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Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosis.

Sugihara K, Kobayashi Y, Suzuki A, Tamura N, Motamedchaboki K, Huang CT, Akama TO, Pecotte J, Frost P, Bauer C, Jimenez JB, Nakayama J, Aoki D, Fukuda MN - Nat Commun (2014)

Bottom Line: Endometriosis is a common gynaecological disease associated with pelvic pain and infertility.Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display.When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan.

ABSTRACT
Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with nonsteroidal anti-inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display. As most endometriosis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the z13 receptor as the cyclic nucleotide-gated channel β3, a sorting pathway protein. We then linked z13 with an apoptosis-inducing peptide and with an endosome-escaping peptide. When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal endometriosis in humans.

No MeSH data available.


Related in: MedlinePlus

Identification of z13 peptide receptor.(a) Fluorescence micrographs of Ishikawa cells (left) and control A431 cells (right) overlayed with a synthetic z13 peptide tagged with fluorescein isothiocyanate (FITC) and left at 37 °C for 15 min. Scale bar, 50 μm. (b) Visualization and isolation of the z13 peptide receptor. Left: cell surface proteins expressed on Ishikawa cells were biotinylated. Cell lysates were bound to z13 peptide-conjugated agarose beads, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Biotinylated proteins in each eluate were detected by peroxidase-conjugated avidin and a luminescent peroxidase substrate. Right: silver staining of peptide-affinity-purified z13 receptor from endometriosis. Endometriosis tissues isolated from patients were homogenized, and microsome membrane fraction was prepared. Proteins solubilized with detergent were applied to a z13 peptide-conjugated agarose column, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Proteins in each eluate in SDS–PAGE were detected by silver staining. (c) Fluorescence micrographs of HeLa cells transfected with control empty vector (upper row) or with an expression vector encoding CNGB3–MYC (lower row). Binding of FITC-z13 peptide (green) to HeLa cells transfected with mammalian expression vectors (a,d), immunostained with anti-MYC followed by Alexa 549 (red)-conjugated anti-mouse IgG antibody (b,e) and merged images including 4',6-diamidino-2-phenylindole (blue) to indicate nuclear staining (c,f). Scale bar, 20 μm.
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f2: Identification of z13 peptide receptor.(a) Fluorescence micrographs of Ishikawa cells (left) and control A431 cells (right) overlayed with a synthetic z13 peptide tagged with fluorescein isothiocyanate (FITC) and left at 37 °C for 15 min. Scale bar, 50 μm. (b) Visualization and isolation of the z13 peptide receptor. Left: cell surface proteins expressed on Ishikawa cells were biotinylated. Cell lysates were bound to z13 peptide-conjugated agarose beads, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Biotinylated proteins in each eluate were detected by peroxidase-conjugated avidin and a luminescent peroxidase substrate. Right: silver staining of peptide-affinity-purified z13 receptor from endometriosis. Endometriosis tissues isolated from patients were homogenized, and microsome membrane fraction was prepared. Proteins solubilized with detergent were applied to a z13 peptide-conjugated agarose column, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Proteins in each eluate in SDS–PAGE were detected by silver staining. (c) Fluorescence micrographs of HeLa cells transfected with control empty vector (upper row) or with an expression vector encoding CNGB3–MYC (lower row). Binding of FITC-z13 peptide (green) to HeLa cells transfected with mammalian expression vectors (a,d), immunostained with anti-MYC followed by Alexa 549 (red)-conjugated anti-mouse IgG antibody (b,e) and merged images including 4',6-diamidino-2-phenylindole (blue) to indicate nuclear staining (c,f). Scale bar, 20 μm.

Mentions: To confirm z13 peptide-binding activity, we chemically synthesized z13 peptide with an amino-terminal fluorescein isothiocyanate (FITC) tag and added to Ishikawa cells and control A431 cells (Fig. 2a). Fluorescence micrographs showed binding of FITC-z13 to Ishikawa cells, but not to A431 cells. Micrograph of Ishikawa cells showed a punctate cytoplasmic staining pattern, suggesting that z13 is internalized to endosomes.


Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosis.

Sugihara K, Kobayashi Y, Suzuki A, Tamura N, Motamedchaboki K, Huang CT, Akama TO, Pecotte J, Frost P, Bauer C, Jimenez JB, Nakayama J, Aoki D, Fukuda MN - Nat Commun (2014)

Identification of z13 peptide receptor.(a) Fluorescence micrographs of Ishikawa cells (left) and control A431 cells (right) overlayed with a synthetic z13 peptide tagged with fluorescein isothiocyanate (FITC) and left at 37 °C for 15 min. Scale bar, 50 μm. (b) Visualization and isolation of the z13 peptide receptor. Left: cell surface proteins expressed on Ishikawa cells were biotinylated. Cell lysates were bound to z13 peptide-conjugated agarose beads, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Biotinylated proteins in each eluate were detected by peroxidase-conjugated avidin and a luminescent peroxidase substrate. Right: silver staining of peptide-affinity-purified z13 receptor from endometriosis. Endometriosis tissues isolated from patients were homogenized, and microsome membrane fraction was prepared. Proteins solubilized with detergent were applied to a z13 peptide-conjugated agarose column, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Proteins in each eluate in SDS–PAGE were detected by silver staining. (c) Fluorescence micrographs of HeLa cells transfected with control empty vector (upper row) or with an expression vector encoding CNGB3–MYC (lower row). Binding of FITC-z13 peptide (green) to HeLa cells transfected with mammalian expression vectors (a,d), immunostained with anti-MYC followed by Alexa 549 (red)-conjugated anti-mouse IgG antibody (b,e) and merged images including 4',6-diamidino-2-phenylindole (blue) to indicate nuclear staining (c,f). Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Identification of z13 peptide receptor.(a) Fluorescence micrographs of Ishikawa cells (left) and control A431 cells (right) overlayed with a synthetic z13 peptide tagged with fluorescein isothiocyanate (FITC) and left at 37 °C for 15 min. Scale bar, 50 μm. (b) Visualization and isolation of the z13 peptide receptor. Left: cell surface proteins expressed on Ishikawa cells were biotinylated. Cell lysates were bound to z13 peptide-conjugated agarose beads, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Biotinylated proteins in each eluate were detected by peroxidase-conjugated avidin and a luminescent peroxidase substrate. Right: silver staining of peptide-affinity-purified z13 receptor from endometriosis. Endometriosis tissues isolated from patients were homogenized, and microsome membrane fraction was prepared. Proteins solubilized with detergent were applied to a z13 peptide-conjugated agarose column, and bound proteins were eluted with irrelevant peptide (lane 1) or z13 peptide (lane 2). Proteins in each eluate in SDS–PAGE were detected by silver staining. (c) Fluorescence micrographs of HeLa cells transfected with control empty vector (upper row) or with an expression vector encoding CNGB3–MYC (lower row). Binding of FITC-z13 peptide (green) to HeLa cells transfected with mammalian expression vectors (a,d), immunostained with anti-MYC followed by Alexa 549 (red)-conjugated anti-mouse IgG antibody (b,e) and merged images including 4',6-diamidino-2-phenylindole (blue) to indicate nuclear staining (c,f). Scale bar, 20 μm.
Mentions: To confirm z13 peptide-binding activity, we chemically synthesized z13 peptide with an amino-terminal fluorescein isothiocyanate (FITC) tag and added to Ishikawa cells and control A431 cells (Fig. 2a). Fluorescence micrographs showed binding of FITC-z13 to Ishikawa cells, but not to A431 cells. Micrograph of Ishikawa cells showed a punctate cytoplasmic staining pattern, suggesting that z13 is internalized to endosomes.

Bottom Line: Endometriosis is a common gynaecological disease associated with pelvic pain and infertility.Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display.When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan.

ABSTRACT
Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with nonsteroidal anti-inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display. As most endometriosis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the z13 receptor as the cyclic nucleotide-gated channel β3, a sorting pathway protein. We then linked z13 with an apoptosis-inducing peptide and with an endosome-escaping peptide. When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal endometriosis in humans.

No MeSH data available.


Related in: MedlinePlus