Investigation Of Deguelin For Regulation Of MCM3-CDC45Expression In Zebrafish Early Development And Related Cancer Research

Objective:Deguelin， an extraction of natural isoflavonoids from leguminous plants, has caught moreattention since it was reported its anti-tumor effect. Deguelin was found to inhibit a variety oftumor cell proliferation. A range of molecular targets, including cell proliferation signals(NF-kappa B, of IκBα, Bcl-2, Bcl-xl and survivin), the nucleophosmin and nucleoporinspathways(Nup88, Nup98, Nup214), as well as through the regulation of steroid receptorco-activator SRC-3and DNA Topoisomerase. Through the model organism zebrafish embryos,our group found that deguelin could significantly delay the embryonic development. Itsspecific mechanism in vivo of zebrafish embryo is not clear. In this study the effect ofapoptosis treated by deguelin on early developing embryos was detected by acridine orangestaining and Tunel assay. Immunohistochemical of phospho histon3detected the inhibition ofcell proliferation treated with deguelin. Microarray is quick and reliable technology toassess the biological effects of small molecules, and its biological processes, as well asmechanisms of activation and signaling pathways of developmental changes. Zebrafish cDNAmicroarray detected transcriptome expression changes before and after dosing. Relevant targetgenes was screened and filted out through the study of Gene Ontology (GO, Gene Ontology)enrichment and signaling pathways (pathway) Analysis. Real-time PCR and the whole mountin situ hybrization study tested and verified the target gene minichromosome maintenanceMCM3and CDC45Temporal and spatial expression change. Further study of growthinhibition of deguelin on MCF-7breast cancer cells and H129lung cancer cells wat detectedby MTT. Expression change of MCM3and CDC45mRNA was studied by real-time PCR toverify the anti-tumor effect of deguelin and provide a new theoretical basis for anti-tumormechanism of prediction deguelin.Results:(1) Deguelin delay embryonic development in the concentration range of0.3μM~0.8μM. It islow toxicity and inhibit zebrafish early embryonic development in a dose-dependent manner.0.6μM deguelin can make embryonic development stagnation, after the dosing period ofabout4~5hours. Tunel staining and the Phospho Histon3immunohistochemistryexperiments show that deguelin significantly inhibits cell proliferation and induce apoptosisin the embryonic head, spinal cord neurons, retinal precursor cell proliferation inhibition andapoptosis was particularly notable.(2) Zebrafish cDNA microarray analysis transcriptome changes before and afterdosing.Significantly changes in gene CDC45, MCM3, CDC16and fgfr4was validated by real-time PCR.(p <0.01). ccnd1, fgfr2, and fgfr3gene expression downregulated with thecontrol group was statistically changed (p <0.05).(3) Spatial and temporal expression of MCM3was stood by whole mount in situ hybrization.MCM3expressed in cell proliferation regional in the various periods of embryonicdevelopment. As a DNA replication licensing factor, MCM3can be used as markers directlyreflect the state of cell proliferation in the zebrafish body.(4) Expression change of MCM3in deguelin treated and un-treated embryos was tested by insitu hybrization. During12to24hpf (hours post fertilization) deguelin inhibited MCM3expression in zebrafish embryos. At24hpf deguelin significantly decrease the MCM3expression in the CNS(central nervous system), wing brain, and retina area and embryonic tailMCM3expression completely disappeared. In deguelin of0.6μM treated embryos pax2expression of brain, ear room, spinal cord neurons and renal tubular was downregulated.Myod expression that marks the body somite was significantly reduced.(5) MCF-7and H1299cells were treated with different concertration of deguelin for48hours,72hours and96hours. after, resulted in the inhibition of cell proliferarion in dose-dependentmanner. Using qRT-PCR to analysis the alterations of MCM3and CDC45transcript levels inthe two tumor cells treated with deguelin, the data showed that MCM3and CDC45weredownregulated at the mRNA levels in MCF-7and H1299cells(p <0.01).Conclusion:(1) Deguelin can induce apoptosis, inhibit cell proliferation during early development inzebrafish embryos.(2) Deguelin inhibits the expression MCM3and CDC45in early development of zebrafishembryos.(3) Deguelin inhibits the proliferation of MCF-7breast cancer cells and H1299lung cancercells and downregulate the expression of MCM3and CDC45.