Chrysin enhances anticancer drug-induced toxicity mediated by the reduction of claudin-1 and 11 expression in a spheroid culture model of lung squamous cell carcinoma cells.

The aberrant expression of claudins (CLDNs), which are tight junctional proteins, is seen in various solid tumors, but the regulatory mechanisms and their pathophysiological role are not well understood. Both CLDN1 and CLDN11 were highly expressed in human lung squamous cell carcinoma (SCC). Chrysin, found in high concentration in honey and propolis, decreased CLDN1 and CLDN11 expression in RERF-LC-AI cells derived from human lung SCC. The phosphorylation level of Akt was decreased by chrysin, but those of ERK1/2 and c-Jun were not. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited the phosphorylation of Akt and decreased the expression levels of CLDN1 and CLDN11. The association between phosphoinositide-dependent kinase 1 (PDK1) and Akt was inhibited by chrysin, but the phosphorylation of PDK1 was not. Immunoprecipitation and quartz-crystal microbalance assays revealed that biotinylated-chrysin binds directly to Akt. The knockdown of CLDN1 and CLDN11 using small interfering RNAs increased the transepithelial flux of doxorubicin (DXR), an anthracycline anticancer drug. Similarly, both chrysin and LY-294002 increased DXR flux. Neither CLDN1 knockdown, CLDN11 knockdown, nor chrysin changed the anticancer drug-induced cytotoxicity in a two-dimensional culture model, whereas they enhanced cytotoxicity in a spheroid culture model. Taken together, chrysin may bind to Akt and inhibit its phosphorylation, resulting in the elevation of anticancer drug-induced toxicity mediated by reductions in CLDN1 and CLDN11 expression in RERF-LC-AI cells. We suggest that chrysin may be useful as an adjuvant chemotherapy in lung SCC.

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

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.

Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells.

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.

Elevation of sensitivity to anticancer agents of human lung adenocarcinoma A549 cells by knockdown of claudin-2 expression in monolayer and spheroid culture models.

Claudins, tight junctional proteins, regulate the paracellular permeability of ions and small molecules. Claudin-2 is highly expressed in human lung adenocarcinoma cells and is involved in the up-regulation of cell proliferation. However, the effect of claudin-2 on cellular sensitivity to anticancer agents has not been clarified. The cytotoxicity of anticancer agents such as cisplatin, gefitinib and doxorubicin (DXR) was increased by claudin-2 knockdown in A549 cells. Claudin-2 knockdown also significantly decreased the expression level of multidrug resistance-associated protein/ABCC2. The expression levels of other drug efflux transporters were unchanged. The intracellular accumulation of 5-chloromethylfluorescein diacetate (CMFDA) and DXR, substrates of ABCC2, was increased by claudin-2 knockdown, whereas the efflux was decreased. MK-571, an inhibitor of ABCC2, enhanced the cytotoxicity of anticancer agents. Claudin-2 knockdown decreased the levels of p-c-Jun and nuclear Sp1. SP600125, an inhibitor of c-Jun, and mithramycin, an inhibitor of Sp1, decreased the level of ABCC2. The promoter activity of ABCC2 was decreased by claudin-2 knockdown, SP600125 and mithramycin treatments, suggesting that claudin-2 is involved in the up-regulation of ABCC2 expression at the transcriptional level. Claudin-2 knockdown increased the paracellular permeability of DXR in a 2D monolayer culture model. In addition, the accumulation of DXR into spheroids was enhanced by claudin-2 knockdown, resulting in a reduction in cell viability. We suggest that claudin-2 may be a novel therapeutic target in lung adenocarcinoma, because claudin-2 knockdown increased the accumulation of anticancer agents in cancer cells and spheroids.