MLN4924导致的成熟脂肪细胞衰老促进乳腺癌细胞的迁移与侵袭

Senescence of mature adipocytes induced by MLN4924 promotes the migration and invasion of breast cancer cells

目的目前化疗药物致细胞衰老的研究鲜有关注终末分化细胞,脂肪组织在乳房中占比很高,且肥胖与乳腺癌的发病密切相关。文章旨在探讨一种潜在的新型化疗药物MLN4924是否导致成熟脂肪细胞的衰老及衰老的脂肪细胞对乳腺癌细胞的影响。方法将分化成熟的脂肪细胞分为4个组,3天对照组(DMSO处理72 h),3天Mln组(Mln4924处理72 h),6天对照组(DMSO处理72 h,无药培养基中继续维持72 h),6天Mln组(Mln4924处理72 h,无药培养基中继续维持72 h)。用衰老相关标记物β-衰老半乳糖苷酶(SA-β-gal)、p53、p21、p16、Rb检测脂肪细胞的衰老;采用qPCR和ELISA检测炎症相关因子的表达与分泌;将乳腺癌细胞分为对照组和Mln组,用收集的脂肪细胞条件培养基培养乳腺癌细胞48 h,使用CCK8试剂检测乳腺癌细胞的增殖,Transwell小室法检测乳腺癌细胞的迁移与侵袭,Western blot检测处理后乳腺癌细胞上皮-间充质转换(EMT)相关蛋白的表达。结果6天Mln组中被染成绿色的脂肪细胞SA-β-gal阳性细胞率(11.71%)明显多于6天对照组(2.98%),差异有统计学意义(P<0.05)。Western blot的结果显示,Mln组较对照组脂肪细胞中p53、p21蛋白的表达明显增加(P<0.01),p16的表达降低(P<0.01)。3天Mln组和6天Mln组炎症因子IL-6、TGF-β、TNF-α、CCL2及基质金属蛋白酶MMP1、MMP2 mRNA水平皆较其对照组上调(P<0.05)。ELISA结果也显示,3天Mln组IL-6(200.41±13.60)、TGF-β(76.29±4.18)均超过3天对照组(130.56±14.70)、(58.45±2.93),差异有统计学意义(P<0.05),6天Mln组IL-6(179.13±4.94)、TGF-β(125.24±0.09)均超过6天对照组(120.27±31.64)、(32.00±14.76),差异有统计学意义(P<0.05)。Transwell迁移和侵袭实验结果显示Mln组被结晶紫染成紫色的细胞数量明显高于对照组,即Mln组穿过膜的MDA-MB-231细胞更多。在衰老的脂肪细胞中,观察到6天Mln组较6天对照组MMP2和MMP9蛋白表达的显著升高(P<0.01)。在条件培养基培养48 h后的MDA-MB-231细胞中,观察到Mln组的E-cadherin较对照组明显下调(P<0.01),而Vimentin和Fibronectin上调(P<0.01)。结论MLN4924处理导致成熟脂肪细胞衰老;衰老的脂肪细胞分泌炎症因子并促进人乳腺癌细胞的迁移与侵袭。

Objective Most of the current studies on cellular senescence caused by chemotherapeutic agents have focused on proliferating cells,while rare studies have been performed on differentiated cells.Adipose tissue is abundant in the breast,and obesity is closely associated with breast cancer occurrence.This paper investigated whether the potent chemotherapeutic drug MLN4924 caused senescence of mature adipocytes and the senescent adipocytes'effect on breast cancer cells.Methods The mature adipocytes differentiated from adult adipose-derived mesenchymal stem cells were divided into Control group(solvent treatment)and Mln group(MLN4924 treatment).After 72 hours treatment,the cultural medium was changed to drug-free medium for another 72 hours before the cells and medium were collected.Senescence-related markersβ-senescence galactosidase(SA-β-gal),p53,p21,p16,Rb were examined.QPCR and ELISA were used to detect the expression and secretion of inflammation-related factors.Breast cancer cells were cultured with the adipocyte conditioned medium for 48 hours.CCK8 was employed to determined proliferation of breast cancer cells.Transwell chambers were used to detect the migration and invasion of breast cancer cells.Western blot was used to detect the epithelial-mesenchymal transition(EMT)related proteins of breast cancer cells after treatment.Results The SA-β-gal positive rate of adipocytes in Mln group was about 3 times more than that of Control group(P<0.05).The protein expressions of p53 and p21 in Mln group were significantly higher than those in Control group(P<0.01).The qPCR and ELISA results showed that the expression of interleukin-6(IL-6)and other inflammatory factors in Mln group was significantly up-regulated.In adipocytes treated with MLN4924,matrix metalloproteinase 2(MMP2)and matrix metalloproteinase 9(MMP9)increased significantly compared with Control group(P<0.01).With the treatment of conditioned medium from adipocytes,breast cancer cells had no difference in proliferation between Mln group and Control group(P>0.05).In the migration and invasion experiments,the number of cells that penetrated the membrane in the Mln group was significantly higher than that in Control group(P<0.01).The epithelial marker protein E-cadherin in Mln group was significantly decreased in breast cancer cells MDA-MB-231,T47D,and MCF-7(P<0.01).In contrast,the mesenchymal marker Vimentin and Fibronectin were significantly increased in MDA-MB-231(P<0.01).Conclusion MLN4924 treatment leads to the senescence of mature adipocytes,and senescent adipocytes promotes the migration and invasion of human breast cancer cells.