title: Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells.
abstract: OBJECTIVE
  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 anin vitroangiogenesis 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.
  
  
  CONCLUSIONS
  MβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.
authors.affiliation: Cancer Biology Lab, Department of Biochemistry, GIS, GITAM University, Visakhapatnam, India.
authors.forename: Anil
authors.initials: A
authors.lastname: Badana
authors.affiliation: Department of Obstetrics & Gynecology, Andhra Medical College, Visakhapatnam, India.
authors.forename: Madhuri
authors.initials: M
authors.lastname: Chintala
authors.affiliation: Cancer Biology Lab, Department of Biochemistry, GIS, GITAM University, Visakhapatnam, India.
authors.forename: Gayathri
authors.initials: G
authors.lastname: Varikuti
authors.affiliation: Cancer Biology Lab, Department of Biochemistry, GIS, GITAM University, Visakhapatnam, India.
authors.forename: Nagaseshu
authors.initials: N
authors.lastname: Pudi
authors.affiliation: Cancer Biology Lab, Department of Biochemistry, GIS, GITAM University, Visakhapatnam, India.
authors.forename: Seema
authors.initials: S
authors.lastname: Kumari
authors.affiliation: Department of Biochemistry, GITAM Institute of Science, GITAM University, Visakhapatnam, India.
authors.forename: Vijaya Rachel
authors.initials: VR
authors.lastname: Kappala
authors.affiliation: Cancer Biology Lab, Department of Biochemistry, GIS, GITAM University, Visakhapatnam, India.; Department of Biochemistry, GITAM Institute of Science, GITAM University, Visakhapatnam, India.
authors.forename: Rama Rao
authors.initials: RR
authors.lastname: Malla
keywords: Beta-cyclodextrin; Cholesterol; Membrane microdomains; Triple negative breast neoplasms