Antitumor component-Ι in Agkistrodon halys venom inhibits proliferation and migration of cisplatin-resistant gastric cancer cells by downregulating RAI14

doi:10.12122/j.issn.1673-4254.2026.01.12

Abstract

Abstract:

Objective To evaluate the inhibitory effect of antitumor component-Ι in Agkistrodon halys venom (AHVAC-I) on proliferation and migration of cisplatin-resistant gastric cancer cells and explore the underlying mechanism. Methods Cisplatin-resistant MKN-28 (MKN-28/DDP) cells were obtained by continuous exposure of MKN-28 cells to stepwise-increasing concentrations of cisplatin. MKN-28/DDP cells were treated with different concentrations of AHVAC-I, and the changes in proliferation, migration and invasion of the cells were examined with colony-forming assay, CCK-8 assay wound-healing assay, and Transwell assay. Western blotting was performed to examine the effect of AHVAC-I on expressions of epithelial-mesenchymal transition (EMT) markers of MKN-28/DDP cells; the changes in protein and mRNA expression of retinoic acid induced 14 (RAI14) was detected with Western blotting and qRT-PCR. Results Treatment with 2, 4, and 8 μg/mL AHVAC-I significantly inhibited proliferative, migratory and invasion abilities and reduced the expressions of EMT markers in MKN-28/DDP cells. Compared with MKN-28 cells, MKN-28/DP cells showed an increased expression of RAI14, which was significantly lowered after treatment with AHVAC-I. Supplementation with exogenous RAI14 obviously attenuated the inhibitory effect of AHVAC-I on proliferation and migration of MKN-28/DDP cells. Conclusion AHVAC-I decreases proliferation and invasion of MKN-28/DDP cells by downregulating RAI14 expression.

Key words: Agkistrodon halys venom, gastric cancer, cisplatin resistance, invasion, proliferation, retinoic acid induced 14

Yanyu LI, Cheng DAI Chuanjun LI, Runzhi GUO, Haoyu HAN, Linming LU, Fangfang ZHOU, Hui ZHI. Antitumor component-Ι in Agkistrodon halys venom inhibits proliferation and migration of cisplatin-resistant gastric cancer cells by downregulating RAI14[J]. Journal of Southern Medical University, 2026, 46(1): 113-121.

Figures/Tables 7

Tab.1 Toxic effects of AHVAC-I on MKN-28/DDP cells (Mean±SD, %)

Group	AHVAC-I (μg/mL)
Group	1	2	4	8	16	32
Cell viability	104.37±2.68	100.8±5.45	85.65±4.67	85.65±4.67	46.23±4.74	3.01±1.87
t	3.09	0.28	5.82	8.26	23.07	89.64
P	>0.05	>0.05	<0.05	<0.01	<0.001	<0.0001

Fig.1 Cytotoxicity of AHVAC-I in MKN-28/DDP cells after treatment for 72 h. A: Cells viability assessed by CCK8 assay. B: Colony formation assay of MKN-28/DDP cells at 3 weeks after AHVAC-I treatment. C: Quantitative analysis of colonys in each group. Con: Blank control. n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

Fig.2 Inhibitory effects of AHVAC-I on migration and invasion of MKN-28/DDP cells. A: Wounding-healing assay for assessing migration ability of MKN-28/DDP cells (Original magnification:×40). B: Quantitative analysis of migration ability of MKN-28/DDP cells in each group. C: Transwell assay for assessing invasion ability of MKN-28/DDP cells (×100). D: Quantitative analysis of invading cells in each group, n=5, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

Fig.3 AHVAC-I inhibits epithelial-mesenchymal transition (EMT) of MKN-28/DDP cells. A: Immunoblots of E-cad, N-cad, vimentin and snail in MKN-28/DDP cells treated with different concentrations of AHVAC-I. B-E: Protein expression levels of E-cad, N-cad, vimentin and snail, n=3, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

Fig.4 AHVAC-I down-regulates RAI14 expression in MKN-28/DDP cells. A: qRT-PCR for detecting RAI14 mRNA expression in MKN-28 cells and MKN-28/DDP cells (n=6, **P<0.01 vs MKN-28 cells). B: qRT-PCR of RAI14 expression in MKN-28/DDP cells treated with different concentrations of AHVAC-I (n=6, **P<0.01, ***P<0.001 vs blank Control). C: Immunoblots of RAI14, AKT and AKT-p in MKN-28/DDP cells treated with different concentrations of AHVAC-I. D: Protein expression levels of RAI14 in different groups (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control.

Fig.5 RAI14 supplementation promotes proliferation of AHVAC-I-treated MKN-20/DDP (MKN-20/DDPAHVAC-I) cells. A: Immunoblots of AKT and AKT-p in MKN-20/DDPAHVAC-I cells treated with RAI14 (n=3). B, C: Colony-forming assay showing restored proliferation ability of MKN-20/DDPAHVAC-I cells after RAI14 treatment (n=5). **P<0.01 vs AHVAC-1.

Fig.6 RAI14 supplementation promotes migration and invasion ability of MKN-20/DDPAHVAC-I cells. A: Migration ability of MKN-28/DDP cells analyzed by wounding-healing assay (×40). B: Quantitative analysis of migration ability of MKN-28/DDP cells. C: Invasion ability of MKN-28/DDP cells was analyzed by Transwell assay (×100). D: Quantitative analysis of number of invaded cells.n=5, *P<0.05, **P<0.01, ***P<0.001 vs AHVAC-I.

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