Increase in resistance to anticancer drugs involves occludin in spheroid culture model of lung adenocarcinoma A549 cells

Hiroaki Eguchi,Satoshi Endo,Ryohei Maruhashi,Takumi Furuta,Risa Akizuki,Akira Ikari,Toshiyuki Matsunaga,Mitsutoshi Tsukimoto

doi:10.1038/s41598-018-33566-w

Hiroaki Eguchi, Satoshi Endo + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-33566-w

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Abstract

Chemoresistance is a serious issue in the therapy of many cancers, but the molecular mechanism is little understood. The mRNA level of occludin (OCLN), a tight junctional protein, was increased in the cisplatin (CDDP), doxorubicin (DXR), 7-ethyl-10-hydroxy-camptothecin, or gemcitabine-resistant human lung adenocarcinoma A549 cells. Here, we investigated the regulatory mechanism and pathophysiological role of OCLN. OCLN was mainly localized at tight junctions in A549 and CDDP-resistant A549 (A549/CDDP) cells. The level of p-Akt in A549/CDDP cells was higher than that in A549 cells, and the mRNA and protein levels of OCLN were suppressed by a phosphoinositide 3-kinase (PI3K)/Akt pathway inhibitor, LY-294002, suggesting that a PI3K/Akt pathway is involved in the elevation of OCLN expression. The overexpression of OCLN in A549 cells decreased paracellular permeability to DXR. Cytotoxicity to CDDP was unaffected by OCLN-overexpression in 2D culture model. In 3D culture model, the spheroid size, hypoxic level, and cell viability were significantly elevated by CDDP resistance, but not by OCLN-overexpression. The accumulation inside the spheroids and toxicity of DXR were correlated with OCLN expression. Our data suggest that OCLN is not directly involved in the chemoresistance, but it enhances chemoresistance mediated by suppression of accumulation of anticancer drugs inside the spheroids.

Highlights

The pathology of lung cancer can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer
Immunofluorescence measurements showed that OCLN was mainly colocalized with zonula occludens (ZO)-1 and DAPI, indicating that OCLN are distributed in the TJs (Fig. 1C)
To clarify the mechanism of OCLN upregulation by CDDP resistance, we investigated the involvement of mitogen-activated protein kinase kinase (MEK)/ extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways

Summary

Introduction

The pathology of lung cancer can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer. The formation of tumor microenvironment is involved in the development of chemoresistance[4], but the molecular mechanism remain elusive Both malignant and non-malignant cells formed the tumor microenvironment in vivo during developing tumors. OCLN is expressed in human lung adenocarcinomas, but not in squamous cell carcinomas and large cell carcinomas[21]. The mRNA level of OCLN is increased in adenocarcinomas compared to normal lung tissue[22]. OCLN overexpression decreased the accumulation and cytotoxicity of DXR in 3D culture model These results indicate that OCLN may be implicated in the promotion of chemoresistance in A549 spheroid cells

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