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

Abstract

Claudin-2 (CLDN2), a component of tight junction, is involved in the reduction of anticancer drug-induced toxicity in spheroids of A549 cells derived from human lung adenocarcinoma. Fisetin, a dietary flavonoid, inhibits cancer cell growth, but its effect on chemosensitivity in spheroids is unknown. Here, we found that fisetin (20 μM) decreases the protein level of CLDN2 to 22.3%. Therefore, the expression mechanisms were investigated by real-time polymerase chain reaction and Western blotting. Spheroids were formed in round-bottom plates, and anticancer drug-induced toxicity was measured by ATP content. Fisetin decreased the phosphorylated-Akt level, and CLDN2 expression was decreased by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting the inhibition of PI3K/Akt signal is involved in the reduction of CLDN2 expression. Hypoxia level, one of the hallmarks of tumor microenvironment, was reduced by fisetin. Although fisetin did not change hypoxia inducible factor-1α level, it decreased the protein level of nuclear factor erythroid 2-related factor 2, a stress response factor, by 25.4% in the spheroids. The toxicity of doxorubicin (20 μM) was enhanced by fisetin from 62.8% to 40.9%, which was rescued by CLDN2 overexpression (51.7%). These results suggest that fisetin can enhance anticancer drug toxicity in A549 spheroids mediated by the reduction of CLDN2 expression.

Keywords: anticancer drug toxicity; claudin-2; fisetin; lung adenocarcinoma.

Figure 1

Decrease in protein level of CLDN2 by fisetin in A549 cells. (A) A549 cells were incubated with 0, 1, 10, and 20 μM fisetin for 24 h. Cell viability is represented as a percentage of 0 μM. (B,C) The cell lysates were immunoblotted with anti-CLDN1, anti-CLDN2, and anti-β-actin antibodies. After normalization by β-actin expression, the protein levels of CLDN1 and CLDN2 are represented as a percentage of 0 μM. (D) Cells cultured on cover glasses were incubated in the absence (control) and presence of 20 μM fisetin for 24 h. Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and fisetin-treated cells was 0.1%. The cells were stained with rabbit anti-CLDN1 (red) plus mouse anti-ZO-1 (green), or mouse anti-CLDN2 (green) plus rabbit anti-ZO-1 (red) antibodies. Merged images are shown on the right. Scale bar indicates 10 μM. n = 3–6. ** p < 0.01 and ^NS p > 0.05 compared with 0 μM.

Figure 2

Effects of fisetin on the phosphorylation of intracellular signaling molecules. (A–C) Cells were incubated in the absence (control) and presence of 20 μM fisetin for 2 h. Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and fisetin-treated cells was 0.1%. The protein levels of p-Akt (T308), p-Akt (S473), Akt, p-ERK1/2, ERK1/2, p-Stat3, Stat3, p-PI3K, PI3K, p-PDK1, PDK1, and β-actin were examined by Western blotting. The expression levels of p-Akt, p-ERK1/2, p-PI3K, and p-PDK1 were represented as a percentage of control. n = 3–4. ** p < 0.01 and ^NS p > 0.05 compared with control.

Figure 3

Decrease in mRNA level of CLDN2 by fisetin and LY-294002 in A549 cells. (A) A549 cells were incubated in the absence (control) and presence of 20 μM fisetin or 10 μM LY-294002 for 6 h. Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and fisetin-treated cells was 0.1%. Real-time PCR was performed using primer pairs for CLDN1, CLDN2, and β-actin. After normalized by β-actin, the mRNA levels of CLDN1 and CLDN2 are represented as a percentage of control. (B) The cells were transfected with CLDN2 promoter construct and internal control pRL-TK vector. The reporter activity was measured using Dual-Glo luciferase assay kit and represented as a percentage of control. n = 3–4. ** p < 0.01 and ^NS p > 0.05 compared with control.

Figure 4

Effect of fisetin on property of spheroids. A549 cells were cultured in the round-bottom plates for 96 h and the cells formed spheroids. The spheroids were incubated in the absence (control) and presence of 20 μM fisetin for 24 h. Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and fisetin-treated cells was 0.1%. (A) The protein levels of CLDN2 and β-actin were examined by Western blotting. The expression levels of CLDN2 were represented as a percentage of control. (B) Upper images are shown in upper panel. Scale bar indicates 500 μM. Spheroid size is represented as a percentage of control. (C) ATP content and fluorescence intensity of LOX-1 are represented as a percentage of control. (D) The protein levels of HIF-1α, Nrf2, and nucleoporin p62 (p62) were examined by Western blotting. The expression levels of HIF-1α and Nrf2 were represented as a percentage of control. (E) The mRNA levels of ABCB1, ABCC1, ABCC2, and ABCG2 were measured by real-time PCR and represented as a percentage of control. n = 5–6. ** p < 0.01, * p < 0.05, and ^NS p > 0.05 compared with control.

Figure 5

Increase in accumulation and toxicity of DXR by fisetin in spheroids. (A,B) The spheroids were treated with DXR in the absence (control) and presence of 20 μM fisetin for (A) 1 h or (B) 24 h. Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and fisetin-treated cells was 0.1%. The fluorescence intensity of DXR and cell viability are represented as a percentage of 0 μM DXR. (C) The spheroids were treated in the absence (control) and presence of 20 μM fisetin, 20 μM DXR, or both of them for 24 h. The proportions of viable cells and apoptotic and necrotic cell deaths are represented as a percentage of total cells. n = 4–6. ** p < 0.01 and * p < 0.05 compared with 0 μM DXR. ^# p < 0.05 compared with DXR alone. ^NS p > 0.05 compared with 0 μM DXR or control.

Figure 6

Enhancement of anticancer drug-induced toxicity by fisetin in spheroids. In the absence (control) and presence of 20 μM fisetin, the spheroids were treated with CDDP, DOC, and GEF at indicated concentration for 24 h. Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and fisetin-treated cells was 0.1%. The cell viability was measured using a CellTiter-Glo 3D Cell Viability Assay kit and represented as a percentage of 0 μM anticancer drugs. n = 5–6. ** p < 0.01 and * p < 0.05. ^## p < 0.01 and ^# p < 0.05 compared with control. ^NS p > 0.05 compared with 0 μM DXR or control.

Figure 7

Inhibition of elevation of fisetin-induced anticancer drug toxicity by CLDN2 overexpression. The spheroid cells transfected with mock or CLDN2/pTRE2 expression vector were incubated in the absence (control) and presence of 20 μM fisetin for 24 h. Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and fisetin-treated cells was 0.1%. (A) The cells were treated with DXR for 1 h. The fluorescence intensity of DXR in the spheroids is represented as a percentage of 0 μM DXR. (B) The cells were treated with DXR at indicated concentration for 24 h. Cell viability is represented as a percentage of 0 μM DXR. n = 5–6. ** p < 0.01 and * p < 0.05 compared with 0 μM DXR or mock + fisetin. ^## p < 0.01 and ^# p < 0.05 compared with mock + fisetin. ^NS p > 0.05 compared with mock or mock + fisetin.

Figure 8

The relationship between structural characters and function of flavonoids. Representative chemical structure of flavonoids is shown above. The substituents at each position of R1, R2, R3, and R4 on the A-, B-, or C-ring in flavonoids are presented as hydrogen (H), hydroxy (OH), and methoxy (OCH₃). In the CLDN2 expression column, the flavonoids that have the ability to reduce the expression of CLDN2 are represented as “○” and those do not have the ability are represented as “x”. In the double bound in C ring column, the flavonoids that have double bond are represented as “○” and those do not have the double bond are represented as “x” [23,24].