Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells

다국어 초록 (Multilingual Abstract)

Purpose: Lipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-β- cyclodextrin (MβCD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells. Methods: We measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively. Results: The present study found that the cholesterol-depleting agent MβCD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1–0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. MβCD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following MβCD treatment (48 hours). MβCD also caused cell cycle arrest at the G2M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that MβCD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that MβCD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines. Conclusion: MβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.

참고문헌 (Reference)

1 Zidovetzki R, "Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies" 1768 : 1311-1324, 2007

2 Pucadyil TJ, "The sterol-binding antibiotic nystatin differentially modulates ligand binding of the bovine hippocampal serotonin1A receptor" 320 : 557-562, 2004

3 Singer SJ, "The fluid mosaic model of the structure of cell membranes" 175 : 720-731, 1972

4 Fang L, "Targeted cholesterol efflux" 12 : 3345-3346, 2013

5 Marcella SW, "Statin use and fatal prostate cancer: a matched case-control study" 118 : 4046-4052, 2012

6 Caliceti C, "Role of plasma membrane caveolae/lipid rafts in VEGFinduced redox signaling in human leukemia cells" 2014 : 857504-, 2014

7 Rao Malla R, "Regulation of NADPH oxidase (Nox2)by lipid rafts in breast carcinoma cells" 37 : 1483-1493, 2010

8 Maxfield FR, "Plasma membrane microdomains" 14 : 483-487, 2002

9 Ghibelli L, "Organelle cross-talk in apoptotic and survival pathways" 2012 : 968586-, 2012

10 Grossmann J, "Molecular mechanisms of “detachment-induced apoptosis:Anoikis”" 7 : 247-260, 2002

11 Raghu H, "Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells" 10 : 647-, 2010

12 Zhang Q, "Lipidomics in the analysis of malignancy" 54 : 93-98, 2014

13 Staubach S, "Lipid rafts: signaling and sorting platforms of cells and their roles in cancer" 8 : 263-277, 2011

14 Pike LJ, "Lipid rafts: bringing order to chaos" 44 : 655-667, 2003

15 Mollinedo F, "Lipid rafts as major platforms for signaling regulation in cancer" 57 : 130-146, 2015

16 Simons K, "Lipid rafts and signal transduction" 1 : 31-39, 2000

17 George KS, "Lipid raft: a floating island of death or survival" 259 : 311-319, 2012

18 Rao Malla R, "Knockdown of cathepsin B and uPAR inhibits CD151 and α3β1 integrin-mediated cell adhesion and invasion in glioma" 52 : 777-790, 2013

19 Simons K, "Functional rafts in cell membranes" 387 : 569-572, 1997

20 Peters KG, "Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumour angiogenesis" 77 : 51-56, 1998

21 Li YC, "Elevated levels of cholesterolrich lipid rafts in cancer cells are correlated with apoptosis sensitivity induced by cholesterol-depleting agents" 168 : 1107-1118, 2006

22 Malla R, "Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas" 5 : e13731-, 2010

23 Bang B, "Disruption of lipid rafts causes apoptotic cell death in HaCaT keratinocytes" 14 : 266-272, 2005

24 Ohtani Y, "Differential effects of alpha-, beta- and gamma-cyclodextrins on human erythrocytes" 186 : 17-22, 1989

25 Cheung KJ, "Collective invasion in breast cancer requires a conserved basal epithelial program" 155 : 1639-1651, 2013

26 Zhuang L, "Cholesterol-rich lipid rafts mediate akt-regulated survival in prostate cancer cells" 62 : 2227-2231, 2002

27 Mohammad N, "Cholesterol depletion by methyl-β-cyclodextrin augments tamoxifen induced cell death by enhancing its uptake in melanoma" 13 : 204-, 2014

28 Malla RR, "Cathepsin B and uPAR knockdown inhibits tumor-induced angiogenesis by modulating VEGF expression in glioma" 18 : 419-434, 2011

29 Longatto Filho A, "Angiogenesis and breast cancer" 2010 : 576384-, 2010