VH-298 br Fig Nanomolar BPS can trigger the
Fig. 1. Nanomolar BPS can trigger the migration and invasion of NSCLC cells. (A) A549, H1299, and H358 cells were treated with BPS for 48 h, the proliferation was evaluated by use of CCK-8 kit; A549 (B) or H1299 (C) cells were treated with 1 or 10 nM BPS for 24 h, the wound closure of cells was recorded (left) and statistically analyzed (right); (D) The eﬀects of 10 nM BPS on the migration of A549 and H1299 cells were checked by transwell assay; (E) Cells were treated with or without 10 nM BPS for 24 h, the expression of MMP2, vimentin, fibronectin was measured by western blot analysis (left) and statistically analyzed (right). Data are presented as means ± SD of six independent experiments. *p < .05 compared with control; **p < .01 compared with control.
2. Materials and methods
2.1. Chemicals and reagents
Bisphenol S was purchased from Sigma-Aldrich (St. Louis, MO, USA). All inhibitors including SB431542 (an inhibitor of TGF-β type I receptor kinases), ICI 182780 (an inhibitor of estrogen receptor α/β), G15 (an inhibitor of G protein-coupled estrogen receptor), PD98054 (an inhibitor of ERK1/2), SB203580 (an inhibitor of p38-MAPK), and JNK inhibitor II were purchased from Cell Signaling Technology (Danvers, MA, USA). All compounds were solubilized in DMSO. Medium con-taining 0.5% DMSO was used as the control. Neutralization VH-298 for IL-10 and TGF-β were purchased from Bioworld Technology, Inc. (Minneapolis, MN, USA). All primary and horseradish peroxidase-con-jugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.2. Cell culture and treatment
Human NSCLC cell A549 (an adenocarcinomic human alveolar basal epithelial cell, p53+/+, noninvasive, passage 8–20), H1299 (a non-small cell lung cancer epithelial cell, p53−/−, invasive passage 14–25), and H358 (a human bronchiolar lung cancer epithelial cell derived from metastatic site, passage 21–29) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37 °C under 5% CO2. Twenty-four hours before the experiments, cells were cultured in medium containing no phenol red with charcoal-treated FBS (5% DC-FBS).
2.3. Analysis of cell proliferation
Cell proliferation assay was conducted according to the previously
Fig. 2. BPS increases the expression of TGF-β in NSCLC cells. (A) A549 cells were treated with 10 nM BPS for 24 h, the mRNA expression of various cytokines was checked by qRT-PCR; H1299 (B) or H358 (C) cells were treated with 10 nM BPS for 24 h, the mRNA expression of various cytokines was checked by qRT-PCR; (D) The expression of TGF-β in cells treated with 10 nM BPS for 24 h was checked by ELISA; (E) A549 cells were treated with 10 nM BPS for the indicated time periods, the mRNA of TGF-β was checked by qRT-PCR. Data are presented as means ± SD of six independent experiments. *p < .05 compared with control; **p < .01 compared with control.
described procedures (Chevalier et al., 2012). Briefly, cells were seeded in 96-well plates for 24 h before exposure to BPS. After treated with increasing concentrations of BPS for 48 h, cell viability was evaluated by use of the CCK-8 kit (Dojindo Molecular Technologies, Gaithers burg, MD, USA) according to the manufacturer's instructions.
2.4. Wound healing and transwell assay
Wound healing and transwell assay were used to evaluate the in vitro migration ability of NSCLC cells treated with BPS according to the previous study (Chen et al., 2015). For wound healing assay, cells were cultured in 6-well plates to generate the confluent monolayer. Then, cells were scratched and washed with PBS to remove cell debris. The wound closure was recorded by a microscope under 4× magnification at 0 and 24 h after scratch. The migrated distance was calculated and normalized to the initial wound distance at time zero.