Stellera chamaejasme L. Inhibiting Cell Proliferation by Reducing YAP1 Expression in Hepatocellular Carcinoma

doi:10.13560/j.cnki.biotech.bull.1985.2022-1418

Abstract

Abstract:

This study is to investigate the effect and mechanism of Stellera chamaejasme L.（SCL）on inhibiting proliferation of hepatocellular carcinoma cells, providing basis for the pathological mechanism study and clinical treatment of primary hepatocellular carcinoma. Firstly, CCK-8 assay was performed to illustrate the ability of tumor growth in the HepG2215 cells treated with SCL serum containing blood. Western blot was used to detect the expression of YAP1（yes-associated protein 1）protein in the HepG2215 cells. Secondly, Yap1^flox/flox and Yap1^LKO mice were induced to form liver tumors in situ by DEN（diethylnirtosamine）/TCPOBOP（3,5-dichloro-2-[4-（3,5-dichloropyridin-2-yl）oxyphenoxy]pyridine）. SCL decoction was given to the stomach of the mice for 7 d, the liver was taken out, and the general shape of the liver tumor was observed. H&E was performed to observe the pathological morphology in tumor tissue. Western blot was to detect the expressions of YAP1 in the liver tumor tissues. The results from cell experiment demonstrated that SCL inhibited the proliferation of HepG2215 cells and decreased YAP1 expression. Moreover, animal experiment revealed that SCL reduced tumor volume and YAP1 expression both in Yap1^flox/flox and Yap1^LKO mice with tumors in liver. The hepatocellular carcinoma tumor volume of Yap1^LKOmice in NS group reduced when compared with Yap1^flox/floxmice. Yap1 knockdown reduced the effect of SCL inhibiting tumor growing in SCL group when compared with Yap1^flox/floxmice. LC-MS was used to detect the active components of SCL drug-containing serum and water decoction, and molecular docking was carried out with YAP1 protein. LC-MS results showed that the active ingredients were caffeic acid 3-sulfate, 5-benzyloxolan-2-one, 3,4,5-trihydroxy-6-[（2-oxo-2h-chromen-5-yl）oxy]oxane-2-carboxylic acid and isoscopoletin in SCL. Among them, caffeic acid 3-sulfate, 3,4,5-trihydroxy-6-[（2-oxo-2h-chromen-5-yl）oxy]oxane-2-carboxylic acid and isoscopoletin directly formed stable docking with YAP1 protein. These results suggest that SCL inhibits liver tumor cell proliferation by reducingYAP1 expression.

Key words: Stellaria chamaejasme L., HepG2215 cells, hepatocellular carcinoma, YAP1

ZHOU Wen-han, ZHENG Kang-ning, LI Yong-min. Stellera chamaejasme L. Inhibiting Cell Proliferation by Reducing YAP1 Expression in Hepatocellular Carcinoma[J]. Biotechnology Bulletin, 2023, 39(7): 316-324.

Figures/Tables 8

Fig. 1 Effects of low concentration of SCL serum on the proliferation of normal liver cells L02 A: The effect of SCL decoction on the morphological appearance of liver.B: Proliferative effect of low concentration of SCL containing serum on L02 cells（*P<0.05）. NS serum: Normal saline serum group; SCL serum : Stellera chamaejasme L. serum group

Fig. 2 Proliferation of HepG2215 cells inhibited by different concentrations of SCL serum A: The effects of different concentrations of SCL serum on the morphologyies of HepG2215 cells. Bar = 50 μm. B: The effects of different concentrations of SCL serum on the survival rates of HepG2215 cells. C: The IC50 values of HepG2215 cells treated with SCL serum for 24 h. *P<0.05, NS serum: Normal saline serum group. SCL serum: Stellera chamaejasme L. serum group

Fig. 3 Expression of YAP1 decreased by SCL serum in HepG2215 cells Expression of YAP1 in HepG2215 cells（*P<0.05）. NS serum: Normal saline serum group. SCL serum: Stellera chamaejasme L. serum group

Fig. 4 SCL inhibiting tumor growth in the mice with hepatic carcinoma in situ A: Morphological appearance of the liver and the volume of liver tumors（*P<0.05）. B: H&E staining of liver tumors. Bar = 100 μm. NS: Normal saline group, SCL: Stellera chamaejasme L. decoction group

Fig. 5 Expression of YAP1 decreased by SCL in the mice with tumors in liver Expression of YAP1 in the mice with tumors in liver（*P<0.05）. NS: normal saline group. SCL: Stellera chamaejasme L. decoction group

Fig. 6 Venn diagram of the number of compounds of different groups detected by LC-MS A: Number of compounds detected by LC-MS negative electrode in different groups. B: Number of compounds detected by LC-MS positive electrode in different groups. NS serum: Normal saline serum group. SCL serum: Stellera chamaejasme L. serum group. SCL decoction: Stellera chamaejasme L. decoction group

Table 1 Active ingredients of SCL

化合物 Compound	峰ID ID	代谢物Metabolite	离子模式 Mode	加合离子模式 Adducts	分子式 Formula	Metlin数据库得分 Fragmentation score	HMDB 数据库得分 Theoretical fragmentation score	瑞香狼毒组 M_5	瑞香狼毒水煎液组W_6
2.89_240.9800m/z	neg_1438	3-硫酸咖啡酸Caffeic acid 3-sulfate	neg	M-H₂O-H	C₉H₈O₇S	-	87.8	42 837.95	4 520 349
6.01_213.0311m/z	neg_2060	5-苄恶唑-2-酮5-Benzyloxolan-2-one	neg	M+K-2H	C₁₁H₁₂O₂	-	73.3	7 321.147	7 124.684
2.33_337.0552m/z	neg_5643	3,4,5-三羟基-6-[（2-氧代-2H-铬-5-基）氧]氧烷-2-羧酸 3,4,5-Trihydroxy-6-[（2-oxo-2H-chromen-5-yl）oxy]oxane-2-carboxylic acid	neg	M-H	C₁₅H₁₄O₉	-	54.7	56 091.29	34 451.94
2.51_193.0492m/z	pos_1816	异东莨菪内酯Isoscopoletin	pos	M+H	C₁₀H₈O₄	89.9	-	227.2022	5 515 259

Fig. 7 Schematic diagram of molecular docking between the active ingredients of Stellera chamaejasme L. and YAP1 A: Caffeic acid 3-sulfate and YAP1. B: 3,4,5-trihydroxy-6-[（2-oxo-2H-chromen-5-yl）oxy]oxane-2-carboxylic acid and YAP1. C: Isoscopoletin and YAP1.D：Verteporfin and YAP1

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