Limits...
Procyanidins Negatively Affect the Activity of the Phosphatases of Regenerating Liver.

Stadlbauer S, Rios P, Ohmori K, Suzuki K, Köhn M - PLoS ONE (2015)

Bottom Line: Increasing the number of catechin units in procyanidins to more than three does not further enhance the potency.As PRL overexpression induces cell migration compared to control cells, the effect of procyanidins on this phenotype was studied.Together, our results show that procyanidins negatively affect PRL activity, suggesting that PRLs could be targets in the polypharmacology of natural polyphenols.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117, Heidelberg, Germany.

ABSTRACT
Natural polyphenols like oligomeric catechins (procyanidins) derived from green tea and herbal medicines are interesting compounds for pharmaceutical research due to their ability to protect against carcinogenesis in animal models. It is nevertheless still unclear how intracellular pathways are modulated by polyphenols. Monomeric polyphenols were shown to affect the activity of some protein phosphatases (PPs). The three phosphatases of regenerating liver (PRLs) are close relatives and promising therapeutic targets in cancer. In the present study we show that several procyanidins inhibit the activity of all three members of the PRL family in the low micromolar range, whereas monomeric epicatechins show weak inhibitory activity. Increasing the number of catechin units in procyanidins to more than three does not further enhance the potency. Remarkably, the tested procyanidins showed selectivity in vitro when compared to other PPs, and over 10-fold selectivity toward PRL-1 over PRL-2 and PRL-3. As PRL overexpression induces cell migration compared to control cells, the effect of procyanidins on this phenotype was studied. Treatment with procyanidin C2 led to a decrease in cell migration of PRL-1- and PRL-3-overexpressing cells, suggesting the compound-dependent inhibition of PRL-promoted cell migration. Treatment with procyanidin B3 led to selective suppression of PRL-1 overexpressing cells, thereby corroborating the selectivity toward PRL-1- over PRL-3 in vitro. Together, our results show that procyanidins negatively affect PRL activity, suggesting that PRLs could be targets in the polypharmacology of natural polyphenols. Furthermore, they are interesting candidates for the development of PRL-1 inhibitors due to their low cellular toxicity and the selectivity within the PRL family.

No MeSH data available.


Related in: MedlinePlus

A) Cytotoxicity of procyanidin B3 (8) in HEK293 cells. Cells were treated with 50, 75 or 100 μM of 8 in the growth medium for 24 h. Cells were harvested, resuspended in PBS buffer and incubated with propidium iodide (1 mg/mL) for 5 min on ice. 10,000 events were set for the number of living cells. The number of living cells was divided by the number of total cells counted and given as percentage of cell survival. Data represent means ± standard errors of the mean (n = 3). B) Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 8 or medium only. Cells were seeded at a concentration of 800,000 cells per insert and procyanidin B3 (8) was added at a concentration of 50 and 75 μM. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 40–77). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520450&req=5

pone.0134336.g007: A) Cytotoxicity of procyanidin B3 (8) in HEK293 cells. Cells were treated with 50, 75 or 100 μM of 8 in the growth medium for 24 h. Cells were harvested, resuspended in PBS buffer and incubated with propidium iodide (1 mg/mL) for 5 min on ice. 10,000 events were set for the number of living cells. The number of living cells was divided by the number of total cells counted and given as percentage of cell survival. Data represent means ± standard errors of the mean (n = 3). B) Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 8 or medium only. Cells were seeded at a concentration of 800,000 cells per insert and procyanidin B3 (8) was added at a concentration of 50 and 75 μM. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 40–77). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05.

Mentions: Seeing the selectivity in cells that we observed in vitro would corroborate our findings that procyanidins affect PRL activity in cells. Therefore, we studied compound 8, a catechin dimer with reported cell permeability [46] and with a much stronger significant difference in inhibitor potency between PRL-1 and PRL-3 in vitro. Again we examined first the cytotoxicity of 8 toward all three cell lines with flow cytometry after treatment with different concentrations of compound 8 for 24 h (Fig 7A). Treatment with 50, 75 and 100 μM of compound 8 did not show any cytotoxic effects in the three cell lines. Next we applied dimer 8 in a concentration of 50 and 75 μM to all three cell lines in a wound healing assay. Treatment with 50 μM of 8 did not significantly increase migration behavior in the empty vector and the PRL-3 overexpressing cell lines. In contrast, treatment with 50 μM of dimer significantly decreased the number of migrating cells in the PRL-1 overexpressing cell line (see Fig 7B). Treatment with 75 μM of 8 further decreased migration in the PRL-1 overexpressing cell line, whereas it did not significantly increase the migration speed of the other two cell lines. Unlike compound 9, compound 8 did not induce cell migration. Thus, dimer 8 is able to suppress cell migration selectively in PRL-1 overexpressing cells without affecting the empty vector and PRL-3 overexpressing cell lines, demonstrating that it acts on PRL-1.


Procyanidins Negatively Affect the Activity of the Phosphatases of Regenerating Liver.

Stadlbauer S, Rios P, Ohmori K, Suzuki K, Köhn M - PLoS ONE (2015)

A) Cytotoxicity of procyanidin B3 (8) in HEK293 cells. Cells were treated with 50, 75 or 100 μM of 8 in the growth medium for 24 h. Cells were harvested, resuspended in PBS buffer and incubated with propidium iodide (1 mg/mL) for 5 min on ice. 10,000 events were set for the number of living cells. The number of living cells was divided by the number of total cells counted and given as percentage of cell survival. Data represent means ± standard errors of the mean (n = 3). B) Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 8 or medium only. Cells were seeded at a concentration of 800,000 cells per insert and procyanidin B3 (8) was added at a concentration of 50 and 75 μM. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 40–77). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520450&req=5

pone.0134336.g007: A) Cytotoxicity of procyanidin B3 (8) in HEK293 cells. Cells were treated with 50, 75 or 100 μM of 8 in the growth medium for 24 h. Cells were harvested, resuspended in PBS buffer and incubated with propidium iodide (1 mg/mL) for 5 min on ice. 10,000 events were set for the number of living cells. The number of living cells was divided by the number of total cells counted and given as percentage of cell survival. Data represent means ± standard errors of the mean (n = 3). B) Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 8 or medium only. Cells were seeded at a concentration of 800,000 cells per insert and procyanidin B3 (8) was added at a concentration of 50 and 75 μM. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 40–77). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05.
Mentions: Seeing the selectivity in cells that we observed in vitro would corroborate our findings that procyanidins affect PRL activity in cells. Therefore, we studied compound 8, a catechin dimer with reported cell permeability [46] and with a much stronger significant difference in inhibitor potency between PRL-1 and PRL-3 in vitro. Again we examined first the cytotoxicity of 8 toward all three cell lines with flow cytometry after treatment with different concentrations of compound 8 for 24 h (Fig 7A). Treatment with 50, 75 and 100 μM of compound 8 did not show any cytotoxic effects in the three cell lines. Next we applied dimer 8 in a concentration of 50 and 75 μM to all three cell lines in a wound healing assay. Treatment with 50 μM of 8 did not significantly increase migration behavior in the empty vector and the PRL-3 overexpressing cell lines. In contrast, treatment with 50 μM of dimer significantly decreased the number of migrating cells in the PRL-1 overexpressing cell line (see Fig 7B). Treatment with 75 μM of 8 further decreased migration in the PRL-1 overexpressing cell line, whereas it did not significantly increase the migration speed of the other two cell lines. Unlike compound 9, compound 8 did not induce cell migration. Thus, dimer 8 is able to suppress cell migration selectively in PRL-1 overexpressing cells without affecting the empty vector and PRL-3 overexpressing cell lines, demonstrating that it acts on PRL-1.

Bottom Line: Increasing the number of catechin units in procyanidins to more than three does not further enhance the potency.As PRL overexpression induces cell migration compared to control cells, the effect of procyanidins on this phenotype was studied.Together, our results show that procyanidins negatively affect PRL activity, suggesting that PRLs could be targets in the polypharmacology of natural polyphenols.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117, Heidelberg, Germany.

ABSTRACT
Natural polyphenols like oligomeric catechins (procyanidins) derived from green tea and herbal medicines are interesting compounds for pharmaceutical research due to their ability to protect against carcinogenesis in animal models. It is nevertheless still unclear how intracellular pathways are modulated by polyphenols. Monomeric polyphenols were shown to affect the activity of some protein phosphatases (PPs). The three phosphatases of regenerating liver (PRLs) are close relatives and promising therapeutic targets in cancer. In the present study we show that several procyanidins inhibit the activity of all three members of the PRL family in the low micromolar range, whereas monomeric epicatechins show weak inhibitory activity. Increasing the number of catechin units in procyanidins to more than three does not further enhance the potency. Remarkably, the tested procyanidins showed selectivity in vitro when compared to other PPs, and over 10-fold selectivity toward PRL-1 over PRL-2 and PRL-3. As PRL overexpression induces cell migration compared to control cells, the effect of procyanidins on this phenotype was studied. Treatment with procyanidin C2 led to a decrease in cell migration of PRL-1- and PRL-3-overexpressing cells, suggesting the compound-dependent inhibition of PRL-promoted cell migration. Treatment with procyanidin B3 led to selective suppression of PRL-1 overexpressing cells, thereby corroborating the selectivity toward PRL-1- over PRL-3 in vitro. Together, our results show that procyanidins negatively affect PRL activity, suggesting that PRLs could be targets in the polypharmacology of natural polyphenols. Furthermore, they are interesting candidates for the development of PRL-1 inhibitors due to their low cellular toxicity and the selectivity within the PRL family.

No MeSH data available.


Related in: MedlinePlus