CXCL8 enhances proliferation and growth and ...

Human Reproduction, Vol.27, No.7 pp. 2107– 2116, 2012 Advanced Access publication on May 4, 2012 doi:10.1093/humrep/des132

ORIGINAL ARTICLE Reproductive biology

Ming-Qing Li 1,†, Xue-Zhen Luo 1,†, Yu-Han Meng 1, Jie Mei1, Xiao-Yong Zhu 1, Li-Ping Jin 1, and Da-Jin Li1,2,* 1 Laboratory for Reproductive Immunology, Hospital & Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, Shanghai 200011, China 2Department of Obstetrics and Gynecology, The Affiliated Hospital, Hainan Medical College, Haikou 571101, China

*Correspondence address. Tel/Fax: +86-21-63457331; E-mail: [email protected]

Submitted on November 10, 2011; resubmitted on February 26, 2012; accepted on March 20, 2012

background: Chemokine CXCL8 (also known as IL-8) has been identified as a potential regulator of endometrial stromal cells (ESCs), but it is unclear how CXCL8 regulates the survival of ESCs in the pathogenesis of endometriosis. methods: We assessed the secretion of CXCL8 by enzyme-linked immunosorbent assays and the expression of its receptors, CXCR1 and CXCR2, by in-cell Western assay and immunhistochemistry. The effects of CXCL8 on the activation or expression of various cell mediators were also investigated by in-cell Western assay. The effects of CXCL8 on the proliferation, growth and apoptosis of ESCs in vitro were assessed by BrdU assays, cell counts and annexin V labeling, respectively. results: Secretion of CXCL8 and expression of CXCR1 in the eutopic ESCs from women with endometriosis were significantly higher than that in control ESCs, but the expression of CXCR2 showed no significant difference between these two cell types. CXCL8 stimulated proliferation and growth and reduced apoptosis of ESCs in an autocrine manner, and these effects were abolished by anti-human CXCL8 and CXCR1 neutralizing antibodies and by a PI3K/Akt inhibitor. Moreover, CXCL8 up-regulated the expression of the anti-apoptotic proteins, survivin and Bcl-2, inhibited the expression of the Phosphatase and tensin homolog (PTEN) and activated the phosphorylation of Akt.

conclusions: This study suggests that CXCL8 and CXCR1 are involved in the pathogenesis of endometriosis by up-regulating proliferation and growth and restricting apoptosis in ESCs by activating the PTEN/Akt pathway and mediating the expression of survivin and Bcl-2. Key words: CXCL8 / ESCs / proliferation / apoptosis / endometriosis

Introduction Endometriotic lesions are commonly defined by the presence of both endometrial stromal and epithelial cells, but most research focuses on the former cell type (Clement et al., 1990). The pathogenesis of endometriosis remains controversial despite extensive research. The most widely accepted theory is that the exfoliated menstrual endometrial †

cells attach to the peritoneal serous membrane, and their subsequent proliferation and invasion into the underlying tissue result in endometriotic lesions (Witz et al., 1999; Nisolle et al., 2000). A growing body of evidence indicates that the primary defect in endometriosis may be located in the eutopic endometrium. Abnormalities inherent to the eutopic endometrium, which are not found in the endometrium of disease-free women, might therefore contribute to the ectopic

These authors contributed equally to this work.

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CXCL8 enhances proliferation and growth and reduces apoptosis in endometrial stromal cells in an autocrine manner via a CXCR1-triggered PTEN/AKT signal pathway

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Materials and Methods Tissue collection, and cell isolation and culture All tissue samples were obtained with informed consent in accordance with the requirements of the Research Ethics Committee in the Obstetrics and Gynecology, Fudan University Shanghai Medical College. Samples of the endometriotic peritoneal lesions (n ¼ 5) and ovarian lesions (n ¼ 5) were obtained from women in the age of 28 – 45 years undergoing laparoscopy for pain or other benign indications. Eutopic endometrial tissues were obtained from fertile women (aged 22 – 48 years) with (n ¼ 12) or without (n ¼ 18) endometriosis. None of the women had received hormonal medication in the 3 months prior to the surgical procedure. All the samples were confirmed histologically according to established criteria. The endometrial tissues were collected under sterile conditions and transported to the laboratory on ice in DMEM (Dulbecco’s modified Eagle’s medium)/F-12 (Gibco, USA) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT, USA). The ESCs were isolated according to methods described previously (Li et al., 2011). The endometrial tissues were digested with collagenase type IV (0.1%; Sigma, USA) for 30 min at 378C with constant agitation for recovering ESCs. The tissue pieces were filtered through sterile gauzes pads (pore diameter sizes: 200 mesh) to remove cellular debris. Following gentle centrifugation, the supernatant

was discarded and the cells were resuspended in DMEM/F-12. The ESCs were separated from epithelial cells by passing them over sterile gauzes pads (pore diameter sizes: 400 mesh). The filtrated suspension was layered over Ficoll and centrifuged at 800g for 20 min to further remove leukocytes and erythrocytes, and the middle layer was collected and then washed with D-Hanks solution. The ESCs were placed in a culture flask and allowed to adhere for 20 min. The adherent stromal cells were cultured as a monolayer in flasks with DMEM/F-12 supplemented with 10% FCS and 20 mmol/l HEPES and incubated in a humidified incubator with 5% CO2 at 378C. This method supplied 95% vimentinpostive and cytokeratin-negative ESCs.

Enzyme-linked immunosorbent assay for determination of CXCL8 ESCs (2 × 105cells/well) derived from the endometrium from women with (n ¼ 10) or without (n ¼ 10) endometriosis were seeded in 24-well plates, and the supernatant was collected after 24, 48 and 72 h in culture, respectively. The culture supernatant was harvested, centrifuged to remove cellular debris and then stored at 2808C until being assayed by ELISA. The CXCL8 concentration in the supernatant was quantified by ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions. Each experiment was carried out in triplicate.

In-cell Western assay According to the description by Egorina et al. (2006) and our previous procedure (Li et al., 2010), we used a newly set-up assay called in-cell Western to determine the in-cell protein level of interest. Normal ESCs (n ¼ 6) or eutopic ESCs with endometriosis (n ¼ 6) were seeded at a density of 2 × 104 cells/well in a 96-well plate for 48 h, then an in-cell Western assay was performed to evaluate the expression of CXCR1 and CXCR2 in normal and endometriosis-associated eutopic ESCs. The procedure for subsequent studies was as follows. First 2 × 104 cells/well eutopic ESCs were seeded in a 96-well plate, and then the cells were then starved for 12 h with DMEM containing 1% FBS before treatment. After stimulation with recombinant human CXCL8 (100 ng/ ml; R&D Systems, USA) for 24 h, the cells were treated with anti-CXCL8 neutralizing antibody (R&D Systems) or with a mouse isotype control (1 mg/ml; Sino-America Co., Ltd), or with the PI3K/Akt inhibitor, LY294002 (50 mmol/l; Cell Signaling Technology, USA), for another 24 h. The cells were then immediately fixed with 4% formaldehyde for 20 min at room temperature. After washing with 0.01% Triton, the cells were blocked by adding 150 ml of LI-COR Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, Nebraska, USA) for 90 min at room temperature. The cells were incubated with mouse anti-human CXCR1 or CXCR2 (20 mg/ml, R&D Systems), mouse anti-human phospho-Erk1/2 (1:50, Santa Cruz Biotechnology, USA), rabbit anti-human Erk1/2 (1:80, Santa Cruz Biotechnology), rabbit anti-human phospho-Akt (1:50, Santa Cruz Biotechnology), goat anti-human Akt (1:80, Santa Cruz Biotechnology), mouse anti-human phospho-JNK (1:50, Santa Cruz Biotechnology), rabbit anti-human JNK (1:80, Santa Cruz Biotechnology), mouse antihuman phospho-p38 (1:50, Santa Cruz Biotechnology), rabbit anti-human p38 (1:80, Santa Cruz Biotechnology), rabbit anti-human PTEN or survivin (20 mg/ml, R&D Systems) or goat anti-human Bcl-2 (20 mg/ml; Cell Signaling Technology, USA) antibody, with anti-actin antibody as the internal control. After overnight treatment at 48C, the wells were incubated with the corresponding secondary antibodies: IRDyeTM 700DX-conjugated affinity purified (red fluorescence) anti-mouse antibody and IRDyeTM 800DX-conjugated affinity purified (green fluorescence) anti-rabbit antibody. However, for Akt and Bcl-2 detection groups, the wells were incubated with the corresponding secondary antibodies: IRDyeTM 700DX-conjugated affinity purified (red fluorescence) anti-rabbit antibody


growth outside the uterine cavity (Ulukus et al., 2006; Szymanowski, 2007). Spontaneous apoptosis of eutopic and ectopic endometrial tissues is impaired in women with endometriosis, and this reduced susceptibility to apoptosis might permit the growth, survival and invasion of endometriotic tissue (Gebel et al., 1998; Imai et al., 2000; Szymanowski, 2007). Chemokine CXCL8 (also named IL-8) is the ligand of CXCR1 and CXCR2, and is produced by many cell types, such as peripheral blood monocytes (Yoshimura et al., 1987), mesothelial cells (Arici et al., 1996) and endometrial cells (Arici et al., 1993). In addition to its chemotactic (Matsushima et al., 1988), angiogenic (Koch et al., 1992) and activating properties for granulocytes, CXCL8 is also found to stimulate the proliferation of many other cells types (Tuschil et al., 1992; Schadendorf et al., 1993) and to inhibit apoptosis in human breast cancer cells (Ginestier et al., 2010). Our previous work has shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can coordinate with estradiol to stimulate the secretion of pro-inflammatory cytokine CXCL8 by endometriotic-associated cells [such as endometrial stromal cell (ESC), human pelvic mesothelial cells (HPMCs) and the human monocytic cell line U937] in vitro, and further evoke and aggravate the inflammatory response (Shi et al., 2006). Moreover, Arici et al. (1998) have found that CXCL8 can directly promote the proliferation of ESCs. However, there are still questions of whether and how CXCL8 regulates the proliferation, growth and apoptosis of ESCs in an autocrine dependent manner. Therefore, the present study was undertaken to analyze the regulatory roles of CXCL8 in proliferation and apoptosis of ESCs and to investigate the possible mechanism. Our current results illustrate that CXCL8 can enhance the survival and growth of ESCs by inhibiting PTEN expression and activating Akt signal pathways. Hence, we propose that the abnormal over-expression of CXCL8 and its receptor CXCR1 in ESCs may play an important role in the origin and development of endometriosis.

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Roles of IL-8 in the endometriotic milieu and IRDyeTM 800DX-conjugated affinity purified (green fluorescence) antigoat fluorescent labeled antibody recommended by the manufacturer (Rockland, Inc., Gibertsvile, PA, USA). This procedure was carried out in the dark. Images of the target genes were obtained using the Odyssey Infrared Imaging System (LI-COR Biosciences GmbH Bad, Homburg, Germany). The expression levels of the corresponding molecules were calculated as the ratio of the intensity of the target genes to actin or as the total molecules (such as total Akt or Erk1/2). The experiments were carried out in triplicate and repeated three times.

Immunohistochemistry

BrdU cell proliferation assay, cell number count and apoptosis assay The BrdU cell proliferation assay and an annexin V-FITC assay were utilized to evaluate the effects of CXCL8 on cell proliferation and apoptosis, respectively, while cell growth was assessed by counting cell numbers. ESCs from women with endometriosis were resuspended in DMEM/ F-12 supplemented with 10% FBS and seeded at a density of 1 × 104 cells/well in 96-well flat-bottom microplates for the BrdU cell proliferation assay, or at 1 × 105 cells/well in 24-well flat-bottom microplates for the apoptosis assay, or at 1 × 105 cells/well in 12-well flat-bottom microplates for counting cell numbers. Thereafter, the cells were starved with DMEM containing 1% FBS for 12 h before treatment. After stimulation with recombinant human CXCL8 protein (100 ng/ml) for 24 h, the cells in wells were treated with an anti-CXCL8, CXCR1 or CXCR2 neutralizing antibodies or with LY294002 (50 mmol/l) for another 24 h. Alternatively, mouse isotype (1 mg/ml; Sino-America Co., Ltd) or vehicle was added as the negative control. Before and after treatment, the cells in 12-well flat-bottom microplates were counted at a magnification of ×100. The results were observed under the microscope (Olympus 1X71; Olympus, Tokyo, Japan) fitted with a digital camera (Olympus DP72; Olympus). The cells were counted in five predetermined fields. Each experiment was carried out in triplicate and repeated three times. The ability of ESCs to proliferate was assessed using the BrdU cell proliferation assay (Millipore, USA) according to the manufacturer’s instruction. Each experiment was performed in triplicate and repeated four times. As a marker of apoptosis, phosphatidylserine externalization was quantified by flow cytometry by using a commercially available annexin V-FITC apoptosis detection kit (Invitrogen, USA) according to the manufacturer’s guideline. ESCs were trypsinized and collected. The culture medium was also retained and pooled with the adherent cells. Cells were centrifuged and the supernatant was discarded. The cells were resuspended in PBS, and washed twice and resuspended in the kit-binding buffer (100 ml/ pellet) containing annexin V solution (5 ml/pellet) and propidium iodide (2.5 mg/ml). Samples were incubated in the dark for 15 min, and the percent of annexin V-positive cells in ESCs was determined by

Statistics All values are shown as the mean + SD. Data were analyzed by using one-way analysis of variance and least significant difference (equal variances assumed), or Tamhane’s test (equal variances not assumed) was used post hoc for multiple comparisons by Statistical Package for the Social Sciences software version 11.5. Differences were considered as statistically significant at P , 0.05.

Results CXCL8 and CXCR1 expression is elevated in the eutopic ESCs associated with endometriosis The secretion of CXCL8 and the expression of CXCR1 and CXCR2 in the primary eutopic ESCs from women with or without endometriosis were evaluated by ELISA and in-cell Western assay, respectively. As depicted in Fig. 1, the secretion of CXCL8 was markedly increased in the eutopic ESCs associated with endometriosis compared with that of the normal ESCs at 48 and 72 h (P , 0.05; Fig. 1a). The expression of CXCR1 in the eutopic ESCs associated with endometriosis was higher than that in the normal ESCs (P , 0.05; Fig. 1b). However, there was no difference in CXCR2 expression between these two groups (P . 0.05; Fig. 1b). Moreover, immunohistochemistry was used to localize CXCL8 and CXCR1 proteins in the paraffin sections. In contrast to normal endometrium, the eutopic endometrium associated with endometriosis and endometriotic tissues showed both epithelial and stromal cells that were strongly strained for CXCL8 and CXCR1 (Fig. 1c). These results suggest that higher expression of CXCL8 and CXCR1 in the eutopic ESCs derived from women with endometriosis may be associated with their unique biological characteristics.

CXCL8 induces the proliferation and growth and reduces apoptosis of ESCs in an autocrine manner To clarify the influence of CXCL8 on ESC survival and growth, we first investigated its effect on proliferation, growth and apoptosis of ESCs. Data presented in Fig. 2 demonstrated that rhCXCL8 notably promotes proliferation (P , 0.05; Fig. 2a left) and growth (P , 0.05; Fig. 2a right), and inhibits apoptosis in eutopic ESCs (P , 0.05; Fig. 2c). Moreover, anti-CXCL8, anti-CXCR1 or the combination of anti-CXCR1 and anti-CXCR2 neutralizing antibodies decreased proliferation (P , 0.05; Fig. 2a left) and growth (P , 0.05; Fig. 2a right), and promoted apoptosis (P , 0.05 or P , 0.01; Fig. 2c) in ESCs, but anti-CXCR2 antibody alone had no effect. The increase in proliferation (P , 0.05; Fig. 2b left) and growth (P , 0.05; Fig. 2b right), and the decrease in apoptosis (P , 0.05 or P , 0.01; Fig. 2d) of the eutopic ESCs caused by CXCL8 could be significantly reversed by anti-CXCL8, anti-CXCR1 or the combination of anti-CXCR1 and anti-CXCR2 neutralizing antibodies, but not anti-CXCR2 antibody alone. Our results indicate that CXCL8 can enhance proliferation


Paraffin sections (5 mm) of the endometriotic and eutopic endometrial tissues, from women with or without endometriosis and from the proliferative or secretory phase of the cycle, were dehydrated in Tris-buffered saline (TBS) and incubated with hydrogen peroxide in 1% bovine serum albumin/TBS to block endogenous peroxidase. The samples were then incubated with mouse anti-human CXCL8 monoclonal antibody (25 mg/ml), mouse anti-human CXCR1 antibody (25 mg/ml) or mouse IgG isotype antibody overnight at 48C in a humid chamber. After washing three times with TBS, the sections were overlaid with peroxidaseconjugated goat anti-mouse IgG antibody (SP-9002; Golden Bridge International, Inc., China), and the reaction was developed with 3,3-diaminobenzidine, and counterstained with hematoxylin. The experiments were repeated five times.

FACSCalibur flow cytometry. The experiments were performed in triplicate and repeated three times.

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Figure 1 CXCL8 and CXCR1 expression is elevated in the eutopic ESCs with endometriosis. The secretion of CXCL8 was determined by ELISA (a), and expression of CXCR1 and CXCR2 was analyzed by in-cell Western assay (b), respectively. The relative protein levels of CXCR1 and CXCR2 in ESCs were compared between the endometriosis-associated eutopic ESCs and normal ESCs (b). The expression of CXCL8 and CXCR1 in the eutopic endometrium from women with or without endometriosis and in endometriotic tissues were analyzed, respectively, by immunohistochemistry (c). Results were highly reproducible in three independent experiments. Normal ESC is ESC from endometrium from women without endometriosis; eutopic ESC is ESC from the eutopic endometrium from women with endometriosis. Original magnification ×200. Error bars depict the standard error of the mean. *P , 0.05 compared with the normal ESC control.

Roles of IL-8 in the endometriotic milieu

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Figure 2 CXCL8 induces proliferation and growth and inhibits apoptosis of ESCs in an autocrine manner. Eutopic ESCs [1 × 104cell/well, (a) left,

(b) left; 1 × 105cell/well, (a) right, (b) right, (c, d)] from women with endometriosis were treated, respectively, with recombinant human CXCL8 (100 ng/ml) for 24 h, and neutralizing antibodies to CXCL8 (15 mg/ml), CXCR1 (15 mg/ml) or CXCR2 (15 mg/ml) or vehicle as controls for another 24 h. Thereafter, BrdU proliferation assays (a left, b left), cell number counts (a right, b right) and annexin V-FITC apoptosis detection assays (c, d) were used to analyze proliferation, growth and apoptosis of ESCs, respectively. Results were highly reproducible in three independent experiments. Error bars depict the standard error of the mean. *P , 0.05, **P , 0.01 compared with the vehicle control. #P , 0.05, ##P , 0.01 compared with the CXCL8 treatment.

2112 and reduce apoptotic behavior of ESCs in an autocrine dependent manner involving the CXCR1 receptor.

CXCL8 down-regulates PTEN expression and activates phosphorylation of Akt

CXCL8 up-regulates survivin and Bcl-2 expression by activating the Akt signal pathway To further analyze the possible mechanism of CXCL8 on the survival of the eutopic ESCs, we used in-cell Western assay to measure the effect of CXCL8 on the survival-related molecules in ESCs, such as PCNA (Beliard et al., 2004), survivin (Ueda et al., 2002) and Bcl-2 (Meresman et al., 2000; Beliard et al., 2004). As shown in Fig. 4, CXCL8 notably increased the expression of survivin and Bcl-2 (P , 0.01; Fig. 4a and b), but did not change the expression of PCNA (P . 0.05; Fig. 4a and b). Furthermore, these effects were completely inhibited by the pI3-kinase/Akt inhibitor, LY294002 (P , 0.05 or P , 0.01; Fig. 4a and b). As expected, the phosphorylation of Akt was also inhibited by LY294002. Our results indicate that CXCL8 may up-regulate the expression of survivin and Bcl-2 in ESCs by activating the Akt signal pathway.

The effect of CXCL8 on proliferation, growth and apoptosis of ESCs is partly mediated by the Akt signal pathway In order to evaluate whether CXCL8 regulates survival of ESCs through the Akt signal pathway, after treatment with CXCL8 for 24 h, the eutopic ESCs from women with endometriosis were

Figure 3 CXCL8 down-regulates PTEN expression and activates the Akt signal pathway. Eutopic ESCs from women with endometriosis were treated with recombinant human CXCL8 and/or neutralizing antibody to CXCL8 (15 mg/ml) for 48 h, or with vehicle as controls, and then in-cell Western assays were performed to analyze the phosphorylation levels of Akt, p38, JNK and Erk1/2, and the expression of PTEN, respectively. Phospho-Akt, phospho-p38, phospho-JNK, phospho-Erk1/2 and actin (red); Akt, p38, JNK, Erk1/2 and PTEN (green). These pictures are representatives of three individual experiments. Error bars depict the standard error of the mean. *P , 0.05, **P , 0.01 compared with the vehicle control. ## P , 0.01 compared with the CXCL8 treatment.


PI3K/Akt and MAPK signaling pathways are involved in the modulation of cell survival and growth, and CXCL8/CXCR1 can inhibit apoptosis in human breast cancer cells via activation of the FAK/Akt signal pathway (Ginestier et al., 2010). Therefore, we investigated whether CXCL8 regulates the survival and growth of the eutopic ESCs via the PI3K/Akt and or MAPK signaling pathways. First, we detected the effect of CXCL8 on these signal pathways. As shown in Fig. 3, CXCL8 markedly stimulated the conversion of Akt to phosphor-Akt (P , 0.01; Fig. 3a and b) and down-regulated the expression of PTEN (P , 0.05; Fig. 3c), and these effects were abolished by antiCXCL8 neutralizing antibody (P , 0.05 or P , 0.01; Fig. 3a, b, c). However, CXCL8 or anti-CXCL8 neutralizing antibody did not influence the phosphorylation levels of Erk1/2, JNK and p38 MAP kinase compared with the vehicle control (P . 0.05). Therefore, there exists the possibility that CXCL8 modulates ESCs survival through inhibition of PTEN expression and by activating the Akt signal pathway.

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riosis were treated, respectively, with recombinant human CXCL8 for 24 h, these cells were treated with or without LY294002 for another 24 h, or with vehicle as controls, and then in-cell Western assays were conducted to analyze the Akt phosphorylation levels, and expression of PCNA, survivin and Bcl-2 (a and b). Phospho-Akt, PCNA, actin for the survivin and Bcl-2 group (red); Akt, survivin, Bcl-2, actin for the PCNA group (green). These pictures are representatives of three individual experiments. Error bars depict the standard error of the mean. *P , 0.05, **P , 0.01 compared with the vehicle control. #P , 0.05, ##P , 0.01 compared with the CXCL8 treatment.

subsequently treated with or without LY294002 for another 24 h. As shown, LY294002 not only directly inhibited proliferation, growth and increased apoptosis in the ESCs (P , 0.05 or P , 0.01; Fig. 5a left, 5a right, 5b), but also reversed the increase in proliferation and growth and decrease in apoptosis induced by CXCL8 (P , 0.05 or P , 0.01; Fig. 5a left, 5a right, 5b). Our observations suggest that the effects of CXCL8 on stimulating proliferation and growth and inhibiting apoptosis of ESCs might be mediated by the Akt signal pathway.

Discussion Despite more than a century of intensive study, the pathophysiology of endometriosis remains unclear. A large body of evidence suggests that immune system alterations play critical roles in the development and maintenance of this enigmatic disorder, in addition to hormonal,

genetic and environmental factors (Noble et al., 1996; Lebovic et al., 2001; Moutsatsou and Sekeris, 2003; Bischoff and Simpson, 2004; Dmowski and Braun, 2004; Louis et al., 2005). IL-8 (CXCL8) is one of the pro-inflammatory chemokines secreted by several cell types, such as trophoblasts and endometrial cells and especially immune cells (Yoshimura et al., 1987). Several studies have found that human eutopic endometrium of healthy women expresses IL-8 (Shi et al., 2006; Ulukus et al., 2009), which may be involved in physiological reproductive processes as pathological processes, such as endometriosis, through regulating the proliferation and invasion of ESCs (Arici et al., 1998; Ulukus et al., 2005). Analogous to other studies (Arici 2002; Ulukus et al., 2005), the present study has also demonstrated that the expression of IL-8 and its receptor, CXCR1, in the eutopic ESCs from women with endometriosis is higher than that of the normal ESCs from women without


Figure 4 CXCL8 up-regulates survivin and Bcl-2 expression by activating the Akt signal pathway. After eutopic ESCs from women with endomet-

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from women with endometriosis were treated, respectively, with recombinant human CXCL8 for 24 h, these cells were treated with or without LY294002 for another 24 h, or with vehicle as control. Thereafter, proliferation (a, left), cell number (a, right) and apoptosis (b) of ESCs were detected by BrdU proliferation assays, cell number counts or annexin V-FITC apoptosis assays, respectively. Results were highly reproducible in three independent experiments. Error bars depict the standard error of the mean. *P , 0.05, **P , 0.01 compared with the vehicle control. # P , 0.05, ##P , 0.01 compared with the CXCL8 treatment.

endometriosis, but the expression of CXCR2 shows no difference between these two groups. Subsequently, we have further found that recombinant human CXCL8 can enhance proliferation and growth and inhibit apoptosis of ESCs, and these effects can be inhibited by both CXCL8 and CXCR1 neutralizing antibodies. These findings indicate that the ESC-derived CXCL8 regulates the biological functions of ESCs at least partly through binding with the CXCR1 receptor. However, CXCR2 blocking has no this effect on ESCs. The underlying mechanism of this differential effect on ESC survival mediated by CXCR1 and CXCR2 is still unclear, and requires further research. Gebel et al. (1998) found for the first time that the pattern of apoptosis in the eutopic endometrium of women with endometriosis is

lower than that of the normal eutopic endometrium from healthy controls. Apoptosis of both endometrial stroma and glands in endometrium associated with endometriosis was lower compared with that in endometrium from women without endometriosis (Szymanowski, 2007). Taking into account our results, we hypothesize that in women with endometriosis an abnormally high level of CXCL8 secretion may reduce apoptosis in the eutopic and ectopic endometrial tissue, which favors ectopic growth, survival and invasion. Interestingly, we have further demonstrated in the present work that CXCL8 regulates the expression of survival-relative molecules, survivin and Bcl-2, inhibits the expression of PTEN and modulates the proliferation, growth and apoptosis of ESCs by activating the Akt signal pathway, but not by influencing the MAPK signal pathway.


Figure 5 The effect of CXCL8 on proliferation, growth and apoptosis of ESCs is partly mediated by the Akt signal pathway. After the eutopic ESCs

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Authors’ roles M.Q.L. and X.Z.L. conducted all experiments and prepared the figures and the manuscript. Y.H.M and J.M. assisted with in-cell Western analyses. X.Y.Z. examined patients, obtained specimens and generated clinical data. L.P.J. critically reviewed and corrected the manuscript. D.J.L. initiated and supervised the project and edited the manuscript.

Funding This study was supported by Major International Joint Research Project of National Natural Science Foundation of China (NSFC) (30910103909) to D.-J.L.; National and Shanghai Leading Academic Discipline Project (211XK22) to D.-J.L.; Program for Outstanding Medical Academic Leader of Shanghai to D.-J.L.; NSFC 31101064 to M.-Q.L.; Ministry of Education Research Fund for Doctoral Program to M.-Q.L. (20110071120092); Research Program of Shanghai Health Bureau to M-Q.L. (2011Y081) and X-Z.L. (2010Y015);

Youth Scientific Research Foundation of Shanghai Medical College to X-Z.L. and Talented Person Development Foundation of Shanghai No. 2009008 to X-Y.Z.

Conflict of interest None declared.

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Survivin is an inhibitor of apoptosis expressed during fetal development and in cancer tissues. Several studies have demonstrated that survivin and Bcl-2 play an important role in physiological homeostasis during the normal menstrual cycle, and up-regulation of survivin and Bcl-2 may contribute to survival and or invasion of ESCs in the progression of endometriosis (Konno et al., 2000; Ueda et al., 2002). Therefore, our current results led us to propose that an increase in survivin and Bcl-2 expression, via the activation of the Akt signal pathway, mediated by IL-8, may enhance the survival of ESCs. Ginestier et al. (2010) established that the effects of CXCR1 blockade on viability of breast cancer stem cells (CSCs) and FASL production were mediated by the FAK/Akt/FOXO3a pathway . In addition, as a small-molecule CXCR1 inhibitor, selectively depleted the CSC population in human breast cancer cell lines in vitro, repertaxin was able to specifically target the CSC population in human breast cancer xenografts, retarding tumor growth and metastasis. These data suggest that CXCR1 blockage may provide a novel means of targeting and eliminating breast CSCs. Endometriosis exhibits a great degree of variability not only in the symptomatic presentation, but also in terms of age of onset, progression of the disease, response to treatment and recurrence. Our present study shows that the chemokine CXCL8 may be a stimulatory factor to the survival of ESCs in the pathogenesis of endometriosis, and a CXCR1 blocker can effectively impair these roles. Therefore, CXCR1 blockage is potential for therapeutic treatment of endometriosis. In conclusion, our findings suggest that the increase of CXCL8 and CXCR1 expression in the eutopic ESCs of women with endometriosis may promote the expression of survivin and Bcl-2, and enhance ESC survival via regulating PTEN/Akt signal pathways. Our previous work has established that estrogen and TCDD can stimulate secretion of IL-8 in HPMC-U937 co-culture or ESC-HPMC co-culture, and up-regulate the expression of CXCR1 in ESCs (Shi et al., 2006). These integral effects will promote survival of the retrograde endometrial cells in the peritoneal cavity in autocrine and paracrine manners, and further give rise to the formation of endometriotic lesions. Therefore, interference with cytokine regulatory loops such as CXCL8 and CXCR1 may represent a novel therapeutic strategy to reduce the growth of endometriosis.

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cell invasiveness in the endometriotic milieu. J Mol Endocrinol 2011; 47:195 – 208. Louis GMB, Weiner JM, Whitcomb BW, Sperrazza R, Schisterman EF, Lobdell DT, Crickard K, Greizerstein H, Kostyniak PJ. Environmental PBC exposure and risk of endometriosis. Hum Reprod 2005;20:279–285. Matsushima K, Morishita K, Yoshimura T, Lavu S, Kobayshi Y, Lew W, Appella E, Kung HF, Leonard EJ, Oppenheim JJ. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med 1988;167:1883 – 1893. Meresman GF, Vighi S, Buquet RA, Contreras-Ortiz O, Tesone M, Rumi LS. Apoptosis and expression of Bcl-2 and Bax in eutopic endometriumfrom women with endometriosis. Fertil Steril 2000;74:760–766. Moutsatsou P, Sekeris CE. Steroid receptors in the uterus: implications in endometriosis. Ann N Y Acad Sci 2003;997:209 – 222. Nisolle M, Casanas-Roux F, Donnez J. Early-stage endometriosis: adhesion and growth of human menstrual endometrium in nude mice. Fertil Steril 2000;74:306 – 312. Noble LS, Simpson ER, Johns A, Bulun SE. Aromatase expression in endometriosis. J Clin Endocrinol Metabol 1996;81:174 – 179. Schadendorf D, Moller A, Algermissen B, Worm M, Sticherling M, Czarnetzki BM. IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor. J Immunol 1993; 151:2667– 2675. Shi YL, Lou XZ, Zhu XY, Hua KQ, Zhu Y, Li DJ. Effects of combined 17b-estradiol with TCDD on secretion of chemokine IL-8 and

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