Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF-κB signaling pathway

Abstract

Endometriosis is a chronic gynecological inflammatory disorder in which immune system dysregulation is thought to play a role in its initiation and progression. Due to altered sex steroid receptor concentrations and other signaling defects, eutopic endometriotic tissues have an attenuated response to progesterone. This progesterone-resistance contributes to lesion survival, proliferation, pain, and infertility. The current agency-approved hormonal therapies, including synthetic progestins, GnRH agonists, and danazol are often of limited efficacy and counterproductive to fertility and cause systemic side effects due to suppression of endogenous steroid hormone levels. In the current study, we examined the effects of curcumin (CUR, diferuloylmethane), which has long been used as an anti-inflammatory folk medicine in Asian countries for this condition. The basal levels of proinflammatory and proangiogenic chemokines and cytokines expression were higher in primary cultures of stromal cells derived from eutopic endometrium of endometriosis (EESC) subjects compared with normal endometrial stromal cells (NESC). The treatment of EESC and NESC with CUR significantly and dose-dependently reduced chemokine and cytokine secretion over the time course. Notably, CUR treatment significantly decreased phosphorylation of the IKKα/β, NF-κB, STAT3, and JNK signaling pathways under these experimental conditions. Taken together, our findings suggest that CUR has therapeutic potential to abrogate aberrant activation of chemokines and cytokines, and IKKα/β, NF-κB, STAT3, and JNK signaling pathways to reduce inflammation associated with endometriosis.

Keywords: curcumin; endometriosis; human; stromal cell.

Figure 1

The intracellular uptake of CUR in NESCs and cells derived from EESCs, and their survival status. Cells were cultured and treated with or without CUR (1, 5, 10, 20, and 40 µg/ml) for 24, 48, and 72 hr in DMEM/Ham’s F‐12 media with 5% exosome‐depleted fetal bovine serum. (a) ESCs were fixed and stained with Hoechst 33248 to identify nuclei. Data represent the percentage of cells displaying morphological alteration of apoptosis based on quantification of nuclear morphologic changes. At least 250–300 cells were counted for each data point. The bar graph represents the mean ± SEM of results from three independent experiments. Significant (p ≤ 0.05) differences are represented with star “*” and compared to the parallel control group. (b) To assess if morphological changes occur in cells, live cell photographs were taken under an inverted epifluorescence microscope to image the green fluorescence signals for the CUR autofluorescence or the control (untreated) group alone along with phase contrast pictures at ×200 magnification at 48 hr posttreatment. Inset images are at a higher magnification, demonstrating CUR autofluorescence. Data are representative of three individual experiments (n = 3) from eutopic endometrial biopsies from three subjects with and three without evidence of endometriosis that were performed for each of the two patient groups. CUR: curcumin; DMEM: Dulbecco’s modified Eagle’s medium; EESC: eutopic endometrium of endometriosis subjects; ESCs: endometrial stromal cells; NESCs: normal human endometrial stromal cells [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

CUR attenuated proinflammatory interleukin secretion from human NESCs and cells derived from EESCs, but not IL‐10 or IL‐12. Cells were treated with or without CUR (5 µg/ml or 10 µg/ml) for 24 and 48 hr in DMEM/Ham’s F‐12 media with 5% exosome‐depleted fetal bovine serum. Basal levels of chemokines and cytokines were measured and analyzed in the supernatants using Bio‐Plex Pro Human Cytokine, Chemokine, and Growth Factor Magnetic Bead‐Based Assays, coupled with the Luminex 200 system. All bar graphs represent the mean ± SEM of results from three individual experiments (n = 3) from eutopic endometrial biopsies from three subjects with and three without evidence of endometriosis. The superscript “a” represents significant differences (p ≤ 0.05) in EESCs groups compared with respective NESCs groups at 24 and 48 hr. Star (*) represents significant differences (p ≤ 0.05) in EESCs groups treated with CUR compared with respective NESCs groups treated with CUR at 24 and 48 hr. CUR: curcumin; DMEM: Dulbecco’s modified Eagle’s medium; EESCs: eutopic endometrium of endometriosis subjects; NESCs: normal endometrial stromal cells

Figure 3

CUR attenuated proinflammatory chemokines and cytokines secreted by human NESCs and cells derived from EESCs. Cells were treated with or without CUR (5 µg/ml or 10 µg/ml) for 24 and 48 hr in DMEM/Ham’s F‐12 media with 5% exosome‐depleted fetal bovine serum. Concentrations of proinflammatory chemokines and cytokines were measured and analyzed in the supernatants using Bio‐Plex Pro Human Cytokine, Chemokine, and Growth Factor Magnetic Bead‐Based Assays, coupled with the Luminex 200 system (R&D System Inc., Minneapolis, MN). All bar graphs represent the mean ± SEM of results from three individual experiments (n = 3) from eutopic endometrial biopsies from three subjects with and three without evidence of endometriosis. The superscript “a” represents significant differences (p ≤ 0.05) in EESCs groups compared with respective NESCs groups at 24 and 48 hr. Star (*) represents significant differences (p ≤ 0.05) in EESCs groups treated with CUR compared with respective NESCs groups treated with CUR at 24 and 48 hr. CUR: curcumin; CCL11: chemokine eotaxin; DMEM: Dulbecco’s modified Eagle’s medium; EESC: eutopic endometrium of endometriosis subjects; FGF: fibroblast growth factors; G‐CSF: granulocyte‐colony stimulating factor; GM‐CSF: granulocyte‐macrophage colony stimulating factor; IFNγ: interferon γ; IP‐10/CXCL10: interferon γ‐induced protein 10; MCP‐1/CCL2: monocyte chemotactic protein‐1; MIP‐1a/CCL3: macrophage inflammatory proteins 1a; NESC: normal endometrial stromal cells; PDGF: platelet‐derived growth factor; TNF‐α: tumor necrosis factor‐α; VEGF: vascular permeability factor/vascular endothelial growth factor

Figure 4

Effects of CUR on phosphorylation and total expression of IKKα, IKKβ, and NF‐κB proteins in human NESCs and cells derived from EESCs subjects. Cells were treated with or without curcumin (CUR, 5 µg/ml or 10 µg/ml) for 24 and 48 hr in DMEM/Ham’s F‐12 media with 5% exosome‐depleted fetal bovine serum. Total protein was isolated, followed by equal amounts of protein (25 μg) from each sample separated by one‐dimensional gel electrophoresis and analyzed for phospho‐IKKα, phospho‐IKKβ, and phospho‐NF‐κB; and total IKKα, IKKβ, and NF‐κB protein. (a) Representative western blot analysis of protein for phospho‐ and total IKKα, IKKβ, and NF‐κB levels in NESCs and EESCs treated with or without CUR. α Tubulin was used as an internal constitutive control. (b) Bar diagrams represent the densitometric analyses of protein in WBs of three independent experiments (n = 3) as mean ± SEM that were performed for each individual group. The bar graphs represent the ratios of phospho‐IKKα, phospho‐IKKβ, and phospho‐NF‐κB protein levels normalized to total IKKα, IKKβ, and NF‐κB, respectively, and the ratios of total IKKα, IKKβ, and NF‐κB protein levels, normalized to α tubulin. The superscript “a” represents significant differences (p ≤ 0.05) in EESCs groups compared with respective NESCs groups at 24 and 48 hr. Star (*) represents significant differences (p ≤ 0.05) in EESCs groups treated with CUR compared with respective NESCs groups treated with CUR at 24 and 48 hr. CUR: curcumin; DMEM: Dulbecco’s modified Eagle’s medium; EESCs: eutopic endometriotic stromal cells; IKKα/β: inhibitor of nuclear factor kappa‐B kinase subunit α/β; JNK: c‐Jun N‐terminal kinases; NESCs: normal endometrial stromal cells; NF‐κB: nuclear factor κ‐light‐chain‐enhancer of activated B cells; STAT3: signal transducer and activator of transcription 3; WBs: western blots

Figure 5

Effects of CUR on phosphorylation and total expression of STAT3 and JNK proteins in human NESCs and cells derived from EESCs subjects. Cells were treated with or without curcumin (CUR, 5 µg/ml or 10 µg/ml) for 24 and 48 hr in DMEM/Ham’s F‐12 media with 5% exosome‐depleted fetal bovine serum. Total protein was isolated, followed by equal amounts of protein (25 μg) from each sample separated by one‐dimensional gel electrophoresis and analyzed for phospho‐STAT3 and phospho‐JNK; and total STAT3 and JNK protein. (a) Representative WBs analysis of protein for phospho‐ and total STAT3 and JNK levels in NESCs and EESCs treated with or without CUR. α Tubulin was used as an internal control. (b) Bar diagrams represent the densitometric analyses of specific protein bands in WBs of three independent experiments (n = 3) as mean ± SEM that were performed for each individual group. The bar graphs represent the ratios of phospho‐STAT3 and phospho‐JNK protein levels normalized to total STAT3 and JNK, respectively, and the ratios of total STAT3 and JNK protein levels normalized to α tubulin. The superscript “a” represents significant differences (p ≤ 0.05) in EESCs groups compared with respective NESCs groups at 24 and 48 hr. Star (*) represents significant differences (p ≤ 0.05) in EESCs groups treated with CUR compared with respective NESCs groups treated with CUR at 24 and 48 hr. CUR: curcumin; DMEM: Dulbecco’s modified Eagle’s medium; EESCs: eutopic endometrium of endometriosis; JNK: c‐Jun N‐terminal kinases; NESCs: normal endometrial stromal cells; STAT3: signal transducer and activator of transcription 3; WBs: western blots

Figure 6

A schematic model showing the antiangiogenic and anti‐inflammatory functions of CUR in human ESCs. Schematic representation of CUR‐dependent inhibition of proangiogenic and proinflammatory chemokine and cytokines may be through inhibited or eliminated phosphorylated forms of JNK and STAT3, along with IKKα, IKKβ, and NF‐κB in human ESCs. CUR: curcumin; ESCs: endometrial stromal cells; IKKα/β: inhibitor of nuclear factor κ‐B kinase subunit α/β; JNK: c‐Jun N‐terminal kinases; NF‐κB: nuclear factor κ‐light‐chain‐enhancer of activated B; STAT3: signal transducer and activator of transcription 3; p—phosphorylated form; arrow represents promotion and blunt arrow represents inhibition [Color figure can be viewed at wileyonlinelibrary.com]