The Growth Inhibition Effects Of Oridonin Combined With DNR On Jurkat Cells And Its Mechanism

ObjectiveOridonin is an active diterpenoid compound extracted from the Rabdosia rubescens,which is found to have significant activity against inflammation、bacterium and tumor.Oridonin has inhibitory effects against many cancer cells to some certain,But little research on acute T cell leukemia. In this study, using acute T lymphoblasticleukemia jurkat cells as the research objects in vitro, to explore the growth inhibition andapoptotic inducing effects of oridonin and DNR alone or in combination on jurkat cells,which provide certain experimental evidence for application to acute T lymphoblasticleukemia of oridonin.MethodsThe jurkat cells in logarithmic growth phase were incubated with differentconcentrations of oridonin(1μg/ml、2μg/ml、4μg/ml、8μg/ml、16μg/ml) and DNR（0.04μg/ml、0.08μg/ml、0.16μg/ml、0.32μg/ml、0.64μg/ml）alone or in combinationCCK-8assay was used to detect the proliferation inhibitory effects of jurkat cells aftertreated with different doses of oridonin and DNR each alone or in combination for24h、48h、72h; inverted phase contrast microscope、Giemsa stain and Rh123stain were appliedto observe the morphologic changes of jurkat cells induced by different concentrations oforidonin for48h; the apoptosis rate of jurkat cells after treated with oridonin alone or incombination with DNR(1μg/ml) for48h was analyzed by flow cytometry(FCM); in orderto further investigate the partial apoptotic mechanism in the second part of experiment,FCM was used to analyze the cell cycle diffusion of jurkat cells after treated with differentconcentrations of oridonin for48h; to detect fluorescence intensity of jurkat cells aftertreated with different concentrations of oridonin for48h by Rho123stain; to investigate the caspase-3activity of jurkat cells after treated with different concentrations of oridonin for48h.Results(1) The proliferation inhibitory effects of jurkat cells detected by CCK-8assayCCK-8assay showed that the absorbance value gradually decreased withconcentration of oridonin increasing under the same time condition and also decreased asthe time go on under the same concentration conditions, which accounted for the growthinhibitory effects of oridonin on jurkat cells was in a dose-and time-dependent manner.After incubated with oridonin (1μg/ml) in combination with different concentrations oforidonin for48h, the inhibitory effects of combination treatment markedly enhanced thanthat of oridonin and DNR each alone group, which showed synergistic effects by the Kim’sformula, and there was a significant difference.(2) Cell morphologic changesInverted phase contrast microscope: with the drug concentration increasing, we canfound that jurkat cell density gradually reduced、fringe vague、brightness weakened andsome cells presented the morphologic changes of apoptosis, such as cell shrinkage、nuclearcracked; morphologic changes observed by giemsa’s staining: with the drug concentrationincreasing, some cells also presented the changes of cell shrinkage、nuclear condensationand even apoptotic bodies, while the control group had no obvious changes; jurkat cellsalso presented cell body shrinkage、fluorescence intensity weakened and so on by Rho123stain.(3) The apoptotic rate of jurkat cells by AnnexinV-FITC/PIFCM showed that the apoptotic rate of jurkat cells treated with differentconcentrations of oridonin(1μg/ml、2μg/ml、4μg/ml、8μg/ml、16μg/ml) is7.74±0.96%、13.26±1.49%、17.42±1.24%、25.13±2.12%、29.07±0.59%respectively, and there was asignificant difference between oridonin treatment groups and control group. When jurkatcells were incubated with oridonin (4μg/ml) and DNR (0.04μg/ml) in combination, theapoptotic rate is higher than that of oridonin (4μg/ml) or DNR (0.04μg/ml) each alone, andthere was also a significant difference.(4) The change of cell cycle diffusionAfter jurkat cells incubated with different concentrations of oridonin for48h, FCMshowed that the ratio of G1phase was33.96±1.15%、36.04±0.30%、37.05±0.18%、37.59±0.76%、38.77±1.58%、47.71±4.94%respectively with the increasing of drug concentration. There was a statistical difference between oridonin treatment groups (8μg/ml、16μg/ml) and control group. The experiment showed that oridonin influenced the diffusion of cell cycle and more cells can be arrested in G1phase by oridonin.5、The change of mitochondrial membrane potentialFCM showing: with the increasing of oridonin concentration, the fluorescenceintensity of oridonin treatment groups was3806.79±186.30、3516.43±223.86、3072.41±28.52、665.82±85.39、193.08±3.70respectively, which reduced in adose-dependent manner. There was a statistical difference (F=302.584，P<0.05) betweencontrol group and oridonin treatment groups except the first group(1μg/ml). The testshowed that oridonin can induce mitochondrial membrane potential to decrease in adose-dependent manner, which was one of the apoptotic mechanisms induced by oridonin.6、The change of caspase-3activity induced by oridoninJurkat cells were incubated with different concentrations of oridonin(1μg/ml、2μg/ml、4μg/ml、8μg/ml、16μg/ml) for48h. Along with the increasing of concentration, theabsorbance value was0.162±0.005、0.169±0.004、0.179±0.015、0.190±0.008、0.203±0.155respectively. There was a significant difference between oridonin treatment groups andcontrol group, which showed absorbance value increased in a dose-dependent manner andalso indirectly reflected the caspase-3activity gradually increased.Conclusions1、Oridonin inhibit the proliferation of jurkat cells in a dose-and time-dependentmanner; the inhibitory effects of jurkat cells treated with oridonin and DNR in combinationhad synergistic effects, which prompted that oridonin can inhibit leukemia cells growthand induce apoptosis as a new drug against tumor at the same time, and also enhance thesensitivity of chemotherapeutics to leukemia cells、reduce drug doses and adverse effects.2、The killer effects of oridonin against to jurkat cells were mainly realized byinducing apoptosis, the relative mechanism as follows:Oridonin influenced the diffusion of cell cycle and arrested cells in G1phase in adose-dependent manner, which can effectively control proliferation of cells;The decrease of mitochondrial membrane potential induced by oridonin was in adose-dependent manner and in parallel with the damage degree of mitochondrialmembrane, which reflected cells stepped into irreversible apoptotic procedure as an earlyapoptotic event. The activity of caspase-3increased in a dosed manner and induced apoptosis inapoptotic cascade reaction, while caspase-3is the powerful effective factor and finallyapoptotic executor.