The Effect Of Koumine On Different Species Cells And The Antitumor Activity Of Koumine On Different Tumor Cells

Gelsemium elegans Benth(GEB)is a small genus of the family Loganiaceae.It is known as Duan Chang Cao,Zhu Ren Shen or Da Cha Yao.Alkloids are the main ingredients in the GEB,including Koumine,Gelsemine,Kouminicine,Kouminidine and Sempervirine.The alkalbids play an important role in antitumor,analgesic activity,anti-inflammatory,immunomodulatory,and anxiolytic activity.Koumine(Kou),the most abundant alkaloid from GEB,has similar function but less toxic commpared with total alkoloids.GEB has differential effects on pig and human and has antitumor activities.In this study,the effects of Kou on pig and human cells and the antitumor mechanism of Kou were investigated.In addition,by using Tet-on inducible system,Vero-TR parental cell line and Vero cell lines that inducibly express or knock down DEC1 were generated,which will serve as tools for future studies.This thesis contains following contents:1.The differential effects of Kou on pig and human cellsFirst,morphological changes in Kou-treated pig cells(PK15 and IPEC-J2)and human cells(H1299,RKO and MCF7)were observed under the microscope.Second,cell cycle was determined by fluorescence-activated cell sorting(FACS)and cell growth was analyzed by cell proliferation assay and colony formation assay.Third,PARP1 expression was analyzed by Western Blot.Results showed that huamn cells but not pig cells exhibited obvious morphological changes upon Kou treatment.FACS anlaysis showed that Kou induced G1 arrest in PK15 and IPEC-J2 cells,but G2/M arrest in H1299,RKO and MCF7 cells.In addition,Kou inhibited cell growth in human cells and this inhibition was irreversible,but slightly inbihited cell growth in pig cells and the inhibition was reversible.Finally,cleaved PARP1 was detected in RKO and MCF7 cells,but not in pig cells.2.The antitumor activities of Kou in different tumor cellsIt has been shown that Kou is capable of inducing tumor cell apoptosis and thus believed to have antitumor activity.The levels of Bax,Bcl-2,Caspase 7 and PARP1 were examined in different tumor cells by Western Blot.The subcellular localization of cytochrome C was determined by cell fractionation assay of Kou-treated tumor cells.Results showed tlhat Kou increased Bax but decreased Bcl-2 expression.In addition,upon Kou treatment,cytochrome C was released from mitochondrial membrane into cytoplasm in RKO and MCF7 cells.Moreover,Caspase 7 and PARP1 proteins were found to be cleaved in RKO and MCF7 cells,but not in pig cells.Therefore,it is likely that Kou induces apoptosis in RKO and MCF7 cells through the mitochondrial pathway.3.Generation of Vero cell lines that inducibly regulate DECI expression by TetracyclinepcDNA6 expressing Tet repressor protein(TetR)was transfected into Vero cells,and positive clones that stably express TetR were selected by Blasticidin and designated Vero-TR parental cell line.In addition,pcDNA4 expressing DEC1 or the pBabe-Hl expressing DEC1 shRNA were transfected into Vero-TR,respectively.The positive cell clones that inducibly express or knock down DEC1 were screened by Zeocin or Puromycin,and designated Vero-DEC1 or Vero-siDEC1.Moreover,the efficacy of expression or knockdown of DEC1 was analyzed by Western Blot.Results showed that the stable cell lines of Vero-TR,Vero-DEG1 and Vero-siDEC1 were successfully generated.Together,this study suggested that Kou had differential effects in human and pig cells and Kou induced cell death in RKO and MCF7 cells.The Vero-TR parental cell line provided an important material for generating cell lines that inducibly express or knock down any other host cell genes.The Vero-DEC1 and Vero-siDEC1 cell lines obtained in this study lay a critical foundation for studying biological functions of DEC1 under normal or stress conditions.