Anwulignan inhibits malignant phenotypes of gastric cancer cells by antagonizing activation of the JAK1/STAT3 signaling pathway

doi:10.12122/j.issn.1673-4254.2026.09.22

Abstract

Abstract:

Methods To evaluate the inhibitory effect of anwulignan (ANW) on malignant biological behaviors of gastric cancer cells and explore its underlying molecular mechanism. Methods The inhibitory effect of ANW on viability and proliferation of gastric cancer HGC-27 and SGC-7901 cells were evaluated using CCK-8 assay, EdU staining and colony formation assay. In a nude mouse model bearing gastric cancer xenografts, the effect of ANW on tumor cell proliferation was examined using immunohistochemistry for Ki-67. The changes in cell cycle, apoptosis, migration, invasion, and expressions of MMPs and epithelial-mesenchymal transition (EMT) markers following ANW treatment were detected using flow cytometry, TUNEL staining, Western blotting, Transwell assay, and qPCR. The role of the JAK1/STAT3 signaling pathway in mediating the anti-tumor activity of ANW was validated by detecting JAK1/STAT3 phosphorylation, pathway activation and siRNA-mediated JAK1 knockdown experiments. Results In HGC-27 and SGC-7901 cells, ANW dose-dependently suppressed cell proliferation and colony formation, induced G1 cell cycle arrest, and downregulated the expressions of cyclin D1/CDK2 and P53. ANW treatment significantly inhibited tumor growth and decreased Ki-67 positivity in the mouse xenografts. ANW effectively promoted cell apoptosis, suppressed Bcl-2 expression, upregulated cleaved caspase-3 and Bax expressions, inhibited migration and invasion, lowered MMP-2/MMP-9 expressions, and blocked EMT progression in gastric cancer cells. Mechanistically, ANW significantly inhibited JAK1/STAT3 phosphorylation, while the JAK1/STAT3 activator RO8191 reversed the anti-tumor effect of ANW; JAK1 knockdown mimicked and enhanced the inhibitory effect of ANW on gastric cancer cells. ANW significantly inhibited EMT in the mouse xenografts, and this effect was attenuated by RO8191 treatment. Conclusion ANW inhibits malignant phenotype of gastric cancer cells by inhibiting the activation of the JAK1/STAT3 signaling pathway, thereby inducing cell cycle arrest, promoting apoptosis, and suppressing EMT and MMP-mediated invasion and migration, suggesting the potential of ANW as a therapeutic agent for gastric cancer.

Key words: gastric cancer, anwuligan, malignant behavior, epithelial-mesenchymal transition, JAK1/STAT3 signaling pathway

Xinyue LIU, Keni ZHANG, Tong QIAO, Lin YIN, Longtao ZHANG, Xinke CHENG, Ben WANG, Jinlin CAO, Zhijun GENG, Hao ZHAO. Anwulignan inhibits malignant phenotypes of gastric cancer cells by antagonizing activation of the JAK1/STAT3 signaling pathway[J]. Journal of Southern Medical University, 2026, 46(9): 2242-2257.

Figures/Tables 15

Tab.1 Primer sequences for RT-qPCR

Gene	Forward (5'-3')	Reverse (5'-3')
Vimentin	TCTATACCACTTCACAAGTCGGA	GAATTGCCATTGCACAACTCTTT
CDH1	GATAGAGAACGCATTGCCACATA	ACCTTCCATGACAGACCCCTTAA
GAPDH	TGAAGGTCGGAGTCAACGGAT	CTGGAAGATGGTGATGGGATT

Fig.1 Effect of ANW on proliferation of gastric cancer cells in vitro. A, B: Cell viability detected by CCK-8 assay. C-E, H: Colony formation assay. F, G, I, J: EdU assay for evaluating cell proliferation. *P<0.05 vs Control (0 μmol/L). ANW: Anwulignan; Con: Control; EdU: 5-ethynyl-2'-deoxyuridine.

Fig.2 Inhibitory effect of ANW on xenograft tumor growth in nude mice. A-C: Tumor volume and weight. D: Representative images and quantitative analysis of Ki-67 staining. *P<0.05 vs Control (0 μmol/L).

Fig.3 Effect of ANW on cell cycle in gastric cancer cells'. A-D: Flow cytometric analysis of cell cycle distribution. E-H: Western blotting analysis of cyclin D1, CDK2, and P53 protein expression in gastric cancer cell lines HGC 27 and SGC 7901. I, J: Western blotting analysis of cyclin D1, CDK2, and P53 protein expression in mouse xenografts. *P<0.05 vs Control (0 μmol/L). Cyclin-D1: G1/S-specific cyclin-D1; CDK2: Cyclin-dependent kinase 2; P53: Tumor protein p53; β-actin: Beta actin.

Fig.4 Flow cytometric analysis of the effects of ANW on apoptosis in gastric cancer cells. A-D: Cell aspoptosis rates detected by flow cytometry. *P<0.05 vs Control (0 μmol/L).

Fig.5 Western blotting and TUNEL analysis of the effects of ANW on apoptosis in gastric cancer cells. A-D: Western blotting analysis of apoptosis-related proteins gastric cancer cells in vitro. E, F: Western blotting analysis of apoptosis-related proteins in mouse xenografts. G, H: TUNEL staining of xenograft tissues. *P<0.05 vs Control (0 μmol/L).

Fig.6 Transwell and wound healing assays of migration and invasion in ANW-treated gastric cancer cells. A-C, E: Transwell migration and invasion assay. D, F, G, H: Wound healing assay. *P<0.05 vs Control (0 μmol/L).

Fig.7 Western blotting for evaluating the effects of ANW on migration and invasion in gastric cancer cells. A, B: Western blotting and quantification of MMP2 and MMP9 expressions in gastric cancer cells in vitro. C: Western blotting and quantification of MMP2 and MMP9 expressions in mouse xenografts. *P<0.05 vs Control (0 μmol/L). MMP2: Matrix Metalloproteinase 2; MMP9: Matrix Metalloproteinase 9.

Fig.8 Transwell analysis of the effects of RO8191 on the inhibitory action of ANW on malignant phenotype and EMT in gastric cancer cells. A-C: Transwell migration and invasion assay. *P<0.05 vs Control; #P<0.05 vs ANW.

Fig.9 Western blotting analysis of the effects of RO8191 on the inhibitory action of ANW on malignant phenotype and EMT in gastric cancer cells. A-H: The expression of proteins related to migration, invasion, apoptosis, and EMT. *P<0.05 vs Control; #P<0.05 vs ANW.

Fig.10 Effect of ANW on epithelial-mesenchymal transition in gastric cancer cells. A-C: Western blotting of EMT-related proteins in vitro and in vivo. D: RT-qPCR analysis of EMT-related gene expression. *P<0.05 vs Control (0 μmol/L). Snail+slug: Snail family zinc finger transcription factors; E-cad: Epithelial cadherin; VIM: Vimentin; CDH1: Cadherin 1.

Fig.11 Effect of ANW on the JAK1/STAT3 signaling pathway. A, B: Western blotting of p-JAK1, JAK1, p-STAT3, and STAT3 in vitro. C: Western blotting analysis of pathway proteins in vivo. *P<0.05vs Control (0 μmol/L). p-STAT3: Phosphorylated signal transducer and activator of transcription 3; STAT3: Signal transducer and activator of transcription 3; p-JAK1: Phosphorylated Janus kinase 1; JAK1: Janus kinase 1.

Fig.12 Transwell analysis of the effects of JAK1/STAT3 pathway silencing on the inhibitory action of ANW on malignant phenotype and EMT in gastric cancer cells. A-C: Transwell migration and invasion assay. *P<0.05 vs Control; #P<0.05 vs ANW.

Fig.13 Western blotting of the effects of JAK1/STAT3 pathway silencing on the inhibitory action of ANW on malignant phenotype and EMT in gastric cancer cells. A-H: Expression of proteins related to migration, invasion, apoptosis, and EMT. *P<0.05 vs Control; #P<0.05 vs ANW.

Fig.14 Validation of pathway mechanism in nude mice. A-C: Tumor volume and weight. D: Expression of proteins related to EMT. *P<0.05 vs Control; #P<0.05 vs ANW.

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