Di-(2-ethylhexyl) Phthalate Triggers Proliferation, Migration, Stemness, and Epithelial–Mesenchymal Transition in Human Endometrial and Endometriotic Epithelial Cells via the Transforming Growth Factor-β/Smad Signaling Pathway

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is a frequently used plasticizer that may be linked to the development of endometriosis, a common gynecological disorder with a profound impact on quality of life. Despite its prevalence, vital access to treatment has often been hampered by a lack of understanding of its pathogenesis as well as reliable disease models. Recently, epithelial–mesenchymal transition (EMT) has been suggested to have a significant role in endometriosis pathophysiology. In this study, we found that DEHP treatment enhanced proliferation, migration, and inflammatory responses, along with EMT and stemness induction in human endometrial and endometriotic cells. The selective transforming growth factor-β (TGF-β) receptor type 1/2 inhibitor LY2109761 reversed the DEHP-induced cell proliferation and migration enhancement as well as the increased expression of crucial molecules involved in inflammation, EMT, and stemness, indicating that DEHP-triggered phenomena occur via the TGF-β/Smad signaling pathway. Our study clearly defines the role of DEHP in the etiology and pathophysiological mechanisms of endometriosis and establishes an efficient disease model for endometriosis using a biomimetic 3D cell culture technique. Altogether, our data provide novel etiological and mechanistic insights into the role of DEHP in endometriosis pathogenesis, opening avenues for developing novel preventive and therapeutic strategies for endometriosis.

Endometrial Cells Acutely Exposed to Phthalates In Vitro Do Not Phenocopy Endometriosis

“…Specifically, no statistically significant alterations in the viability of the three cell types were observed after in vitro DEHP exposure for 24–72 h. These results contradict those published by Kim et al, who described a significant increase in the viability of human EnSC and Ishikawa cells after 72 or 48 h, respectively, of DEHP exposure at 0.01 and 1 µM [ 17 ], respectively, and recently by Kim et al, who observed increases in the proliferation of Ishikawa, and endometriotic and unaffected endometrial epithelial lines 48 h after 25 μM of DEHP exposure [ 20 ]. However, our results are supported by the observations of Cho et al and Huang et al [ 18 , 19 ], who also did not observe alterations in the viability of endometrial cells after DEHP exposure during 24–72 h. Doses of 0.01, 0.1, and 1 nM and 0.2, 2, 20, and 200 µM were used in the studies of Cho et al and Huang et al, respectively [ 18 , 19 ].…”

“…Our observations conflict with previous studies with an experimental design comparable to ours, with acute exposure (24–48 h) to DEHP doses in the picomolar to micromolar range. In these previous studies, the authors essentially observed an increase in the inflammatory response and invasiveness of the exposed cells [ 16 , 17 , 19 , 20 , 21 ], which we intended to replicate in our study. Although we were able to recapitulate the invasiveness increase after acute DEHP exposure, we were not able to induce the inflammatory, angiogenic, morphogenic, epigenetic, and steroid receptor expression alterations described in endometrial cells from women with this pathology.…”

“…As endometriosis has an important pro-inflammatory component [ 1 ], different inflammatory molecules have been studied as endometriosis markers after acute DEHP exposure (24–48 h). It has been reported that acute DEHP exposure, at doses ranging from picomolar to micromolar, during 24–48 h, increases NF-κβ signaling and the activation of downstream molecules, such as IL-1β, IL-8, COX-2, and NOS in EnSC [ 19 , 23 ] and IL-1β, IL-6, IL-8 TNF-α, INF-γ, MCP-1, RANTES, and COX-2 in EnEC [ 20 ]. Our results show that the expression of IL-6 , a pro-inflammatory cytokine downstream of NF-κβ signaling, was unaffected after acute DEHP exposure, at doses ranging from 0.1–10 uM over 48 h, in both primary EnSC and EnEC and in Ishikawa cells, suggesting that acute phthalate exposure alone could not induce an inflammatory response in these cells.…”

“…Several studies have reported cellular aberrations in endometrial cells obtained from both disease free women and those suffering from endometriosis after acute DEHP exposure [ 16 , 17 , 18 , 19 , 20 ]. Acute DEHP exposure was found to increase cell viability, oxidative stress, and invasive capacity, altering the inflammatory response and steroid signaling in endometrial cells; these abnormalities are shared with both eutopic and ectopic endometrial cells in endometriosis [ 16 , 17 , 18 , 19 , 20 ]. In addition, in mouse models of endometriosis, exposure to DEHP seemed to increase the size and the proliferation of the ectopic endometrial implants [ 16 ].…”

Tumor-Associated Macrophage Targeting of Nanomedicines in Cancer Therapy

“…Furthermore, TAMs promote tumor cell invasion by mediating the degradation of the extracellular matrix through the involvement of cathepsins and matrix metalloproteinases (MMPs) such as MMP7, MMP2, and MMP9 [ 80 ]. Macrophages are also implicated in driving epithelial-mesenchymal transition (EMT) through factors like transforming growth factor-β (TGF-β) and IL-8 [ 81 ] ( Figure 2 ).…”