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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.


Related in: MedlinePlus

4-HPR inhibits the migration and invasion of bladder cancer cells through suppression of MMP-2. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of MMP-2 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 MMP-2 by western blotting with an anti-MMP-2 antibody. (C) EJ cells were treated with SP600125, an inhibitor of c-Jun N-terminal kinase, and then incubated with 5 µM 4-HPR for 48 h. Next, the protein extracts were analyzed by western blotting for MMP-2. 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; MMP-2, matrix metalloproteinase-2.
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f4-ol-0-0-4908: 4-HPR inhibits the migration and invasion of bladder cancer cells through suppression of MMP-2. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of MMP-2 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 MMP-2 by western blotting with an anti-MMP-2 antibody. (C) EJ cells were treated with SP600125, an inhibitor of c-Jun N-terminal kinase, and then incubated with 5 µM 4-HPR for 48 h. Next, the protein extracts were analyzed by western blotting for MMP-2. 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; MMP-2, matrix metalloproteinase-2.

Mentions: The evidence for MMPs as active contributors to cancer progression comes from previous animal studies (23). In previous transplantation assays, relatively benign cancer cells acquired malignant properties when MMP expression was upregulated (23). Conversely, highly malignant cells became less aggressive when MMP expression or activity was reduced (12). It is well established that MMP-2 is involved in bladder cancer migration and invasion. In the present study, 4-HPR decreased the expression of MMP-2 in a dose-dependent manner, when the migration and invasion of EJ cells were inhibited (Fig. 4A). Furthermore, this suppressed expression of MMP-2 was abrogated by a suppressed expression of Wnt5a, compared with control siRNA transfection (Fig. 4B). These results indicate that 4-HPR inhibits the migration and invasion of EJ cells through inhibiting MMP-2 expression, which is regulated by Wnt5a signaling.


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 bladder cancer cells through suppression of MMP-2. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of MMP-2 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 MMP-2 by western blotting with an anti-MMP-2 antibody. (C) EJ cells were treated with SP600125, an inhibitor of c-Jun N-terminal kinase, and then incubated with 5 µM 4-HPR for 48 h. Next, the protein extracts were analyzed by western blotting for MMP-2. 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; MMP-2, matrix metalloproteinase-2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4998198&req=5

f4-ol-0-0-4908: 4-HPR inhibits the migration and invasion of bladder cancer cells through suppression of MMP-2. (A) EJ cells were incubated with the indicated concentrations of 4-HPR for 48 h, and the protein levels of MMP-2 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 MMP-2 by western blotting with an anti-MMP-2 antibody. (C) EJ cells were treated with SP600125, an inhibitor of c-Jun N-terminal kinase, and then incubated with 5 µM 4-HPR for 48 h. Next, the protein extracts were analyzed by western blotting for MMP-2. 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; MMP-2, matrix metalloproteinase-2.
Mentions: The evidence for MMPs as active contributors to cancer progression comes from previous animal studies (23). In previous transplantation assays, relatively benign cancer cells acquired malignant properties when MMP expression was upregulated (23). Conversely, highly malignant cells became less aggressive when MMP expression or activity was reduced (12). It is well established that MMP-2 is involved in bladder cancer migration and invasion. In the present study, 4-HPR decreased the expression of MMP-2 in a dose-dependent manner, when the migration and invasion of EJ cells were inhibited (Fig. 4A). Furthermore, this suppressed expression of MMP-2 was abrogated by a suppressed expression of Wnt5a, compared with control siRNA transfection (Fig. 4B). These results indicate that 4-HPR inhibits the migration and invasion of EJ cells through inhibiting MMP-2 expression, which is regulated by Wnt5a signaling.

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