Down-Regulation of Claudin-2 Expression by Cyanidin-3-Glucoside Enhances Sensitivity to Anticancer Drugs in the Spheroid of Human Lung Adenocarcinoma A549 Cells

Abstract

Claudin-2 (CLDN2), an integral membrane protein located at tight junctions, is abnormally expressed in human lung adenocarcinoma tissues, and is linked to drug resistance in human lung adenocarcinoma A549 cells. CLDN2 may be a target for the prevention of lung adenocarcinoma, but there are few compounds which can reduce CLDN2 expression. We found that cyanidin-3-glucoside (C3G), the anthocyanin with two hydroxyl groups on the B-ring, and cyanidin significantly reduce the protein level of CLDN2 in A549 cells. In contrast, pelargonidin-3-glucoside (P3G), the anthocyanin with one hydroxyl group on the B-ring, had no effect. These results suggest that cyanidin and the hydroxyl group at the 3-position on the B-ring play an important role in the reduction of CLDN2 expression. The phosphorylation of Akt, an activator of CLDN2 expression at the transcriptional level, was inhibited by C3G, but not by P3G. The endocytosis and lysosomal degradation are suggested to be involved in the C3G-induced decrease in CLDN2 protein expression. C3G increased the phosphorylation of p38 and the p38 inhibitor SB203580 rescued the C3G-induced decrease in CLDN2 expression. In addition, SB203580 rescued the protein stability of CLDN2. C3G may reduce CLDN2 expression at the transcriptional and post-translational steps mediated by inhibiting Akt and activating p38, respectively. C3G enhanced the accumulation and cytotoxicity of doxorubicin (DXR) in the spheroid models. The percentages of apoptotic and necrotic cells induced by DXR were increased by C3G. Our data suggest that C3G-rich foods can prevent the chemoresistance of lung adenocarcinoma A549 cells through the reduction of CLDN2 expression.

Keywords: chemosensitivity; claudin-2; cyanidin-3-glucoside; lung adenocarcinoma.

Figure 1

Effects of anthocyanins on the protein levels of CLDN1 and CLDN2 in A549 cells. Cells were incubated with 0, 1, 10, or 50 µM C3G (A), P3G (B), or cyanidin (C) for 24 h. Cell lysates were immunoblotted with anti-CLDN1, anti-CLDN2, and anti-β-actin antibodies. The protein levels of CLDN1 and CLDN2 are represented as a percentage relative to 0 µM. n = 3–4. ** p < 0.01 and NS p > 0.05 compared with 0 µM.

Figure 2

Effects of anthocyanins on the cellular localization of CLDN1 and CLDN2. Cells cultured on cover glasses were incubated in the absence (control) and presence of 50 µM C3G or P3G for 24 h. (A) The cells were stained with anti-CLDN1 (green), anti-ZO-1 (red), and nuclear marker DAPI (blue). (B) The cells were stained with anti-CLDN2 (red), anti-ZO-1 (green), and DAPI (blue). Merged images are shown in the right panel. The scale bar represents 10 µm. The TJ localizations of CLDN1 and CLDN2 are shown as percentage of control (n = 132-220 cells). ** p < 0.01 and NS p > 0.05 compared with control.

Figure 3

Effects of anthocyanins on the viability and mRNA levels of CLDN1 and CLDN2. (A) Cells were incubated in the absence (control) and presence of 50 µM anthocyanins for 6 h. The mRNA levels were measured by quantitative real-time PCR and represented as a percentage relative to control. (B) Cells were incubated with 0, 1, 10, 20, or 50 µM anthocyanins for 24 h, followed by the cell viability assays. n = 4. ** p < 0.01, * p < 0.05 and NS p > 0.05 compared with control or 0 µM.

Figure 4

Effects of anthocyanins on intracellular signaling pathways. Cells were incubated in the absence (control) and presence of 50 µM anthocyanins for 1 h. (A) Cell lysates were immunoblotted with anti-p-Akt (Thr308), anti-p-Akt (ser473), and anti-Akt antibodies. (B) Cell lysates were immunoblotted with anti-p-ERK1/2, anti-ERK1/2, anti-p-Stat3, and anti-Stat3 antibodies. (C) Cell lysates were immunoblotted with anti-p-PI3K p85 (Tyr458), and anti-p-PI3K p85, anti-p-PDK1, and anti-PDK1 antibodies. The protein levels were represented as a percentage relative to control. n = 4. * p < 0.05 and NS p > 0.05 compared with control.

Figure 5

Decrease in the protein stability and reporter activity of CLDN2 by C3G. (A) Cells were incubated with 10 µM CHX in the absence and presence of 50 µM C3G for 0, 2, 4, and 6 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-β-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to control. (B) Cells were incubated in the absence (control) and presence of 50 µM anthocyanins for 6 h, and then the reporter activity was measured by the Dual-Luciferase Reporter Assay System. The reporter activity is represented as a percentage relative to control. (C) Cells were incubated with 1 µg/mL actinomycin D in the absence and presence of 50 µM C3G for 0, 3, and 6 h. The mRNA level of CLDN2 was measured by quantitative real-time PCR and represented as a percentage relative to 0 h. n = 3–5. ** p < 0.01, * p < 0.05 and NS p > 0.05 compared with control or 0 h.

Figure 6

Effect of C3G on the degradation and endocytosis of the CLDN2 protein. (A) Cells were incubated in the absence (control) and presence of 50 µM C3G, 5 µM MDC, or 10 µM MβCD for 24 h. (B) Cells were incubated in the absence (control) and presence of 50 µM C3G, 20 µM CHL, or 5 µM MG132 for 24 h. (C) Cells were incubated in the absence (control) and presence of 50 µM C3G for 24 h. CAN (0.5 µM) was added into the medium 9 h before collection. Cell lysates were immunoblotted with anti-CLDN2 and anti-β-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to control. n = 3–5. ** p < 0.01 compared with control. ## p < 0.01, and NS p > 0.05 compared with vehicle.

Figure 7

Effect of C3G on the levels of p-p38 and CLDN2. (A) Cells were incubated with 10 µM CHX in the absence (control) and presence of 10 µM LY-294002 for 0, 2, 4, and 6 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-β-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to 0 h. (B) Cells were incubated in the absence and presence of 50 µM C3G for 1 h. Cell lysates were immunoblotted with anti-p-p38 and anti-p38 antibodies. The p-p38 levels are represented as a percentage relative to control. (C) Cells were incubated in the absence and presence of 50 µM C3G and 10 µM SB203580 (SB) for 24 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-β-actin antibodies. The protein levels are represented as a percentage relative to control. (D) Cells were incubated with 0, 5, and 10 µM anisomycin for 24 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-β-actin antibodies. The protein levels are represented as a percentage relative to 0 µM. n = 3–4. ** p < 0.01 and NS p > 0.05 compared with control. ## p < 0.01 compared with vehicle.

Figure 8

Effects of anisomycin and SB203580 on CLDN2 expression. (A) Cells were incubated in the absence (vehicle) and presence of 10 µM SB203580 (SB) for 24 h. Cell lysates were immunoblotted with anti-CLDN2 and anti-β-actin antibodies. The protein levels of CLDN2 are represented as a percentage relative to vehicle. (B) Cells were incubated with 10 µM CHX in the absence (control) and presence of 10 µM SB203580 (SB) for 0, 2, 4, and 6 h. The protein levels are represented relative to 0 h. (C) Cells were incubated in the absence (control) and presence of 10 µM C3G and 10 µM SB203580 for 6 h. The mRNA levels are represented as a percentage relative to control. n = 3–4. ** p < 0.01 and * p < 0.05 compared with 0 µM or control. NS p > 0.05 compared with vehicle.

Figure 9

Effect of anthocyanins on the barrier function of TJs. Cells were cultured on transwell inserts and treated with 0 µM (control), 50 µM C3G, or 50 µM P3G for 24 h. TER was measured using a volt-ohmmeter. DXR and LY were applied to the apical compartment. The buffer in the basal compartment was collected after 30 min, and fluorescence intensity was measured. Papp was calculated as described in the method using equation 1. n = 3–4. ** p < 0.01 and NS p > 0.05 compared with control.

Figure 10

Increase in DXR-induced toxicity by C3G in spheroid cells. Cells cultured on PrimeSurface 96U multi-well plates were treated with 0 µM (control) and 50 µM anthocyanins for 24 h. (A) The spheroid size and fluorescence intensity of LOX-1 are represented as a percentage relative to control. (B) After treatment with anthocyanins, the cells were incubated with DXR at the indicated concentrations for 1 h. The accumulation of DXR is represented as a percentage relative to 0 µM DXR. (C,D) The cells were incubated with DXR at the indicated concentrations for 24 h. The spheroid size and cell viability are represented as a percentage relative to 0 µM DXR. n = 4–6. ** p < 0.01, * p < 0.05, and NS p > 0.05 compared with control.

Figure 11

Increase in the rates of DXR-induced apoptotic and necrotic cell death by C3G. After being treated in the absence (control) and presence of 50 µM anthocyanins for 24 h, the spheroid cells were incubated with 0 or 20 µM DXR for 24 h. The rates of apoptotic (A) and necrotic cell death (B) were examined using a BZ-X800 fluorescence microscope. n = 4. ** p < 0.01, * p < 0.05, and NS p > 0.05 compared with control.