Salkin Hasan, Yay Arzu, Gokdemir Nur Seda, Gönen Zeynep Burçin, Özdamar Saim, Yakan Birkan
Department of Medical Services and Techniques, Program of Pathology Laboratory Techniques, Vocational School, Beykent University Istanbul, Turkey.
Department of Histology and Embryology, Faculty of Medicine, Erciyes University Kayseri, Turkey.
Am J Stem Cells. 2022 Dec 25;11(5):64-78. eCollection 2022.
This study aimed to investigate the effect of TGF-B1-transfected adipose-derived mesenchymal stem cell (AD-MSC) conditional medium (TGF-B1-CM) on CD44 expression and biological activities in MCF-7 and MDA-MB-231 cells.
In the study, the experimental groups were created as a standard medium, AD-MSC-CM, TGF-B1-CM, and TGF-B1 recombinant protein. The medium and proteins specified in these groups were applied to MCF-7 and MDA-MB-231 cells separately at 24, 48 and 72 hours. Western blot and immunofluorescent staining were performed with antibodies suitable for CD44 and canonical smad signaling pathway analyses between groups. Cellular proliferation in MCF-7 and MDA-MB-231 cells was measured by MTT. Biological activity analyses such as apoptosis, cell cycle, proliferation, DNA damage, and membrane depolarization between groups were tested on the Muse Cell Analyzer using appropriate kits. Cellular migration between groups was determined by showing cells that migrated to the scar area with in vitro scar formation. Statistics were performed with GraphPad Prism 8.02 software.
It was determined that TGF-B1-CM activates the smad signaling pathway in MCF-7 and MDA-MB-231 cells. TGF-B1-CM increased pSMAD2/3 expression and decreased SMAD4 expression in breast cancer cells. A decrease in CD44 expression was found at points of increase in pSMAD2/3 expression. Decreased expression of SMAD4 in breast cancer cells with TGF-B1-CM was associated with decreased expression of CD44. In MCF-7 and MDA-MB-231 cells, TGF-B1-CM was found to increase apoptosis, decrease proliferation, disrupt membrane depolarization, and arrest cells at G0/G1 stage. TGF-B1-CM suppressed MCF-7 and MDA-MB-231 migrations.
SMAD4-targeted therapeutic strategies may be considered to suppress CD44 expression in breast cancer cells. Both the anti-tumorigenic factors released by AD-MSCs and the secretomes obtained as a result of supporting these factors with the overexpression of TGF-B1, severely suppressed breast cancer cells. With this study, it was planned to obtain a targeted biological product that suppresses breast cancer cells .
本研究旨在探讨转化生长因子-β1（TGF-β1）转染的脂肪来源间充质干细胞（AD-MSC）条件培养基（TGF-β1-CM）对MCF-7和MDA-MB-231细胞中CD44表达及生物学活性的影响。
在本研究中，设置标准培养基、AD-MSC-CM、TGF-β1-CM和TGF-β1重组蛋白作为实验组。这些组中指定的培养基和蛋白分别在24、48和72小时应用于MCF-7和MDA-MB-231细胞。用适合CD44和经典smad信号通路分析的抗体进行蛋白质印迹和免疫荧光染色，以比较各组之间的差异。采用MTT法检测MCF-7和MDA-MB-231细胞的增殖情况。使用合适的试剂盒在Muse细胞分析仪上检测各组之间的凋亡、细胞周期、增殖、DNA损伤和膜去极化等生物学活性分析。通过体外形成瘢痕来显示迁移到瘢痕区域的细胞，从而确定各组之间的细胞迁移情况。使用GraphPad Prism 8.02软件进行统计学分析。
研究发现，TGF-β1-CM可激活MCF-7和MDA-MB-231细胞中的smad信号通路。TGF-β1-CM可增加乳腺癌细胞中pSMAD2/3的表达，降低SMAD4的表达。在pSMAD2/3表达增加的点处，发现CD44表达降低。TGF-β1-CM处理的乳腺癌细胞中SMAD4表达的降低与CD44表达的降低相关。在MCF-7和MDA-MB-231细胞中，TGF-β1-CM可增加细胞凋亡、降低细胞增殖、破坏膜去极化，并使细胞停滞在G0/G1期。TGF-β1-CM可抑制MCF-7和MDA-MB-231细胞的迁移。
可考虑采用靶向SMAD4的治疗策略来抑制乳腺癌细胞中CD44的表达。AD-MSCs释放的抗肿瘤因子以及通过TGF-β1过表达支持这些因子而获得的分泌产物，均能严重抑制乳腺癌细胞。通过本研究，计划获得一种抑制乳腺癌细胞的靶向生物制品。
