Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells

Abstract

Objectives: Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues).

Materials and methods: We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation.

Results: We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages.

Conclusions: Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks.

Figure 1

Effect of hyperforin on embryonic stem ( ES ) cell and fibroblast viability. Employing mouse ES cells (a) and NIH/3T3 cells (b) were treated with hyperforin at indicated concentrations for 7 days. Cell viability was measured by CellTiter‐Glo luminescent cell viability assay. Cell cultures exposed to 0 μm drug were considered to be 100% viable. Cell viability of each drug‐treated sample was presented as a percentage of viability of cultures treated with 0 μm drug. Data are mean ± SEM of results from at least three independent experiments. *P < 0.05, compared to 0 μm.

Figure 2

Effects of hyperforin on embryonic stem ( ES ) cell apoptosis and proliferation. (a, b) Employing mouse ES (mES) cells were treated with hyperforin at indicated concentrations for 24 h (a) or 72 h (b). Cell viability was measured by CellTiter‐Glo luminescent cell viability assay. Cell cultures exposed to 0 μm drug were considered to be 100% viable. Cell viability of each drug‐treated sample was presented as percentage of that of cultures treated with 0 μm drug. (c, d) mES cells were treated with hyperforin at indicated concentrations for 24 h (c) or 72 h (d). Apoptosis was measured by cell death detection ELISA assay. Apoptotic level in each drug‐treated sample was presented as fold‐change compared to that in cultures treated with 0 μm drug. (e, f) mES cells were treated with hyperforin at indicated concentrations for 24 h (e) or 72 h (f). Uptake of BrdU was measured by ELISA. BrdU incorporation in each drug‐treated sample was presented as fold‐change compared to that in cultures treated with 0 μm drug. Data are the mean ± SEM of results from at least three independent experiments. *P < 0.05, compared to 0 μm.

Figure 3

Effects of hyperforin on fibroblast apoptosis. (a) NIH/3T3 cells were treated with hyperforin at indicated concentrations for 72 h. Cell viability was measured by CellTiter‐Glo luminescent cell viability assay. Cell cultures exposed to 0 μm drug were considered to be 100% viable. Cell viability of each drug‐treated sample was presented as percentage of that of cultures treated with 0 μm drug. (b) NIH/3T3 cells were treated with hyperforin at indicated concentrations for 72 h. Apoptosis was measured by cell death detection ELISA assay. Apoptotic level in each drug‐treated sample was presented as fold‐change compared to that in cultures treated with 0 μm drug. (c–d) NIH/3T3 cells were treated with hyperforin at indicated concentrations for 72 h. Caspase‐3/7 (c), ‐8 (d) and ‐9 (e) activities were determined using Caspase‐Glo Assays. Data are expressed as fold‐increases relative to respective untreated samples (RLU/60 min/μg protein). (f) NIH/3T3 cells were treated with hyperforin at indicated concentrations for 72 h. Uptake of BrdU was measured by ELISA. BrdU incorporation in each drug‐treated sample was presented as fold‐change compared to that in cultures treated with 0 μm drug. Data are expressed as mean ± SEM of results from at least four independent experiments. *P < 0.05, compared to 0 μm.

Figure 4

Effect of hyperforin on embryonic stem ( ES ) cell differentiation. Analysis of expression levels for undifferentiated state and tissue‐specific differentiation markers. Expression levels of undifferentiated markers Oct3/4 (a) and Sox2 (b), endodermal markers GATA6 (c) and TTR (d), mesodermal markers BMP4 (e) and ANF (f) and ectodermal markers nestin (g) and GFAP (h) were quantified at each concentration of hyperforin with real‐time RT‐PCR. Each experiment was performed in triplicate. Data are expressed as mean ± SEM of results from at least three independent experiments. *P < 0.05, compared to 0 μm. un, undifferentiated ES cell. (e) The frequencies of cardiomyocytes, identified by their distinctive beating movement, derived from ES cells were quantified at each concentration of hyperforin.