6-O-Angeloylenolin Induces Cell Circle Arrest And Apoptosis Of Human Clear Cell Renal Cell Carcinoma Through The PI3K/AKT Signaling Pathway

Objective :In this study,we investigate the effect and mechanism of a sesquiterpene lactone6-O-angeloylplenolin(6-OAP)on human Clear Cell Renal Cell Carcinoma(cc RCC),and may provide a theoretical basis of a new drug for renal carcinoma.Methods :1.The cck-8 assay was used to observe the effects of 6-O-angeloylplenolin on proliferation and viability of 786-o cells and caki-1 cells.2.The Hoechst33258 staining method was used to observe the induction of apoptosis after treatment.The Annexin V-FITC/PI double-staining assay was used to assess on apoptosis of cc RCC cells,and the cell cycle analysis was assessed by Flow cytomety.3.Determination of AKT,BAX and BCL-2 by real-time fluorescence quantitative PCR.4.The determination of PI3 K,AKT,p-AKT,PTEN,BCL-2,BAX,CDC 25 C,Cyclin B1 protein was measured by Western-blot for exploring the mechanism of 6-OAP on CCRCC cells.Results :1.The cck-8 assay demonstrated the cell viability of 6-Oangeloylplenolin with concentration gradients(0?g/ml,5?g/ml,10?g/ml,15?g/ml,20?g/ml,25?g/ml,30?g/ml)for different treating time(12h,24 h,48h,72h)on cc RCC cells.The results showed that the rate of viability of the two cells was significantly inhibited by 6-O-angeloylplenolin,and increased with drug concentration and time(P<0.05).Based on the preliminary results and relevant literature,we chose the concentration gradient of 6-Oangeloylplenolin are 5?g/ml,10?g/ml,15?g/ml for later experiments.2.The Hoechst33258 staining method was used for observing apoptosis with thefluorescence inverted microscope,which could be observed more nuclear fragmentation and pyknosis in 15?g/ml group than control group.Meanwhile,Annexin-V-FITC/PI double staining assessed the percentage of apoptosis cells increased with the drug concentration(P<0.05).With quantitative real-time PCR,we found that the m RNA expression of bcl-2 decreased with the increase of drug concentration,whereas the expression of BAX increased with the increase of drug concentration(P<0.05).Simultaneously,western-blot further verified that the protein expressions of bcl-2 and BAX were consistent with the m RNA expressions(P<0.05).3.The flow cytometry analysis showed that treatment with 6-O-angeloylplenolin led to a decrease in a proportion of G1 phase cells,and increased the proportion of G2 phase cells(P<0.05).Meanwhile,Western blot showed that the determination of CDC 25 C and Cyclin B1 proteins were down-regulated.4.The Western blot indicated that determinations of PI3 K and p-AKT proteins were down-regulated,and PTEN protein was up-regulated in the experimental group.The compound 6-OAP could promote apoptosis by negatively modulate the PI3K/AKT signaling pathway.Conclusion:Our results demonstrate that 6-O-angeloylplenolin could induce the cell apoptosis and cell cycle arrest of cc RCC cells,which may exert an anti-tumor effect by negative regulation of the PIK3/AKT signaling pathway.