Elevation of Chemosensitivity of Lung Adenocarcinoma A549 Spheroid Cells by Claudin-2 Knockdown through Activation of Glucose Transport and Inhibition of Nrf2 Signal

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.

Harnessing graphene oxide nanocarriers for siRNA delivery in a 3D spheroid model of lung cancer

“…This finding is consistent with previous studies that showed a decreased protein expression intensity in many cell lines, including A549, when cultured in 3D with respect to 2D. 42,45 The reduced expression in 3D cultures might be closer to the in vivo situations, since 2D conditions generally promote abnormal cellular proliferation, metabolism and cell interactions, which are not similar to the in vivo cancer tissue behaviour. 42 In conclusion, spheroids demonstrated to be a potential powerful cell culture tool for further experiments.…”

“…However, even if it was reported that the cell internalization of GO is size-dependent in different cell lines; 55,56 interestingly, the literature is not in total consensus. 29,45,57…”

Increase in Anticancer Drug-Induced Toxicity by Fisetin in Lung Adenocarcinoma A549 Spheroid Cells Mediated by the Reduction of Claudin-2 Expression

“…Furthermore, the effect of fisetin was cancelled by the overexpression of CLDN2 ( Figure 7 ). In the electron microscope observation and immunofluorescence analysis, CLDN2 is abundantly expressed in the outer layer of spheroids and forms TJ [ 40 ]. Fisetin may also enhance the anticancer drug toxicity mediated by the increase in penetration of anticancer drugs in the spheroids.…”

Elevation of Anticancer Drug Toxicity by Caffeine in Spheroid Model of Human Lung Adenocarcinoma A549 Cells Mediated by Reduction in Claudin-2 and Nrf2 Expression

“…Caffeine may suppress chemoresistance mediated by the reduction in ROS stress. We recently reported that CLDN2 decreases the intracellular content of glucose in A549 spheroid cells [ 40 ]. Glucose deprivation shifts energy production from glucose metabolism to mitochondrial respiration.…”