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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.

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Related in: MedlinePlus

Bladder cancer cell growth, migration and invasion were inhibited by increasing doses of 4-HPR. (A) Cell proliferation, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was significantly inhibited at all the 4-HPR concentrations tested after 96-h exposure. (B) Cell motility through uncoated filters was measured 24 h after plating in the absence or presence of the indicated concentrations of 4-HPR for 48 h. Then, cells in the lower chamber were stained with crystal violet and photographed with a light microscope (magnification, ×100), followed by calculation of the percentage of migrating cells. (C) Invasion assays adopted the same method than migration assays, with the exception of the use of coated filters for 48 h. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. C, control; 4-HPR, N-(4-hydroxyphenyl) retinamide.
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f1-ol-0-0-4908: Bladder cancer cell growth, migration and invasion were inhibited by increasing doses of 4-HPR. (A) Cell proliferation, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was significantly inhibited at all the 4-HPR concentrations tested after 96-h exposure. (B) Cell motility through uncoated filters was measured 24 h after plating in the absence or presence of the indicated concentrations of 4-HPR for 48 h. Then, cells in the lower chamber were stained with crystal violet and photographed with a light microscope (magnification, ×100), followed by calculation of the percentage of migrating cells. (C) Invasion assays adopted the same method than migration assays, with the exception of the use of coated filters for 48 h. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. C, control; 4-HPR, N-(4-hydroxyphenyl) retinamide.

Mentions: The action of 4-HPR on bladder cancer cell proliferation was assessed by treating EJ cells with a range of 4-HPR concentrations. All the concentrations tested inhibited cell growth, with statistically significant differences only after 96-h exposure (Fig. 1A). Migration of cancer cells is one of the key factors responsible for cancer metastasis (10). To metastasize, cancer cells must migrate from the original growth site, invade surrounding tissues and locate to other parts of the body through the blood or the lymphatic system (10). The effect of 4-HPR on migration and invasion of EJ cells upon exposure (48 h) to micromole concentrations of 4-HPR was then examined. All the concentrations tested inhibited cell migration and invasion, particularly when cells were treated with 4-HPR at 10 µM (Fig. 1B and C).


4-HPR impairs bladder cancer cell migration and invasion by interfering with the Wnt5a/JNK and Wnt5a/MMP-2 signaling pathways
Bladder cancer cell growth, migration and invasion were inhibited by increasing doses of 4-HPR. (A) Cell proliferation, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was significantly inhibited at all the 4-HPR concentrations tested after 96-h exposure. (B) Cell motility through uncoated filters was measured 24 h after plating in the absence or presence of the indicated concentrations of 4-HPR for 48 h. Then, cells in the lower chamber were stained with crystal violet and photographed with a light microscope (magnification, ×100), followed by calculation of the percentage of migrating cells. (C) Invasion assays adopted the same method than migration assays, with the exception of the use of coated filters for 48 h. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. C, control; 4-HPR, N-(4-hydroxyphenyl) retinamide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4998198&req=5

f1-ol-0-0-4908: Bladder cancer cell growth, migration and invasion were inhibited by increasing doses of 4-HPR. (A) Cell proliferation, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was significantly inhibited at all the 4-HPR concentrations tested after 96-h exposure. (B) Cell motility through uncoated filters was measured 24 h after plating in the absence or presence of the indicated concentrations of 4-HPR for 48 h. Then, cells in the lower chamber were stained with crystal violet and photographed with a light microscope (magnification, ×100), followed by calculation of the percentage of migrating cells. (C) Invasion assays adopted the same method than migration assays, with the exception of the use of coated filters for 48 h. Data were expressed as the mean ± standard deviation from three independent experiments. *P<0.05, **P<0.01 vs. control. C, control; 4-HPR, N-(4-hydroxyphenyl) retinamide.
Mentions: The action of 4-HPR on bladder cancer cell proliferation was assessed by treating EJ cells with a range of 4-HPR concentrations. All the concentrations tested inhibited cell growth, with statistically significant differences only after 96-h exposure (Fig. 1A). Migration of cancer cells is one of the key factors responsible for cancer metastasis (10). To metastasize, cancer cells must migrate from the original growth site, invade surrounding tissues and locate to other parts of the body through the blood or the lymphatic system (10). The effect of 4-HPR on migration and invasion of EJ cells upon exposure (48 h) to micromole concentrations of 4-HPR was then examined. All the concentrations tested inhibited cell migration and invasion, particularly when cells were treated with 4-HPR at 10 µM (Fig. 1B and C).

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