Study On The Function And The Molecular Mechanism Of Osthole Inhibiting Cholangiocarcinoma Via PI3K/Akt Pathway

Objective 1.Investigate the anticancer effect of osthole in intrahepatic cholangiocarcinoma in vitro.2.Researchthe mechanism of anticancer effect of osthole in intrahepatic cholangiocarcinoma.3.Investigate the anticancer effect of osthole in intrahepatic cholangiocarcinoma in vivo.Methods1.Investigate the anticancer effect of osthole in intrahepatic cholangiocarcinomain vitro by using RBE and HCCC-9810 as tool cell lines.(1)The effect of osthole on the viability of ICC cells and was evaluated using the CCK-8 assay.The ability of ICC cells to form clones was investigated using a colony formation assay.(2)The morphological changes of ICC cells treated with osthole was observed by Hoechst 33342 staining using a fluorescence microscope.The apoptosis rate of ICC cells was determined using flow cytometric analysis after Annexin V/PI staining.2.Research the mechanism of anticancer effect of osthole in intrahepatic cholangiocarcinoma.(1)The changes of the mitochondrial membrane potential(??m)was determined through Rhodamine 123 staining using flow cytometric analysis.(2)The changes of apoptosis related protein was measured by western blot assays,such as cleaved caspase-3,caspase-3,cleaved caspase-9,caspase-9,cleaved PARP,Bcl-2 and Bax.We further investigated the expression level of PI3 K,Akt,and p-Akt.(3)Tofurther investigate theimpactof Akt activation onostholeanticancereffectionin ICC,We evaluated the cell viability using CCK-8 assays after transfection of WT-Akt and CA-Akt plasmid.(4)Weinvestigatedtheapoptosis of transfected ICC cells through Annexin V/PI using flow cytometric analysis.(5)The expression levels of Akt,p-Akt,Bcl-2 and Bax in transfected ICC cells were measured by Western blot assays.3.In vivo tumor xenograft study.(1)Using nude mice as an animal model to investigate the effect of osthole on ICC in vivo.After intraperitoneal injection withdeferent concentration osthole,thesizeof xenograft tumorswere measured.(2)Extract total protein of xenograft tumors.Western blot assays were performed to investigate the changes of Akt,p-Akt,Bcl-2,Bax and cleaved PARP.Results1.Osthole inhibits the proliferation and induces apoptosis of ICC cells in vitro.(1)The results of CCK-8 assays showed that the proliferation of these ICC cell lines was inhibited by osthole in a dose-and time-dependent manner.The results of colony formation assay showed the number and size of the colonies derived from osthole-treated cells were markedly smaller than those of the colonies in the control group.(2)Osthole induces apoptosis in ICC cells.The results of Hoechst 33342 showed that the cells treated with osthole exhibited markedly increased chromatin condensation and fragmentation relative to the cells in the control group,which were round and homogeneously stained.The results of flow cytometric analysis of apoptosis showed that compared with the rates in the control group,the early and late apoptosis rates of cells treated with osthole significantly increased in a dose-dependent manner.2.Osthole induces mitochondria-dependent apoptosis via PI3K/Akt pathway.(1)The results of Rhodamine 123 staining showed that apoptosis cells through mitochondria-dependent pathway increased in a dose-dependent manner.(2)Western blot assay showed the expression level of the mitochondrial anti-apoptotic protein Bcl-2 was significantly decreased,while the expression levels of pro-apoptotic factors including Bax and the cleaved forms of caspase-3,caspase-9,and PARP were upregulated.The expression level of PI3 K and p-Akt was decreased in a dose-depend manner.(3)CCK-8 assay after transfection with WT-Akt and CA-Akt plasmid showed overexpression of WT-Akt or CA-Akt abolished osthole cytotoxicity in HCCC-9810 and RBE cells.(4)The apoptosis rate was significantly decreased in cells transfected with WT-Akt and CA-Akt,especially the early apoptosis rate.(5)The expression level of Bcl-2 was significantly increased in cells transfected with WT-Akt and CA-Akt while the expression level of Bax was decreased in cells transfected with WT-Akt and CA-Akt.3.Osthoel exert anticancer effect in vivo.(1)After intraperitoneal injection withdeferent concentration(50mg/kg and 100mg/kg)osthole for 26 days,the tumor size and tumor weight were significantly lower in mice treated with osthole.(2)Bcl-2 and p-Akt expression levels were strikingly lower and Bax and cleaved-PARP expression levels were significantly higher in osthole-treated groups than in the control group.Conclusion 1.Osthole can suppress proliferation and induce apoptosis in HCCC-9810 and RBE cell lines in vitro.2.Osthole induces mitochondria-dependent apoptosis via PI3K/Akt pathway.3.Osthole improves the expression level of Bax and cleaved PARP,inhibit the expression of p-Akt and Bcl-2.Osthole suppresses tumor growth in vivo by induces apoptosis.