Alteration in endometrial proteins during early- and mid-secretory phases of the cycle in women with unexplained infertility.

BACKGROUND: Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility. METHODS: 2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women. RESULTS: Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women. CONCLUSIONS: De-regulation of the expression of Sorcin, Cofilin-1, Apo-A1 and Ran, during early- to mid-secretory phase may have physiological significance and it may be one of the causes for altered differentiation and/or maturation of endometrium, in women with unexplained infertility

Immunohistochemical localization of Sorcin, Cofilin-1, Apolipoprotein-A1 (Apo-A1) and Ran GTP-binding nuclear protein (Ran) in infertile women.

<p>Representative images (upper panel) showing immunohistochemical localization of of Sorcin, Cofilin-1, Apo-A1 and Ran in early-secretory (LH+2) and mid-secretory (LH+7) phase endometrium from women with unexplained infertility. Lower panel shows image analysis of Sorcin, Cofilin-1, Apo-A1 and Ran in early-secretory and mid-secretory phase endometrium of infertile women. Staining intensity of all these proteins were quantified by image analysis software Image-Pro Plus 4.0 (Maryland, USA). Anti-mouse/anti-goat IgG was used as negative control and shown in inset (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111687#pone-0111687-g004" target="_blank">Fig. 4 A, E, I, M</a>). Magnification X 400, bar = 25 µm, p-values are (a) p<0.001, (b) p<0.01 and (c) p<0.05 versus early-secretory phase (LH+2). Values are expressed as mean ± SE. n = 4 in all the groups. S = stroma, GE = Glandular epithelium, LE = Luminal epithelium.</p

Validation of Sorcin, Cofilin-1, Apolipoprotein-A1 (Apo-A1) and Ran GTP-binding nuclear protein (Ran) in infertile women.

<p>Expression of Sorcin, Cofilin-1, Apo-A1 and Ran were analyzed by western blot analysis in early-secretory (LH+2) and mid-secretory phase (LH+7) endometrium of infertile women, which had not been previously analyzed in the 2D-PAGE analysis. The results matched the findings of 2D-PAGE analysis. Representative images (upper panel) of immunoblot of Sorcin, Cofilin-1, Apo-A1 and Ran have been shown. β-actin served as a loading control for normalization. Quantification of band intensity (lower panel) was performed by densitometric analysis by using Quantity One software (v. 4.5.1) and a Gel Doc imaging system (Bio-Rad). P values are (b) p<0.01 and (c) p<0.05 versus early-secretory phase (LH+2). Values are expressed as mean± SE; n = 4. ES = early-secretory phase, MS = mid-secretory phase, LH+2 = 2 days after luteinizing hormone surge, LH+7 = 7 days after luteinizing hormone surge.</p

Various differentially expressed proteins.

<p>Bar diagram showing 9 differentially expressed proteins between early-secretory (LH+2) and mid-secretory (LH+7) phase endometrium from women with unexplained infertility. Values were expressed as mean ± SE, n = 8. P values are (b) p<0.01 and (c) p<0.05 versus early-secretory phase (LH+2). ES = early-secretory phase, MS = mid-secretory phase, LH+2 = 2 days after luteinizing hormone surge, LH+7 = 7 days after luteinizing hormone surge.</p

2D-PAGE of early-secretory (LH+2) and mid-secretory (LH+7) phase endometrium of women with unexplained infertility.

<p>Representative gel images are shown here. The first dimension was performed by IEF on IPG strips over a range of pI 3–10, the second dimension on 12.5% SDS-PAGE gels and the proteins were visualized by silver staining. 9 differentially altered protein spots were identified by image master 2D platinum software and number denotes spot ID (0–23). LH+2 = 2 days after luteinizing hormone surge, LH+7 = 7 days after luteinizing hormone surge.</p

Details of differentially expressed proteins identified by LC-MS analysis and comparison thereof with previous reports.

<p>Alteration in endometrial proteins during early- and mid-secretory phases of the cycle in women with unexplained infertility.</p><p>Details of differentially expressed proteins identified by LC-MS analysis and comparison thereof with previous reports.</p

Immunoblot analysis of Sorcin, Cofilin-1, Apolipoprotein-A1 (Apo-A1) and Ran GTP-binding nuclear protein (Ran) in fertile women.

<p>Expression of Sorcin, Cofilin-1, Apo-A1 and Ran were checked in early-secretory (LH+2) and mid-secretory (LH+7) phase endometrium of fertile women. Representative images (upper panel) of immunoblot of Sorcin, Cofilin-1, Apo-A1 and Ran have been shown. β-actin served as a loading control for normalization. Quantification of band intensity (lower panel) was performed by densitometric analysis by using Quantity One software (v. 4.5.1) and a Gel Doc imaging system (Bio-Rad). p-values are (b) p<0.01, (c) p<0.05 and (d) p>0.05 versus early-secretory phase (LH+2). Values are expressed as mean± SE; n = 4, ES = early-secretory phase, MS = mid-secretory phase, LH+2 = 2 days after luteinizing hormone surge, LH+7 = 7 days after luteinizing hormone surge.</p

Chemotherapeutic Potential of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2H-Benzo(b)pyran in Estrogen Receptor- Negative Breast Cancer Cells: Action via Prevention of EGFR Activation and Combined Inhibition of PI-3-K/Akt/FOXO and MEK/Erk/AP-1 Pathways

<div><p>Inhibition of epidermal growth factor receptor (EGFR) signaling is considered to be a promising treatment strategy for estrogen receptor (ER)-negative breast tumors. We have investigated here the anti-breast cancer properties of a novel anti-proliferative benzopyran compound namely, 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) in ER- negative and EGFR- overexpressing breast cancer cells. The benzopyran compound selectively inhibited the EGF-induced growth of MDA-MB 231 cells and ER-negative primary breast cancer cell culture. The compound significantly reduced tumor growth in xenograft of MDA-MB 231 cells in nude mice. The compound displayed better binding affinity for EGFR than inhibitor AG1478 as demonstrated by molecular docking studies. CDRI-85/287 significantly inhibited the activation of EGFR and downstream effectors MEK/Erk and PI-3-K/Akt. Subsequent inhibition of AP-1 promoter activity resulted in decreased transcription activation and expression of c-fos and c-jun. Dephosphorylation of downstream effectors FOXO-3a and NF-κB led to increased expression of p27 and decreased expression of cyclin D1 which was responsible for decreased phosphorylation of Rb and prevented the transcription of E2F- dependent genes involved in cell cycle progression from G1/S phase. The compound induced apoptosis via mitochondrial pathway and it also inhibited EGF-induced invasion of MDA-MB 231 cells as evidenced by decreased activity of MMP-9 and expression of CTGF. These results indicate that benzopyran compound CDRI-85/287 could constitute a powerful new chemotherapeutic agent against ER-negative and EGFR over-expressing breast tumors.</p></div