Cancer Biology Research Laboratory, Department of Biochemistry, GIS, GITAM University, Visakhapatnam, India.
Department of Obstetrics & Gynecology, Andhra Medical College, Visakhapatnam, India.
Biomed Pharmacother. 2018 Jan;97:359-368. doi: 10.1016/j.biopha.2017.10.045. Epub 2017 Nov 6.
Triple negative breast cancer is a clinically challenging subtype due to lack of biomarker for rational targeted therapy. Lipid rafts are cholesterol enriched rigid platforms, which colocalize signalling molecules of cancer progression. This study explores the effect of lipid rafts disruption by cholesterol depleting agent, MβCD on induction of apoptosis and expression of WNT receptor LRP6, survivin and common apoptotic markers in TNBC cell lines. The in vitro effect of lipid rafts disruption on viability, single cell reproductive ability, proliferation and migration were evaluated by MTT, clonogenic, BrdU incorporation and wound scratch assays, respectively. The morphological changes were assessed by tryphan blue, Wright and Giemsa staining; nuclear changes by Hoechst staining. The induction of apoptosis was evaluated by AO/EtBr staining, DNA damage and DNA fragmentation assays. Expression of Caveolin-1, LRP6, β-Catenin, Survivin, Bcl2, BAX, Caspase-3, Ki67 and c-myc were analyzed by PCR and Western blotting techniques. The lipid raft disruption resulted in reduction of the proliferation of MDA-MB 231 and MDA-MB 468 cells by 56.3 and 42.0%; survival fraction by 54.7 and 59.4%; migration by 44.3 and 48.4%, respectively. It also induced apoptosis by causing cell shrinkage, membrane blebbing, nuclear condensation, chromatin cleavage, oligonucleotide fragmentation with an apoptotic index of 59.1 and 46.6% in MDA-MB 231 and 468 cells, respectively. Further, it downregulated the expression of caveolin-1, LRP6, β-catenin, survivin, Bcl2, ki67, c-myc and upregulated BAX, caspase-3. The cholesterol supplementation enhanced the clonogenic potential and upregulated the expression of caveolin-1 and LRP6. The results underline a potential effect of lipid rafts disruption on induction of apoptosis in TNBC cells.
三阴性乳腺癌是一种临床挑战性的亚型，因为缺乏合理的靶向治疗生物标志物。脂筏是富含胆固醇的刚性平台，其聚集了癌症进展的信号分子。本研究探讨了胆固醇耗竭剂 MβCD 破坏脂筏对三阴性乳腺癌细胞系中凋亡诱导和 WNT 受体 LRP6、survivin 和常见凋亡标志物表达的影响。通过 MTT、集落形成、BrdU 掺入和划痕实验分别评估脂筏破坏对细胞活力、单细胞生殖能力、增殖和迁移的体外影响。通过台盼蓝、Wright 和吉姆萨染色评估形态变化；通过 Hoechst 染色评估核变化。通过 AO/EB 染色、DNA 损伤和 DNA 片段化分析评估细胞凋亡诱导。通过 PCR 和 Western blot 技术分析 Caveolin-1、LRP6、β-Catenin、Survivin、Bcl2、BAX、Caspase-3、Ki67 和 c-myc 的表达。脂筏破坏导致 MDA-MB 231 和 MDA-MB 468 细胞增殖减少 56.3%和 42.0%；存活分数减少 54.7%和 59.4%；迁移减少 44.3%和 48.4%。它还通过引起细胞收缩、膜泡形成、核浓缩、染色质裂解、寡核苷酸片段化，导致 MDA-MB 231 和 MDA-MB 468 细胞的凋亡指数分别为 59.1%和 46.6%，诱导凋亡。此外，它下调了 Caveolin-1、LRP6、β-Catenin、Survivin、Bcl2、ki67、c-myc 的表达，上调了 BAX、Caspase-3 的表达。胆固醇补充增强了集落形成能力，并上调了 Caveolin-1 和 LRP6 的表达。这些结果强调了脂筏破坏对三阴性乳腺癌细胞诱导凋亡的潜在影响。
