A significant increase of cell surface (1.89-fold) was measured the 10 M of HNK was present in the medium. ROS production (Shen et al., 2010). On the TBP basis of the aforementioned evidences, HNK is a promising compound to be exploited to attenuate cisplatin-induced renal toxicity and to improve clinical safety of cisplatin for patients who undergo cancer treatments. In this study, we aim to evaluate OS evaluation, after required treatments, cells were fixed and stained with anti-8-OHdG antibody (1:1000). Positive cells were manually counted and the percentages of positive cells were calculated accordingly. For signal correlation analysis between Occludin and E-cadherin, 10 images were randomly taken from each group, Pearsons correlation coefficients were calculated using ImageJ (NIH1). 2D Polarized Transwell? Culture and Signal Dispersion Analysis Madin Darby Canine Kidney cells were seeded in the Transwell? (pore size 0.4 m) at the density of 80,000 cells/well and were grown for overnight to achieve 100% confluence of monolayer. The cells were serum-starved and treatments were added in both the upper and the lower chamber. Immunofluorescent staining on polarized cells was carried out as in regular cultured cells described above. After staining, the Transwell? membranes were excised and placed on glass slides for microscope evaluation. To generate 3D information on monolayer images from 2D Transwell? culture, polarized cells were scanned with Leica TCS SP5 II confocal scanning microscopy using 100X object with a step size of 0.3 m. For junction protein dispersion analysis, the z-stack images were re-sliced along mTOR inhibitor-2 the 0.05. Results Honokiol Attenuated Cisplatin-Induced Disorganization of Occludin and E-Cadherin To investigate the effects of HNK, we first examined HNK effect on protein expression and cellular localization of E-Cadherin and Occludin, two proteins located at the adhesion and tight junction of kidney epithelial cells, respectively. As showed in Figure ?Figure1A1A, up to 10 M of cisplatin and 15 M of HNK, no cytotoxicity was detected, we thereafter applied 10 M concentrations for both compounds in all subsequent experiments. Moreover, we observed no changes in cell viability under 10 M HNK/10 M cisplatin combination in 24 h- MTT assay (data not showed) indicated no apparent cytotoxicity from this combined incubation. We detected no significant changes on protein expression level for both E-Cadherin and Occludin upon cisplatin or HNK treatments (Figure ?Figure1B1B); however, when cells were grown in polarized 2D Transwell? system, an apparent redistribution of both proteins was noted (Figure ?Figure1C1C). Occludin (in green) and E-Cadherin (in red) redistributed from the apical or lateral side of MDCK cells toward the cytosol upon cisplatin treatment; however, when HNK was co-present with cisplatin, the disorganized signals of both proteins were partially inhibited (Figure ?Figure1C1C). Quantification analyses further confirmed our observation that when compared with control or HNK-treated cells, Occludin and E-Cadherin were 1.42- and 1.84- fold more dispersed into the cytosol in those of cisplatin-treated cells. Moreover, HNK co-incubation with cisplatin significantly reduced the dispersion of both proteins (Figure ?Figure1D1D, stripped bars). In agreement with our observations, co-localization analysis on epi-fluorescent images indicated that cisplatin treatment reduced the co-localization of Occludin and E-Cadherin signals (Figures ?Figures2A2ACC); however, when HNK was present in the cisplatin-containing medium, co-localization of two proteins and Pearsons correlation can partly be restored (Figure ?Figure2D2D). Pearsons correlation analyses showed a restoration value from 0.4 to 0.69 with a higher degree of co-localized signal (in yellow) when HNK was co-present in the cisplatin-containing incubation medium (Figures 2D,E). The reduced co-localization of two junction proteins and the increased cytosol mTOR inhibitor-2 detection of both Occludin mTOR inhibitor-2 and E-Cadherin suggested the internalization of both proteins. Open in a separate window FIGURE 1 Cytotoxicity analyses and the effects of mTOR inhibitor-2 cisplatin and honokiol (HNK) on mTOR inhibitor-2 protein expression and cellular localization of Occludin and E-Cadherin. (A) To determine cell toxicity of cisplatin and HNK, cell viability assay namely MTT [(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)] assay was carried out. Up to 10 M of cisplatin and 15 M of HNK, no cytotoxicity was.
