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Published on: August 19, 2019
Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of
Risa Akizuki1, Ryohei Maruhashi1, Hiroaki Eguchi1
1Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Japan.
Abstract:
Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.
Insights
Chemotherapy resistance in lung cancer involves claudin-1 (CLDN1) and claudin-3, which are upregulated in resistant cells. This upregulation, driven by a PI3K/Akt/NF-κB pathway, reduces anticancer drug penetration, contributing to treatment failure.
Area of Science:
- Oncology
- Cell Biology
- Molecular Medicine
Background:
- Chemotherapy resistance is a significant obstacle in cancer treatment, with mechanisms often poorly understood.
- Claudins (CLDN1 and CLDN3), tight junction proteins, have been implicated in various cellular processes, including drug resistance.
Purpose of the Study:
- To investigate the expression mechanism and functional role of claudin-1 (CLDN1) and claudin-3 in cisplatin-resistant lung adenocarcinoma cells.
- To elucidate the signaling pathways involved in the upregulation of CLDN1 and CLDN3 in chemoresistant cells.
Main Methods:
- Comparative analysis of CLDN1 and CLDN3 expression in cisplatin-sensitive (A549) and resistant (A549R) human lung adenocarcinoma cells.
- Assessment of protein phosphorylation in key signaling pathways (Src, MEK, ERK, Akt).
- Inhibition studies using phosphoinositide 3-kinase (PI3K) and NF-κB inhibitors, and small interfering RNA (siRNA) for CLDN1.
- Spheroid culture models to evaluate drug penetration, accumulation, and toxicity.
Main Results:
- Claudin-1 (CLDN1) and claudin-3 expression were significantly upregulated in cisplatin-resistant A549R cells, which also exhibited cross-resistance to doxorubicin (DXR).
- Upregulation of CLDN1 and CLDN3 was linked to increased phosphorylation in Src, MEK, ERK, c-Fos, and Akt signaling pathways.
- Inhibition of PI3K and NF-κB pathways reduced CLDN1 and CLDN3 expression.
- Overexpression of CLDN1 and CLDN3 decreased DXR paracellular permeability, and increased hypoxia in spheroid models, leading to reduced DXR accumulation and toxicity within spheroids. CLDN1 siRNA reversed these effects in A549R cells.
Conclusions:
- Claudin-1 (CLDN1) is upregulated in response to cisplatin resistance, mediated by the activation of the PI3K/Akt/NF-κB pathway.
- Increased CLDN1 expression contributes to reduced anticancer drug penetration into tumor spheroids, thereby promoting chemotherapy resistance.
- Targeting CLDN1 or its associated pathways may offer a therapeutic strategy to overcome chemotherapy resistance in lung adenocarcinoma.
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