川陈皮素调节FAK/AKT信号通路对喉鳞状细胞癌细胞增殖和凋亡的影响

doi:10.11958/20250433

摘要/Abstract

摘要：

目的探究川陈皮素对喉鳞状细胞癌（LSCC）细胞增殖和凋亡的影响，及其机制是否与黏着斑激酶（FAK）/蛋白激酶B（AKT）信号通路有关。方法将LSCC TU177细胞分为对照（Control）组、L-川陈皮素组（10 mg/L）、M-川陈皮素组（20 mg/L）、H-川陈皮素组（40 mg/L）和H-川陈皮素+ZINC40099027组（40 mg/L川陈皮素+2.5 g/L FAK激活剂ZINC40099027）。采用CCK-8法检测细胞活性，流式细胞术检测细胞凋亡，划痕实验检测细胞迁移，Transwell法检测细胞侵袭，Western blot检测细胞凋亡相关蛋白及FAK/AKT通路相关蛋白。结果 L、M、H-川陈皮素组较Control组细胞活性、划痕愈合率、侵袭细胞数、B细胞淋巴瘤-2（Bcl-2）、p-FAK/FAK、p-AKT/AKT蛋白表达呈剂量依赖性降低，细胞凋亡率、Bcl-2相关X蛋白（Bax）、胱天蛋白酶-3（caspase-3）蛋白表达呈剂量依赖性升高（P<0.05）；H-川陈皮素+ZINC40099027组较H-川陈皮素组OD值、划痕愈合率、侵袭细胞数、Bcl-2、p-FAK/FAK、p-AKT/AKT蛋白表达增加，细胞凋亡率、Bax、caspase-3蛋白表达降低（P<0.05）。结论川陈皮素可能通过抑制FAK/AKT信号通路抑制LSCC细胞增殖并促进其凋亡。

关键词: 喉肿瘤, 癌，鳞状细胞, 川陈皮素, 黏着斑蛋白酪氨酸激酶类, 原癌基因蛋白质c-akt, 细胞增殖, 细胞凋亡

Abstract:

Objective To investigate the effect of nobiletin on the proliferation and apoptosis of laryngeal squamous cell carcinoma (LSCC) cells and its mechanism related to the focal adhesion kinase (FAK)/protein kinase B (AKT) signaling pathway. Methods TU177 cells of LSCC were randomly assigned into the control group, the L-nobiletin group (10 mg/L) group, the M-nobiletin group (20 mg/L), the H-nobiletin group (40 mg/L) and the H-nobiletin+FAK activator ZINC40099027 group (40 mg/L nobiletin+2.5 g/L ZINC40099027). CCK-8 method was used to detect cell viability. Flow cytometry was used to detect cell apoptosis. Scratch experiment was used to detect cell migration. Transwell method was used to detect cell invasion. Western blot assay was used to detect apoptosis and FAK/AKT pathway related proteins. Results Compared with the control group, the cell viability, scratch healing rate, number of invasive cells, the protein expressions of Bcl-2, p-FAK/FAK and p-AKT/AKT were decreased in a dose-dependent manner in the L-nobiletin group, the M-nobiletin group and the H-nobiletin group, while the cell apoptosis rate and protein expressions of Bax and caspase-3 were increased in a dose-dependent manner (P<0.05). The OD value, scratch healing rate, number of invasive cells, Bcl-2, p-FAK/FAK and p-AKT/AKT protein expression levels were higher in the H-nobiletin+ZINC40099027 group than those of the H-nobiletin group, and cell apoptosis rate, Bax and caspase-3 protein expression were lower compared to the H-nobiletin group (P<0.05). Conclusion Nobiletin may inhibit the proliferation and promote the apoptosis of LSCC cells by suppressing the FAK/AKT signaling pathway.

Key words: laryngeal neoplasms, carcinoma, squamous cell, nobiletin, focal adhesion protein-tyrosine kinases, proto-oncogene proteins c-akt, cell proliferation, apoptosis

中图分类号:

R739.65

图/表 9

表1 不同剂量组的川陈皮素对TU177细胞活性、凋亡率的影响（n=6，$\bar{x} \pm s$）

Tab.1 The effects of nobiletin on the cell viability and apoptosis rate of TU177 cells

组别	OD值	凋亡率/%
Control组	1.65±0.17	8.67±0.91
L-川陈皮素组	1.27±0.13^a	13.86±1.41^a
M-川陈皮素组	0.86±0.09^ab	18.12±1.86^ab
H-川陈皮素组	0.51±0.06^abc	23.52±2.40^abc
H-川陈皮素+ZINC40099027组	1.31±0.14^d	10.43±1.09^d
F	75.798^＊＊	81.980^＊＊

图1 流式细胞术检测川陈皮素对TU177细胞凋亡的影响 A：Control组；B：L-川陈皮素组；C：M-川陈皮素组；D：H-川陈皮素组；E：H-川陈皮素+ZINC40099027组；图2—5同。

Fig.1 The effect of nobiletin on the apoptosis of TU177 cells detected by flow cytometry

图2 划痕实验检测川陈皮素对TU177细胞迁移的影响（×200）

Fig.2 The effect of nobiletin on the migration of TU177 cells detected by scratch assay (×200)

图3 Transwell法检测川陈皮素对TU177细胞侵袭的影响（结晶紫染色，×200）

Fig.3 The effect of nobiletin on the invasion of TU177 cells detected by Transwell assay (crystal violet staining, ×200)

表2 不同剂量组的川陈皮素对TU177细胞迁移、侵袭的影响（n=6，$\bar{x} \pm s$）

Tab.2 The effect of nobiletin on the migration and invasion of TU177 cells

组别	划痕愈合率/%	侵袭细胞数/个
Control组	83.62±8.53	186.32±19.12
L-川陈皮素组	68.51±6.92^a	143.68±14.51^a
M-川陈皮素组	51.37±5.15^ab	119.62±12.33^ab
H-川陈皮素组	39.50±4.01^abc	80.55±8.17^abc
H-川陈皮素+ZINC40099027组	70.34±7.11^d	145.27±14.66^d
F	41.968^＊＊	44.681^＊＊

图4 Western blot检测川陈皮素对TU177细胞凋亡相关蛋白的影响

Fig.4 The effect of nobiletin on apoptosis-related proteins in TU177 cells detected by Western blot assay

表3 不同剂量组的川陈皮素对TU177细胞凋亡相关蛋白的影响（n=6，$\bar{x} \pm s$）

Tab.3 The effect of various concentrations of nobiletin on apoptosis-related proteins in TU177 cells

组别	Bax/GAPDH	Bcl-2/GAPDH	caspase-3/GAPDH
Control组	0.62±0.07	1.42±0.15	0.29±0.03
L-川陈皮素组	0.89±0.09^a	1.13±0.12^a	0.68±0.07^a
M-川陈皮素组	1.08±0.11^ab	0.92±0.10^ab	0.85±0.09^ab
H-川陈皮素组	1.33±0.14^abc	0.61±0.07^abc	0.99±0.11^abc
H-川陈皮素+ ZINC40099027组	0.81±0.09^d	1.21±0.13^d	0.52±0.06^d
F	41.665^＊＊	41.384^＊＊	76.449^＊＊

图5 Western blot检测川陈皮素对FAK/AKT通路相关蛋白的影响

Fig.5 The effect of nobiletin on the proteins related to the FAK/AKT pathway detected by Western blot assay

表4 不同剂量组的川陈皮素对FAK/AKT通路相关蛋白的影响（n=6，$\bar{x} \pm s$）

Tab.4 The effects of nobiletin in different dose groups on proteins related to FAK/AKT pathway

组别	p-FAK/FAK	p-AKT/AKT
Control组	0.88±0.09	0.81±0.09
L-川陈皮素组	0.65±0.07^a	0.59±0.06^a
M-川陈皮素组	0.43±0.05^ab	0.40±0.05^ab
H-川陈皮素组	0.31±0.04^abc	0.18±0.02^abc
H-川陈皮素+ZINC40099027组	0.70±0.08^d	0.63±0.07^d
F	65.017^＊＊	88.877^＊＊

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