香菇多糖抑制Akt通路和微管蛋白聚合诱导肿瘤细胞凋亡的分子机制  

Molecular mechanism of Lentinus edodes polysaccharide to induce tumor cell apoptosis by inhibiting Akt pathway and tubulin polymerization

作　　者：张琪琳[1] 杜兆松 王凯平[3] 张玉[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院药剂科,湖北武汉430022 [2]武汉市儿童医院药学部,湖北武汉430016 [3]华中科技大学同济医学院药学院,湖北武汉430030

出　　处：《中国医院药学杂志》2016年第23期2046-2051,共6页Chinese Journal of Hospital Pharmacy

基　　金：国家自然科学基金项目(编号:81373300)

摘　　要：目的:研究香菇多糖(SLNT1)诱导H22和MCF-7细胞凋亡的分子机制。方法:体外MTT法检测SLNT1对H22和MCF-7细胞的生长抑制作用;AO/EB染色考察SLNT1对H22凋亡细胞形态学变化;微管红色荧光探针试剂盒观察SLNT1对MCF-7细胞微管蛋白聚合作用的影响;体内建立H22荷瘤小鼠模型,给予不同剂量香菇多糖(50,100,200 mg·kg-1)溶液治疗,空白组(不接种不给药),阳性对照组(腹腔注射环磷酰胺,25 mg·kg-1),给药10 d,末次给药后剥离肿瘤组织,保存于-80℃冰箱中。Western blot法检测H22肿瘤组织中p-Akt、p53、Bcl-2、Bax、cleaved caspase-3蛋白表达情况。结果:SLNT1能显著抑制H22和MCF-7细胞的体外生长增殖。AO/EB染色表明给药SLNT1后,出现典型的H22凋亡细胞,大部分细胞被染成橙红色,细胞核固缩、破裂并出现凋亡小体,且SLNT1能抑制微管蛋白聚合诱导MCF-7细胞发生凋亡。Western blot结果显示SLNT1显著降低H22肿瘤组织中p-Akt和Bcl-2的表达,上调肿瘤抑制蛋白p53和Bax,cleaved caspase-3的表达,并增加Bax/Bcl-2的值。结论:香菇多糖的诱导肿瘤细胞凋亡作用可能是通过抑制Akt通路进而促进线粒体凋亡途径和抑制微管蛋白聚合来实现的。OBJECTIVE To clarify the molecular mechanism of polysaccharide SLNT1 to induce H22 and MCF-7 cells apoptosis.METHODS MTT colorimetric method was employed to test the growth inhibiting effects of SLNT1 on H22 and MCF-7cells in vitro.AO/EB staining was used to observe morphological changes of H22 apoptotic cells.Furthermore,effects of SLNT1 on microtubule protein polymerization of MCF-7 cells were detected by microtubules red fluorescent probe kit.H22-bearing mouse models were established in vivo and were randomly divided into five groups with10 mice in each group：negative control group,mice were intraperitoneally injected with the same volume of 0.9% normal saline（NS）;positive control group,intraperitoneally injected with cytoxan（CTX,25 mg·kg-1）dissolved in 0.9% normal saline;experimental groups,intraperitoneally injected with different concentrations of SLNT1 at 50,100 and 200 mg·kg-1 once daily for 10 days.All mice were killed by cervical dislocation the next day after the last treatment,and then tumors were removed and stored at-80℃.Western blot assay was used to detect expression of signal transduction proteins in H22 tumor tissues,including p-Akt,p53,Bcl-2,Bax and cleaved caspase-3.RESULTS SLNT1 had direct inhibitory effects on proliferation of H22 and MCF-7 cells.AO/EB staining demonstrated that SLNT1-treated H22 cells were dyed orange with cells shrinking and nucleus fragmentation.Moreover,SLNT1 prevented MCF-7 cell mitosis through inhibiting tubulin polymerization to induce MCF-7 cells apoptosis.Western blot showed that SLNT1 treatment upregulated expressions of Bax,p53 and cleaved caspase-3,while levels of p-Akt and Bcl-2were reduced,and ratio of Bax/Bcl-2 was increased.CONCLUSION Tumor cell apoptosis of SLNT1 may be induced through inhibiting Akt signal,promoting mitochondrial apoptosis pathway and inhibiting tubulin polymerization.

关 键 词：香菇多糖 抗肿瘤活性 凋亡 AKT 微管蛋白聚合 

分 类 号：R285.5[医药卫生—中药学]