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

Hiroaki Eguchi,Toshiyuki Matsunaga,Saki Onuma,Akira Ikari,Haruka Matsunaga,Yuta Yoshino

doi:10.3390/ijms22020499

Hiroaki Eguchi, Toshiyuki Matsunaga + Show 4 more

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https://doi.org/10.3390/ijms22020499

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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.

Highlights

Cyanidin is an abundant polyphenolic pigment present in vegetation
We previously reported that the expression level of CLDN2 is increased in human lung adenocarcinoma tissues [15], and is involved in the increased proliferation [16], migration [17], and drug-resistance [18] of human lung adenocarcinoma A549 cells
We examined the effects of C3G, cyanidin and pelargonidin-3glucoside (P3G) on CLDN2 expression by Western blotting and real-time polymerase chain reaction (PCR) in A549 cells

Summary

Introduction

Cyanidin is an abundant polyphenolic pigment present in vegetation. C3G, known as chrysanthemin, is one of the most widely distributed anthocyanins with glucose bound to the cyanidin at the 3-position on the C-ring. It can be detected in red to blue fruits and vegetables, such as blackberry and black olive [1]. The mechanisms of pharmacokinetics of anthocyanins have been investigated in rodents and humans [2,3]. Anthocyanins are quickly absorbed from the small intestine after oral administration and exist in unchanged forms (glycosides) or metabolized forms (glucuronidated, sulphated, and methylated derivatives). These forms are distributed in the digestive system, and in all other tissues. C3G has potent superoxide radical-scavenging activity, with an IC50 of

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