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4-HPR impairs bladder cancer cell migration and invasion by interfering with the Wnt5a/JNK and Wnt5a/MMP-2 signaling pathways

View Article: PubMed Central - PubMed

ABSTRACT

In order to identify the anti-invasive and anti-metastatic effect of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on the human bladder cancer EJ cell line, and to study its impact on the expression of wingless-type mouse mammary tumor virus integration site family, member 5a (Wnt5a), the phosphorylation of c-Jun N-terminal kinase (JNK), the expression levels of matrix metalloproteinase-2 (MMP-2), and the migration and invasion of EJ cells, migration and Matrigel invasion assays, as well as western blot analyses, were used in the present study. The results of the migration and Matrigel invasion assays indicated that the inhibitor of JNK SP600125 could inhibit the effect of 4-HPR on EJ cells. The expression of Wnt5a and MMP-2, and the phosphorylation of JNK, were analyzed by western blotting. The data revealed that 4-HPR inhibited the migration and invasion of bladder cancer cells through stimulating Wnt5a activation, causing the downregulation of MMP-2 expression and enhancing the phosphorylation of JNK in these cells. However, JNK signaling did not appear to have a direct effect on the expression of MMP-2. The present study demonstrated that 4-HPR may be a potent anti-invasive and anti-metastatic agent that functions via the Wnt5a/JNK and Wnt5a/MMP-2 signaling pathways.

No MeSH data available.


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4-HPR inhibits the migration and invasion of EJ cells by stimulating Wnt5a activity and JNK phosphorylation. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of JNK and pJNK were measured by western blotting. GAPDH was used as an internal control. (B) EJ cells were treated with 5 µM 4-HPR for 48 h, and then transfected with control or Wnt5a small interfering RNA. Cell lysates were assayed for JNK and pJNK by western blotting with anti-JNK, anti-pJNK and anti-GAPDH antibodies. (C-E) EJ cells were treated with SP600125, an inhibitor of JNK, and then incubated with 5 µM 4-HPR for 48 h. Cell migration and invasion were measured by (C) migration and (D) invasion assays (magnification, ×100), (E) and the protein extracts were analyzed by western blotting for JNK and pJNK. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. 4-HPR, N-(4-hydroxyphenyl) retinamide; siRNA, small interfering RNA; Wnt5a, wingless-type mouse mammary tumor virus integration site family, member 5a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ctrl, control; JNK, c-Jun N-terminal kinase; pJNK, phosphorylated c-Jun N-terminal kinase.
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f3-ol-0-0-4908: 4-HPR inhibits the migration and invasion of EJ cells by stimulating Wnt5a activity and JNK phosphorylation. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of JNK and pJNK were measured by western blotting. GAPDH was used as an internal control. (B) EJ cells were treated with 5 µM 4-HPR for 48 h, and then transfected with control or Wnt5a small interfering RNA. Cell lysates were assayed for JNK and pJNK by western blotting with anti-JNK, anti-pJNK and anti-GAPDH antibodies. (C-E) EJ cells were treated with SP600125, an inhibitor of JNK, and then incubated with 5 µM 4-HPR for 48 h. Cell migration and invasion were measured by (C) migration and (D) invasion assays (magnification, ×100), (E) and the protein extracts were analyzed by western blotting for JNK and pJNK. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. 4-HPR, N-(4-hydroxyphenyl) retinamide; siRNA, small interfering RNA; Wnt5a, wingless-type mouse mammary tumor virus integration site family, member 5a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ctrl, control; JNK, c-Jun N-terminal kinase; pJNK, phosphorylated c-Jun N-terminal kinase.

Mentions: As shown in Fig. 3A, treatment of EJ cells with 4-HPR for 48 h upregulated the phosphorylation of JNK in a dose-dependent manner, as detected by western blot analysis of extracts from EJ cells. To further confirm whether the phosphorylation of JNK could be induced following Wnt5a stimulation, EJ cells were exposed to 4-HPR for 48 h to stimulate the activity of Wnt5a, and the phosphorylation of JNK was induced upon stimulation with 4-HPR, as assessed by western blot analysis. In addition, the phosphorylation of JNK was enhanced by 4-HPR, and this 4-HPR-induced enhancement was abrogated by Wnt5a siRNA (Fig. 3B). Additionally, knocking down Wnt5a expression abrogated the 4-HPR-induced migration and invasion of EJ cells, further demonstrating the critical roles of phosphorylation of JNK, induced by Wnt5a signaling, in the migration and invasion of EJ cells (Fig. 3C and D).


4-HPR impairs bladder cancer cell migration and invasion by interfering with the Wnt5a/JNK and Wnt5a/MMP-2 signaling pathways
4-HPR inhibits the migration and invasion of EJ cells by stimulating Wnt5a activity and JNK phosphorylation. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of JNK and pJNK were measured by western blotting. GAPDH was used as an internal control. (B) EJ cells were treated with 5 µM 4-HPR for 48 h, and then transfected with control or Wnt5a small interfering RNA. Cell lysates were assayed for JNK and pJNK by western blotting with anti-JNK, anti-pJNK and anti-GAPDH antibodies. (C-E) EJ cells were treated with SP600125, an inhibitor of JNK, and then incubated with 5 µM 4-HPR for 48 h. Cell migration and invasion were measured by (C) migration and (D) invasion assays (magnification, ×100), (E) and the protein extracts were analyzed by western blotting for JNK and pJNK. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. 4-HPR, N-(4-hydroxyphenyl) retinamide; siRNA, small interfering RNA; Wnt5a, wingless-type mouse mammary tumor virus integration site family, member 5a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ctrl, control; JNK, c-Jun N-terminal kinase; pJNK, phosphorylated c-Jun N-terminal kinase.
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f3-ol-0-0-4908: 4-HPR inhibits the migration and invasion of EJ cells by stimulating Wnt5a activity and JNK phosphorylation. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of JNK and pJNK were measured by western blotting. GAPDH was used as an internal control. (B) EJ cells were treated with 5 µM 4-HPR for 48 h, and then transfected with control or Wnt5a small interfering RNA. Cell lysates were assayed for JNK and pJNK by western blotting with anti-JNK, anti-pJNK and anti-GAPDH antibodies. (C-E) EJ cells were treated with SP600125, an inhibitor of JNK, and then incubated with 5 µM 4-HPR for 48 h. Cell migration and invasion were measured by (C) migration and (D) invasion assays (magnification, ×100), (E) and the protein extracts were analyzed by western blotting for JNK and pJNK. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. 4-HPR, N-(4-hydroxyphenyl) retinamide; siRNA, small interfering RNA; Wnt5a, wingless-type mouse mammary tumor virus integration site family, member 5a; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ctrl, control; JNK, c-Jun N-terminal kinase; pJNK, phosphorylated c-Jun N-terminal kinase.
Mentions: As shown in Fig. 3A, treatment of EJ cells with 4-HPR for 48 h upregulated the phosphorylation of JNK in a dose-dependent manner, as detected by western blot analysis of extracts from EJ cells. To further confirm whether the phosphorylation of JNK could be induced following Wnt5a stimulation, EJ cells were exposed to 4-HPR for 48 h to stimulate the activity of Wnt5a, and the phosphorylation of JNK was induced upon stimulation with 4-HPR, as assessed by western blot analysis. In addition, the phosphorylation of JNK was enhanced by 4-HPR, and this 4-HPR-induced enhancement was abrogated by Wnt5a siRNA (Fig. 3B). Additionally, knocking down Wnt5a expression abrogated the 4-HPR-induced migration and invasion of EJ cells, further demonstrating the critical roles of phosphorylation of JNK, induced by Wnt5a signaling, in the migration and invasion of EJ cells (Fig. 3C and D).

View Article: PubMed Central - PubMed

ABSTRACT

In order to identify the anti-invasive and anti-metastatic effect of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on the human bladder cancer EJ cell line, and to study its impact on the expression of wingless-type mouse mammary tumor virus integration site family, member 5a (Wnt5a), the phosphorylation of c-Jun N-terminal kinase (JNK), the expression levels of matrix metalloproteinase-2 (MMP-2), and the migration and invasion of EJ cells, migration and Matrigel invasion assays, as well as western blot analyses, were used in the present study. The results of the migration and Matrigel invasion assays indicated that the inhibitor of JNK SP600125 could inhibit the effect of 4-HPR on EJ cells. The expression of Wnt5a and MMP-2, and the phosphorylation of JNK, were analyzed by western blotting. The data revealed that 4-HPR inhibited the migration and invasion of bladder cancer cells through stimulating Wnt5a activation, causing the downregulation of MMP-2 expression and enhancing the phosphorylation of JNK in these cells. However, JNK signaling did not appear to have a direct effect on the expression of MMP-2. The present study demonstrated that 4-HPR may be a potent anti-invasive and anti-metastatic agent that functions via the Wnt5a/JNK and Wnt5a/MMP-2 signaling pathways.

No MeSH data available.


Related in: MedlinePlus