Capsaicin inhibits VEGF transcriptional activation by targeting HIF-1α in a p53-dependent manner.

<p>(A) Time-dependent expression profiles of HIF-1α mRNA and -protein were determined by Western blot and RT-PCR, respectively, in capsaicin-treated Hy-A549 cells (<i>left panel</i>). Capsaicin-treated Hy-A549 cells were examined for time-dependent variation in the expression profiles of HIF-1α by quantitative real time PCR analysis and represented graphically (<i>right panel</i>). (B) Hy-A549 cells, transiently transfected with a non-targeting control-siRNA or HIF-1α-siRNA, were incubated with/without capsaicin for 24 h; the cells were then analyzed to determine VEGF expression at protein and mRNA levels (<i>left panel</i>). Immunoblot showing transfection efficiency of HIF-1α (<i>right panel</i>) (C) Control-siRNA/HIF-1α-siRNA-transfected Hy-A549 cell-supernatants were used to assess HUVEC migration by wound healing assay after capsaicin-treatment (37.5 µM; 24 h) and represented graphically. (D) Time-dependent expression profile of p53-mRNA and -protein was determined by Western blotting and RT-PCR in capsaicin-treated Hy-A549 cells (<i>left panel</i>). p53 phosphorylation at Serine-15 position was also evaluated (<i>right panel</i>). (E) Hy-A549 cells, transfected with control-shRNA/p53-shRNA were incubated with capsaicin for 24 h and HIF-1α VEGF-mRNA and -protein were determined by Western blot and RT-PCR (<i>left panel</i>). Transfection efficiency was checked by analyzing p53 expression level (<i>right panel</i>). (F) HIF-1α was immunoprecipitated from capsaicin-treated Hy-A549 cell lysates and immunoblotted with anti-Ub antibody to assay HIF-1α ubiquitination. The ladder of bands represented ubiquitinated HIF-1α. In parallel experiment, immunoprecipitates were assayed for HIF-1α levels by Western blot. Comparable protein input was confirmed by direct Western blotting with anti-α-actin using 20% of the cell lysates that were used for immunoprecipitation. (G) Control and MG-132 drug-pretreated Hy-A549 cells were subjected to capsaicin-treatment for 24 h and then were examined for expression of HIF-1α/VEGF by Western blotting. α-Actin/GAPDH was used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p

Cell free tumor supernatant leads to CD4⁺ T cell depletion by inducing apoptosis.

<p>A, Purified human peripheral CD4⁺ T cells were cultured in the presence of media alone or cell-free MCF-7-spent media (±theaflavins, doses from 6.25 µg/ml to 50 µg/ml). After 48 hours, viable cell numbers were scored by Trypan Blue exclusion method. B, Graphical representation of percent apoptosis of CD4⁺ T cells (<i>left panel</i>) and Jurkat T cells (<i>right panel</i>). CD4⁺ T cells labelled with Annexin V-PE and 7AAD were analyzed flow cytometrically. Annexin V/7AAD-positive cells were regarded as apoptotic cells. Values are mean±S.E.M. of five independent sets of experiments.</p

Stat-5A transfection confers resistance to CD4⁺ T cells from tumor-induced death.

<p>A, Stat-5A (<i>left panel</i>) and Stat-5B (<i>right panel</i>) isoforms were immunoprecipitated from cell lysates using specific antibodies and then Western blotted with anti-phospho-tyrosine or anti-Stat-5A/Stat-5B antibodies to determine phosphorylation status of specific proteins. B, Jurkat T cells were transfected with control vector, wild-type <i>Stat-5A/Stat-5B</i>, C-terminal truncated <i>Stat-5A</i>₇₁₃/<i>Stat-5B</i>₇₁₈ or constitutively active <i>Stat-5A1*6</i> genes and were cultured in the presence of media alone or MCF-7 spent media (±theaflavins) for 48 h. Percent cell death (Annexin-V-PE⁺/7AAD⁺) was determined flow cytometrically. Values are mean±S.E.M. of three independent sets of experiments.</p

Capsaicin inhibits the nuclear localization of HIF-1α by down regulating Cox-2 in a p53-dependent manner.

<p>(A) Immunoblot representing nuclear and cytosolic levels of HIF-1α in capsaicin-treated/p53-shRNA-transfected Hy-A549 cells. (B) HIF-1α expression was monitored in capsaicin-treated Hy-A549 cells by confocal microscopy (magnification 60x). (C) Time-dependent expression of Cox-2-mRNA and -protein in capsaicin-treated Hy-A549 cells was determined by Western blot and RT-PCR. (D) Control siRNA-/Cox-2-siRNA-transfected Hy-A549 cells were treated with capsaicin (37.5 µM; 24 h) and nuclear translocation of HIF-1α was assessed by Western blot analysis (<i>left panel</i>). Transfection efficiency was determined by analyzing the expression of Cox-2 (<i>right panel</i>). (E) Control vector-/Cox-2 cDNA transfected Hy-A549 cells were treated with capsaicin (37.5 µM; 24 h) and analyzed for nuclear and cytosolic expression of HIF-1α (<i>left panel</i>). Transfection efficiency of Cox-2 was also verified (<i>right panel</i>). (F) Control-shRNA-/p53-shRNA-transfected Hy-A549 cells were treated with capsaicin (37.5 µM; 24 h) and expression profiles of Cox-2 both at protein and mRNA level were examined. (G) Time-dependent expression profiles of SMAR1-protein and mRNA in capsaicin-treated Hy-A549 cells were determined by Western blot and RT-PCR (<i>left panels</i>). Control-/p53-shRNA transfected Hy-A549 cells were treated with capsaicin and expression levels of SMAR1 were checked (<i>right panel</i>). (H) Control-/SMAR1-shRNA transfected Hy-A549 cells were treated with capsaicin and immune-blotted with p-Ser¹⁵-p53. α-Actin/H1-Histone/GAPDH were used as internal loading control. Values are mean ± SEM of three independent experiments in each case or representative of typical experiment.</p

Effect of capsaicin on lung cancer cell spent medium-induced endothelial cell migration and network formation.

<p>(A) Migration of HUVECs in presence or absence of spent media of NME, WI-38, A549 and Hy-A549 (CoCl₂-stimulated to mimic hypoxic condition required for tumor-induced angiogenesis) were subjected to bi-directional wound healing assay for 24 h (<i>left panel</i>). The number of cells migrated in the wound area are represented graphically (<i>right panel</i>). (B) Representative images of HUVEC migration upon incubation with capsaicin-treated (0–50 µM) Hy-A549 cell spent medium (<i>left panel</i>). Percent cell migrated in the wound area has been represented graphically (<i>right panel</i>). (C) Hy-A549 cells were treated with capsaicin in a dose-dependent manner for 24 h and cell viability was scored by trypan blue dye-exclusion assay (<i>left panel</i>). Hy-A549 cells, treated with capsaicin (37.5 µM), were subjected to Annexin-V-FITC/PI binding and analyzed flow cytometrically for the determination of percent early apoptosis (<i>right panel</i>). (D) Cytotoxic effect of different doses of capsaisin on HUVEC cells were measured by trypan blue dye-exclusion assay. (E) Graphical representation of HUVEC migration upon incubation with spent media from capsaicin-treated (37.5 µM) HBL-100, HCT-15, HeLa and A549. (F) Representative images of capillary-like sprout formation by HUVECs in presence of media alone or spent media of WI-38/A549/Hy-A549/capsaicin-treated Hy-A549. Values are mean ± SEM of three independent experiments in each case or representative of typical experiment.</p

Schematic diagram representing molecular mechanisms of capsaicin-mediated down-regulation of pro-angiogenic factor, VEGF.

<p>Schematic diagram representing molecular mechanisms of capsaicin-mediated down-regulation of pro-angiogenic factor, VEGF.</p

Capsaicin suppresses VEGF expression by SMAR1-mediated down regulation of Cox-2.

<p>(A) Control-/SMAR1-shRNA (<i>left panel</i>) or control-vector/SMAR1-cDNA (<i>right panel</i>) transfected Hy-A549 cells were treated with capsaicin were assayed for Cox-2 expression by Western blot and RT-PCR. Transfection efficiency was verified by Western blot (<i>bottom panels</i>). (B) Cox-2 promoter activity was checked in Hy-A549. Schematic representation of the Cox-2 promoter showing the probable SMAR1-binding sites predicted by using the MARWIZ software, PCR run with eight different sets of primers designed for each probable MAR binding site spreading over four regions (−141 bp to −421 bp; −631 bp to −1121 bp; −1262 bp to −1331 bp; −1471 bp to −1891 bp). ChIP assay with anti-SMAR1 was performed on MAR-binding regions of Cox-2 promoter. Input and control IgG was used as internal control and negative control. (C) The relative abundance of SMAR1 on Cox-2 promoter was analyzed in control and capsaicin treated Hy-A549 cells at binding sites 6 & 7 after ChIP of Cox-2 promoter with anti-SMAR1(<i>left panel</i>) and represented graphically (<i>right panel</i>). (D) Control-/SMAR1-shRNA-transfected Hy-A549 cells were treated with capsaicin and analyzed for reporter HIF-1α and VEGF gene expression by RT-PCR. α-Actin/GAPDH were used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p

Re-confirmation of PGE₂ as the molecule behind tumor-induced perturbation in CD4⁺ T cell survival signaling.

<p>A, MCF-7 cells were treated with theaflavins or celecoxib or transfected with Cox-2-siRNA and the levels of Cox-2 and GAPDH (internal control) mRNA were determined by RT-PCR (<i>upper panel</i>). Western blot analysis was performed for the determination of levels of Cox-2 or α-Actin (internal control) proteins (<i>middle panel</i>). In parallel experiments the amount of tumor-secreted PGE₂ in the cell-free supernatant was determined by ELISA (<i>lower panel</i>). B, Purified CD4⁺ T cells were cultured in the presence of media alone or MCF-7-spent media (tumors were either pre-treated with 25 µg/ml theaflavins/3.5 ng/ml PGE₂/50 µM celecoxib or transfected with 300pmole Cox-2-siRNA) for 48 h. Expression levels of IL2Rγc and Bcl-2 as well as phosphorylation status of Jak-3/-Stat-5 were determined by Western blotting in which α-Actin was used as internal control (<i>upper panel</i>). In parallel experiments, flowcytometric determination of percent cell death (<i>lower panel</i>) was established. Values are mean±S.E.M. of three independent experiments.</p

Tumor-shed PGE₂ is responsible for CD4⁺ T cell apoptosis.

<p>A, Tumor-secreted PGE₂ over time in cell-free spent media of MCF-7 cells (control (○), Cox-2-siRNA-transfected (Δ) or theaflavin-treated (•) was determined by ELISA. B, Percent CD4⁺ T cell death (Annexin-V-PE⁺/7AAD⁺), induced by the spent media as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007382#pone-0007382-g002" target="_blank">Fig. 2A</a>, was plotted over time. Values are mean±S.E.M. of three independent sets of experiments.</p

Capsaicin selectively inhibits VEGF secretion to retard Hy-A549 cell-induced HUVEC cell migration.

<p>(A) Representative phase contrast photomicrographs demonstrating HUVEC migration upon incubation with spent media of Brefeldin-A-pretreated, capsaicin (37.5 µM)-treated Hy-A549 cells (<i>left panel</i>). Percent cell migrated in the wound area has been represented graphically (<i>right panel</i>). (B) Immunoblots showing expression profiles of pro-angiogenic factors VEGF, bFGF, EGF, TGF-β, in presence or absence of capsaicin. (C) Secreted VEGF from cell-free supernatant of Hy-A549 was quantified by ELISA assay (<i>left panel</i>). Time-dependent expression profiles of VEGF-mRNA/-protein in capsaicin-treated Hy-A549 cells were determined by Western blot and RT-PCR respectively (<i>middle panel</i>). Capsaicin-treated Hy-A549 cells were examined for time-dependent variation in the expression profiles of VEGF by quantitative real time PCR analysis and represented graphically (<i>right panel</i>). (D) Immuno-fluorescent images (60x magnification) showing time-dependent pattern of VEGF protein (TRITC-fluorescent) in capsaicin-treated Hy-A549 cells were represented along with nuclear staining (DAPI: blue). (E) Representative images of HUVEC migration upon incubation with (i) recombinant VEGF-supplemented control media, or VEGF-supplemented spent media of capsaicin-treated Hy-A549 cells, or with (ii) anti-VEGF-treated Hy-A549 spent media (<i>left panel</i>). Percent cell migrated in the wound area is being represented graphically (<i>right panel</i>). (F) Representative images of capillary-like sprout formation by HUVECs upon incubation with recombinant VEGF-supplemented spent media of capsaicin-treated Hy-A549 cells or with anti-VEGF-treated Hy-A549 spent media. GAPDH/α-Actin was used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p