Abstract
Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in a three-dimensional spheroid culture model of human lung adenocarcinoma A549 cells. However, the mechanism has not been fully clarified. We found that the knockdown of CLDN2 expression by siRNA in the spheroid reduces the expression of glucose transporters and metabolic enzymes. In a two-dimensional culture model, the expression of these proteins was increased by glucose deprivation or fasentin, an inhibitor of glucose transporter. In addition, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant enzymes including heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and a glutamate–cysteine ligase modifier subunit were increased by fasentin. The fluorescence intensities of JC-1, a probe of mitochondrial membrane potential, and MitoROS 580, a probe of mitochondrial superoxide production, were increased by fasentin. These results suggest that mitochondrial production of reactive oxygen species is increased by glucose deficiency. The knockdown of CLDN2 enhanced the flux of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), a fluorescent deoxyglucose derivative, in a transwell assay, and the accumulation of glucose and 2-NBDG in spheroid cells. The expression of Nrf2 was decreased by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane, a typical Nrf2 activator, in spheroid cells. The sensitivity of spheroid cells to doxorubicin, an anthracycline antitumor antibiotic, was enhanced by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane. We suggest that CLDN2 induces chemoresistance in spheroid cells mediated through the inhibition of glucose transport and activation of the Nrf2 signal.
Highlights
A solid tumor microenvironment is created by cancer cells, extracellular matrix, and non-cancer cells such as fibroblasts, macrophages, and vascular endothelial cells in the body [1]
Glucose is transported into the cancer cells mediated through glucose transporters such as GLUTs
Our results indicate that mitochondrial membrane potential and reactive oxygen species (ROS) production were increased by glucose deprivation and fasentin in 2D monolayer cells (Figure 6), whereas they were decreased by CLDN2 knockdown in spheroid cells (Figure 7C)
Summary
A solid tumor microenvironment is created by cancer cells, extracellular matrix, and non-cancer cells such as fibroblasts, macrophages, and vascular endothelial cells in the body [1]. The cells in the central region of the microenvironment are commonly exposed to hypoxic, oxidative and nutritional stress conditions. These stress conditions correlate with therapeutic resistance and metastasis, suggesting that stress adaptation is a critical survival advantage for cancer cells. 2-related factor 2 (Nrf2) play important roles in the hypoxic and oxidative stress responses, respectively. The improvement of stress conditions may be useful to prevent the tumor progression. The pharmacological targeting of the HIF-1 and Nrf signaling pathways was considered as a new strategy for cancer therapy in recent years [2,3]
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