AsA inhibits glioma cells induced chemotactic motility of endothelial cells.

<p><b>A & B.</b> HUVEC or HBMEC (3×10⁴ per well) were seeded in the upper chamber of a Transwell plate with 0.5% serum media. In the lower chamber, LN18 cells (3×10⁴ cells per well) were treated with DMSO or AsA 20 µM in 0.5% serum media as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. HUVEC and HBMEC migrated through the matrigel layer were stained and quantified after 10 h and 22 h of their plating in the upper chamber respectively. Cell invasion data shown are mean ± standard deviation of three samples for each treatment. *, p≤0.001; $, p≤0.05.</p

AsA reduces VEGF level (both cellular and secreted) in glioma cells and strongly inhibits VEGF-stimulated angiogenesis <i>in vivo.</i>

<p><b>A.</b> LN18 and U87-MG cells were treated with DMSO or AsA (20 µM) for 24 and 48 h. After each treatment time, cell lysates were prepared and analyzed for VEGF by Western blotting as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. Membranes were stripped and reprobed with anti- α-tubulin antibody to confirm equal protein loading. Densitometric values presented below the bands are ‘fold change’ compared to respective control after normalization with loading control. <b>B.</b> In LN18 and U87-MG cells, media was collected 24 h after AsA treatment (20 µM) and analyzed for secreted VEGF level by Western blotting and ELISA as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. <b>C.</b> Nude mice were subcutaneously injected with 0.5 mL Matrigel containing 100 ng/mL VEGF, 100 units of heparin and different doses of AsA (12.5, 25 and 50 µg/mL). Matrigel plugs were removed after 5 days and representative pictures are shown. *, p≤0.001; #, p ≤ 0.01.</p

AsA inhibits human glioma cell-induced angiogenesis <i>in vitro.</i>

<p><b>A.</b> LN18 and U87-MG human glioma cells were treated with AsA at 20 µM doses for 24 h, media was removed, washed with 0.5% serum media and incubated for an additional 12 h in 0.5% serum media without the presence of DMSO or AsA. Subsequently, both LN18 and U87-MG cells were trypsinized, and counted using haemocytometer as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. <b>B.</b> VEGF expression in CCM (control conditioned media) and AsA20CM (AsA20 conditioned media) or total cell lysates collected from LN18 and U87-MG cells (detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’) was analyzed by Western blotting. The loading volume for conditioned media in each case was normalized with respective cell number. Densitometric values presented below the bands are ‘fold change’ compared to respective controls. AsA20-T/W refers to the group, where glioma cells were treated with AsA 20 µM dose for 24 h and then AsA was washed-out and cell lysates were prepared after 12 h. <b>C & D.</b> HUVEC or HBMEC (4×10⁴ per well) were seeded in 24-well plates coated with matrigel and treated with CCM or AsA20CM from LN18 cells or U87-MG cells for 10 h and tube formation assay was performed as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. In this experiment, HUVEC or HBMEC incubated with 0.5% serum containing LN18 or U87-MG media served as a negative control. Tubular structures were photographed at 100x magnification and tube length was measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. Tube length data is presented as mean ± standard deviation of three samples for each treatment. The volume for CCM/AsA20CM used in tube formation assay was normalized with respective cell number shown in panel A. *, p≤0.001.</p

AsA inhibits VEGF-stimulated cell growth and capillary tube formation in HUVEC.

<p>HUVEC were grown under 0.5% serum conditions and treated with or without VEGF (10 ng/mL) and various doses of AsA (5–20 µM) for 12 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>. After 12 h, both adherent and non-adherent cells were collected and processed for determination of total cell number (<b>A</b>) and dead cells percentage (<b>B</b>) as mentioned in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. <b>C.</b> HUVEC with or without VEGF (10 ng/mL) in 0.5% serum media were placed in 24-well plates coated with Matrigel and treated with AsA at indicated doses. After 10 h tubular structures were photographed at 100x magnification and tube length was measured as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. These results (A–C) were similar in 2–3 independent experiments. Each bar is representative of mean ± standard deviation of three samples for each treatment. *, p≤0.001; #, p≤0.01.</p

AsA inhibits motility and capillary-structure formation in HUVEC.

<p><b>A.</b> Effect of AsA treatment on the migratory potential of HUVEC was analyzed through wound healing assay. Representative photomicrographs of initial and final wounds are shown at 100x magnification and migration distance was measured as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. Cell migration distance data shown are mean ± standard deviation of three samples for each treatment. <b>B.</b> Effect of AsA treatment on the invasive potential of HUVEC was examined using invasion chambers as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. Cell invasion data shown are mean ± standard deviation of three samples for each treatment. <b>C.</b> Effect of AsA on the tube formation of HUVEC was examined by plating HUVEC on the matrigel. After 6 h, tubular structures were photographed at 100x magnification and tube length was measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. Tube length data is presented as mean ± standard deviation of three samples for each treatment. <b>D.</b> Effect of AsA on the pre-formed tubes in HUVEC was analyzed and tube length was measured as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. Tube length data shown are mean ± standard deviation of three samples for each treatment. These results (A–D) were similar in 2–3 independent experiments. *, p≤0.001.</p

AsA inhibits growth and induces apoptosis in HUVEC.

<p><b>A & B.</b> HUVEC (4×10⁴ cells per well) were treated with DMSO or different doses of AsA (5–20 µM) in complete HUVEC media for 6, 12, 24 and 48 h. At each treatment time, both adherent and non-adherent cells were collected and processed for the determination of viable cell number and dead cells percentage as mentioned in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. <b>C.</b> HUVEC were treated with DMSO or AsA (5–20 µM) for 24 h and analyzed for apoptotic cell population using annexin V/PI staining as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. In panels A, B, & C, each bar represents the mean ± standard deviation of three samples. These results were almost similar in two independent experiments. *, p≤0.001; #, p≤0.01; $, p≤0.05. <b>D.</b> HUVEC were treated with DMSO or AsA (20 µM) for 24 and 48 h. After each treatment time, total cell lysates were prepared and analyzed for cleaved PARP, cleaved caspase 3, cleaved caspase 9, Bad, survivin, pAkt-ser473, and total Akt by Western blotting as detailed in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022745#s4" target="_blank">Materials and Methods</a>’. In each case, membrane was also stripped and reprobed with anti-α-tubulin antibody to confirm equal protein loading.</p

Effects of fisetin, acacetin and doxorubicin on cytotoxicity IC50 values of A549 and H1299 cells.

<p>Effects of fisetin, acacetin and doxorubicin on cytotoxicity IC50 values of A549 and H1299 cells.</p

Expression of MDR1 gene by RT-PCR.

<p>A549 and H1299 cells were treated with respective treatments for 72 h and processed by RNA isolation. Semi quantitative PCR reaction of amplified MDR1 product were analysed by 1% agarose gel electrophoresis. (A) and (B) are representing the RNA expression of MDR1 gene in A549 and H1299 cells, respectively. (C) and (D) are bar diagram of densitometric value of bands representing the relative fold change in RNA expression. GAPDH was used as loading control.</p

Combinatorial effects of fisetin or acacetin with doxorubicin on cell viability in NSCLC cells.

<p>A549 (A, B) and H1299 cells (C, D) were treated with 10 and 25 μM of Fs and Ac in combination with Dox (10 nM for A549 and 25 nM for H1299 cells) for 48 and 72 h. At the end of treatments, cells were processed for trypan blue assay. The data are presented as means of triplicate samples for each treatment. Fs, fisetin; Ac, acacetin; Dox, doxorubicin. Bars, SE; P<0.0001 (#), P<0.001 (***), P<0.01 (**), P< 0.05 (*).</p

Flow cytometry analysis of doxorubicin uptake in A549 and H1299 cells.

<p>(A) A549 and (B) H1299 cells were pre-treated with 25 μM Ac and 25 μM SB for 6 h and post-incubated with 10 μM Dox. The cells were then collected at different time points and proceed for FACS analysis. (C) Representative figure of flow cytometry histogram of fluorescence intensity after respective treatments. The data is presented as means of triplicate samples for each treatment. Groups at different time points have been compared with respective Dox alone treatment. Ac, acacetin; SB, silibinin; Dox, doxorubicin. P<0.001 (***), P<0.01 (**), P< 0.05 (*).</p