Synthesis of steroidal 16α-triazoles by CuAAc.

<p>Synthesis of steroidal 16α-triazoles by CuAAc.</p

Effects of 5f and 5g (10 μM) on the expression of phosphorylated (upper panel) and total (lower panel) stathmin protein in HeLa cells after incubation for 48 h, determined by western blot analysis.

<p>Results are mean values ± SEM of the data on two separate measurements, n = 6. ns indicates p > 0.05, *** indicates p < 0.001 as compared with the untreated control cells. Panels below are representative membrane pictures.</p

Induction of caspase-3, caspase-8 and caspase-9 activities after incubation with compounds 5f and 5g for 24 h.

<p>White, gray and black columns denote 3, 10 and 30 μM of the given agent. * and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control condition.</p

Effects of compounds 5f, 5g, 5h and 4i on the HeLa cell cycle distribution after incubation for 24 (left panels) and 48 h (right panels).

<p>Gray and black columns indicate 3 and 10 μM, respectively. * and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control cells.</p

Fluorescent microscopy images of Hoechst 33258-PI double staining.

<p>Two separate pictures from the same field were taken for the two markers. MRC5 cells were treated with vehicle (control), or with <b>5f</b> and <b>5g</b> at the indicated concentrations. The blue fluorescence (left panels) indicates Hoechst 33258, and the red fluorescence (right panels) is a consequence of PI accumulation. The bar in the PI control picture indicates 100 μm.</p

Expression of CDK1, cyclin B1, cyclin B2 and cdc25B at the mRNA level after incubation with 3 (gray columns) or 10 (black columns) μM of compounds 5f or 5g.

<p>*, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively, as compared with the control condition.</p

Effects of compounds 5f and 5g on the Bax/Bcl-2 ratio after incubation of HeLa cells for 24 h.

<p>* and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control condition.</p

TGF-β1 and activated B16/F10 conditioned medium modulate TEER, melanoma-endothelial cell adhesion and transendothelial migration of melanoma cells.

<p><b>(a) TGF-β1 and activated B16/F10 conditioned medium reduce TEER of RBECs</b>. Both TGF-β1 and activated B16/F10 conditioned medium caused a significant, time dependent decline of the transendothelial electric resistance (TEER) of the RBEC monolayer. SB-431542 (60 min) pretreatment and treatment of RBECs inhibited or reduced the decline of TEER upon stimulation with either stimulus. The graph summarizes the results of 3 independent measurements for each treatment, average and SE values are presented. Comparing the TEER between TGF and control (*), ACM and control (*), TGF+SB and TGF (**), ACM+SB and ACM (***) we obtained p<0.05 in all three time points, except for ACM vs. control at 2 hrs where the significance was lower (p<0.01, ****), as assessed by ANOVA and Bonferroni’s post hoc test. <b>(b) TGF-β-dependent adhesion of B16/F10 melanoma cells to RBECs</b>. Confluent RBECs were pretreated with TGF-β1, B16/F10 conditioned media (B16 CM) or B16/F10 activated conditioned media (B16 ACM) in the presence or absence of SB-431542 (SB) for 5 hrs. Fluorescently labeled B16/F10 melanoma cells (5x10⁴/well) were plated onto confluent RBECs and left for 70 min. After washing of non-adherent cells, attached melanoma cells were counted. TGF-β1 pretreatment led to a marked increase of melanoma cells attached to the endothelial monolayer. Similarly to TGF-β1, B16/F10 ACM pre-treatment also enhanced the attachment of B16/F10 melanoma cells onto the endothelial monolayer, in a TGF-β- dependent manner, since this effect was mitigated in the presence of the TGF-β inhibitor SB-431542 (p<0.05 in case of TGF vs. control (*), B16 ACM vs. control (*) and B16 ACM+SB vs. B16 ACM (**), as assessed by ANOVA and Bonferroni’s post hoc test). <b>(c) Enhanced TGF- β-dependent transendothelial migration of melanoma cells</b>. RBECs were cultured until confluence in 12 well plates and treated with TGF-β1 or B16/F10 ACM for 5 hrs. Fluorescently labelled B16/F10 melanoma cells (2 x 10⁴/well) were plated onto the monolayer. Cells were monitored for 10 hrs and then transmigrating melanoma cells were counted. Both stimuli enhanced the number of transmigrating melanoma cells. Preincubation of RBECs with 10 μM SB-431542 for 60 min inhibited ACM induced transendothelial migration of melanoma cells (*- p<0.01 for TGF vs. control and B16 ACM vs control, **- p<0.01 for B16 ACM+ SB vs. B16 ACM, ANOVA and Bonferroni’s post hoc test).</p

TGF-β1 induces EndMT in primary rat BECs.

<p><b>(a)</b> BECs were subjected to 48 hrs TGF-β1 treatment, then fixed and stained for claudin-5 and VE-cadherin. Control cells showed claudin-5 and VE-cadherin at their intercellular borders, which was depleted in TGF-β1 treated cells (400x magnification). BECs were treated with TGF-β1 for 48 hrs, and were analyzed for endothelial, mesenchymal and myogenic marker expression by Western blot. TGF-β1 treatment led to the <b>(b)</b> down-regulation of tight and adherens junction protein expression (claudin-5, occludin, VE-cadherin), it induced β1-integrin expression and <b>(c)</b> and led to a marked increase in N-cadherin expression (2.18±0.48 fold based on three independent experiments, where N-cadherin expression increased 2.11, 1.52 and 2.89 fold respectively, as compared to controls). TGF-β1 treated cells showed a robust expression of fibronectin <b>(d)</b>, SMA and calponin <b>(e)</b>. SMA expression was observed by immunofluorescence microscopy (400x magnification) under similar conditions as well <b>(f)</b>.</p

Activated breast cancer cell conditioned medium induces SMA expression through TGF-β signaling in HUVECs.

<p>FBS-free M199 medium was conditioned with MDA-MB231 <b>(a)</b> or SK-BR3 <b>(b)</b> human breast cancer cells for 24 hrs. A half of the conditioned media were subjected heat activation, and then both conditioned (CM) and activated conditioned media (ACM) were supplemented with FBS. Cells were pre-treated with 10 μM SB-431542 for 60 min and then treated with CM or ACM for 72 hrs, in the presence of the inhibitor. Samples were analyzed by Western blot. SB-431542 inhibited MDA-MB231 or SK-BR3 ACM induced SMA expression. <b>(c) Co-culture with melanoma cells leads to expressional changes characteristic to EndMT in HUVECs</b>. Gene expression profiles of HUVECs and HUVECs co-cultured with 1205Lu human metastatic melanoma cells were analyzed and compared. In HUVECs co-cultured with melanoma cells there was a marked decrease in expression levels of several endothelial markers (KRT7, KRT18, TJP2), as well as a severe decrease in FST expression. In parallel, co-cultured HUVECs exhibited elevated expression levels of EndMT markers (FN1, COL3A1, S100A4, MMP2, COL1A2) and transcriptional regulators (ZEB1, Wnt5a, TWIST1, Snai2).</p