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Dienogest enhances autophagy induction in endometriotic cells by impairing activation of AKT, ERK1/2, and mTOR  JongYeob Choi, Ph.D., MinWha Jo, M.S.,

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Presentation on theme: "Dienogest enhances autophagy induction in endometriotic cells by impairing activation of AKT, ERK1/2, and mTOR  JongYeob Choi, Ph.D., MinWha Jo, M.S.,"— Presentation transcript:

1 Dienogest enhances autophagy induction in endometriotic cells by impairing activation of AKT, ERK1/2, and mTOR  JongYeob Choi, Ph.D., MinWha Jo, M.S., EunYoung Lee, M.S., Dong-Yun Lee, M.D., Ph.D., DooSeok Choi, M.D., Ph.D.  Fertility and Sterility  Volume 104, Issue 3, Pages e1 (September 2015) DOI: /j.fertnstert Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

2 Figure 1 Effects of progesterone and dienogest on autophagy induction and the activation (phosphorylation) of protein kinase B (AKT), extracellular-signal-regulated kinase 1/2 (ERK), and S6K in endometriotic cyst stromal cells (ECSCs). (A) Representative immunoblots of microtubule-associated protein light chain 3 (LC3) and p62 in ECSCs. (B) Densitometric quantification of the levels of LC3-II and p62 in ECSCs. Experiments were repeated four times, and data are expressed as mean ± standard error (SE). ∗Statistically significant differences (P<.05) when compared with the estrogen-only group. Est = estrogen; Prog = progesterone; Die = dienogest. (C) Representative immunofluorescence images of ECSCs cultured in vitro. LC3 is visualized via green fluorescence. LC3-I exhibited a diffuse distribution within the cytoplasm, whereas LC3-II was present in punctate structures. (D) Transmission electron microscope images of luteal cells cultured with estrogen alone (left), estrogen + progesterone (middle), or estrogen + dienogest (right). Arrows indicate representative autophagosomes. Scale bars: 2 μm. N = nucleus. (E) Representative flow cytometry plots of acridine orange (AO) staining in estrogen alone (left), estrogen + progesterone (middle), and estrogen + dienogest (right) groups. FL1-H indicates the intensity of green fluorescence, and FL3-H indicates the intensity of red fluorescence. Cells in the upper left and right quadrants (AO red-positive) were considered to be autophagic cells. (F) Percentages of autophagic ECSCs, as determined by flow cytometry analysis of AO-stained cells. (G) Representative immunoblots of AKT, ERK1/2, and S6K in ECSCs. (H) Densitometric quantification of the levels of phosphorylated AKT (p-AKT), ERK1/2 (p-ERK1/2), and S6K (p-S6K) in ECSCs. Experiments were repeated four times, and data are expressed as mean ± SE. ∗Statistically significant differences (P<.05) when compared with the estrogen-only group. Est = estrogen; Prog = progesterone; Die = dienogest. Fertility and Sterility  , e1DOI: ( /j.fertnstert ) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

3 Figure 2 Effects of pharmacologic inhibition of protein kinase B (AKT) on S6K phosphorylation and the induction of autophagy in endometriotic cyst stromal cells (ECSCs). (A) Representative immunoblot of AKT, S6K, and microtubule-associated protein light chain 3 (LC3) in ECSCs cultured with estrogen after the addition of either dienogest or an AKT inhibitor. (B) Densitometric quantification of the levels of p-AKT, p-S6K, and LC-II in ECSCs. Experiments were repeated four times, and data are expressed as mean ± standard error (SE). ∗Statistically significant differences (P<.05) when compared with the estrogen-only group. Est = estrogen; Die = dienogest; AKT in = AKT inhibitor. (C) Immunofluorescence staining of p-AKT and LC3 in ECSCs. Phosphorylated AKT (p-AKT) and LC3 are visualized via red and green fluorescence, respectively. LC3-I exhibited a diffuse cytoplasmic distribution, whereas LC3-II was found in punctate structures. Intense staining of p-AKT was observed in ECSCs cultured with estrogen alone (left), and a few punctate cytoplasmic LC3-II structures were detected. In contrast, only weak staining of p-AKT was observed in ECSCs cultured with estrogen + progesterone (middle) or estrogen + an AKT inhibitor (right). Moreover, the punctate LC3-II structures had accumulated. Fertility and Sterility  , e1DOI: ( /j.fertnstert ) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

4 Figure 3 Effects of extracellular-signal-regulated kinase 1/2 (ERK1/2) inhibition on S6K phosphorylation and autophagy induction in endometriotic cyst stromal cells (ECSCs). (A) Representative immunoblot showing the levels of ERK1/2, S6K, and microtubule-associated protein light chain 3 (LC3) in ECSCs cultured with estrogen after the addition of dienogest or an inhibitor of ERK1/2. (B) Densitometric quantification of the levels of p-ERK1/2, p-S6K, and LC-II. Experiments were repeated four times, and data are expressed as mean ± standard error (SE). ∗Statistically significant differences (P<.05) when compared with the estrogen-only group. Est = estrogen; Die = dienogest. (C) Immunofluorescence staining of p-ERK1/2 and LC3 in ECSCs. Phosphorylated ERK1/2 (p-ERK1/2) and LC3 are visualized via red and green fluorescence, respectively. LC3-I exhibited a diffuse cytoplasmic distribution, whereas LC3-II was found in punctate structures. Intense staining of p-ERK1/2 was observed in ECSCs cultured with estrogen alone (left), and a few punctate cytoplasmic LC3-II structures were detected. In contrast, only weak staining of p-ERK1/2 was observed in ECSCs cultured with estrogen + progesterone (middle) or estrogen + an ERK1/2 inhibitor (right). Moreover, an accumulation of punctate LC3-II structures was observed. Fertility and Sterility  , e1DOI: ( /j.fertnstert ) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

5 Figure 4 Effects of dienogest-mediated autophagy induction on apoptosis in endometriotic cyst stromal cells (ECSCs). (A) Representative immunoblot showing the levels of microtubule-associated protein light chain 3 (LC3) and cleaved caspase-3 in ECSCs cultured with estrogen alone, estrogen + dienogest, or estrogen + dienogest + 3-MA. (B) Densitometric quantification of the levels of LC-II and cleaved caspase-3. Experiments were repeated four times, and data are expressed as mean ± standard error (SE). ∗Statistically significant differences (P<.05) when compared with the estrogen-only group. (C) Representative flow cytometry plots from cells treated with estrogen alone (left), estrogen + dienogest (middle), and estrogen + dienogest + 3-MA (right). Lower right quadrant, annexin-V+/propidium iodide (PI)−; upper right quadrant, annexin-V+/PI+ (apoptotic). (D) Percentages of apoptotic cells as assessed by flow cytometry. Data are given as mean ± SE of three independent experiments. ∗Statistically significant differences (P<.05) when compared with estrogen alone. Est = estrogen; Die = dienogest. Fertility and Sterility  , e1DOI: ( /j.fertnstert ) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

6 Supplemental Figure 1 Schematic diagram of potential regulatory mechanism for dienogest-induced autophagy in endometriotic cells. Dienogest suppresses protein kinase B (AKT) and extracellular-signal-regulated kinase 1/2 (ERK1/2) activity followed by mammalian target of rapamycin (mTOR) inhibition in endometriotic cells, which results in increased autophagy induction. Fertility and Sterility  , e1DOI: ( /j.fertnstert ) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

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