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

doi:10.12122/j.issn.1673-4254.2024.08.06

Abstract

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

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.

Figures/Tables 6

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.

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).

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.

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.

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.

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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