Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells

REN Yuliang; OU Hongling; WU Hui; WAN Fang; LIU Ying; SI Yuan

doi:10.13422/j.cnki.syfjx.202202423

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Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells

更新时间：2023-09-08

- Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 19, Pages: 126-135(2023)
- 作者机构：
  
  1.湖北医药学院基础医学院，湖北十堰 442000
  2.湖北医药学院武当特色中药研究湖北省重点实验室，湖北十堰 442000
  3.湖北医药学院胚胎干细胞研究湖北省重点实验室，湖北十堰 442000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.202202423
  CLC： R22;R242;R2-031;R285.5
- Published：05 October 2023，
  
  Published Online：03 November 2022，
  
  Received：23 July 2022，
- 稿件说明：
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任宇亮,欧虹灵,吴慧等.重楼皂苷Ⅰ激活Hippo信号诱导结直肠癌细胞凋亡及自噬的作用机制[J].中国实验方剂学杂志,2023,29(19):126-135.

REN Yuliang,OU Hongling,WU Hui,et al.Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(19):126-135.
任宇亮,欧虹灵,吴慧等.重楼皂苷Ⅰ激活Hippo信号诱导结直肠癌细胞凋亡及自噬的作用机制[J].中国实验方剂学杂志,2023,29(19):126-135. DOI： 10.13422/j.cnki.syfjx.202202423.

REN Yuliang,OU Hongling,WU Hui,et al.Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(19):126-135. DOI： 10.13422/j.cnki.syfjx.202202423.

摘要

目的

研究重楼皂苷Ⅰ（PPI）抑制结直肠癌细胞生长的作用及其分子机制。

方法

培养RKO细胞，分为空白组和浓度为0.6、0.8、1.0 μmol·L

-1

的PPI组，培养HRT18细胞，分为空白组，浓度为1.2、1.4、1.6 μmol·L

-1

的PPI组，细胞增殖抑制实验、平板克隆形成实验及共聚焦高内涵细胞成像分析系统检测PPI对结直肠癌增殖及形态的影响；流式细胞术检测结直肠癌细胞凋亡率；pQCXIP-GFP-LC3质粒转染实验检测PPI处理后，结直肠癌细胞自噬体的形成情况；蛋白免疫印迹法检测凋亡相关蛋白胱天蛋白酶（Caspase）-3、Caspase-8和多腺苷二磷酸核糖聚合酶（PARP）的表达、自噬相关蛋白微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）的表达及Hippo信号通路蛋白大肿瘤抑制激酶1（LATS1）、Yes相关蛋白（YAP）的表达及磷酸化水平；plvx-Flag-YAP质粒转染实验，过表达YAP后，用PPI处理，细胞增殖毒性实验检测结直肠癌细胞增殖，蛋白免疫印迹法检测结直肠癌细胞LC3Ⅱ、PARP的表达；对SwissADME预测PPI的药代动力学参数。

结果

与空白组比较，PPI组的结直肠癌细胞的存活率及克隆形成能力显著降低（

0.01），并且PPI组的结直肠癌细胞的细胞面积显著降低，圆度显著升高（

0.01）；与空白组比较，PPI组的结直肠癌细胞凋亡率显著增加（

0.01），并且PPI组的结直肠癌细胞中凋亡蛋白Caspase-3及Caspase-8蛋白前体的表达降低，PARP切割显著增加（

0.01）；与空白组比较，PPI组的结直肠癌细胞中自噬相关蛋白LC3Ⅱ的表达水平明显增加，并且促进自噬体的形成（

0.01）；与空白组比较，PPI组的结直肠癌细胞中YAP蛋白的表达明显降低，并且磷酸化LATS1、YAP的表达显著升高（

0.01）；与空白组比较，过表达YAP能显著拮抗PPI对结直肠癌细胞的凋亡自噬活化作用及增殖抑制作用（

0.01）；SwissADME模拟结果显示PPI具有较好的类药活性。

结论

PPI能通过靶向激活Hippo信号通路诱导结直肠癌细胞发生凋亡和自噬，进而抑制其增殖。

Abstract

Objective

To study the inhibitory effect of polyphyllin Ⅰ （PPI） on the growth of colorectal cancer cells and its molecular mechanism.

Method

RKO cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 0.6， 0.8， 1.0 μmol·L

-1

， respectively. HRT18 cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 1.2， 1.4， 1.6 μmol·L

-1

， respectively. The effects of PPI on the proliferation and morphology of colorectal cancer were detected by cell proliferation toxicity assay， trypan blue exclusion assay， plate clone formation assay， and confocal high-intension cell imaging analysis system. Flow cytometry was used to detect the apoptosis rate of colorectal cancer cells. The pQCXIP-GFP-LC3 plasmid transfection assay was used to detect the formation of autophagosomes in colorectal cancer cells after PPI treatment. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3， Caspase-8， and poly ADP ribose polymerase （PARP）， the expression of autophagy related protein LC3Ⅱ， and the expression and phosphorylation of Hippo signaling pathway proteins LATS1 and YAP. In the plvx-Flag-YAP plasmid transfection assay， YAP was overexpressed and treated with PPI， and the proliferation of colorectal cancer cells was detected by cytotoxicity assay. The expression of LC3Ⅱ and PARP in colorectal cancer cells was detected by Western blot. SwissADME predicted pharmacokinetic parameters of PPI.

Result

As compared with the blank group， the survival rate and clone formation ability of colorectal cancer cells in the PPI group were significantly decreased （

0.01）， the cell area of colorectal cancer cells in the PPI group was significantly decreased， and the roundness of colorectal cancer cells was significantly increased （

0.01）. As compared with the blank group， the apoptosis rate of colorectal cancer cells in PPI treatment groupw was significantly increased （

0.01）， the expression of apoptotic proteins Caspase-3 and Caspase-8 protein precursor in PPI treatment groups was decreased， and the cleavage of PARP was increased （

0.01）. As compared with the blank group， the expression level of autophagy-related protein LC3Ⅱ in colorectal cancer cells in PPI treatment groups was significantly increased， and the formation of autophagosomes was promoted （

0.01）. As compared with the blank group， the expression of YAP protein in colorectal cancer cells in PPI treatment groups was significantly decreased， and the expressions of phosphorylated LATS1 and YAP were significantly increased （

0.01）. As compared with the blank group， overexpression of YAP could significantly antagonize the effect of PPI on apoptosis， autophagy activation， and proliferation inhibition of colorectal cancer cells. SwissADME simulation results showed that PPI had good drug like activity.

Conclusion

PPI can induce apoptosis and autophagy of colorectal cancer cells through targeted activation of Hippo signaling pathway， thereby inhibiting their proliferation.

关键词

重楼皂苷Ⅰ结直肠癌凋亡自噬Hippo信号通路

Keywords

polyphyllin Ⅰcolorectal cancerapoptosisautophagyHippo signaling pathway

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