Effect of Emulsifier and Co-emulsifier Combination on Oxidation Stability of Flaxseed Oil Nanoemulsion

The purpose of this study was to investigate the effects of sodium starch octenyl succinate (SSOS) as emulsifier and soy protein isolate (SPI) as co-emulsifier on the oxidative stability of nanoemulsion. The ultrasonic emulsification method was used to prepare flaxseed oil nanoemulsion. The results showed that the optimum preparation conditions were as follows: ultrasonic power of 600 W, core-to-wall ratio of 1:1.5, pulse mode of 8 s, and ultrasonication time of 20 min. The nanoemulsion obtained under these conditions was characterized by high embedding rate ((75.1 ± 4.9)% to (74.6 ± 4.2)%), low particle size ((244.0 ± 3.0) to (246.8 ± 4.5) nm) and high absolute zeta-potential value ((−67.2 ± 3.0) to (−69.3 ± 4.0) mV). With the addition of SPI, the peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) value of the nanoemulsion were significantly reduced, and the oxidative stability was remarkably improved. This could result from the fact that SPI could increase the hydrophobicity of the mixed emulsifier, preventing flaxseed oil from contacting with oxygen radicals in the aqueous phase. This study will provide a new idea for improving the water solubility and oxidative stability of flaxseed oil, and provide a theoretical basis for extending the shelf life of flaxseed oil and promoting its application in the food field

PI3K and ERK-Induced Rac1 Activation Mediates Hypoxia-Induced HIF-1α Expression in MCF-7 Breast Cancer Cells

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1

Dvl2-Dependent Activation of Daam1 and RhoA Regulates Wnt5a-Induced Breast Cancer Cell Migration

The Dishevelled (Dvl) and Dishevelled-associated activator of morphogenesis 1 (Daam1) pathway triggered by Wnt5a regulates cellular polarity during development and tissue homoeostasis. However, Wnt5a signaling in breast cancer progression remains poorly defined.We showed here that Wnt5a activated Dvl2, Daam1 and RhoA, and promoted migration of breast cancer cells, which was, however, abolished by Secreted Frizzled-related protein 2 (sFRP2) pretreatment. Dominant negative Dvl2 mutants or Dvl2 siRNA significantly decreased Wnt5a-induced Daam1/RhoA activation and cell migration. Ectopic expression of N-Daam1, a dominant negative mutant, or Daam1 siRNA remarkably inhibited Wnt5a-induced RhoA activation, stress fiber formation and cell migration. Ectopic expression of dominant negative RhoA (N19) or C3 exoenzyme transferase, a Rho inhibitor, decreased Wnt5a-induced stress fiber formation and cell migration.Taken together, we demonstrated for the first time that Wnt5a promotes breast cancer cell migration via Dvl2/Daam1/RhoA

RhoA activation is essential for Wnt5a-induced MDA-MB-231 cell migration.

<p>(A, B) RhoA activation was induced by Wnt5a (A) and blocked by Dvl2 mutants or siRNA (B). Serum-starved MDA-MB-231 cell monolayers were incubated with 500 ng/mL rWnt5a for 0–60 min (A), or transiently transfected with Dvl2 mutants or siRNA, and then incubated with 500 ng/mL rWnt5a for 30 min (B). Cell lysates were assayed for active RhoA by pulldown assays. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 3 independent experiments in (A) and (B). (D) Expression of empty vector, WT-RhoA, V14-RhoA or N19-RhoA was verified using total protein from cells and immunoblotted using anti-GFP antibody. (C, E) Wnt5a-induced cell migration was abolished by C3 exoenzyme transferase (C) or N19-RhoA, a dominant negative mutant of RhoA (E). MDA-MB-231 cells were preincubated with Rho inhibitor C3 (10 ng/µL) for 1 h (C), or transiently transfected with empty vector, WT-RhoA, V14-RhoA, or N19-RhoA (E), and then incubated with 500 ng/mL rWnt5a for 4 h. Cell migration rate was determined by wound healing assay. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 5 independent experiments in (C) and (E).</p

Daam1 activation is required for Wnt5a-induced RhoA activation.

<p>(A) ΔDAD-Daam1 and <i>C</i>-Daam1 induced while dominant negative mutant and Daam1 siRNA blocked RhoA activation. MDA-MB-231 cells were transiently transfected with Daam1 mutants or siRNAs, and then treated with 500 ng/mL rWnt5a for 30 min. Cells were lysed and quantitated for protein and equal amounts of lysates were assayed for active RhoA by pulldown assays. (B) RhoA did not change the activation of Daam1 with or without Wnt5a treatment. MDA-MB-231 cells were transiently transfected with WT-RhoA, V14-RhoA and N19-RhoA, and then treated with 500 ng/mL rWnt5a for 30 min. Equal amounts of lysates were assayed for active Daam1 by pulldown assays. (C) Overexpression of N19-RhoA abolished the Daam1-dependent cell migration. MDA-MB-231 cells were transiently co-transfected with N19-RhoA and ΔDAD-Daam1. Cells were subjected to a wound healing assay in the absence or presence of 500 ng/mL rWnt5a. (D) Expression of empty vector or ΔDAD-Daam1 was verified using total protein from MCF-7 cells and immunoblotted using anti-GFP antibody. (E) ΔDAD-Daam1 induced RhoA activation in MCF-7 cells. MCF-7 cells were transiently transfected with ΔDAD-Daam1. Cells were lysed and quantitated for protein and equal amounts of lysates were assayed for active RhoA by pulldown assays. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 3 independent experiments in (A), (B) and (E). (F) MCF-7 cells were stimulated by ΔDAD-Daam1. MCF-7 cells were transiently transfected with ΔDAD-Daam1 or empty vector, and migration was quantified by Boyden chamber assays after 48 h. Values are mean ± s.d. of 5 independent observations in (C) and (F).</p

Wnt5a promotes MDA-MB-231 and MCF-7 cell migration.

<p>(A, B) MDA-MB-231 cells were stimulated by rWnt5a at the indicated doses for 4 h. The cell motility rate was measured by wound healing assays (A) or Boyden chamber assays (B). Magnification, ×100 (A) and ×200 (B). (C) MDA-MB-231 cells were preincubated with Wnt5a inhibitor (sFRP2) for 1 h at the indicated doses, and migration in response to rWnt5a (500 ng/mL, for 4 h) was measured by wound healing assays. (D) MCF-7 cells were preincubated with sFRP2 for 1 h at the indicated doses, and migration was measured by Boyden chamber assays after 48 h. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 5 independent experiments in (A) to (D).</p

Dvl2 activation is required for Wnt5a-induced MDA-MB-231 cell migration.

<p>(A, B) Dvl2 activation was induced by Wnt5a (A) and blocked by sFRP2 (B). Serum-starved MDA-MB-231 cell monolayers were incubated with 500 ng/mL rWnt5a for 0–60 min (A), or treated with 1000 ng/mL sFRP2 for 1 h prior to 500 ng/mL rWnt5a treatment for 5 min (B). Cell lysates were assayed for phosphorylated Dvl2 by immunoblotting analyses with anti-Dvl2 and β-actin antibodies. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 3 independent experiments in (A) and (B). (C) The domain structures of Dvl2 and two mutants. Schematic representations of wild-type Dvl2, ΔPDZ-Dvl2 (lacking the PDZ domain), and ΔDEP-Dvl2 (lacking the DEP domain). Residue numbers above domains denote the domain boundaries. (D) Expression of empty vector, WT-Dvl2, ΔPDZ-Dvl2 or ΔDEP-Dvl2 was verified using total protein from cells and immunoblotted using anti-GFP antibody. (E) Overexpression of ΔPDZ-Dvl2 and ΔDEP-Dvl2 in cells abolished Wnt5a-induced cell migration. MDA-MB-231 cells transiently transfected with EGFP-tagged empty vector, WT-Dvl2, ΔPDZ-Dvl2 or ΔDEP-Dvl2 were incubated in the absence or presence of 500 ng/mL rWnt5a. The cell migration rate was determined by wound healing assays. (F) Efficiency of gene knockdown was analyzed by RT-PCR (left) and immunoblotting (right) for Dvl2. MDA-MB-231 cells were transfected with control (Mock) or Dvl2 siRNAs. Total mRNA or protein extracts from MDA-MB-231 transfected with control (Mock) or Dvl2 siRNA were analyzed by RT-PCR and immunoblotting for Dvl2. The same assay was performed with GAPDH as a loading standard. (G) Dvl2 siRNA significantly inhibited cell migration. MDA-MB-231 cells transfected with control (Mock) or Dvl2 siRNA were subjected to a wound healing assay in the absence or presence of 500 ng/mL rWnt5a. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 5 independent experiments in (E) and (G).</p