| Objective: To observe the effect on three triterpene glycosides from Cimicifugafoetida, named Compound4,5,10(hereinafter referred to as A, DA, KCY4), explored theeffects induced by cyclinD1,APC, for there seems to key effector β-catenin expression hasshown through Wnt/β-catenin signaling pathways inducting the self-renewal for the drugtargeted in acute myeloid leukemia (AML), which seek to the supports for experiments.Inaddition, providing in a hope of a advantage of the clinical efficacy of leukemia and newideas and methods.Methods:1.In vitro:(1) Cultured the human acute myeloid leukemia KG-1a, whichMidiMACS magnetic separated CD34~+/CD123~+expression of leukemia stem cells, whichdetected that the cell purity was more than98%using Flow cytometry (FCM). Standingcultured of the separated cells.(2) Cellular viability was measure by CCK-8assay, and thecells were incubated in the absence or presence of increasing concentration of A、DA andKCY4for different time，which determined the IC50of each group.（3）Apoptosis and cellcycle distribution were monitored by FCM.(4) The protein expression levels of each groupof β-catenin, APC, cyclin D1were assessed by western-blot analysis.(5) The levels ofAPC, Cyclin D1and β-catenin mRNA were tested by real-time fluorescent quantitativePCR analysis.2.In vivo:(1) Establishment and evaluation of human acute myeloidLeukemia KG-1a Model in NOD/SCID Mice, and mice were treated of A,DA and KCY4.(2)We observed the survival status, then monitoring the changes of peripheral blood beforeand after treatment with daily i.g. of A,DA and KCY4, in addition we evaluated the dyingmouse organization by microscopy, the application of G-banding method karyotypeanalysis of the bone marrow of mice in each group.(3)At the end of the experiment, the mice were killed by institutionally approved method after3weeks.The apoptosis and thecell cycle progression of spleen cells were examined using FCM. And the changes ofβ-catenin,APC,cyclin D1protein expression were detected using immunohistochemistry.Results:1.In vitro:(1)CCK-8assay showed that inhibited cell growth and decreasedcell viability in a dose-and time-dependent manner, each group of IC50was0.33μg/mL,0.61μg/mL and7.08μg/mL.Therefore,in our study,we choose with a medium concentrationrespectively was0.2μg/mL,0.4μg/mL,5μg/mL (lower than IC50) at48h.(2) The studydemonstrated that after treatment for48h, the three groups apoptosis and the cell cycleprogression of these LSCs were compared with control group,obviously, a significantincrease was observed in KCY4(P<0.01) higher than that in the others.(3) As shown bywestern blot analysis, as depicted showed that compared with controls, the three groupselevated APC protein level and degraded Cyclin D1, β-catenin protein level (P<0.01), therewere significant differences. In addition, the findings demonstrated that β-catenin andcyclinD1were positively correlated (r=0.92), β-catenin and APC were negativelycorrelated(r=-0.896).(4) We evaluated the level of each gene mRNA expression byreal-time fluorescent quantitative PCR analysis, respectively, and found that the level ofAPC, Cyclin D1and β-catenin mRNA were concordant with the protein levels. Overall,ourresults implied that after drugs treatments, the expression of the protein at the levels ofgene transcription.2.In vivo: Transplantation was always successful,and results showedthat the three groups treatments increased the average survival time (P<0.01).After drugstreatments,immunohistochemistry showed that cleaved of APC level was increased andCyclinD1,β-catenin level were decreased compared with controls, the protein expressionsof the three groups were concordant with the vitro. KCY4in vivo mechanism of action issuperior to the others.Conclusion: These findings indicate that the study on three triterpene glycoside fromC. foetida in vitro and in vivo have varying degrees of anti-cancer activity, which as atarget of cancer treatment for experimental and theoretical basis,wherein KCY4is betterthan the others. |
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