Claudin-1 enhances chemoresistance of human lung adenocarcinoma A549 cells mediated by forming an amino acid barrier

Claudin-1 (CLDN1) is highly expressed in human lung adenocarcinoma-derived A549 cells and is involved in the augmentation of chemoresistance. However, the mechanism of chemoresistance is not fully understood. In the tumor microenvironment, cancer cells are exposed to stress conditions such as hypoxia and malnutrition. Here, we investigated the effect of CLDN1 expression on amino acid (AA) flux and chemoresistance using A549 cells. L-type AA transporters, LAT1 and LAT3, were highly expressed in three-dimensional spheroid cells compared with in two-dimensional (2D) cultured cells. The expression of these transporters was increased by AA deprivation in 2D cultured cells. The paracellular AA flux except for Ser, Thr, and Tyr was enhanced by CLDN1 silencing. The expression of AA transporters and AA contents in spheroids were decreased by CLDN1 silencing. These results suggest that CLDN1 forms a paracellular AA barrier, leading to a compensatory increase in LAT1/3 expression in spheroids. The production of reactive oxygen species in the mitochondria and cytosol was decreased by CLDN1 silencing in spheroids, resulting in downregulation of the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target antioxidant genes. CLDN1 silencing enhanced the cytotoxicity of doxorubicin, an anthracycline antitumor agent, which was blocked by sulforaphane, an Nrf2 activator. In 2D cultured cells, the cytotoxicity of doxorubicin was attenuated by a reduction in extracellular AA concentration or treatment with sulforaphane. We suggest that CLDN1 forms an AA barrier in spheroids, leading to the augmentation of Nrf2-dependent chemoresistance in A549 cells.