华蟾素通过下调PIM3的表达抑制高转移胃癌细胞的实验研究

Experimental Study of Bufothionine Inhibits Highly Metastatic Gastric Cancer Cells by Down Regulating PIM3 Expression

目的:胃癌(GC)是全球第四大常见癌症。丁硫磷是华蟾酥的主要活性成分,从蟾蜍的皮肤和腮腺毒腺中提取,在体外具有抗癌活性。然而,丁硫磷是否对胃癌有抗癌作用尚不清楚。本研究旨在评价丁硫磷对GC的抑制作用。方法:以高转移性MKN28 GC细胞为细胞模型,研究华蟾素的抗癌作用。使用CCK-8测定细胞活力,LDH检测丁硫磷对细胞膜完整性的影响。Hoechst/PI双染色和Annexin V-FITC/PI染色检测细胞凋亡,随后使用流式细胞仪进行分析。western blotting检测相关蛋白的表达水平。通过iTRAQ分析,确定丁硫磷处理后GC细胞的差异表达基因。结果:丁硫磷能抑制高转移性GC MKN28细胞的生长,破坏细胞膜,促进GC细胞凋亡。i TRAQ分析表明PIM3是丁硫磷素抗癌作用的分子靶点。研究还发现,PIM3基因敲除显著增强了丁硫磷对GC细胞的抗生长和促凋亡作用。相反,异位PIM3的表达明显抑制了丁硫磷的抗肿瘤活性。结论:华蟾素对高转移性的GC具有抗癌活性是通过下调PIM3表达而实现的。

Objective: Gastric cancer(GC) is the fourth most common cancer globally. Bufothionine is a major active constituent of Cinobufacini, which is extracted from the skin and parotid venom gland of the toad Bufo bufogargarizans Cantor. It exhibits anti-cancer activities in vitro. However, whether bufothionine exerts anti-cancer activities against GC is unknown. To evaluate the inhibitory effect of bufothionine on GC. Methods: MKN28 GC cells were chosen as cell models to study the anti-cancer effect of bufothionine. Cell viability was determined by CCK-8 assay, while the effect of bufothionine on cell membrane integrity was examined by LDH assay. Cell apoptosis was detected by Hoechst/PI staining and Annexin V-FITC/PI staining followed by flow cytometry analysis. The expression levels of proteins involved were examined using western blotting. i TRAQ analysis was conducted to identify the differentially expressed genes in AGS cells following bufothionine treatment. Results: The results revealed that bufothionine prevented the growth, destroyed cell membrane, and promoted apoptotic cell death of GC cells. i TRAQ analysis revealed that PIM3 might be a molecular target responsible for the anti-cancer effects of bufothionine. PIM3 knockdown significantly augmented the anti-growth and pro-apoptotic effects of bufothionine in GC cells. In contrast, ectopic PIM3 expression markedly dampened the anti-neoplastic activities of bufothionine.Conclusion:Bufothionine exhibits anti-cancer activities in vitro GC cells via downregulating PIM3.