| Background: Primary liver cancer has insidious onset,strong aggressiveness and rapid development,and has become the second leading cause of cancer death in the world.Hepatocellular carcinoma(HCC)is the most common type of liver cancer,accounting for more than 90% of primary liver cancer.For patients at different stages of early,middle and advanced HCC,multiple treatment methods including surgery,radiotherapy and chemotherapy and targeted drug therapy may be adopted,however,no significant reduction in mortality was observed in HCC patients.With the progress of science and the continuous study of traditional Chinese medicine,the pharmacological effect of traditional Chinese medicine with high efficiency and low toxicity has been widely concerned by scholars.In recent years,the anticancer effect of ginseng has attracted increasing attention.Ginseng contains many active ingredients,ginsenoside CK has a good anti-tumor effect,and compared with chemotherapy drugs,the toxic and side effects are very small,has further development potential.BC1-2 associated transcription factor 1(Bclaf1)is a protein involved in regulating gene transcription and plays an important role in the development and progression of tumors.Mitochondria are energy supply stations and are involved in regulating a variety of biological behaviors including cell apoptosis.Mitochondria associated apoptosis is associated with extracellular regulated protein kinases(ERK).When the cell receives external stimuli,such as gene activation and DNA damage,the cell membrane potential drops,the membrane permeability changes,the apoptotic factors are activated,and the cell apoptosis occurs.Extracellular regulated protein kinases(ERK)are involved in cell proliferation,differentiation and canceration.The ERK signaling pathway can be activated by external stimulation to participate in the transcriptional of certain genes and then regulate the biological behavior of cells.Therefore,this study aims to investigate the role of ginsenoside CK in regulating liver cancer cells mitochondrial apoptosis induced by ERK signaling pathway through Bclaf1.Objective: In this study,we investigated the effect of ginsenoside CK on mitochondrial apoptosis induced by ERK signaling pathway in SMMC-7721 and BEL-7404 human hepatoma cells and the regulatory mechanism of Bclaf1 through in vitro and in vivo experiments.Methods: CCK-8 assay was used to detect the effect of ginsenoside CK(0,20,40,60,80 μmol/L)on the proliferation of human hepatoma cells SMMC-7721 and BEL-7404;the effect of ginsenoside CK on the cell cycle of SMMC-7721 and BEL-7404 were detected by propidium iodide single staining;phosphatidylserine eversion assay was used to detect the apoptosis rate of hepatoma cells under the action of ginsenoside CK;fluorescence probe was used to detect the effect of CK on mitochondrial membrane potential in cells;the localization and nucleation behavior of Bclaf1 in cells were detected by immunofluorescence technique;the protein expressions of Cyt-c,Cleaved caspase 3,Bcl-2,Bax,ERK,p-ERK and Bclaf1 in HCC cells were detected by western blot,after ERK pathway inhibitor U0126 was added,Western blot verified the effect of ERK on apoptosis of hepatoma cells induced by ginsenoside CK;after CRISPR/Cas9 knocked out the Bclaf1 gene,phosphatidylserine eversion assay was used to detect the effect of CK on apoptosis induced by Bclaf1 knockout hepatoma cells;mitochondrial apoptosis and expression of ERK pathway related proteins were detected by western Blot assay.To determine the regulatory mechanism of Bclaf1 on ERK-mediated mitochondrial apoptosis;to establish a xenograft model of human hepatoma cell line SMMC-7721 in nude mice,they were divided into blank control group,ginsenoside CK(0,10 and 20 mg/kg)administration group,positive control 5-FU administration group to detected the effect of ginsenoside CK on transplanted tumor;the expressions of Bclaf1,Bcl-2and Bax were detected by immunohistochemistry;Western blot detected the expression of ERK and phosphorylation of ERK.Results: CCK-8 assay showed that ginsenoside CK could inhibit the proliferation of SMMC-7721 and BEL-7404 cells in vitro and had time and concentration dependence;the results of propidium iodide single staining showed that ginsenoside CK blocked the G0/G1 phase of HCC cells;phosphatidylserine eversion analysis showed that the apoptosis rate of HCC cells increased with the increase of ginsenoside CK;ginsenoside CK could decrease mitochondrial membrane potential in hepatoma cells by fluorescent probe method;western blot results showed that ginsenoside CK could up-regulate mitochondrial apoptosis-related proteins Cyt-c and Cleaved caspase3,and down-regulate Bcl-2/Bax;western blot analysis showed that ginsenoside CK inhibited the expression of phosphorylation of ERK;after the addition of ERK inhibitor U0126,ginsenoside CK could further inhibit the expression of phosphorylation of ERK,the Bcl-2/Bax declined;immunofluorescence assay showed that Bclaf1 was expressed in both the nucleus and cytoplasm,western blot analysis showed that ginsenoside CK inhibited the expression of Bclaf1;compared with normal liver cancer cells,in the stable knockout of Bclaf1 liver cancer cells,the apoptotic rate was significantly increased and mitochondrial apoptosis was more obvious after ginsenoside CK treatment,the mitochondrial membrane potential decreased significantly,the expression levels of mitochondrial apoptosis-related proteins Cyt-C and Cleaved caspase3 increased,Bcl2/Bax down-regulated,phosphorylation of ERK declined;to construct human hepatocellular carcinoma cell line SMMC-7721 xenograft model in nude mice,the results showed that ginsenoside CK inhibited tumor growth;immunohistochemistry and western blot analysis showed that the expression of Bclaf1 and Bcl-2 decreased and the expression of Bax increased after CK treatment,the expression of phosphorylation of ERK was decreased.Conclusions: Ginsenoside CK inhibited the proliferation of human hepatoma cells through ERK mediated mitochondrial apoptosis,and this effect was related to the regulation of Bclaf1. |
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