Lack of Association between Bax Promoter (-248G>A) Single Nucleotide Polymorphism and Susceptibility towards Cancer: Evidence from a Meta-Analysis

<div><p>Background</p><p>The Bcl-2-associated X protein (Bax) is a proapoptotic member of the Bcl-2 family known to be activated and upregulated during apoptosis. Single nucleotide polymorphisms (SNPs) in Bax promoter may participate in the process of carcinogenesis by altering its own expression and the cancer related genes. Bax-248G>A polymorphism has been implicated to alter the risk of cancer, but the listed results are inconsistent and inconclusive. In the present study, we performed a meta-analysis to systematically summarize the possible association of this polymorphism with the risk of cancer.</p> <p>Methodology</p><p>We conducted a search of case-control studies on the associations of Bax-248G>A polymorphism with susceptibility to cancer in Pub Med, Science Direct, Wiley Online Library and hand search. Data from all eligible studies based on some key search terms, inclusion and exclusion criteria were extracted for this meta-analysis. Hardy-Weinberg equilibrium (HWE) in controls, power calculation, heterogeneity analysis, Begg’s funnel plot, Egger’s linear regression test, forest plot and sensitivity analysis were performed in the present study.</p> <p>Results</p><p>Cancer risk associated with Bax-248G>A polymorphism was estimated by pooled odds ratios (ORs) and 95% confidence intervals (95% CIs). The pooled ORs were calculated in allele contrast, homozygous comparison, heterozygous comparison, dominant and recessive model. Statistical significance was checked through Z and p-value in forest plot. A total of seven independent studies including 1772 cases and 1708 controls were included in our meta-analysis. Our results showed that neither allele frequency nor genotype distributions of this polymorphism were associated with risk for cancer in any of the genetic model. Furthermore, Egger’s test did not show any substantial evidence of publication bias.</p> <p>Conclusions/Significance</p><p>This meta-analysis suggests that the Bax-248G>A polymorphism is not an important cancer risk factor. Nevertheless, additional well-designed studies with larger sample size focusing on different ethnicities and cancer types are required to further validate the results.</p> </div

Funnel plots of the Egger’s test to detect publication bias.

<p>Each point represents a separate study. The OR was plotted on a logarithmic scale against the precision (the reciprocal of the SE) of each study.</p

Flow diagram of articles selection.

<p>This is based on publication search, inclusion and exclusion criteria.</p

Forest plots of meta-analysis for Bax-248G>A polymorphism and cancer risk.

<p>The squares and horizontal lines correspond to the study specific odds ratios (ORs) and 95% confidence intervals (CI) respectively. The area of the squares reflects the study specific weight (inverse of the variance). The diamond represents the pooled ORs and 95%CI.</p

Additional file 1: of Cytotoxicity and cell cycle arrest induced by andrographolide lead to programmed cell death of MDA-MB-231 breast cancer cell line

Andrographolide-induced externalization of phosphatidyl serine and apoptosis in MCF-7 cells. Figure S1. Effect of andrographolide treatment on apoptosis in MCF-7 cells. Cells were treated with IC50 concentration of andrographolide for 48 h, double stained with annexin V-FITC/PI and analyzed in a FACSVerse™ (Becton Dickinson, USA) flow cytometer. The percentage Annexin V-positive population refers apoptosis induction (region 2 and 4). Data are representative of three independent experiments. (PDF 88 kb

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

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

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