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Finding ATF4/p75NTR/IL-8 signal pathway in endothelial-mesenchymal transition by safrole oxide.

Ge D, Jing Q, Zhao W, Yue H, Su L, Zhang S, Zhao J - PLoS ONE (2014)

Bottom Line: Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h.Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly.The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO.

View Article: PubMed Central - PubMed

Affiliation: Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan, China.

ABSTRACT
Targeting the endothelial-to-mesenchymal transition (EndoMT) may be a novel therapeutic strategy for cancer and various diseases induced by fibrosis. We aimed to identify a small chemical molecule as an inducer of EndoMT and find a new signal pathway by using the inducer. Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h. To understand the underlying molecular mechanism, we performed microarray, quantitative real-time PCR and western blot analysis to find key factors involved in SFO-induced EndoMT and demonstrated the involvement of the factors by RNAi. The expression of activating transcription factor 4 (ATF4), p75 neurotrophin receptor (p75NTR), and interleukin 8 (IL-8) was greatly increased in SFO-induced EndoMT. Knockdown of ATF4 inhibited the SFO-induced EndoMT completely, and knockdown of p75NTR or IL-8 partially inhibited the EndoMT, which suggests that all three factors were involved in the process. Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly. The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO. Our findings support potential novel targets for the therapeutics of cancer and fibrosis disease.

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ATF4, p75NTR and IL-8 were increased by SFO in HUVECs and MS1 cells.HUVECs were exposed to 50 µg/ml of SFO at 3, 6 and 12 h, then the relative mRNA level of the three genes was detected by qPCR (A to C) and western blot analysis (D and E) or ELISA (F). Data below the western blot bands were used to quantify the density of the bands. β-actin was a normalization control. MS1 cells were exposed to 50–150 µg/ml SFO at 6 h (The effective time and concentration used in this study was according to our preliminary experiment), the relative mRNA level and protein level of ATF4, p75NTR and IL-8 were detected by qPCR (G to I) and western blot analysis (J and K). IL-8 secretion was detected by ELISA (L). β-actin was used as a normalization control (*p<0.05; **p<0.01 vs. Ctr, n = 3).
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pone-0099378-g004: ATF4, p75NTR and IL-8 were increased by SFO in HUVECs and MS1 cells.HUVECs were exposed to 50 µg/ml of SFO at 3, 6 and 12 h, then the relative mRNA level of the three genes was detected by qPCR (A to C) and western blot analysis (D and E) or ELISA (F). Data below the western blot bands were used to quantify the density of the bands. β-actin was a normalization control. MS1 cells were exposed to 50–150 µg/ml SFO at 6 h (The effective time and concentration used in this study was according to our preliminary experiment), the relative mRNA level and protein level of ATF4, p75NTR and IL-8 were detected by qPCR (G to I) and western blot analysis (J and K). IL-8 secretion was detected by ELISA (L). β-actin was used as a normalization control (*p<0.05; **p<0.01 vs. Ctr, n = 3).

Mentions: To understand the molecular mechanism by which SFO induces EndoMT, we first found the candidates that were implicated in the EndoMT. On microarray assay, 717 genes showed changed expression (>1.5-fold) during EndoMT induced by treatment with SFO, 50 µg/ml, for 6 h; 13 genes showed > eight-fold changed expression (Fig. 3). The greatly increased expression of ATF4, p75NTR and IL-8 (CXCL1 in mouse) was verified by qPCR and western blot assay in HUVECs and endothelial MS1 cells (Fig. 4).


Finding ATF4/p75NTR/IL-8 signal pathway in endothelial-mesenchymal transition by safrole oxide.

Ge D, Jing Q, Zhao W, Yue H, Su L, Zhang S, Zhao J - PLoS ONE (2014)

ATF4, p75NTR and IL-8 were increased by SFO in HUVECs and MS1 cells.HUVECs were exposed to 50 µg/ml of SFO at 3, 6 and 12 h, then the relative mRNA level of the three genes was detected by qPCR (A to C) and western blot analysis (D and E) or ELISA (F). Data below the western blot bands were used to quantify the density of the bands. β-actin was a normalization control. MS1 cells were exposed to 50–150 µg/ml SFO at 6 h (The effective time and concentration used in this study was according to our preliminary experiment), the relative mRNA level and protein level of ATF4, p75NTR and IL-8 were detected by qPCR (G to I) and western blot analysis (J and K). IL-8 secretion was detected by ELISA (L). β-actin was used as a normalization control (*p<0.05; **p<0.01 vs. Ctr, n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048316&req=5

pone-0099378-g004: ATF4, p75NTR and IL-8 were increased by SFO in HUVECs and MS1 cells.HUVECs were exposed to 50 µg/ml of SFO at 3, 6 and 12 h, then the relative mRNA level of the three genes was detected by qPCR (A to C) and western blot analysis (D and E) or ELISA (F). Data below the western blot bands were used to quantify the density of the bands. β-actin was a normalization control. MS1 cells were exposed to 50–150 µg/ml SFO at 6 h (The effective time and concentration used in this study was according to our preliminary experiment), the relative mRNA level and protein level of ATF4, p75NTR and IL-8 were detected by qPCR (G to I) and western blot analysis (J and K). IL-8 secretion was detected by ELISA (L). β-actin was used as a normalization control (*p<0.05; **p<0.01 vs. Ctr, n = 3).
Mentions: To understand the molecular mechanism by which SFO induces EndoMT, we first found the candidates that were implicated in the EndoMT. On microarray assay, 717 genes showed changed expression (>1.5-fold) during EndoMT induced by treatment with SFO, 50 µg/ml, for 6 h; 13 genes showed > eight-fold changed expression (Fig. 3). The greatly increased expression of ATF4, p75NTR and IL-8 (CXCL1 in mouse) was verified by qPCR and western blot assay in HUVECs and endothelial MS1 cells (Fig. 4).

Bottom Line: Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h.Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly.The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO.

View Article: PubMed Central - PubMed

Affiliation: Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan, China.

ABSTRACT
Targeting the endothelial-to-mesenchymal transition (EndoMT) may be a novel therapeutic strategy for cancer and various diseases induced by fibrosis. We aimed to identify a small chemical molecule as an inducer of EndoMT and find a new signal pathway by using the inducer. Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h. To understand the underlying molecular mechanism, we performed microarray, quantitative real-time PCR and western blot analysis to find key factors involved in SFO-induced EndoMT and demonstrated the involvement of the factors by RNAi. The expression of activating transcription factor 4 (ATF4), p75 neurotrophin receptor (p75NTR), and interleukin 8 (IL-8) was greatly increased in SFO-induced EndoMT. Knockdown of ATF4 inhibited the SFO-induced EndoMT completely, and knockdown of p75NTR or IL-8 partially inhibited the EndoMT, which suggests that all three factors were involved in the process. Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly. The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO. Our findings support potential novel targets for the therapeutics of cancer and fibrosis disease.

Show MeSH
Related in: MedlinePlus