| Research Background: Cholangiocarcinoma is the second most common primary malignant tumor in the liver.Cholangiocarcinoma has the characteristics of high malignancy,poor prognosis,and poor sensitivity to radiotherapy and chemotherapy,and lacks an effective treatment plan.Crowberry is a small evergreen creeping shrub.Studies have reported that crowberry extract has many pharmacological effects such as anti-tumor,anti-fungal and anti-inflammatory,among them,2′,4′,β-trihydroxydihydrochalcone,2′,4′-dihydroxydihydrochalcone and 2′-methoxy-4′-hydroxydihydrochalcone are proven to have anti-cancer effects.DEK oncogene is a proto-oncogene on the chromatin framework.Its ability to bind nucleic acids leads to the regulation of many cellular processes,including regulation of hematopoiesis,overall heterochromatin integrity,DNA replication,gene transcription and DNA repair,and is related to the formation and development of tumors.Serine-threonine kinase/Mammalian target of rapamycin signaling pathway is one of the key ways to regulate cell growth and survival in cancer biology.Studies have confirmed that the abnormal activation of Akt/m TOR signaling pathway is one of the main mechanisms for the development and malignant progression of cholangiocarcinoma.So far,there is no research report on the application of crowberry crude extract in cholangiocarcinoma.This study aims to explore the inhibitory effect of crowberry crude extract on the proliferation and metastasis of cholangiocarcinoma cells,and apply it to human cholangiocarcinoma cells and nude mice transplanted tumor models to clarify the crowberry crude extract from both in vivo and in vitro.The effect of substances on the proliferation of cholangiocarcinoma and its molecular mechanism.Objective: To explore the inhibitory effect of crowberry crude extract on the proliferation and metastasis of cholangiocarcinoma cells and it’s molecular mechanism.Methods: Chemical profiles of crowberry crude extract were qualitatively analyzed used high performance liquid chromatography and HPLC mass spectrometry.Varying concentrations of crowberry crude extract(0,40,80,160 μg/m L and 0,100,150,225μg/m L)were used to treat on Hu CCT1 and QBC939 cells,The consequence of crowberry crude extract on the proliferation of cholangiocarcinoma cells Hu CCT1 and QBC939 were detected by MTT,colony formation assays,and Edu assays.The morphological changes of cell nuclei was observed by Hoechst 33342 staining.The effect of crowberry crude extract on apoptosis of CCA cells was detected by flow cytometry.The effect of crowberry crude extract on CCA cells migration was assessed by wound healing and migration assays.The expression level of apoptosis associated proteins,EMT markers,key members of Akt/m TOR signaling pathwayassociated proteins and DEK protein were detected by western bloting assay.The effect of crowberry crude extract on tumors in vivo was evaluated by nude mouse tumor-bearing experiment.The positive rate of ki67 and the expression level of DEK in tumors of nude mice were detected by immunohistochemical staining.Results: 1.The qualitative analysis results of high performance liquid chromatography and HPLC mass spectrometry showed that crowberry crude extract contained monomeric compounds mainly composed of 2’-methoxy-4’-hydroxydihydrochalcone,2’,4’-dihydroxy-dihydrochalcone and 2’,4’-dihydroxychalcone.2.Varying concentrations of crowberry crude extract(0,40,80,160 μg/m L and 0,100,150,225 μg/m L)were used to treat on Hu CCT1 and QBC939 cells,the ability of proliferation of the crowberry crude extract treatment group decreased in a timeconcentration dependent manner(p<0.05);3.The results of colony formation assays and Edu assays showed that crowberry crude extract can significantly inhibit the colony forming ability and proliferation ability of Hu CCT1 and QBC939 cells(p<0.05);4.The number of bright blue apoptotic bodies increased gradually in drug group by hoechst33342 staining;5.The results of flow cytometry showed that the apoptotic rate of crowberry crude extract-treated cells were significantly increased(p<0.05);6.The transwell migration assay and wound healing demonstrated that the horizontal and longitudinal migration ability were supressed obviously in the crowberry crude extract-treated group(p <0.05);7.Western blot showed that the ratio of Bax/Bcl-2 is significantly increased and the expression of apoptosis marker proteins,Cytochrome C,Cl-caspase 9,Cl-PARP and Cl-caspase 3 were significantly increased(p<0.05);The expression levels of E-cadherin were significantly upregulated,and the expression levels of Vimentin and Snail were down-regulated(p<0.05);The phosphorylation levels of Akt/m TOR signaling pathway-related proteins Akt,4EBP1,and S6 were significantly inhibited in the crowberry crude extract-treated group(p<0.05).8.Western blot showed that DEK was overexpressed in CCA cells compared with HIBEpic cell,the expression level of DEK in cholangiocarcinoma cells was significantly down-regulated in the crowberry crude extract-treated group(p<0.05);9.Silenced DEK significantly inhibited the viability,horizontal and longitudinal migration ability of CCA cells by MTT assay,migration assay and wound healing assay(p<0.05);10.DEK silencing upregulated E-cadherin expression and downregulated the expression levels of Vimentin and Snail,additionally,the phosphorylation levels of Akt/m TOR signaling pathway-related proteins Akt,4EBP1,and S6 were significantly inhibited(p<0.05);11.The results of the nude mouse tumor-bearing experiment showed that there was no significant difference in the weight of the nude mice in the crowberry crude extract drug group compared with control group,and the tumor volume began to be significantly different on the 18 th day(p<0.05),the results of immunohistochemistry showed that the positive rate of ki67 and DEK protein expression in tumors were significantly reduced compared with the control group.Conclusions: 1.Crowberry crude extract can significantly inhibits the proliferation and migration of cholangiocarcinoma cells,promote apoptosis and inhibit EMT process.2.Crowberry crude extract inhibits the proliferation and migration of cholangiocarcinoma and closely related to the inhibition of Akt/m TOR signaling pathway by targeting DEK. |
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