Effects of Asperuloside on the Activity of Hepatocellular Carcinoma Cells and the Growth of Transplanted Tumors in Nude Mice by Regulating RAS/RAF/MEK/ERK Signaling Pathway
Objective To investigate the effects of asperuloside on the activity of hepatocellular carcinoma(HCC)cells and the growth of transplanted tumors in nude mice regulating Ras protein(RAS)/Raf protein kinase(RAF)/mito-gen-activated protein kinase kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling pathway.Methods In vitro cultured human HCC-LM3 were screened for the best action concentration of asperuloside in vitro by cell counting kit 8(CCK-8).HCC-LM3 cells were randomly divided into control group(normal cell culture,no other intervention),asperuloside group(intervention with 3 mmol/L asperuloside),ML-098 group(intervention with 0.5 μmol/L RAS activator ML-098),and asperuloside+ML-098 group(combined intervention with asperuloside 3 mmol/L and RAS activator ML-098 final concentration of 0.5 μmol/L).The activity and apoptosis rate of HCC-LM3 cells in each group were detected by CCK-8 method and flow cytometry,respectively;the proliferation and apoptosis of HCC-LM3 cells and the expression of RAS/RAF/MEK/ERK signaling pathway-related proteins were detected by Western blot.The HCC-LM3 nude mice in situ cancer model was prepared and randomly divided into control group(5 ml/kg normal saline),low-dose group of asperuloside(25 mg/ml asperuloside),middle-dose group of asperuloside(50 mg/ml asperuloside),and high-dose group of asperuloside(100 mg/ml asperuloside).The liver-to-body ratio and tumor length of nude mice in each group were detected.The proliferation of tumor cells in nude mice in each group was detected by Ki67 immunohistochemical staining.The expression of proteins related to RAS/RAF/MEK/ERK signaling pathway in nude mice in each group was detected by Western blot.Results Compared with the control group,the apoptosis rate of HCC-LM3 cells,the expression of Cleaved caspase-3,Bcl-2 associated X protein(Bax),and cysteine containing aspartate specific protein 9(caspase-9)were all increased in the asperuloside group(all P＜0.05),and the cell activity,the expression of proliferating cell nuclear antigen(PCNA)and RAS protein,p-RAF/RAF,p-MEK/MEK and p-ERK/ERK were decreased(all P＜0.05);the effect of ML-098 on HCC-LM3 cells was oppo-site to that of asperuloside(P＜0.05).Compared with asperuloside group,the apoptosis rate of HCC-LM3 cells,the ex-pression of Cleaved caspase-3,Bax and caspase-9 protein in asperuloside+ML-098 group were decreased(all P＜0.05),and the cell activity,the expression of PCNA and RAS protein,p-RAF/RAF,p-MEK/MEK and p-ERK/ERK were in-creased(all P＜0.05).Compared with control group,the liver-to-body ratio,tumor length,tumor cell proliferation in-dex,RAS protein expression,p-RAF/RAF,p-MEK/MEK,p-ERK/ERK in the low,medium and high dose groups of asperuloside were decreased(all P＜0.05),and there was a dose-dependent manner(all P＜0.05).Conclusion Asperu-loside can inhibit the activation of RAS/RAF/MEK/ERK signaling pathway,reduce the cell activity of HCC cells in vitro and promote their apoptosis,and delay the growth of transplanted tumors in nude mice.
AsperulosideHepatocellular carcinomaCell activityTransplanted tumorsRas protein/Raf protein kinase/mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway
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