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.

Several anticancer drugs including cisplatin (CDDP) induce hypomagnesemia. However, it remains fully uncertain whether Mg2+ deficiency affects chemosensitivity of cancer cells. Here, we investigated the effect of low Mg2+ concentration (LM) on proliferation and chemosensitivity using human lung adenocarcinoma A549 cells. Cell proliferation was reduced by continuous culture with LM accompanied with the elevation of G1 phase proportion. The amounts of reactive oxygen species (ROS) and stress makers such as phosphorylated-ataxia telangiectasia mutated and phosphorylated-p53 were increased by LM. Cell injury was dose-dependently increased by anticancer drugs such as CDDP and doxorubicin (DXR), which were suppressed by LM. Similar results were obtained by roscovitine, a cell cycle inhibitor. These results suggest that LM induces chemoresistance mediated by ROS production and G1 arrest. The mRNA and protein levels of ATP binding cassette subfamily B member 1 (ABCB1) were increased by LM and roscovitine. The LM-induced elevation of ABCB1 and nuclear p38 expression was suppressed by SB203580, a p38 MAPK inhibitor. PSC833, an ABCB1 inhibitor, and SB203580 rescued the sensitivity to anticancer drugs. In addition, cancer stemness properties were suppressed by SB203580. We suggest that Mg2+ deficiency reduces the chemotherapy sensitivity of A549 cells, although it suppresses cell proliferation.

Background Osteoarthritis (OA) of the hip rarely develops in the early second decade. As the incidence of this disease is low, no treatment method has been established. We report two patients with unilateral OA in their early teens in whom the anteversion angle of the femoral neck on the affected side was greater than that on the unaffected side. Case presentation Case 1 was an 11-year-old girl with left coxalgia and limited range of motion. There was no history of femoroacetabular impingement (FAI) or developmental dysplasia of the hip (DDH). Plain X-rays revealed the disappearance of the Y cartilage, joint space narrowing of the left hip, and acetabular/femoral head osteosclerosis. In CT images, the anteversion angle of the femoral neck (lt/rt) was 45/35 degrees. As osteoarthritis was severe, proximal femoral flexional derotational varus osteotomy (PFFDVO) and triple pelvic osteotomy (TPO) were performed. Case 2 was a 13-year-old girl with left coxalgia and limited range of motion. There was no history of FAI or DDH. Plain X-ray revealed irregularity of the left anterolateral femoral head, and a subcartilaginous cyst. In CT images, the anteversion angle of the femoral neck (lt/rt) was 30/20 degrees. As osteoarthritis was severe, PFFDVO was performed. In addition, we resected bone spurs on the femoral head because flexion was limited owing to the presence of osteophytes. In both patients, coxalgia and claudication/gait disorder resolved postoperatively, and joint space narrowing and osteosclerosis improved. However, in Case 1, there was a 3-cm difference in the leg length, and in Case 2, range-of-motion limits remained. Conclusions We present the findings in two patients with unilateral OA in their early second decade in whom the femoral anteversion angle on the affected side was greater than that on the unaffected side. PFFDVO + TPO was performed in Case 1, and PFFDVO + bone spur resection on the femoral head was performed in Case 2. Coxalgia resolved, and plain X-ray demonstrated improvements in OA; however, a difference in the leg length and range-of-motion limits remained.

Claudin-2 (CLDN2), a component of tight junctions, is abnormally expressed in human lung adenocarcinoma tissue. CLDN2 contributes to chemoresistance in human lung adenocarcinoma-derived A549 cells, and it may be a target for cancer therapy. Here, we found that coffee ingredients, namely caffeine and theobromine, decreased the protein level of CLDN2 in human lung adenocarcinoma-derived A549 cells. In contrast, other components, such as theophylline and chlorogenic acid, had no effect. These results indicate that the 7-methyl group in methylxanthines may play a key role in the reduction in CLDN2 expression. The caffeine-induced reduction in the CLDN2 protein was inhibited by chloroquine, a lysosome inhibitor. In a protein-stability assay using cycloheximide, CLDN2 protein levels decreased faster in caffeine-treated cells than in vehicle-treated cells. These results suggest that caffeine accelerates the lysosomal degradation of CLDN2. The accumulation and cytotoxicity of doxorubicin were dose-dependently increased, which was exaggerated by caffeine but not by theophylline in spheroids. Caffeine decreased nuclear factor-erythroid 2-related factor 2 (Nrf2) levels without affecting hypoxia-inducible factor-1α levels. Furthermore, caffeine decreased the expression of Nrf2-targeted genes. The effects of caffeine on CLDN2 expression and anticancer-drug-induced toxicity were also observed in lung adenocarcinoma RERF-LC-MS cells. We suggest that caffeine enhances doxorubicin-induced toxicity in A549 spheroids mediated by the reduction in CLDN2 and Nrf2 expression.

No abstract