Research On Autophagy And Apoptosis Induced By Raddeanin A In Colorectal Cancer Cells By Regulating PI3K/AKT/mTOR

Research Purpose:There are several studies have confirmed that Chinese medicine extract of anemone radde anemone,A(Raddeanin A,RA)can effectively inhibit liver cancer,ovarian cancer,breast cancer and other malignant tumor cell line proliferation,and our tearm in the early stage also confirmed RA could inhibit high,medium and low degree of differentiation of gastric cancer cell proliferation,but failed to observe antitumor effect of RA in vivo.Thus,This experiment was to observer the effect of RA on autophagy and apoptosis in colorectal cancer both in vitro and in vivo,and to explore the possible mechanism further,whic might provide new theoretical basis for better treatment of colorectal cancer.Research Methods:Research methods in vitro1 The proliferation inhibition of different concentrations of RA(2,4,8,16 μM)in HCT116 cells different time(12 h,24 h,36 h,48 h)was detected by MTT assay.2 The effect of RA on the morphological changes of HCT116 cells was observed by inverted phase contrast microscope.3 Transmission electron microscopy(TEM)was used to observe the autophagosome.4 Hoechst33258 staining and Flow Cytometry(FCM)were worked for observe the effect of RA on apoptosis of HCT116 cells.5 The expression of related genes about autophagy,apoptosis and PI3K/AKT/mTOR signaling pathway were tested by Real Time-PCR(RT-PCR)assay.6 The expression of autophagy associated proteins LC3 was tested by immunofluorescence assay.7 The expression of related proteins about autophagy,apoptosis and PI3K/AKT/mTOR signaling pathway were tested by Western Blot(WB).8 The PI3K/AKT/mTOR signaling pathway was blocked by PI3K inhibitor LY294002,and the effects of RA on autophagy and apoptosis of HCT116 cells were observed separately.9 The relationship between autophagy and apoptosis which were induced by RA in HCT116 cells were observed by using autophagy inhibitor Hydroxychloroquine(HCQ)and autophagic agonist Rapamycin(RAPA).Research methods in vivo1 The model of subcutaneous graft tumor of nude mice was established by HCT116,and intraperitoneal injection was used to observe the anti-tumor effect of RA in nude mice.2 Hematoxylin-eosin(HE)staining was used to observe the effect of RA on morphological changes of tumor tissues in nude mice.3 Autophagosome was detected by TEM.4 TdT-mediated dUTP Nick-End Labeling(TUNEL)staining assay was used to find the apoptotic cells that induced by RA.5 Western Blot,Immunohistochemistry(IHC)assay were used to detect the related molecules of RA on autophagy,apoptosis and PI3K/AKT/mTOR signaling pathway respectively in xenograft tumor model.Research Results:Results in vitro1 MTT assay showed that,the cells of control grew well,while at the same time,with the increase of concentration of RA(2,4,8,16 pM)in HCT116 cells,the inhibition rate increased gradually;The same concentration of RA in HCT116 cells 12 h,24 h,36 h,48 h respectively,the inhibition rate increased with the extension of function time,the proliferation inhibition of RA in HCT116 cells has good concentration and time dependence.2 To observe the effects of RA in HCT116 cell morphological changes,by inverted microscope we found the connects of control cells were closely,the rank was regular,the shape was clear.However,the number of cells which were treated with RA were gradually reduce,the shape were trend to round and solid,the gap became wider,and with the increase of concentration of RA,the cells lose its basic morphology,even appeared floating cells.3 It was found that there was no autophagy related structure in control cells by TEM.While the double membrane autophagosome was found in the cytoplasm of HCT116 cells treated with RA(4 pM).4 HCT116 cells were treated with RA(4,8 μM)for 12 h,Hoechst33258 staining observed the nuclei of cells were pyknotic and cataclastic.The results of flow cytometry showed that compared with the control group,with the increase concentration of RA,the number of apoptotic cells no matter in the early stage or in the late stage both increased gradually.5 RT-PCR assay results showed that compared with the control group,the expression of related genes Beclin-1,ATG5,ATG12 of autophagy increased with the increase of concentration of RA;the expression of BAX gene up-regulated,while Bcl-2 gene down-regulated.6 After being treated with 4 μM RA,Immunofluorescence results showed that the expression of LC3 protein increased significantly compared with the control group.7 Western Blot results found that the expression of autophagy related proteins Beclin 1,ATG 5,ATG12 increased with the increase of concentration of RA,and with the increase of concentration of RA,the signals intensity of LC3I protein reduced while LC3Ⅱ protein increased.Besides,the expression of Cleaved-caspase-3 and Cleaved-PARP proteins were lower in control group,while they were higher erexpression in RA group.Moreover,the expression of Cleaved-caspase-9 and Cleaved-caspase-8 proteins which were crucial for endogenous pathway and exogenous pathway respectively aslo increased.In addition,the expression of PI3K/AKT/mTOR pathway related proteins p-PI3K,p-AKT and p-mTOR decreased with the increase concentration of RA.8 When HCT116 cells were processed by RA(4 μM)and PI3K inhibitor LY294002(10 μM)together,Western Blot results showed that the expression of autophagy related protein LC3Ⅱ,Beclin-1 increased compared with the group treated with RA alone;FCM found that the apoptosis rate of the group of RA and LY294002 was higher than the the group of RA.In addition,the expression of apoptosis related proteins Cleaved-caspase-3,Cleaved-PARP also increases.9 When autophagy inhibitor HCQ(10 μM)was added to HCT116 cells with RA(4 μM)together,autophagy process was suppressed,and the expression of LC3Ⅱ,Beclin-1 proteins decreased.At the same time,the apoptosis rate was also reduced,in addition,the expression of BAX,Cleaved-caspase-3,Cleaved-PARP also down-regulated.However,when RA combined autophagy agonists RAPA(100 nM)worked in HCT116 cells,the expression of LC3Ⅱ,Beclin-1 proteins increased,cell apoptosis rate was also increased compared with the group of RA alone,the density of BAX,Cleaved-caspase-3,Cleaved-PARP also increased.Results in vivo1 Compared with the control group,the results of mouse xenograft model experimentalshowed that the volume and weight of the tumor of RA group were decreased.2 HE staining showed that the area of tumor necrosis of the RA(4 mg/kg)group was over 1/2,and a lot of inflammatory cells were found,while the control group was less than 1/4 and litter inflammatory cells were found.3 The double membrane autophagosome was found in tissue cells that treated with RA by TEM.4 TUNEL staining revealed that the control group had few staining apoptotic cells,while the RA treatment group showed a large number of staining apoptotic cells.5 Compared with control group,WB and IHC showed the expression of autophagy protein LC3 and Beclin-1 of RA group increased;WB results reminded that the expression of Cleaved-caspase-3 and Cleaved-PARP protein increased,the expression of PI3K/AKT/mTOR pathway related proteins p-PI3K,p-AKT and p-mTOR also decreased;while the expression of caspase-3 and PARP decreased by immunohistochemistry.Research Conclusion:1 RA can effectively inhibit the proliferation of colorectal cancer HCT116 cells,which is in a concentration-time dependence.2 RA can induce autophagy and apoptosis in HCT116 cells in vitro,and the mechanism may be achieved by regulating the PI3K/AKT/mTOR pathway.3 There is a certain relationship between autophagy arid apoptosis of HCT116 cells which induced by RA,by promoting autophagy can increase apoptosis.4 RA has a good inhibitory effect on the growth of HCT116 cells-xenograft tumor.5 The mechanism of RA can inhibit the proliferation of tumor cells in vivo might through inducing autophagy and apoptosis.