This study deepens the understanding of the effects of p53 on the responses of CRC cells to antimitotic agents,providing the basis for the potential development of lead compounds targeting microtubule for p53-positive CRC treatment.In summary,our results delineate that CS1 and PMN inhibit mitosis via the inhibition of microtubule polymerization,providing a basis for the development of CS1 and PMN as antineoplastic lead compounds and a pharmacophore for rational design of novel microtubule destabilizing agents.CS1 inhibts sister chromatid resolution and induces unique phosphorylation of histones,opening opportunities for the development of CS1 for probing mitotic progression.