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 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 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 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

Chemical structure of silybin and its derivatives.

<p>(<b>a–h</b>) Chemical structures of silybin, silybin A, silybin B, 2,3-dehydrosilybin, 7-<i>O</i>-methylsilybin, 7-<i>O</i>-galloylsilybin, 7,23-disulphate silybin, 7-<i>O</i>-palmitoylsilybin, and 23-<i>O</i>-palmitoylsilybin.</p

Anti-Cancer Efficacy of Silybin Derivatives - A Structure-Activity Relationship

<div><p>Silybin or silibinin, a flavonolignan isolated from Milk thistle seeds, is one of the popular dietary supplements and has been extensively studied for its antioxidant, hepatoprotective and anti-cancer properties. We have envisioned that potency of silybin could be further enhanced through suitable modification/s in its chemical structure. Accordingly, here, we synthesized and characterized a series of silybin derivatives namely 2,3-dehydrosilybin (DHS), 7-<i>O</i>-methylsilybin (7OM), 7-O-galloylsilybin (7OG), 7,23-disulphatesilybin (DSS), 7-<i>O</i>-palmitoylsilybin (7OP), and 23-<i>O</i>-palmitoylsilybin (23OP); and compared their anti-cancer efficacy using human bladder cancer HTB9, colon cancer HCT116 and prostate carcinoma PC3 cells. In all the 3 cell lines, DHS, 7OM and 7OG demonstrated better growth inhibitory effects and compared to silybin, while other silybin derivatives showed lesser or no efficacy. Next, we prepared the optical isomers (A and B) of silybin, DHS, 7OM and 7OG, and compared their anti-cancer efficacy. Isomers of these three silybin derivatives also showed better efficacy compared with respective silybin isomers, but in each, there was no clear cut silybin A versus B isomer activity preference. Further studies in HTB cells found that DHS, 7OM and 7OG exert better apoptotic activity than silibinin. Clonogenic assays in HTB9 cells further confirmed that both the racemic mixtures as well as pure optical isomers of DHS, 7OM and 7OG were more effective than silybin. Overall, these results clearly suggest that the anti-cancer efficacy of silybin could be significantly enhanced through structural modifications, and identify strong anti-cancer efficacy of silybin derivatives, namely DHS, 7OM, and 7OG, signifying that their efficacy and toxicity should be evaluated in relevant pre-clinical cancer models in rodents.</p> </div

Schematic of possible therapeutic targets for silibinin in CEES-induced skin injury pathways identified in our studies.

<p>Possible targets of silibinin (red green arrows); increase or decrease by silibinin (up or down arrows, respectively).</p

Silibinin reverses CEES-induced activation of NF-κB and AP-1 transcription factors in SKH-1 hairless mouse skin.

<p>Dorsal skin of mice was exposed topically to either 200 µL of acetone or CEES (2 mg) in 200 µL acetone, or treated with either silibinin alone or with 0.5/1 mg silibinin 30 min after CEES exposure. Mice were sacrificed and dorsal skin tissue samples were collected and snap frozen in liquid nitrogen at 24 h following the above exposures and treatment. Nuclear lysates were prepared from the skin tissue and analyzed for NF-κB (<b>A</b>) and AP-1 (<b>B</b>) DNA binding activity by EMSA as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046149#s2" target="_blank">Materials and Methods</a>. Nuclear extract from CEES exposed skin tissue samples were used for cold competition with cold NF-κB (<b>A</b>) or AP-1 (<b>B</b>) consensus oligo, and super shift assay was performed as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046149#s2" target="_blank">Materials and Methods</a>. The results obtained were quantified by densitometric analysis. VC, vehicle (acetone); SB, silibinin; CEES+0.5SB, 0.5 mg silibinin treatment 30 min after CEES exposure; CEES+1SB, 1 mg silibinin treatment 30 min after CEES exposure.</p