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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

Effect of polyphenolic compounds 8 (A), 9 (B), 10 (C) and 11 (D) on the phosphatase activity of PRL-1, PRL-2, PRL-3, PP1, and PP2A.Proteins were incubated for 30 min with 0–1000 μM of the corresponding procyandin 8–11. Phosphatase activity was measured in the presence of DiFMUP at 25°C using the concentration corresponding to the KM of each protein. The KM for PRLs was determined separately (PRL-3 = 21 μM, PRL-2 and PRL-1 = 24 μM). The KM for PP1 (91 μM) and for PP2A (100 μM) was taken from the literature [38,39]. Protein concentrations are 50 nM for all PRLs and 2 mU for PP1 and 0.05 U for PP2A (see the Experimental Procedures). Phosphatase activity in the absence of inhibitors was set as 100%. Data represent means ± standard errors of the mean (n = 3–5).
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pone.0134336.g002: Effect of polyphenolic compounds 8 (A), 9 (B), 10 (C) and 11 (D) on the phosphatase activity of PRL-1, PRL-2, PRL-3, PP1, and PP2A.Proteins were incubated for 30 min with 0–1000 μM of the corresponding procyandin 8–11. Phosphatase activity was measured in the presence of DiFMUP at 25°C using the concentration corresponding to the KM of each protein. The KM for PRLs was determined separately (PRL-3 = 21 μM, PRL-2 and PRL-1 = 24 μM). The KM for PP1 (91 μM) and for PP2A (100 μM) was taken from the literature [38,39]. Protein concentrations are 50 nM for all PRLs and 2 mU for PP1 and 0.05 U for PP2A (see the Experimental Procedures). Phosphatase activity in the absence of inhibitors was set as 100%. Data represent means ± standard errors of the mean (n = 3–5).

Mentions: Biochemical phosphatase activity measurements were carried out in the presence of different concentrations of various polyphenols, compounds 1–11 (Fig 1), using recombinant PRL-3 and 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a fluorogenic substrate (see the experimental procedures). Results are shown in Table 1 and Fig 2. We observed that the monomeric epicatechins EGC and EC inhibit PRL-3 only weakly, with a slightly better activity of EGC, which bears three hydroxyl groups at the B-ring. This is in agreement with the findings of He et al. who observed that the hydroxyl groups at position 4’ (at the B-ring) and 7 (at the A-ring) (see numbering scheme in Fig 1) are important for exhibiting inhibitor activity toward PRL-3 [37]. An about two-fold increase in inhibition was observed for compounds 2 and 4 bearing a gallate ester at the 3-position of the molecule compared to 1 and 3, respectively. However, gallic acid (7) itself did not show any inhibition up to 1000 μM. This indicates that the gallate moiety in combination with the flavan skeleton is beneficial for activity. In general, as a basic structure activity-relationship the following trend was observed: a higher number of hydroxyl groups at the B-ring increased the activity slightly. However, attachment of a gallate ester (D-ring) at position 3 of the flavan skeleton significantly increased the inhibitor activity. Therefore, combination of a pyrogallol ring as the B-ring and a gallate ester at position 3, exhibited the highest activity in these series. The influence of the stereochemistry at C(2)–C(3) was studied by using GCG (the trans-epimer of EGCG). The results indicated that both epimers suppress the activity of PRL-3 similarly. The same observation was made by using (+)-catechin (6, IC50 = 456 μM vs. 514 μM for EC(1)). Thus the C(2)–C(3) stereochemistry does not have significant influence on the inhibitor activity of the corresponding flavanol on PRL-3.


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

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

Effect of polyphenolic compounds 8 (A), 9 (B), 10 (C) and 11 (D) on the phosphatase activity of PRL-1, PRL-2, PRL-3, PP1, and PP2A.Proteins were incubated for 30 min with 0–1000 μM of the corresponding procyandin 8–11. Phosphatase activity was measured in the presence of DiFMUP at 25°C using the concentration corresponding to the KM of each protein. The KM for PRLs was determined separately (PRL-3 = 21 μM, PRL-2 and PRL-1 = 24 μM). The KM for PP1 (91 μM) and for PP2A (100 μM) was taken from the literature [38,39]. Protein concentrations are 50 nM for all PRLs and 2 mU for PP1 and 0.05 U for PP2A (see the Experimental Procedures). Phosphatase activity in the absence of inhibitors was set as 100%. Data represent means ± standard errors of the mean (n = 3–5).
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Related In: Results  -  Collection

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pone.0134336.g002: Effect of polyphenolic compounds 8 (A), 9 (B), 10 (C) and 11 (D) on the phosphatase activity of PRL-1, PRL-2, PRL-3, PP1, and PP2A.Proteins were incubated for 30 min with 0–1000 μM of the corresponding procyandin 8–11. Phosphatase activity was measured in the presence of DiFMUP at 25°C using the concentration corresponding to the KM of each protein. The KM for PRLs was determined separately (PRL-3 = 21 μM, PRL-2 and PRL-1 = 24 μM). The KM for PP1 (91 μM) and for PP2A (100 μM) was taken from the literature [38,39]. Protein concentrations are 50 nM for all PRLs and 2 mU for PP1 and 0.05 U for PP2A (see the Experimental Procedures). Phosphatase activity in the absence of inhibitors was set as 100%. Data represent means ± standard errors of the mean (n = 3–5).
Mentions: Biochemical phosphatase activity measurements were carried out in the presence of different concentrations of various polyphenols, compounds 1–11 (Fig 1), using recombinant PRL-3 and 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a fluorogenic substrate (see the experimental procedures). Results are shown in Table 1 and Fig 2. We observed that the monomeric epicatechins EGC and EC inhibit PRL-3 only weakly, with a slightly better activity of EGC, which bears three hydroxyl groups at the B-ring. This is in agreement with the findings of He et al. who observed that the hydroxyl groups at position 4’ (at the B-ring) and 7 (at the A-ring) (see numbering scheme in Fig 1) are important for exhibiting inhibitor activity toward PRL-3 [37]. An about two-fold increase in inhibition was observed for compounds 2 and 4 bearing a gallate ester at the 3-position of the molecule compared to 1 and 3, respectively. However, gallic acid (7) itself did not show any inhibition up to 1000 μM. This indicates that the gallate moiety in combination with the flavan skeleton is beneficial for activity. In general, as a basic structure activity-relationship the following trend was observed: a higher number of hydroxyl groups at the B-ring increased the activity slightly. However, attachment of a gallate ester (D-ring) at position 3 of the flavan skeleton significantly increased the inhibitor activity. Therefore, combination of a pyrogallol ring as the B-ring and a gallate ester at position 3, exhibited the highest activity in these series. The influence of the stereochemistry at C(2)–C(3) was studied by using GCG (the trans-epimer of EGCG). The results indicated that both epimers suppress the activity of PRL-3 similarly. The same observation was made by using (+)-catechin (6, IC50 = 456 μM vs. 514 μM for EC(1)). Thus the C(2)–C(3) stereochemistry does not have significant influence on the inhibitor activity of the corresponding flavanol on PRL-3.

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