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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2020, Vol. 46 Issue (6): 667-676    DOI: 10.3785/j.issn.1008-9209.2020.01.021
Biological sciences & biotechnology     
Effect of saponins extracted from tea (Camellia sinensis) flower on the proliferation of ovarian cancer stem like cells and its mechanism
Lianfu CHEN1(),Ning REN1,Yi Charlie CHEN2(),Youying TU1()
1.Department of Tea Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
2.Alderson Broaddus University, Philippi 26416, West Virginia, USA
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Abstract  

The effects of tea flower saponin, which was extracted and purified by macroporous resin and preparative liquid chromatography, on the proliferation and traits of human ovarian cancer stem like cells (OCSLCs) and its mechanism were studied. The stem cell marker aldehyde dehydrogenase (ALDH) activity, cell vitality, colony formation capacity, tumor sphere formation capacity and the expression of stemness-related proteins were measured to investigate the effects of tea flower saponin on OCSLCs of A2780/CP70 cells obtained by serum-free suspension culture. Western blot assay was used to investigate the effects of tea flower saponin on Wnt/β-catenin signaling pathway proteins. The results showed that tea flower saponin could decrease the proliferation of OCSLCs by suppressing their cell activity and colony formation ability. Tea flower saponin inhibited the formation of tumor sphere and reduced cell self-renewal ability. The ALDH＋ cell proportion and expressions of Oct-4 and Nanog proteins were decreased in OCSLCs treated with tea flower saponin. In addition, tea flower saponin treatment could down-regulate the expression of P-AKT, P-GSK-3β, β-catenin, c-Myc and up-regulate the expression of P-β-catenin to inhibit the Wnt/β-catenin signaling pathway. These results indicate that suppression of the Wnt/β-catenin signaling pathway might be one of the mechanisms by which tea flower saponin inhibits the proliferation of OCSLCs and cancer stem cell traits.

Key words： tea flower      saponins      ovarian cancer      cancer stem cell      Wnt/β-catenin signaling pathway     
Received: 02 January 2020      Published: 31 December 2020
CLC:  S 571.1  
Corresponding Authors: Yi Charlie CHEN,Youying TU     E-mail: c.lianfu@foxmail.com;chenyc@ab.edu;youytu@zju.edu.cn
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Lianfu CHEN
Ning REN
Yi Charlie CHEN
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Cite this article:

Lianfu CHEN,Ning REN,Yi Charlie CHEN,Youying TU. Effect of saponins extracted from tea (Camellia sinensis) flower on the proliferation of ovarian cancer stem like cells and its mechanism. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(6): 667-676.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2020.01.021     OR     http://www.zjujournals.com/agr/Y2020/V46/I6/667


茶树花皂苷对卵巢癌干细胞样细胞增殖的影响及机制

利用大孔树脂、制备液相色谱法等从茶树花中提取纯化皂苷，研究茶树花皂苷对人卵巢癌干细胞样细胞（ovarian cancer stem like cells, OCSLCs）增殖生长与干性特征的作用及其机制；采用无血清悬浮培养法获得A2780/CP70细胞的OCSLCs，通过检测干细胞标志物乙醛脱氢酶（aldehyde dehydrogenase, ALDH）活性、细胞活力、细胞克隆和肿瘤球形成能力以及干细胞相关蛋白的表达，分析茶树花皂苷对OCSLCs的作用；同时，采用免疫印迹法分析茶树花皂苷对Wnt/β-联蛋白信号通路蛋白的影响。结果表明：茶树花皂苷可通过降低OCSLCs的细胞活力和克隆形成能力抑制其增殖，抑制肿瘤球形成能力，减弱其自我更新能力；茶树花皂苷可降低OCSLCs中的ALDH＋细胞比例，使干性相关蛋白Oct-4和Nanog表达量下降；同时，茶树花皂苷通过下调P-AKT、P-GSK-3β、β-联蛋白、c-Myc蛋白的表达，上调P-β-联蛋白的表达，使Wnt/β-联蛋白信号通路被抑制。综上所述，茶树花皂苷对OCSLCs的增殖和干性具有显著抑制作用，抑制Wnt/β-联蛋白信号通路的活化是其作用机制之一。


关键词： 茶树花,  皂苷,  卵巢癌,  肿瘤干细胞,  Wnt/β-联蛋白信号通路 
Fig. 1 UPLC-ultraviolet chromatograms (A) and UPLC-Q-TOF/MS/MS total ion chromatograms (B) of saponins extracted from tea flower

峰

Peak

保留时间

Retention time/min

分子质量

[M-H]－

质谱碎片

MS2 (m/z)

分子式

Formula

化合物

Compound

153.401 245.593 71 083, 1 065, 951, 915, 753, 709, 611C60H94O27未知Unknown[13]
254.451 229.598 31 083, 1 067, 1 049, 789, 611C60H94O26Floratheasaponin D[11,13]
355.571 215.581 61 083, 1 035, 951, 933, 789, 611C59H92O26Floratheasaponin A[10,13]
Table 1 Mass spectrum (MS) data of saponins extracted from tea flower in negative mode
Fig. 2 Morphological observation of ovarian cancer cells cultured in wall and suspension conditions and the proportion of ALDH＋ cells cultured in FBS free medium for different weeksA. Morphology of ADCs and SPCs cultured in FBS free medium; B. Statistical graph of ALDH＋ cell proportion; C. Flow cytometric analysis of ALDH＋ cell. ADCs: Ovarian cancer adherent cells; SPCs-1: The 1st generation ovarian cancer spheroid cells; SPCs-2: The 2nd generation ovarian cancer spheroid cells; SPCs-3: The 3rd generation ovarian cancer spheroid cells. Compared with the control, double asterisks (**) indicate highly significant differences at the 0.01 probability level.
Fig. 3 Expression of stem cell-related proteins in ovarian cancer adherent and spheroid cellsA. Western blot image of protein; B. Statistics histogram of protein quantization. ADCs: Ovarian cancer adherent cells; SPCs-3: The 3rd generation ovarian cancer spheroid cells. Compared with the control, double asterisks (**) indicate highly significant differences at the 0.01 probability level.
Fig. 4 Effect of BTFS on cell viability of OCSLCsCompared with the control, single asterisk (*) indicates significant differences at the 0.05 probability level.
Fig. 5 Effect of BTFS on colony formation ability (A) and sphere formation ability (B) of OCSLCs
Fig. 6 Effect of BTFS on the proportion of ALDH＋ cells in OCSLCsA. Statistical graph of ALDH＋ cell proportion; B. Flow cytometric analysis of ALDH＋ cell. Compared with the control, double asterisks (**) indicate highly significant differences at the 0.01 probability level.
Fig. 7 Effect of BTFS on the expression of stem cell-related proteinsA. Western blot image of protein; B. Statistics histogram of protein quantization. Compared with the control, double asterisks (**) indicate highly significant differences at the 0.01 probability level.
Fig. 8 Effect of BTFS on the expression of Wnt/β-catenin signaling pathway related proteinsA. Western blot image of protein; B. Statistics histogram of protein quantization. Compared with the control, single asterisk (*) indicates significant differences at the 0.05 probability level, and double asterisks (**) indicate highly significant differences at the 0.01 probability level.
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