Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells

Abstract

Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.

Increase in resistance to anticancer drugs involves occludin in spheroid culture model of lung adenocarcinoma A549 cells

“…OCLN may be not directly implicated in the acquisition of resistance to anticancer drugs in lung adenocarcinoma cells, but it suppresses chemosensitivity in spheroid cells. Our recent data indicate that CLDN1 and CLDN2 are also implicated in the development of chemoresistance in 3D spheroid cells 8 , 9 . These tight junctional proteins may be new targets of adjuvant chemotherapy in lung adenocarcinoma.…”

“…These results suggest that OCLN may not be essential to regulate paracellular permeability to ions, but we cannot deny the involvement of other CLDNs. Actually, the expression of CLDN1 was also elevated by CDDP-resistance in A549 cells, which was blocked by LY-294002 9 . The balance of the expression of CLDNs and OCLN may induce this property of paracellular permeability to ions.…”

“…However, the molecular mechanisms of chemoresistance are not entirely elucidated in 3D culture model. We recently reported that claudin-1 (CLDN1) and CLDN2, components of tight junctions (TJs), decrease chemosensitivity to doxorubicin (DXR) in 3D-cultured A549 cells established from human lung adenocarcinoma 8 , 9 .…”

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

“…Cells were cultured on transwell plates (0.4 µm pore size, 12 mm diameter) with polyester membrane inserts (Corning Incorporated, Corning, NY, USA). The barrier function of TJ was estimated by transepithelial electrical resistance (TER) and flux of doxorubicin [22,23].…”

Role of Claudin Proteins in Regulating Cancer Stem Cells and Chemoresistance-Potential Implication in Disease Prognosis and Therapy

“…Another study by a Japanese group of researchers reported a high expression of claudin-4 in the ovarian cancer tissues of platinum-resistant patients [99]. In lung cancer, claudin-1 is a key deciding factor for metastasis and a responsible factor for drug resistance towards cisplatin through the up-regulation of Unc-51 Like Autophagy Activating Kinase 1 (ULK1) phosphorylation [100,101]. It is also known to enhance drug resistance in liver cancer cells by modulating autophagy to achieve drug resistance.…”