Journal: PLOS One

Article Title: Carbonic anhydrase IX downregulation linked to disruption of HIF-1, NFκB and STAT3 pathways as a new mechanism of ibuprofen anti-cancer effect

doi: 10.1371/journal.pone.0323635

Figure Lengend Snippet: κ B protein level and its transcriptional activity. (A) Detection of NFκB precursor p105, NFκB subunits p50 and p60 and other NFκB-related proteins in HCT116, RKO, FaDu and UM-22A cells incubated in hypoxia under IBU treatment (0.5 mM and 1 mM) by Western blot demonstrated decreased NFκB levels and impaired transcriptional activity of NFκB. (B) Detection of NFκB subunits p50 and p65 in nuclear extracts prepared from different cell lines treated with 1 mM IBU or DMSO as control for 48 hours in hypoxia showed decreased level of both subunits in all four treated cell line nuclei compared to levels in control nuclei from untreated cells.

Article Snippet: After 30 min blocking with 5% non-fat dry milk in 0.1% Tween in PBS, membranes were incubated O/N at 4°C with primary antibodies: hybridoma medium containing mouse monoclonal antibody M75 recognizing the PG-domain of CA IX [ ] (prepared at the Institute of Virology, Biomedical Research Center, 1:200 in blocking buffer); anti-β-actin (Cell Signaling, 1:8000); anti-HIF-1α (BD Transduction Laboratories, 1:250); anti-PDHK1 (Abcam, 1:1000); anti-LDHA (Cell Signaling, 1:1000); anti-phospho STAT3 (Cell Signaling, 1:500); anti-STAT3 (Cell Signaling, 1:1000); anti-NFκB p105/p50 (Cell Signaling, 1:1000); anti-NFκB p65 (Santa Cruz, 1:700); anti-IκBα (Cell Signaling, 1:1000); anti-survivin (Cell Signaling, 1:1000); anti-Lamin A (Santa Cruz, 1:700); anti-cleaved PARP (Cell Signaling, 1:1000); anti-COX-2 (Cell Signaling, 1:1000); anti-p-AKT (Ser 473) (Cell Signaling, 1:1000); anti-AKT (Cell Signaling, 1:1000); anti-p-ERK1/2 (Cell Signaling, 1:1000); anti-ERK1/2 (Cell Signaling, 1:1000); anti-pS9-GSK3β (Cell Signaling, 1:1000); anti-LC3B (Cell Signaling, 1:1000).

Techniques: Activity Assay, Incubation, Western Blot, Control

Journal: PLOS One

Article Title: Carbonic anhydrase IX downregulation linked to disruption of HIF-1, NFκB and STAT3 pathways as a new mechanism of ibuprofen anti-cancer effect

doi: 10.1371/journal.pone.0323635

Figure Lengend Snippet: IBU impairs the STAT3 pathway and downregulates HIF-1α protein leading to impaired HIF-1 binding on the CA9 promoter. The disruption of CA IX transcription results in a decreased level of CA IX on cancer cell membrane. Additionally, IBU impairs NFκB transcriptional activity by reducing the translocation of p50 and p65 to the nucleus despite decreased IκBα level. NFκB-mediated gene transcription is also hindered via inhibition of GSK3β. This involvement in crucial pathways results in apoptosis and autophagy of cancer cells. Our study also proved that despite the fact that IBU inhibits COX-2 activity, it paradoxically upregulates COX-2 expression via the PI3K/AKT and ERK1/2 pathways. Created with BioRender.com.

Article Snippet: After 30 min blocking with 5% non-fat dry milk in 0.1% Tween in PBS, membranes were incubated O/N at 4°C with primary antibodies: hybridoma medium containing mouse monoclonal antibody M75 recognizing the PG-domain of CA IX [ ] (prepared at the Institute of Virology, Biomedical Research Center, 1:200 in blocking buffer); anti-β-actin (Cell Signaling, 1:8000); anti-HIF-1α (BD Transduction Laboratories, 1:250); anti-PDHK1 (Abcam, 1:1000); anti-LDHA (Cell Signaling, 1:1000); anti-phospho STAT3 (Cell Signaling, 1:500); anti-STAT3 (Cell Signaling, 1:1000); anti-NFκB p105/p50 (Cell Signaling, 1:1000); anti-NFκB p65 (Santa Cruz, 1:700); anti-IκBα (Cell Signaling, 1:1000); anti-survivin (Cell Signaling, 1:1000); anti-Lamin A (Santa Cruz, 1:700); anti-cleaved PARP (Cell Signaling, 1:1000); anti-COX-2 (Cell Signaling, 1:1000); anti-p-AKT (Ser 473) (Cell Signaling, 1:1000); anti-AKT (Cell Signaling, 1:1000); anti-p-ERK1/2 (Cell Signaling, 1:1000); anti-ERK1/2 (Cell Signaling, 1:1000); anti-pS9-GSK3β (Cell Signaling, 1:1000); anti-LC3B (Cell Signaling, 1:1000).

Techniques: Binding Assay, Disruption, Membrane, Activity Assay, Translocation Assay, Inhibition, Expressing