华法林通过调控Gas6/Axl/PI3K/Akt/NF-κB通路影响肺癌细胞株的增殖和凋亡

doi:10.5246/jcps.2023.03.016

中国药学(英文版) ›› 2023, Vol. 32 ›› Issue (3): 190-199.DOI: 10.5246/jcps.2023.03.016

华法林通过调控Gas6/Axl/PI3K/Akt/NF-κB通路影响肺癌细胞株的增殖和凋亡

阳丽梅¹, 王丽满², 黄旭慧², 庄捷¹^,^*()

1. 福建医科大学省立临床医学院/福建省立医院药学部, 福建福州 350001
2. 福建省立医院南院药学部, 福建福州 350001

收稿日期:2022-10-28 修回日期:2022-11-17 接受日期:2023-01-10 出版日期:2023-03-31 发布日期:2023-03-30
通讯作者: 庄捷
作者简介:
+ Tel.: +86-591-88216353, E-mail: 13860687823@163.com
基金资助:
Startup Fund for scientific research, Fujian Medical University (Grant No. 2017XQ010), Beijing Medical and Health Foundation (Grant No. B183023), and Natural Science Foundation of Fujian (Grant No. 2022J011011), China.

Warfarin affects the proliferation and apoptosis of lung cancer cells by regulating the Gas6/Axl/PI3K/Akt/NF-κB pathway

Limei Yang¹, Liman Wang², Xuhui Huang², Jie Zhuang¹^,^*()

1 Department of Pharmacy, Provincial Clinical College of Fujian Medical University/Fujian Provincial Hospital, Fuzhou 350001, Fujian, China
2 Department of Pharmacy, Fujian Provincial Hospital Jinshan Branch/Fujian Provincial Hospital South Branch, Fuzhou 350001, Fujian, China

Received:2022-10-28 Revised:2022-11-17 Accepted:2023-01-10 Online:2023-03-31 Published:2023-03-30
Contact: Jie Zhuang

摘要/Abstract

摘要：

华法林的抗肿瘤潜力已经在不同的细胞模型中证实了, 然而华法林降低肺癌发生率的可能机制尚不明确。本文的研究目的在于研究华法林对肺癌细胞株H226的增殖、凋亡的影响, 及其可能的机制。选择肺癌细胞株H226作为研究对象, 用浓度为128, 256和512 μmol/L的华法林干预后, 采用CCK8法测定细胞增殖情况, 流式细胞法和Weastern blot法评估华法林对细胞凋亡的影响, 然后分别用Weastern blot法和RT-PCR法检测华法林对Gas6/Axl/PI3K/Akt/NF-κB通路蛋白及基因表达的影响。结果发现, 华法林作用于H226细胞24, 48, 72 h后可抑制细胞的生长, 抑制效应呈剂量和时间相关性。流式细胞检测结果显示, 在128, 256和512 μmol/L华法林干预下, H226细胞凋亡比率显著增加; Bcl-2蛋白的表达下降, Bax蛋白和Capase3蛋白表达增加; 与空白组相比, 差异均有统计学意义(P < 0.05)。与对照组相比, 华法林和Axl抑制剂BGB324可以显著抑制Gas6/Axl通路及其下游通路蛋白的表达(P < 0.05), 并显著抑制Gas6/Axl/PI3K/Akt/NF-κB通路中基因的表达(P < 0.05)。研究发现, 华法林可以抑制肺癌细胞株H226增殖, 诱导H226凋亡, 其可能的机制与华法林抑制Gas6/Axl/PI3K/Akt/NF-κB通路蛋白和基因有关。

关键词: 华法林, 肺癌, 细胞增值, 细胞凋亡, 体外技术

Abstract:

The antitumor potential of warfarin is demonstrated in different experimental cancer model systems. However, the mechanism of warfarin in reducing the incidence of lung cancer is unclear. In the present study, we aimed to investigate the effect and the possible mechanism of warfarin on the proliferation and apoptosis of lung cancer cell line H226. It was an in vitro experiment. The lung cancer cell line H226 was intervened with warfarin at the concentrations of 128, 256, and 512 μmol/L. The cell proliferation was determined by CCK8 assay, and the cell apoptosis was evaluated by flow cytometry and Western blotting analysis. The effects of warfarin on Gas6/Axl/PI3K/Akt/NF-κB protein and gene expression were determined by Western blotting analysis and RT-PCR, respectively. Warfarin inhibited the growth of H226 cells at 24, 48, and 72 h, showing a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of H226 cells was significantly increased when treated with 128, 256, and 512 μmol/L warfarin. Meanwhile, the expression of Bcl-2 protein was decreased, and the expressions of Bax protein and Caspase 3 protein were increased, showing significant differences compared with the blank group (P < 0.05). Compared with the control group, warfarin and Axl inhibitor BGB324 could inhibit the protein expression of the Gas6/Axl pathway and its downstream pathway (P < 0.05) significantly and decrease the gene expression of the Gas6/Axl/PI3K/Akt/NF-κB pathway significantly (P < 0.05). Warfarin could inhibit the proliferation and induce the apoptosis of the lung cell line. The possible mechanism might be related to the inhibitory effect on Gas6/Axl/PI3K/Akt/NF-κB pathway proteins and genes.

Key words: Warfarin, Lung neoplasms, Cell proliferation, Apoptosis, In vitro techniques

Supporting:

阳丽梅, 王丽满, 黄旭慧, 庄捷. 华法林通过调控Gas6/Axl/PI3K/Akt/NF-κB通路影响肺癌细胞株的增殖和凋亡[J]. 中国药学(英文版), 2023, 32(3): 190-199.

Limei Yang, Liman Wang, Xuhui Huang, Jie Zhuang. Warfarin affects the proliferation and apoptosis of lung cancer cells by regulating the Gas6/Axl/PI3K/Akt/NF-κB pathway[J]. Journal of Chinese Pharmaceutical Sciences, 2023, 32(3): 190-199.

图/表 7

Table 1. The sequences of primer for RT-PCR.

Figure 1. Growth inhibition curve on H226 cells by warfarin measured by CCK8 method (n = 6).

Figure 2. Left: H226 cell apoptosis detected by flow cytometry treated with various concentrations of warfarin (a: blank group; b: 128 μmol/L warfarin group; c: 256 μmol/L warfarin group; d: 512 μmol/L warfarin group, n = 3). Right: histogram of apoptotic cell proportion.

Figure 3. H226 cell apoptosis detected by Western blotting analysis treated with various concentrations of warfarin. (Above: apoptosis-related protein expression by Western blotting analysis; below: Histogram of apoptosis-related protein expression; *compared with the normal group, P < 0.05, n = 3).

Figure 4. Protein expression of Gas6/Axl/PI3K/Akt/NF-κB pathway proteins detected by Western blotting analysis treated with various concentrations of warfarin on H226 cells. (Above: Gas6/Axl/PI3K/Akt/NF-κB pathway protein expression by Western blotting analysis; below: Histogram of Gas6/Axl/PI3K/Akt/NF-κB pathway protein expression; *compared with the normal group, P < 0.05, n = 3).

Table 2. Effects of warfarin on gene expressions of Gas6/Axl/PI3K/Akt/NF-κB pathway genes (n = 3).

Figure 5. A possible mechanism underlying the inhibitory effect of warfarin on tumor proliferation, apoptosis, and metastasis.

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华法林通过调控Gas6/Axl/PI3K/Akt/NF-κB通路影响肺癌细胞株的增殖和凋亡

Warfarin affects the proliferation and apoptosis of lung cancer cells by regulating the Gas6/Axl/PI3K/Akt/NF-κB pathway

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