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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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SFO promoted endothelial–mesenchymal transition (EndoMT) in HUVECs.HUVECs were exposed to 10–70 µg/ml SFO in normal medium. (A) qPCR analysis of mRNA expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 3, 6 and 12 h. Expression was normalized to that of GAPDH. *p<0.05;**p<0.01 vs. Ctr, n = 4. (B) Western blot analysis of protein expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 6 and 12 h. Results are representative of 3 independent experiments. (C) HUVECs were treated with ethanol (Control) or 50 µg/ml of SFO for 6 h, then exposed to 30 µM sphingosylphosphorylcholine (SPC) for 1 h, with or without rescue for 1 h, and contraction of cells was calculated. VSMC stimulated with SPC was as a positive control (**p<0.01, n = 3).
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pone-0099378-g002: SFO promoted endothelial–mesenchymal transition (EndoMT) in HUVECs.HUVECs were exposed to 10–70 µg/ml SFO in normal medium. (A) qPCR analysis of mRNA expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 3, 6 and 12 h. Expression was normalized to that of GAPDH. *p<0.05;**p<0.01 vs. Ctr, n = 4. (B) Western blot analysis of protein expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 6 and 12 h. Results are representative of 3 independent experiments. (C) HUVECs were treated with ethanol (Control) or 50 µg/ml of SFO for 6 h, then exposed to 30 µM sphingosylphosphorylcholine (SPC) for 1 h, with or without rescue for 1 h, and contraction of cells was calculated. VSMC stimulated with SPC was as a positive control (**p<0.01, n = 3).

Mentions: To identify SFO as an inducer of EndoMT in the presence of serum and FGF-2, we examined the specific markers of mesenchymal and endothelial cells in HUVECs treated with SFO at different concentrations. α-SMA, the specific marker of smooth muscle cells and EndoMT [22], [23], showed a very low level of expression in untreated cells. In contrast, low concentrations of SFO (10–30 µg/ml) treatment significantly increased α-SMA expression at 6 and 12 h (Fig. 2A and B). High concentrations of SFO (50–70 µg/ml) increased α-SMA expression only before 12 h. At 12 h, it could not increase α-SMA expression again, and this time point was verified to induce apoptosis in our previous report [7], [8]. In contrast, levels of the specific marker of endothelial cells, CD31, and eNOS were decreased by high but not low concentrations of SFO (Fig. 2A and 2B). Along with the morphological changes and our previous findings [7], [8], we concluded that high concentrations of SFO promoted EndoMT within 12 h.


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)

SFO promoted endothelial–mesenchymal transition (EndoMT) in HUVECs.HUVECs were exposed to 10–70 µg/ml SFO in normal medium. (A) qPCR analysis of mRNA expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 3, 6 and 12 h. Expression was normalized to that of GAPDH. *p<0.05;**p<0.01 vs. Ctr, n = 4. (B) Western blot analysis of protein expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 6 and 12 h. Results are representative of 3 independent experiments. (C) HUVECs were treated with ethanol (Control) or 50 µg/ml of SFO for 6 h, then exposed to 30 µM sphingosylphosphorylcholine (SPC) for 1 h, with or without rescue for 1 h, and contraction of cells was calculated. VSMC stimulated with SPC was as a positive control (**p<0.01, n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099378-g002: SFO promoted endothelial–mesenchymal transition (EndoMT) in HUVECs.HUVECs were exposed to 10–70 µg/ml SFO in normal medium. (A) qPCR analysis of mRNA expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 3, 6 and 12 h. Expression was normalized to that of GAPDH. *p<0.05;**p<0.01 vs. Ctr, n = 4. (B) Western blot analysis of protein expression of α-SMA, CD31 and eNOS in HUVECs treated with SFO at 6 and 12 h. Results are representative of 3 independent experiments. (C) HUVECs were treated with ethanol (Control) or 50 µg/ml of SFO for 6 h, then exposed to 30 µM sphingosylphosphorylcholine (SPC) for 1 h, with or without rescue for 1 h, and contraction of cells was calculated. VSMC stimulated with SPC was as a positive control (**p<0.01, n = 3).
Mentions: To identify SFO as an inducer of EndoMT in the presence of serum and FGF-2, we examined the specific markers of mesenchymal and endothelial cells in HUVECs treated with SFO at different concentrations. α-SMA, the specific marker of smooth muscle cells and EndoMT [22], [23], showed a very low level of expression in untreated cells. In contrast, low concentrations of SFO (10–30 µg/ml) treatment significantly increased α-SMA expression at 6 and 12 h (Fig. 2A and B). High concentrations of SFO (50–70 µg/ml) increased α-SMA expression only before 12 h. At 12 h, it could not increase α-SMA expression again, and this time point was verified to induce apoptosis in our previous report [7], [8]. In contrast, levels of the specific marker of endothelial cells, CD31, and eNOS were decreased by high but not low concentrations of SFO (Fig. 2A and 2B). Along with the morphological changes and our previous findings [7], [8], we concluded that high concentrations of SFO promoted EndoMT within 12 h.

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