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.

References 32

1	Smyth EC, Nilsson M, Grabsch HI, et al. Gastric cancer[J]. Lancet, 2020, 396(10251): 635-48.
2	Guan WL, He Y, Xu RH. Gastric cancer treatment: recent progress and future perspectives[J]. J Hematol Oncol, 2023, 16(1): 57-65.
3	Ajani JA, D’Amico TA, Bentrem DJ, et al. Gastric cancer, version 2.2022, NCCN clinical practice guidelines in oncology[J]. J Natl Compr Canc Netw, 2022, 20(2): 167-92.
4	Zeng YJ, Jin RU. Molecular pathogenesis, targeted therapies, and future perspectives for gastric cancer[J]. Semin Cancer Biol, 2022, 86(Pt 3): 566-82.
5	Sexton RE, Al Hallak MN, Diab M, et al. Gastric cancer: a comprehensive review of current and future treatment strategies[J]. Cancer Metastasis Rev, 2020, 39(4): 1179-203.
6	Hu X, Pan GZ, Luo JL, et al. Kuwanon H inhibits melanoma growth through cytotoxic endoplasmic reticulum stress and impaired autophagy flux[J]. J Agric Food Chem, 2023, 71(37): 13768-82.
7	Ma MJ, Luan XY, Zheng H, et al. A mulberry diels-alder-type adduct, kuwanon M, triggers apoptosis and paraptosis of lung cancer cells through inducing endoplasmic reticulum stress[J]. Int J Mol Sci, 2023, 24(2): 1015.
8	Koirala P, Seong SH, Zhou YJ, et al. Structure⁻Activity relationship of the tyrosinase inhibitors kuwanon G, mulberrofuran G, and albanol B from Morus species: a kinetics and molecular docking study[J]. Molecules, 2018, 23(6): 1413-26.
9	Motallebi M, Bhia M, Rajani HF, et al. Naringenin: a potential flavonoid phytochemical for cancer therapy[J]. Life Sci, 2022, 305: 120752-63.
10	Guo C, Huang QX, Wang YS, et al. Therapeutic application of natural products: NAD⁺ metabolism as potential target[J]. Phytomedicine, 2023, 114: 154768.
11	Rong L, Li ZD, Leng X, et al. Salidroside induces apoptosis and protective autophagy in human gastric cancer AGS cells through the PI3K/Akt/mTOR pathway[J]. Biomedecine Pharmacother, 2020, 122: 109726-40.
12	Morana O, Wood W, Gregory CD. The apoptosis paradox in cancer[J]. Int J Mol Sci, 2022, 23(3): 1328-39.
13	Bradley RK, Anczuków O. RNA splicing dysregulation and the hallmarks of cancer[J]. Nat Rev Cancer, 2023, 23(3): 135-55.
14	Pu HC, Qian Q, Wang FL, et al. SchizandrinA induces the apoptosis and suppresses the proliferation, invasion and migration of gastric cancer cells by activating endoplasmic reticulum stress[J]. Mol Med Rep, 2021, 24(5): 787-92.
15	Song CR, Shen T, Kim HG, et al. 20(S)-protopanaxadiol from Panax ginseng induces apoptosis and autophagy in gastric cancer cells by inhibiting src[J]. Am J Chin Med, 2023, 51(1): 205-21.
16	Li X, Ma XL, Nan Y, et al. 18β-glycyrrhetinic acid inhibits proliferation of gastric cancer cells through regulating the miR-345-5p/TGM2 signaling pathway[J]. World J Gastroenterol, 2023, 29(23): 3622-44.
17	Guo QR, Zhou WM, Zhang GB, et al. Jaceosidin inhibits the progression and metastasis of NSCLC by regulating miR-34c-3p/Integrin α2β1 axis[J]. Heliyon, 2023, 9(5): e16158.
18	Park KR, Jeong Y, Lee J, et al. Anti-tumor effects of jaceosidin on apoptosis, autophagy, and necroptosis in human glioblastoma multiforme[J]. Am J Cancer Res, 2021, 11(10): 4919-30.
19	Liu J, Li SM, Tang YJ, et al. Jaceosidin induces apoptosis and inhibits migration in AGS gastric cancer cells by regulating ROS-mediated signaling pathways[J]. Redox Rep, 2024, 29(1): 2313366-75.
20	Wei ZC, Chen J, Zuo F, et al. Traditional Chinese Medicine has great potential as candidate drugs for lung cancer: a review[J]. J Ethnopharmacol, 2023, 300: 115748.
21	Chimento A, de Luca A, D'Amico M, et al. The involvement of natural polyphenols in molecular mechanisms inducing apoptosis in tumor cells: a promising adjuvant in cancer therapy[J]. Int J Mol Sci, 2023, 24(2): 1680-9.
22	Imran M, Rauf A, Abu-Izneid T, et al. Luteolin, a flavonoid, as an anticancer agent: a review[J]. Biomedecine Pharmacother, 2019, 112: 108612-23.
23	Li GZ, Doherty GM, Wang JP. Surgical management of gastric cancer: a review[J]. JAMA Surg, 2022, 157(5): 446-54.
24	Song SY, Su ZL, Xu H, et al. Luteolin selectively kills STAT3 highly activated gastric cancer cells through enhancing the binding of STAT3 to SHP-1[J]. Cell Death Dis, 2017, 8(2): e2612-25.
25	Sokolova O, Naumann M. Matrix metalloproteinases in Helicobacter pylori-associated gastritis and gastric cancer[J]. Int J Mol Sci, 2022, 23(3): 1883-95.
26	Wu LR, Qian CC, Zhang WQ, et al. Ginkgetin suppresses ovarian cancer growth through inhibition of JAK2/STAT3 and MAPKs signaling pathways[J]. Phytomedicine, 2023, 116: 154846-55.
27	Xia PF, Liang J, Jin D, et al. Reversine inhibits proliferation, invasion and migration and induces cell apoptosis in gastric cancer cells by downregulating TTK[J]. Exp Ther Med, 2021, 22(3): 929-43.
28	Wang C, Yang Z, Xu E, et al. Apolipoprotein C-II induces EMT to promote gastric cancer peritoneal metastasis via PI3K/AKT/mTOR pathway[J]. Clin Transl Med, 2021, 11(8): e522-9.
29	Morgos DT, Stefani C, Miricescu D, et al. Targeting PI3K/AKT/mTOR and MAPK signaling pathways in gastric cancer[J]. Int J Mol Sci, 2024, 25(3): 1848-57.
30	Chen G, Zhang HH, Sun HX, et al. Bufalin targeting BFAR inhibits the occurrence and metastasis of gastric cancer through PI3K/AKT/mTOR signal pathway[J]. Apoptosis, 2023, 28(9/10): 1390-405.
31	Lee HJ, Venkatarame Gowda Saralamma V, Kim SM, et al. Pectolinarigenin induced cell cycle arrest, autophagy, and apoptosis in gastric cancer cell via PI3K/AKT/mTOR signaling pathway[J]. Nutrients, 2018, 10(8): 1043-52.
32	Zhang XR, Wang SY, Sun W, et al. Isoliquiritigenin inhibits proliferation and metastasis of MKN28 gastric cancer cells by suppressing the PI3K/AKT/mTOR signaling pathway[J]. Mol Med Rep, 2018, 18(3): 3429-36.

[1]	Yongsheng XIA, Lian WANG, Xiaohua CHEN, Yulu ZHANG, Aofei SUN, Deli CHEN. TSR2 overexpression inhibits proliferation and invasion of gastric cancer cells by downregulating the PI3K/AKT signaling pathway [J]. Journal of Southern Medical University, 2024, 44(5): 913-919.
[2]	LIANG Yihao, LAI Yingjun, YUAN Yanwen, YUAN Wei, ZHANG Xibo, ZHANG Bashan, LU Zhifeng. Screening of differentially expressed genes in gastric cancer based on GEO database and function and pathway enrichment analysis [J]. Journal of Southern Medical University, 2024, 44(3): 605-616.
[3]	SHEN Mengdi, ZHAO Na, DENG Xiaojing, DENG Min. High expression of COX6B2 in gastric cancer is associated with poor long-term prognosis and promotes cell proliferation and cell cycle progression by inhibiting p53 signaling [J]. Journal of Southern Medical University, 2024, 44(2): 289-297.
[4]	ZHANG Nuo, ZHANG Zhen, ZHANG Yulu, SONG Xue, ZHANG Xiaofeng, LI Jing, ZUO Lugen, HU Jianguo. PCID2 is highly expressed in gastric cancer and affects the prognosis by regulating cancer cell cycle and proliferation [J]. Journal of Southern Medical University, 2024, 44(2): 324-332.
[5]	ZHANG Wenjing, ZHANG Nuo, YANG Zi, ZHANG Xiaofeng, SUN Aofei, WANG Lian, SONG Xue, GENG Zhijun, LI Jing, HU Jianguo. Overexpression of BZW1 promotes invasion and metastasis of gastric cancer cells by regulating Wnt/β-catenin signaling and promoting epithelial-mesenchymal transition [J]. Journal of Southern Medical University, 2024, 44(2): 354-362.
[6]	DUAN Ting, ZHANG Zhen, SHI Jinran, XIAO Linyu, YANG Jingjing, YIN Lixia, ZHANG Xiaofeng, GENG Zhijun, LU Guoyu. High expression of CPNE3 correlates with poor long-term prognosis of gastric cancer by inhibiting cell apoptosis via activating PI3K/AKT signaling [J]. Journal of Southern Medical University, 2024, 44(1): 129-137.
[7]	FENG Wen, LAI Yuexing, WANG Jing, XU Ping. Long non-coding RNA ABHD11-AS1 promotes glycolysis in gastric cancer cells to accelerate tumor progression [J]. Journal of Southern Medical University, 2023, 43(9): 1485-1492.
[8]	WANG Lian, XIA Yongsheng, ZHANG Zhen, LIU Xinyue, SHI Jinran, WANG Yueyue, LI Jing, ZHNAG Xiaofeng, GENG Zhijun, SONG Xue, ZUO Lugen. High expression of MRPL13 promotes cell cycle progression and proliferation of gastric cancer cells by inhibiting p53 signaling to affect long-term prognosis [J]. Journal of Southern Medical University, 2023, 43(9): 1558-1566.
[9]	ZUO Lugen, WANG Lian, YANG Zi, LI Junjie, WANG Wenfeng, LI Jing, WANG Yueyue, SONG Xue, ZHNAG Xiaofeng, GENG Zhijun. High expression of CAMSAP2 promotes invasion and metastasis of gastric cancer cells by upregulating TGF-β signaling [J]. Journal of Southern Medical University, 2023, 43(9): 1460-1468.
[10]	WANG Qiusheng, ZHANG Zhen, WANG Lian, WANG Yu, YAO Xinyu, WANG Yueyue, ZHANG Xiaofeng, GE Sitang, ZUO Lugen. High expression of death-associated protein 5 promotes glucose metabolism in gastric cancer cells and correlates with poor survival outcomes [J]. Journal of Southern Medical University, 2023, 43(7): 1063-1070.
[11]	ZHANG Hao, ZHANG Zhen, WANG Qiusheng, WANG Lian, YANG Zi, GENG Zhijun, WANG Yueyue, LI Jing, ZUO Lugen. FJX1 overexpression is associated with poor prognosis and promotes gastric cancer proliferation via the PI3K/AKT signaling pathway [J]. Journal of Southern Medical University, 2023, 43(6): 975-984.
[12]	GE Fei, WAN Mengqi, CHENG Zhenyu, CHEN Xuelei, CHEN Qianyi, QI Zhilin. Aloin inhibits gastric cancer cell proliferation and migration by suppressing the STAT3/HMGB1 signaling pathway [J]. Journal of Southern Medical University, 2023, 43(5): 702-709.
[13]	WU Jiaming, DENG Zhongquan, ZHU Yi, DOU Guangjian, LI Jin, HUANG Liyong. Overexpression of miR-431-5p impairs mitochondrial function and induces apoptosis in gastric cancer cells via the Bax/Bcl-2/caspase3 pathway [J]. Journal of Southern Medical University, 2023, 43(4): 537-543.
[14]	LÜ Chunxin, WU Xiuqi, XIA Qing. Multiple primary malignancies combined with SWI/SNF complex-deficient gastric cancer: a case report and literature review [J]. Journal of Southern Medical University, 2023, 43(3): 495-498.
[15]	GONG Ying, AIMAITI Ailifeire, HE Zongzhong. ARL67156, a small-molecule CD39 inhibitor, enhances natural killer cell cytotoxicity against gastric cancer cells in vitro and in nude mice [J]. Journal of Southern Medical University, 2023, 43(12): 2006-2014.