Research On Apoptosis In Human Renal Clear Cell Carcinoma Cell Line786-0Induced By Emodin In Vitro

Objective：To investigate the effect of emodin on apoptosis in human renal clearcell carcinoma cell line786-0in vitro and to obtain insights into itsmolecular mechanism of action.To provides the experimental basis fortherapy of renal clear cell carcinoma with emodin and explore themechanism， the dose-effect relationship and the time-effectrelationship。Methods:（1） Cells of the contemporary were divided into different groups:blank control group (no drug),the negative control group (0.05%DMSO),emodin group (10、20、40、60and80μmol/L).（2） Ordinary optical microscope were used to observe the inhibitioneffect of emodin on the proliferation of cell786-0and nuclei withHE staining.（3） DAPI staining was used to investigate the morphological changesof apoptosis.（4） Methods CCK-8was used to examine the786-0cells inhibition whichinduced by emodin.（5） RT-PCR was applied to identity the expression of BCL-2, BAX at thetranscription level.（6） Western-Blot was applied to investigate the expressions of Bcl-2and Bax protein.（7） JC-1probe method was used to detected the changes of786-0 misochondrial membrane potential.（8） Caspase-9and Caspase-3Activity Assay Kit was used respectivelyto detect the activity of Caspase-9and Caspase-3.Results:Under the inverted microscope inverted microscope, cells wereepithelioid type, adherent, with clear cell contours, no abnormalparticles was found in cytoplasm in blank control group, obviously lesscells and more intracellular vacuoles, irregular cell shapes, andfloating cells were observed along with the increase of time and emodinconcentration.Rippled or creased cell nucleus，nuclear chromatinconcentration and edge accumulation，apoptotic bodies were seen underthe high-power biological microscope when the cells were stained by HE。Following stained with DAPI，cells produced weaker blue fluorescence andthe nuclei were round in negative control group and lots of darker blueapoptotic cells, with nuclear fragmentation, karyopyknosis or apoptoticbody were investigated in the emodin groups.Different concentrations (10,20,40,60, and80μmol/L) of emodinall could inhibit the proliferation of786-0cells, and the inhibitionrate were concentration dependent and time dependent; half maximalinhibitory concentration (IC50) of emodin were respectively（45.56±2.12）μmol/L,（33.59±1.38）μmol/L,（24.41±1.36）μmol/L,after24h,48h and72h. After24h，the expression levels of Bal-2mRNAand Bal-2protein of786-0in emodin group were decreased and theexpression levels of Bax mRNA and Bax protein were increased，ratio ofBcl-2/Bax decreased significantly。Mitochondria membrane potential wasmarkedly decreased when treated with emodin. The relative ratio of orangefluorescent and green fluorescent indicated and the activity of caspase-9and caspase-3intensified along with the increase of time and emodinconcentration. Conclusion:（1） Emodin can induce cell apoptosis of human renal clear cellcarcinoma cell line786-0in vitro. Cells presentedcharacteristic morphological changes of apoptosis. The effect ofapoptosis-inducing increased dose-dependently andtime-dependently.（2） One of its main passways of apoptosis is to significantlydecrease the ratio of Bcl-2/Bax, then markedly decrease themitochondria membrane potential and intensify the activity ofcaspase-9and caspase-3.