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  • br BPS increases the expression of TGF in NSCLC

    2020-08-18


    3.2. BPS increases the expression of TGF-β in NSCLC cells
    Previous studies indicated that BPA can regulate the expression of cytokine/chemokine production in human HBX-41108 (Chen et al., 2018; Tajiki-Nishino et al., 2018), which are important for cancer migration and invasion. We checked the effects of BPS on the expression of VEGF, interferon (IFN)-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, EGF, and TGF-
    β in A549 cells by qRT-PCR. The data showed that BPS treatment can increase the expression of IL-8 and TGF-β in A549 cells (Fig. 2 A). Consistently, BPS can increase the mRNA expression of IL-10 and TGF-β in H1299 cells (Fig. 2 B). However, BPS only increased the mRNA of TGF-β in H358 cells (Fig. 2 C). The upregulation of TGF-β in NSCLC cells were confirmed by ELISA (Fig. 2 D). Further, BPS can increase the expression of TGF-β in A549 cells via a time dependent manner (Fig. 2 E).
    Smad2/3 are two molecules mediate the TGF-β signaling pathway in human cells (Moustakas and Heldin, 2007). Our data showed that BPS treatment can increase the phosphorylation of Smad2/3 in both
    Fig. 4. Activation of TGF-β/Smad2/3 signal mediates BPS induced migration of NSCLC cells. A549 cells were treated with 10 nM BPS combination with or without anti-IL-10 (200 ng/ml) or anti-TGF-β (200 ng/ml) for 24 h, (A) the wound closure of cells was recorded (left) and statistically analyzed (right), and (B) the expression of MMP-2 was measured by western blot analysis (left) and statistically analyzed (right); A549 (C) or H1299 (D) cells were pre-treated with or without 10 μM SB431542 for 90 min and then further treated with 10 nM BPS for 24 h, the cell migration was checked by transwell assay. Data are presented as means ± SD of three independent experiments. **p < .01 compared with control; NS, no significant.
    Fig. 5. BPS increases the transcription of TGF-β via ERα/β or GPER independent pathway. A549 (A) or H1299 (B) cells were pre-treated with 10 μM ICI 182780 or G15 for 90 min and further incubated with 10 nM BPS for 24 h, the mRNA of TGF-β was checked by qRT-PCR; (C) A549 cells were treated as (A), the phosphorylation and total expression of Smad2/3 were measured by western blot analysis (left) and statistically analyzed (right); (D) A549 cells were transfected with si-NC or si-GPER for 24 h, the expression of GPER was measured by western blot analysis; (E) After transfected with si-NC or si-GPER for 12 h, A549 cells were further incubated with 10 nM BPS for 24 h, the mRNA of TGF-β was checked by qRT-PCR. Data are presented as means ± SD of six independent experiments. **p < .01 compared with control.
    ERK1/2 (PD98059, PD, 10 μM), JNK inhibitor II (10 μM) or p38-MAPK inhibitor (SB203580, SB, 10 μM) for 90 min and then further treated with 10 nM BPS for 24 h, the mRNA of TGF-β was checked by qRT-PCR; (C) A549 cells were treated as (A), the phosphorylation and total expression of Smad2/3 were measured by western blot analysis (left) and statistically analyzed (right); (D) A549 and H1299 cells were treated with 10 nM BPS for 2 h, the phosphorylation and total expression of ERK1/2 were 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 com-pared with control. NS, no significant.
    3.4. Activation of TGF-β/Smad2/3 signal mediates BPS induced migration of NSCLC cells
    We further investigated whether the upregulation of IL-10 and TGF-
    β was involved in BPS induced migration of NSCLC cells. Would closure assay showed that the neutralization antibody of TGF-β, while not IL-10, can suppress the BPS induced migration of A549 cells (Fig. 4 A). Further, anti-TGF-β, while not anti-IL-10, attenuated BPS induced 
    3.5. BPS increases the transcription of TGF-β via ERα/β or GPER independent pathway
    Previous studies suggested that ERα/β and GPER can mediate the biological functions of EDCs such as BPS in cancer cells (Deng et al., 2018; Liu et al., 2015). We then pretreated cells with the specific in-hibitors of ERα/β (ICI 182780) and GPER (G15) and further treated with BPS for 24 h. Our data showed that neither ICI 182780 nor G15 can attenuate BPS induced upregulation of TGF-β in A549 cells (Fig. 5A). Similar results were also observed in H1299 cells (Fig. 5 B). Further, either ICI 182780 or G15 can attenuate BPS induced
    phosphorylation of Smad2/3 in A549 cells (Fig. 5 C). We further knocked down the expression of GPER in A549 cells (Fig. 5 D). Con-sistently, si-GPER had no significant effect on BPS induced transcription of TGF-β in A549 cells (Fig. 5 E). These data suggested that BPS in-creases the transcription of TGF-β via ERα/β or GPER independent pathway.