Apocynin attenuates motility and induces transition from sustained to transient EGF-dependent Akt activation in MCF-7 cells that overexpress adaptor protein Ruk/CIN85

A. V. Bazalii, L. B. Drobot, S. V. Komisarenko © 2016 A. V. Bazalii et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.112


Introduction
A large body of experimental evidence suggests that reactive oxygen species (ROS) at physiological concentrations function as signaling molecules to mediate various responses including the tumor cell proliferation, migration, invasion, and gene expression [1][2][3].NADPH oxidases are a major source of ROS within cells required for space-and time-dependent control of redox signaling [4][5][6].The localized production of H 2 O 2 in the vicinity of specific substrates leads to their reversible oxidized modification and hence to the modulation of signaling responses [6,7].It can be suggested that the adaptor/scaffold proteins consisting of domains and motifs involved in the intermolecular interactions could play key roles in the compartmentalization of ROS-mediated signal transduction as well as could influence its specificity, efficiency and dynamics [8,9].
Our previous studies revealed that adaptor protein Ruk/CIN85, which contains three SH3 domains in its structure, can interact with Pro-rich motifs of adaptor protein Tks4 [10,11] functioning as an organizer subunit of NADPH-oxidase complex mediated by Nox1 [12,13].Additionaly, it has been shown by us that the ROS production by human colorectal adenocarcinoma HT-29 cells is positively correlated with the Ruk/CIN85 expression [14].Recently, we have demonstrated systemic multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22 Phox in Ruk/CIN85-overexpressing human breast adenocarcinoma MCF-7 cells [15] characterized by increased malignant phenotype [16].Using the expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with ISSN 1993-6842 (on-line)

Protein preparation and Western blot analysis
MCF-7 cells were scraped off in lysis buffer [50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1 % Triton X-100, 1 mM ο-vanadate, 50 mM NaF, 2 mM EDTA, 1 mM PMSF, complete inhibitor cocktail tablet (Roche)], mechanically triturated through a 1 ml syringe, kept on ice for 20 min and centrifuged at 14 000 g for 20 min at 4°C.After centrifugation of lysates, the protein content in supernatants was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology).Proteins (30 µg per sample) were separated by electrophoresis on 10 % polyacrylamide gels and transferred to nitrocellulose membranes.The membranes were incubated with monoclonal anti-Ruk/CIN85 [17], anti-Akt (Cell Signaling) and anti-phospho-Akt (Ser473) (Cell Signaling) antibodies overnight at 4°C.Appropriate peroxidase-conjugated secondary antibodies (Sigma) were used at 1:3000 dilutions.The enhanced chemiluminescence kit (Amersham Pharma cia Biotech) was used for detection of the immunoreactive bands.

Transwell migration assay
The cell motility assay was performed essentially as described in [16] using 24-well cell culture inserts with 8 µm pores (Greiner Bio-One).Where indicated, the cells were preincubated with 300 µM apocynin for 24 h. 3 x 10 4 cells were seeded on the upper wells of chambers in the presence of 0.1 % serum and 300 µM apocynin.The lower wells were filled with medium containing 5 % FCS.After incubation for 12 h, the cells that migrated out onto the lower surface of membranes were fixed in 4 % paraformaldehyde.These cells were stained with 1 % crystal violet and counted in five random fields (more than 100 cells were scored in each experiment).

Statistics
Statistical analysis was carried out using SPSS 12.0 software.Paired and unpaired Student's t-tests were performed and the difference was considered to be significant when the P-value was ≤0.05.

Results and Discussion
Many reports of last decade provide an evidence that ROS, produced by NADPH oxidases in mammalian cells in response to the activation of membrane receptors, play important roles in carcinogenesis [1][2][3].Redox-sensitive signaling associated with NADPH oxidases has been shown to occur in different subcellular compartments involved in the control of tumor cells migration and invasion [2,18].The adaptor/scaffold proteins of modular structure not only determine the formation and localization of signaling complexes [8] but also control the specificity, efficiency and amplitude of signal propagation [9].Adaptor/scaffold protein Ruk/CIN85 containing three SH3 domains, proline-rich region and C-terminal coiled-coil domain was shown to be a platform for signaling complexes formation involved in the control of fundamental cellular and signaling events as well as carcinogenesis [19,20].By analyzing samples from breast cancer patients with invasive breast adenocarcinomas, we found high levels of Ruk/CIN85 especially in lymph node metastases and intravascular tumor emboli [16].
Stable expression of Ruk/CIN85 in weakly invasive breast adenocarcinoma MCF-7 cells was followed by their reduced growth rate, decreased cell adhesion, enhanced anchorage-independent growth and increased motility.The stimulation of Ruk/CIN85overexpressing MCF-7 cells with EGF led to a more rapid and prolonged activation of Src, Akt and ERK1/2 while the treatment with Src inhibitor PP2 and PI3K inhibitor LY294002 abolished the Ruk/ CIN85-dependent changes in cell motility [16].
Taking into account our data regarding the direct physical interaction of Ruk/CIN85 with the organizer subunit Tks4 of NOX1-containing NADPH oxidase complex [10,11], a positive correlation between the level of Ruk/CIN85 expression and the ROS production in tumor cells [14, and our unpublished data], co-localization of adaptor protein and H 2 O 2 generation at the edges of certain vesicular structures in MCF-7 cells transiently transfected with the plasmid encoding fluorescent sensor of hydrogen peroxide Hyper fused with Ruk/CIN85 [17], the potential of Ruk/CIN85 to differentially influence NOX genes expression in MCF-7 cells stably overexpressing different levels of Ruk/CIN85 [15], it was the aim of this study to investigate a possible involvement of NADPH oxidases in the control of cell motility and Akt signaling depending on the Ruk/CIN85 expression levels in MCF-7 cells.For this purpose we used apocynin, which is considered to be one of the most specific inhibitor of NADPH oxidases, which blocks the assembly of the active enzyme complex [21].In accordance with our previous results [16], we demonstrated that the motility of MCF-7 G10 cells with a high level of Ruk/CIN85 overexpression was significantly higher than that of control cells (Fig. 1A).The treatment of G10 cells with apocynin resulted in the suppression of their  migratory potential to the level of control cells.As can be seen from Fig. C, EGF induced more potent activation of Akt kinase up to 30 min of stimulation in comparison with the dynamics of Akt activity characteristic of control cells (Fig. 1B).The pretreatment of G10 cells with apocynin resulted in the disappearance of a signal for pAkt at 30 min while in control cells any changes in the Akt activity caused by apocynin were not observed (Fig. 1B, 1C).These data allow us to conclude that in Ruk/CIN85overexpressing MCF-

Fig. 1 .
Fig. 1.Apocynin attenuates motility and induces the transition from sustained to transient EGF-dependent Akt activation in human breast adenocarcinoma MCF-7 cells that overexpress adaptor protein Ruk/CIN85.A. The transwell migration assay was used to study the motility of control (MCF-7 control) and Ruk/ CIN85-overexpressing (MCF-7 G10) cells.The number of migrated control cells was set equal to 100 %.Data are mean ± SEM of three independent experiments.*P ≤ 0.05 compared with control.#P ≤ 0.05 compared with MCF-7 G10.B, C. Western blotting of pAkt, tAkt and Ruk/CIN85 in lysates of control MCF-7 cells (B) and Ruk/CIN85-overexprerssing MCF-7 G10 cells (C) treated with EGF (100 ng/mL) for indicated periods of time.The representative blots of three independent experiments are shown.