ABT-737对M2型TAM来源外泌体处理的卵巢癌细胞SKOV3自噬性凋亡与干性特征的影响
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海南省卫生健康行业科研项目(21A200406)


Effects of ABT-737 on autophagic apoptosis and stemness characteristics of SKOV3 ovarian cancer cells treated with M2 tumor-associated macrophage-derived exosomes
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    摘要:

    探讨Bcl-2小分子抑制剂ABT-737对经肿瘤相关巨噬细胞(TAM)来源外泌体(Exo)处理的卵巢癌细胞自噬性凋亡及干性特征的作用。 方法 利用佛波酯(PMA)与重组人白细胞介素-4(IL-4)诱导人外周血单核细胞THP-1为M2型TAM,流式细胞术检测细胞表面巨噬细胞相关标志物表达;差速离心法提取外泌体,透射电镜与纳米粒子追踪技术观察外泌体形态及径粒分布,Western blot检测外泌体标志蛋白表达,Dil结合PKH67染色检测TAM来源外泌体被卵巢癌细胞系SKOV3摄取情况;实验分组为对照组、Exo组、Exo+ABT-737组,采用TAM来源外泌体与ABT-737按照分组处理SKOV3细胞,流式细胞术检测细胞凋亡率,双荧光mRFP-GFP-LC3实验检测细胞自噬水平,Western blot检测细胞自噬相关蛋白(LC3-Ⅱ/LC-3Ⅰ、Beclin-1、p62)与凋亡相关蛋白(Bax、Bcl-2、Cleaved-caspase3)表达,肿瘤细胞成球实验检测细胞干性,Western blot检测肿瘤干细胞相关蛋白(CD133、OCT4、SOX2)表达。 结果 经PMA与IL-4诱导后,THP-1细胞形态发生改变,M1型巨噬细胞标记物CD86表达比例降低而M2型巨噬细胞标记物CD206表达比例升高(P<0.05)。分离的颗粒物为球形囊泡,大小均匀,粒径峰值约为140 nm,外泌体标志蛋白Alix、CD63、TSG101表达明显,由此判断成功分离到M2型TAM来源Exo,且其能被SKOV3摄取。与对照组比较,Exo组SKOV3细胞凋亡率减少,GFP与mRFP的荧光斑点减少,细胞内LC3-Ⅱ/LC-3Ⅰ、Beclin-1、Bax及Cleaved-caspase3表达均下调,p62与Bcl-2表达均上调,此外,细胞成球数量增加,CD133、OCT4及SOX2表达均上调,差异均具有统计学意义(P<0.05)。与Exo组比较,Exo+ABT-737组SKOV3细胞凋亡率增加,GFP与mRFP的荧光斑点也增加,细胞内LC3-Ⅱ/LC-3Ⅰ、Beclin-1、Bax及Cleaved-caspase3表达均上调,p62与Bcl-2表达均下调,细胞成球数量减少,且CD133、OCT4及SOX2表达均下调,差异均具有统计学意义(P<0.05)。 结论 ABT-737 能够在M2型TAM来源外泌体处理的卵巢癌细胞中发挥促自噬性凋亡的作用,并且可以抑制卵巢癌肿瘤干性特征

    Abstract:

    To investigate the effect of Bcl-2 small molecule inhibitor ABT-737 on autophagy apoptosis and stemness characteristics of ovarian cancer cells treated with tumor-associated macrophage (TAM)-derived exosomes (Exo). Methods Using phorbol ester (PMA) and recombinant human interleukin-4 (IL-4) to induce human peripheral blood mononuclear cells THP-1 to M2 type TAM, the expression of macrophage-related markers on the cell surface was detected by flow cytometry; The exosomes were extracted by differential centrifugation, the morphology and size distribution of exosomes were observed by transmission electron microscopy and nanoparticle tracking technology, and the expression of exosome marker proteins was detected by Western blot, Dil combined with PKH67 staining was used to detect the uptake of TAM-derived exosomes by ovarian cancer cell line SKOV3; The experimental groups were divided into control group, Exo group, and Exo+ABT-737 group. TAM-derived exosomes and ABT-737 were used to treat SKOV3 cells according to the groups, and flow cytometry was used to detect the apoptosis rate, double fluorescent mRFP-GFP-LC3 assay was used to detect the level of autophagy, Western blot was used to detect autophagy-related proteins (LC3-II/LC-3Ⅰ, Beclin-1, p62) and apoptosis-related proteins (Bax, Bcl-2, Cleaved-caspase3) expression, tumor cell spheroidization assay was used to detect cell stemness, and Western blot was used to detect the expression of cancer stem cell-related proteins (CD133, OCT4, SOX2). Results After induction by PMA and IL-4, the morphology of THP-1 cells changed, the expression ratio of M1 macrophage marker CD86 decreased while the expression ratio of M2 macrophage marker CD206 increased (P<0.05). The isolated particles are spherical vesicles with uniform size and a peak particle size of about 140 nm, the exosome marker proteins Alix, CD63, and TSG101 are clearly expressed. It is judged that the M2-type TAM source Exo was successfully isolated and can be taken up by SKOV3. Compared with the control group, the apoptosis rate of SKOV3 cells in the Exo group decreased, the fluorescent spots of GFP and mRFP decreased, the expressions of LC3-II/LC-3I, Beclin-1, Bax and Cleaved-caspase3 in the cells were all down-regulated, and p62 and Bcl-2 were all up-regulated, in addition, the number of cell spheroids increased, and the expressions of CD133, OCT4 and SOX2 were all up-regulated, the differences were statistically significant (P<0.05). Compared with the Exo group, the apoptosis rate of SKOV3 cells in the Exo+ABT-737 group increased, the fluorescent spots of GFP and mRFP also increased, the intracellular expressions of LC3-II/LC-3I, Beclin-1, Bax and Cleaved-caspase3 were all up-regulated, the expressions of p62 and Bcl-2 were down-regulated, the number of cell spheroids decreased, and the expressions of CD133, OCT4 and SOX2 were down-regulated, the differences were statistically significant (P<0.05). Conclusion ABT-737 can promote the autophagy and apoptosis of ovarian cancer cells treated with M2-type TAM-derived exosomes, and inhibit the tumor stemness characteristic of ovarian cancer

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  • 在线发布日期: 2023-05-18
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