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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

Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 9 or medium only.Cells were seeded at a concentration of 800,000 cells per insert and procyanidin C2 (9) was added at a concentration of 25 and 50 μM. A) Pictures of the wound gaps were taken at 0 h and 7 h for both conditions (with and without treatment). B) Quantification of the wound healing experiment. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 38–88). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05. C) Effect of polyphenolic compounds in a transwell assay on the cell migration of HEK293 empty vector and PRL-3 overexpressing cell lines. Cells were seeded at a concentration of 100,000 cells per insert and procyanidin C2 (9) was added at a final concentration of 25 and 50 μM. Migration was allowed for 16 h before harvesting and staining with calcein AM for fluorescent readout. Mean ± SD (n = 60–72) is shown for combined data from different replicates. Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant. D) Quantification of wound healing experiment including blocking PRL-1 and PRL-3 with analog 3. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 63–82). Two-sided t-tests with Welsh correction were performed for p < 0.05.
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pone.0134336.g006: Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 9 or medium only.Cells were seeded at a concentration of 800,000 cells per insert and procyanidin C2 (9) was added at a concentration of 25 and 50 μM. A) Pictures of the wound gaps were taken at 0 h and 7 h for both conditions (with and without treatment). B) Quantification of the wound healing experiment. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 38–88). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05. C) Effect of polyphenolic compounds in a transwell assay on the cell migration of HEK293 empty vector and PRL-3 overexpressing cell lines. Cells were seeded at a concentration of 100,000 cells per insert and procyanidin C2 (9) was added at a final concentration of 25 and 50 μM. Migration was allowed for 16 h before harvesting and staining with calcein AM for fluorescent readout. Mean ± SD (n = 60–72) is shown for combined data from different replicates. Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant. D) Quantification of wound healing experiment including blocking PRL-1 and PRL-3 with analog 3. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 63–82). Two-sided t-tests with Welsh correction were performed for p < 0.05.

Mentions: To study if procyanidin C2 is able to inhibit PRL-1 and PRL-3 in cells, HEK293 cells were treated in a wound-healing assay (see the experimental procedures). The PRL-1 and PRL-3 stably overexpressing HEK293 cells were treated with 25 and 50 μM of 9, since these concentrations did not cause any cytotoxic effects as mentioned above. As a control the empty vector cell line was again utilized. As expected [29,45], compared to the untreated empty vector control cell line the untreated PRL-3 and PRL-1 overexpressing cells showed a significantly higher migration due to PRL-1 and PRL-3 activity. Treatment with 25 μM of 9 slightly increased migration behavior in the empty vector cell line, and treatment with 50 μM of 9 significantly increased the number of migrating cells in the control cell line (see Fig 6A and 6B). In contrast, treatment of the PRL-1 cells with 25 μM of 9 showed a slight drop in cell migration, and for PRL-3 overexpressing cells the migration speed did not decrease significantly at 25 μM. When the concentration of 9 was increased to 50 μM, the cell migration speed of the PRL-1 overexpressing cell line did not change any further compared to 25 μM. At treatment with 50 μM 9, the PRL-3 overexpressing cell line showed a significant decrease in cell migration compared to 25 μM of 9. The migration speed was for all cell lines similar when treated with 50 μM of 9. To further corroborate these findings we performed additionally a transwell assay as an alternative way to study cell migration [45]. Here again the untreated controls showed a significant increase in cell migration for the PRL-3 overexpressing cell line compared to the empty vector cell line. Treatment with 25 μM of 9 did not lead to a significant change in cell migration for neither the empty vector cell line nor the PRL-3 overexpressing cell line. In contrast, treatment with 50 μM of 9 lead to a significant increase in cell migration for the empty vector cell line and a significant decrease in migration for PRL-3 overexpressing cell line (see Fig 6C), with the speed dropping to the level of the untreated control cell line, corroborating the results from the wound healing assay.


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

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

Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 9 or medium only.Cells were seeded at a concentration of 800,000 cells per insert and procyanidin C2 (9) was added at a concentration of 25 and 50 μM. A) Pictures of the wound gaps were taken at 0 h and 7 h for both conditions (with and without treatment). B) Quantification of the wound healing experiment. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 38–88). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05. C) Effect of polyphenolic compounds in a transwell assay on the cell migration of HEK293 empty vector and PRL-3 overexpressing cell lines. Cells were seeded at a concentration of 100,000 cells per insert and procyanidin C2 (9) was added at a final concentration of 25 and 50 μM. Migration was allowed for 16 h before harvesting and staining with calcein AM for fluorescent readout. Mean ± SD (n = 60–72) is shown for combined data from different replicates. Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant. D) Quantification of wound healing experiment including blocking PRL-1 and PRL-3 with analog 3. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 63–82). Two-sided t-tests with Welsh correction were performed for p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134336.g006: Wound healing abilities of PRL-1 or PRL-3 overexpressing or empty vector control HEK293 cell lines were analyzed in the presence of compound 9 or medium only.Cells were seeded at a concentration of 800,000 cells per insert and procyanidin C2 (9) was added at a concentration of 25 and 50 μM. A) Pictures of the wound gaps were taken at 0 h and 7 h for both conditions (with and without treatment). B) Quantification of the wound healing experiment. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 38–88). Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant;***: < 0.001; **: < 0.01; *: < 0.05. C) Effect of polyphenolic compounds in a transwell assay on the cell migration of HEK293 empty vector and PRL-3 overexpressing cell lines. Cells were seeded at a concentration of 100,000 cells per insert and procyanidin C2 (9) was added at a final concentration of 25 and 50 μM. Migration was allowed for 16 h before harvesting and staining with calcein AM for fluorescent readout. Mean ± SD (n = 60–72) is shown for combined data from different replicates. Two-sided t-tests with Welsh correction were performed for p < 0.05. ns = not significant. D) Quantification of wound healing experiment including blocking PRL-1 and PRL-3 with analog 3. Speed of migration is shown for combined data from different replicates, as mean ± SD (n = 63–82). Two-sided t-tests with Welsh correction were performed for p < 0.05.
Mentions: To study if procyanidin C2 is able to inhibit PRL-1 and PRL-3 in cells, HEK293 cells were treated in a wound-healing assay (see the experimental procedures). The PRL-1 and PRL-3 stably overexpressing HEK293 cells were treated with 25 and 50 μM of 9, since these concentrations did not cause any cytotoxic effects as mentioned above. As a control the empty vector cell line was again utilized. As expected [29,45], compared to the untreated empty vector control cell line the untreated PRL-3 and PRL-1 overexpressing cells showed a significantly higher migration due to PRL-1 and PRL-3 activity. Treatment with 25 μM of 9 slightly increased migration behavior in the empty vector cell line, and treatment with 50 μM of 9 significantly increased the number of migrating cells in the control cell line (see Fig 6A and 6B). In contrast, treatment of the PRL-1 cells with 25 μM of 9 showed a slight drop in cell migration, and for PRL-3 overexpressing cells the migration speed did not decrease significantly at 25 μM. When the concentration of 9 was increased to 50 μM, the cell migration speed of the PRL-1 overexpressing cell line did not change any further compared to 25 μM. At treatment with 50 μM 9, the PRL-3 overexpressing cell line showed a significant decrease in cell migration compared to 25 μM of 9. The migration speed was for all cell lines similar when treated with 50 μM of 9. To further corroborate these findings we performed additionally a transwell assay as an alternative way to study cell migration [45]. Here again the untreated controls showed a significant increase in cell migration for the PRL-3 overexpressing cell line compared to the empty vector cell line. Treatment with 25 μM of 9 did not lead to a significant change in cell migration for neither the empty vector cell line nor the PRL-3 overexpressing cell line. In contrast, treatment with 50 μM of 9 lead to a significant increase in cell migration for the empty vector cell line and a significant decrease in migration for PRL-3 overexpressing cell line (see Fig 6C), with the speed dropping to the level of the untreated control cell line, corroborating the results from the wound healing assay.

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