Down-regulation of Claudin-2 Expression and Proliferation by Epigenetic Inhibitors in Human Lung Adenocarcinoma A549 Cells

Abstract

Claudin-2 is highly expressed in lung adenocarcinoma tissues and increases proliferation in adenocarcinoma cells. The chemicals that reduce claudin-2 expression may have anti-cancer effects, but such therapeutic medicines have not been developed. We found that azacitidine (AZA), a DNA methylation inhibitor, and trichostatin A (TSA) and sodium butyrate (NaB), histone deacetylase (HDAC) inhibitors, decrease claudin-2 levels. The effect of AZA was mediated by the inhibition of phosphorylated Akt and NF-κB. LY-294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and BAY 11-7082, an NF-κB inhibitor, decreased claudin-2 levels. The reporter activity of claudin-2 was decreased by AZA and LY-294002, which was blocked by the mutation in a putative NF-κB-binding site. NF-κB bound to the promoter region of claudin-2, which was inhibited by AZA and LY-294002. AZA is suggested to decrease the claudin-2 mRNA level mediated by the inhibition of a PI3K/Akt/NF-κB pathway. TSA and NaB did not change phosphorylated Akt and NF-κB levels. Furthermore, these inhibitors did not change the reporter activity of claudin-2 but decreased the stability of claudin-2 mRNA mediated by the elevation of miR-497 microRNA. The binding of histone H3 to the promoter region of miR-497 was inhibited by TSA and NaB, whereas that of claudin-2 was not. These results suggest that HDAC inhibitors decrease claudin-2 levels mediated by the elevation of miR-497 expression. Cell proliferation was additively decreased by AZA, TSA, and NaB, which was partially rescued by ectopic expression of claudin-2. We suggest that epigenetic inhibitors suppress the abnormal proliferation of lung adenocarcinoma cells highly expressing claudin-2.

Kaempferide Enhances Chemosensitivity of Human Lung Adenocarcinoma A549 Cells Mediated by the Decrease in Phosphorylation of Akt and Claudin-2 Expression

“…The expression of CLDN2 is upregulated by several intracellular signaling pathways, including the PI3K/Akt pathway in A549 cells [10,20]. LY-294002, an inhibitor of PI3K, decreases mRNA levels and promoter activity of CLDN2 [19]. Kaempferide decreased the p-Akt level without affecting the total amount of Akt ( Figure 6).…”

HDAC-4 regulates claudin-2 expression in EGFR-ERK1/2 dependent manner to regulate colonic epithelial cell differentiation

“…Moreover, genetic alteration in claudin-2 expression was sufficient to modulate cyclin-D1 and P-21 expressions in colon cancer cells and modulate growth properties. Similarly, knockdown of claudin-2 by siRNA in lung cancer cells A549 was recently reported to decrease proliferation concomitant with decreases in cyclin-D1 and E1 expression, cell cycle progression factors [ 64 ].…”

“…Studies have further reported causal role of the HDACi in regulating EGFR expression (mRNA transcription) and associated signaling using colon cancer cells [ 41 ]. Among claudin proteins, claudin-2 is the most regulated family member as its expression is regulated by the EGFR, C-Met, Ras and PI-3 Kinase signaling [ 3 , 64 , 68 ]. We were the first to demonstrate the key role of EGFR-activation in regulating colonic claudin-2 expression as claudin-2 expression was specifically downregulated in Waved-2 mice, which are defective in EGFR-tyrosine kinase signaling [ 3 ].…”

Up-regulation of IRF-3 expression through GATA-1 acetylation by histone deacetylase inhibitor in lung adenocarcinoma A549 cells

“…Transcriptional start site of human IRF-3 promoter was set as +1. The truncated human IRF-3 promoter plasmids pGL3-982, pGL3-149, and pGL3-67 were constructed as before [ 9 ]. The software Matlnspector ( http://www.genomatix.de//matinspector.html ) were used to search for IRF-3 promoter sequence for potential transcription factors target sites which are subject to acetylation.…”

“…The molecular mechanism of HDACi in lung cancer cells was currently under investigation. Hichino et al demonstrated that A549 cells proliferation was decreased by TSA, which was partially rescued by ectopic expression of claudin-2 [ 9 ]. Entinostat, as a kind of HDACi drugs, has been investigated in SALL4 positive lung caners [ 10 ].…”