Lipid rafts disruption induces apoptosis by attenuating expression of LRP6 and survivin in triple negative breast cancer.

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, MßCD 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, ß-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, ß-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.

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

PURPOSE: Lipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-ß-cyclodextrin (MßCD)-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 MßCD, 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. MßCD 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 MßCD treatment (48 hours). MßCD 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 MßCD 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 MßCD 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: MßCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.

Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization.

BACKGROUND: The defensive capacities of plant protease Inhibitors (PI) rely on inhibition of proteases in insect guts or those secreted by microorganisms; and also prevent uncontrolled proteolysis and offer protection against proteolytic enzymes of pathogens. METHODS: An array of chromatographic techniques were employed for purification, homogeneity was assessed by electrophoresis. Specificity, Ki value, nature of inhibition, complex formation was carried out by standard protocols. Action of SNTI on insect gut proteases was computationally evaluated by modeling the proteins by threading and docking studies by piper using Schrodinger tools. RESULTS: We have isolated and purified Soap Nut Trypsin Inhibitor (SNTI) by acetone fractionation, ammonium sulphate precipitation, ion exchange and gel permeation chromatography. The purified inhibitor was homogeneous by both gel filtration and polyacrylamide gel electrophoresis (PAGE). SNTI exhibited a molecular weight of 29 kDa on SDS-PAGE, gel filtration and was negative to Periodic Acid Schiff's stain. SNTI inhibited trypsin and pronase of serine class. SNTI demonstrated non-competitive inhibition with a Ki value of 0.75 ± 0.05×10-10 M. The monoheaded inhibitor formed a stable complex in 1:1 molar ratio. Action of SNTI was computationally evaluated on larval gut proteases from Helicoverpa armigera and Spodoptera frugiperda. SNTI and larval gut proteases were modeled and docked using Schrodinger software. Docking studies revealed strong hydrogen bond interactions between Lys10 and Pro71, Lys299 and Met80 and Van Der Waals interactions between Leu11 and Cys76amino acid residues of SNTI and protease from H. Armigera. Strong hydrogen bonds were observed between SNTI and protease of S. frugiperda at positions Thr79 and Arg80, Asp90 and Gly73, Asp2 and Gly160 respectively. CONCLUSION: We conclude that SNTI potentially inhibits larval gut proteases of insects and the kinetics exhibited by the protease inhibitor further substantiates its efficacy against serine proteases.