桑黄酮G通过调控PI3K/AKT/mTOR通路抑制胃癌细胞的生长、迁移和侵袭

doi:10.12122/j.issn.1673-4254.2024.08.06

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (8): 1476-1484.doi: 10.12122/j.issn.1673-4254.2024.08.06

桑黄酮G通过调控PI3K/AKT/mTOR通路抑制胃癌细胞的生长、迁移和侵袭

耿志军¹^,⁴(), 杨晶晶², 牛民主³, 刘馨悦⁴, 施金冉⁴, 刘亦珂⁴, 姚新宇⁴, 张雨路⁴, 张小凤¹^,⁴, 胡建国⁴^,⁵()

^1.蚌埠医科大学第一附属医院，中心实验室，安徽蚌埠 233004
^2.蚌埠医科大学第一附属医院，胃肠外科，安徽蚌埠 233004
^5.蚌埠医科大学第一附属医院，检验科，安徽蚌埠 233004
^3.蚌埠医科大学检验医学院免疫学教研室，安徽蚌埠 233000
^4.炎症相关性疾病基础与转化研究安徽省重点实验室，安徽蚌埠 233004

收稿日期:2024-04-12 出版日期:2024-08-20 发布日期:2024-09-06
通讯作者: 胡建国 E-mail:gengzhijun1213@bbmc.edu.cn;jghu9200@bbmc.edu.cn
作者简介:耿志军，研究实习员，E-mail: gengzhijun1213@bbmc.edu.cn
基金资助:
安徽省高校协同创新项目(GXXT-2020-020);安徽省高等学校自然科研究项目(KJ2020A0563);安徽省卫生健康科研项目(AHWJ2022b088)

Kuwanon G inhibits growth, migration and invasion of gastric cancer cells by regulating the PI3K/AKT/mTOR pathway

Zhijun GENG¹^,⁴(), Jingjing YANG², Minzhu NIU³, Xinyue LIU⁴, Jinran SHI⁴, Yike LIU⁴, Xinyu YAO⁴, Yulu ZHANG⁴, Xiaofeng ZHANG¹^,⁴, Jianguo HU⁴^,⁵()

^1.Central Laboratory,
^2.Department of Gastrointestinal Surgery,
^5.Clinical Laboratory, First Affiliated Hospital of Bengbu Medical University, Bengbu 233003, China
^3.Department of Immunology, School of Laboratory Medicine, Bengbu Medical University, Bengbu 233000, China
^4.Anhui Provincial Key Laboratory of Basic and Translational Research of Inflammation-related Diseases, Bengbu 233003, China

Received:2024-04-12 Online:2024-08-20 Published:2024-09-06
Contact: Jianguo HU E-mail:gengzhijun1213@bbmc.edu.cn;jghu9200@bbmc.edu.cn

摘要/Abstract

摘要：

目的探讨桑黄酮G（KG）对胃癌细胞的增殖、凋亡、迁移和侵袭的作用和分子机制。方法通过CCK-8实验、细胞克隆形成实验、裸鼠背部成瘤和Ki-67免疫组织化学染色评估不同浓度KG对胃癌细胞增殖与生长的影响；应用Annexin V-FITC/PI细胞凋亡检测试剂盒、Western blotting分析凋亡相关蛋白表达和Tunel染色评估KG对胃癌细胞凋亡的影响；使用Transwell迁移和侵袭实验与Western blotting分析基质金属蛋白酶表达检测KG对胃癌细胞迁移和侵袭的作用；利用免疫印迹技术和rescue实验验证PI3K/AKT/mTOR通路在KG调控胃癌细胞的增殖、迁移和侵袭的作用。结果 KG以浓度依赖性抑制胃癌细胞的增殖和减少细胞形成克隆数（P<0.05）；KG上调凋亡细胞比例，促进cleaved caspase-3、Bcl2-Bax表达并下调Bcl2水平（P<0.05）；KG干扰胃癌细胞的迁移和侵袭，并且抑制基质金属蛋白酶2（MMP2）和基质金属蛋白酶9（MMP9）的表达（P<0.05）。机制验证显示，KG抑制PI3K/AKT/mTOR通路的激活，且PI3K/AKT/mTOR通路的激活剂IGF-1逆转了KG对胃癌细胞增殖、迁移和侵袭的影响（P<0.05）。结论 KG至少部分是通过抑制PI3K/AKT/mTOR通路的激活影响胃癌细胞的增殖、凋亡、迁移和侵袭。

关键词: 胃癌, 桑黄酮G, 恶性行为, PI3K/AKT/mTOR通路

Abstract:

Objective To investigate the effects of kuwanon G (KG) on proliferation, apoptosis, migration and invasion of gastric cancer cells and the molecular mechanisms. Methods The effects of KG on proliferation and growth of gastric cancer cells were assessed with CCK-8 assay and cell clone formation assay, by observing tumor formation on the back of nude mice and using immunohistochemical analysis of Ki-67. The effect of KG on cell apoptosis was analyzed using Annexin V-FITC/PI apoptosis detection kit, Western blotting and TUNEL staining. The effects of KG on cell migration and invasion were detected using Transwell migration and invasion assay and Western blotting for matrix metalloproteinase (MMP). The role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in KG-mediated regulation of gastric cancer cell proliferation, migration, and invasion was verified by Western blotting and rescue assay. Results KG significantly inhibited proliferation and reduced clone formation ability of gastric cancer cells in a concentration-dependent manner (P<0.05). KG treatment also increased apoptosis, enhanced the expressions of cleaved caspase-3 and Bax, down-regulated Bcl-2, lowered migration and invasion capacities and inhibited the expression of MMP2 and MMP9 in gastric cancer cells (P<0.05). Mechanistic validation showed that KG inhibited the activation of the PI3K/AKT/mTOR pathway, and IGF-1, an activator of the PI3K/AKT/mTOR pathway, reversed the effects of KG on proliferation, migration and invasion of gastric cancer cells (P<0.05). Conclusion KG inhibits proliferation, migration and invasion and promotes apoptosis of gastric cancer cells at least in part by inhibiting the activation of the PI3K/AKT/mTOR pathway.

Key words: gastric cancer, kuwanon G, malignant behaviors, PI3K/AKT/mTOR pathway

耿志军, 杨晶晶, 牛民主, 刘馨悦, 施金冉, 刘亦珂, 姚新宇, 张雨路, 张小凤, 胡建国. 桑黄酮G通过调控PI3K/AKT/mTOR通路抑制胃癌细胞的生长、迁移和侵袭[J]. 南方医科大学学报, 2024, 44(8): 1476-1484.

Zhijun GENG, Jingjing YANG, Minzhu NIU, Xinyue LIU, Jinran SHI, Yike LIU, Xinyu YAO, Yulu ZHANG, Xiaofeng ZHANG, Jianguo HU. Kuwanon G inhibits growth, migration and invasion of gastric cancer cells by regulating the PI3K/AKT/mTOR pathway[J]. Journal of Southern Medical University, 2024, 44(8): 1476-1484.

图/表 6

图1 KG对胃癌细胞增殖的影响

Fig.1 Effect of KG on proliferation of gastric cancer cells. A, D, E, H: CCK-8 assay for assessing proliferative activity of gastric cancer cells. B, C, F, G: Number of gastric cancer cell clones in colony formation assay. *P<0.05 vs Control, 0 μmol/L.

图3 KG对胃癌细胞迁移和侵袭的影响

Fig.3 KG inhibits migration and invasion of gastric cancer cells. A-D: Transwell migration and invasion assay for analyzing cell migration and invasion. E, F: Western blotting and quantification of MMP2 and MMP9 expressions. *P<0.05 vs Control (0 μmol/L).

图4 KG抑制胃癌细胞增殖、迁移和侵袭与PI3K/AKT/mTOR通路的相关

Fig.4 KG-induced inhibition of gastric cancer cell proliferation, migration and invasion is correlated with activation of the PI3K/AKT/mTOR pathway. A, B: Western blotting and quantification of p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR expressions. C: CCK-8 assay for assessing cell proliferative activity. D, E: Western blotting and quantification of MMP2 and MMP9 expressions. *P<0.05 vs Control, #P<0.05 vs KG.

图5 裸鼠体内验证KG对移植瘤生长的影响

Fig.5 Effect of KG on subcutaneous xenograft growth in nude mice. A, C: Volume and size of the xenografts in different groups. B: Immunohistochemistry for and quantitative analysis of Ki-67 expression in the xenografts. *P<0.05 vs Con.

图6 在体研究KG对胃癌细胞凋亡和侵袭的影响

Fig.6 Effect of KG on apoptosis and invasion of gastric cancer cells in nude mice. A, B: TUNEL staining of the xenografts. C, D: Western blotting and quantification of cleaved-caspase 3, Bax and Bcl2 expressions. E, F: Western blotting and quantification of MMP2 and MMP9 expressions. *P<0.05 vs Control.

图7 在体研究KG对PI3K/AKT/mTOR通路的影响

Fig.7 Effect of KG on the PI3K/AKT/mTOR pathway the xenografts in nude mice. A, B: Western blotting and quantification of p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR expressions. *P<0.05 vs Con.

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桑黄酮G通过调控PI3K/AKT/mTOR通路抑制胃癌细胞的生长、迁移和侵袭

Kuwanon G inhibits growth, migration and invasion of gastric cancer cells by regulating the PI3K/AKT/mTOR pathway

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