Capsaicin-induced activation of p53-SMAR1 auto-regulatory loop down-regulates VEGF in non-small cell lung cancer to restrain angiogenesis

Abstract

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite decades of research, the treatment options for lung cancer patients remain inadequate, either to offer a cure or even a substantial survival advantage owing to its intrinsic resistance to chemotherapy. Our results propose the effectiveness of capsaicin in down-regulating VEGF expression in non-small cell lung carcinoma (NSCLC) cells in hypoxic environment. Capsaicin-treatment re-activated p53-SMAR1 positive feed-back loop in these cells to persuade p53-mediated HIF-1α degradation and SMAR1-induced repression of Cox-2 expression that restrained HIF-1α nuclear localization. Such signal-modulations consequently down regulated VEGF expression to thwart endothelial cell migration and network formation, pre-requisites of angiogenesis in tumor micro-environment. The above results advocate the candidature of capsaicin in exclusively targeting angiogenesis by down-regulating VEGF in tumor cells to achieve more efficient and cogent therapy of resistant NSCLC.

Figure 1. Effect of capsaicin on lung cancer cell spent medium-induced endothelial cell migration and network formation.

(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 (left panel). The number of cells migrated in the wound area are represented graphically (right panel). (B) Representative images of HUVEC migration upon incubation with capsaicin-treated (0–50 µM) Hy-A549 cell spent medium (left panel). Percent cell migrated in the wound area has been represented graphically (right panel). (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 (left panel). 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 (right panel). (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.

Figure 2. Capsaicin selectively inhibits VEGF secretion to retard Hy-A549 cell-induced HUVEC cell migration.

(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 (left panel). Percent cell migrated in the wound area has been represented graphically (right panel). (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 (left panel). Time-dependent expression profiles of VEGF-mRNA/-protein in capsaicin-treated Hy-A549 cells were determined by Western blot and RT-PCR respectively (middle panel). 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 (right panel). (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 (left panel). Percent cell migrated in the wound area is being represented graphically (right panel). (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.

Figure 3. Capsaicin inhibits VEGF transcriptional activation by targeting HIF-1α in a p53-dependent manner.

(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 (left panel). 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 (right panel). (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 (left panel). Immunoblot showing transfection efficiency of HIF-1α (right panel) (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 (left panel). p53 phosphorylation at Serine-15 position was also evaluated (right panel). (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 (left panel). Transfection efficiency was checked by analyzing p53 expression level (right panel). (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.

Figure 4. Capsaicin inhibits the nuclear localization of HIF-1α by down regulating Cox-2 in a p53-dependent manner.

(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 (left panel). Transfection efficiency was determined by analyzing the expression of Cox-2 (right panel). (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 (left panel). Transfection efficiency of Cox-2 was also verified (right panel). (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 (left panels). Control-/p53-shRNA transfected Hy-A549 cells were treated with capsaicin and expression levels of SMAR1 were checked (right panel). (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.

Figure 5. Capsaicin suppresses VEGF expression by SMAR1-mediated down regulation of Cox-2.

(A) Control-/SMAR1-shRNA (left panel) or control-vector/SMAR1-cDNA (right panel) 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 (bottom panels). (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(left panel) and represented graphically (right panel). (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.

Figure 6. Schematic diagram representing molecular mechanisms of capsaicin-mediated down-regulation of pro-angiogenic factor, VEGF.