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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 an endometriosis-targeting peptide by subtractive phage library screening.(a) Illustration of library screening steps. A peptide displaying phage library was selected by subtraction using female mouse peritoneum followed by adhesion to and internalization by Ishikawa cells. (b) Binding efficiency of phage pools obtained after each screening round assessed by plaque-forming assay. (c) Binding of cloned phage to Ishikawa (purple bars) and control A431 (red bars) cells. (d) Z13 phage binding to endometrial cell lines (blue bars) and control non-endometrial cell lines (pink bars). In c,d, each cloned phage was added to a monolayer of each cell line at 37 °C for 30 min. Internalized phage was counted by a plaque-forming assay. Experiments shown in panels b–d were repeated three times. Each error bar represents s.d.
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f1: Identification of an endometriosis-targeting peptide by subtractive phage library screening.(a) Illustration of library screening steps. A peptide displaying phage library was selected by subtraction using female mouse peritoneum followed by adhesion to and internalization by Ishikawa cells. (b) Binding efficiency of phage pools obtained after each screening round assessed by plaque-forming assay. (c) Binding of cloned phage to Ishikawa (purple bars) and control A431 (red bars) cells. (d) Z13 phage binding to endometrial cell lines (blue bars) and control non-endometrial cell lines (pink bars). In c,d, each cloned phage was added to a monolayer of each cell line at 37 °C for 30 min. Internalized phage was counted by a plaque-forming assay. Experiments shown in panels b–d were repeated three times. Each error bar represents s.d.

Mentions: As yet, there is neither an in vitro model nor an in vivo animal model available to study endometriosis, other than non-human primates. To identify a peptide that specifically binds to epithelial cells in endometriosis, we hypothesized that some, if not all, human endometrial adenocarcinoma cell lines would express cell surface proteins expressed in glandular epithelial cells in endometriosis. To devise a probe that specifically binds to the endometriosis surface but not to the surface of other organs facing the peritoneum, we undertook subtractive phage library screening1819. A T7 phage-based library (109 clones, 1011 plaque-forming unit) of linear 9-mer peptides was injected into the peritoneal cavity of a female mouse to allow absorption of phage clones to the peritoneal surface in vivo for 1 h. The precleared library was recovered from peritoneal fluid and added to a monolayer of human endometrial adenocarcinoma Ishikawa cells cultured in vitro. We choose Ishikawa cells as this cell line shares characteristics with mature endometrial epithelial cells2021. We also wanted to identify a peptide internalized by endometrial glandular epithelial cells so that a drug conjugated with that peptide would penetrate target cells. We therefore incubated phage with live Ishikawa cells at 37 °C for 30 min to facilitate phage internalization. Selected phage clones were recovered after solubilizing cells with detergent and amplified in bacteria (Fig. 1a). After three rounds of subtractive library screening, the number of phage clones with Ishikawa cell-binding activity relative to the total number of added phage increased 10,000-fold (Fig. 1b).


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 an endometriosis-targeting peptide by subtractive phage library screening.(a) Illustration of library screening steps. A peptide displaying phage library was selected by subtraction using female mouse peritoneum followed by adhesion to and internalization by Ishikawa cells. (b) Binding efficiency of phage pools obtained after each screening round assessed by plaque-forming assay. (c) Binding of cloned phage to Ishikawa (purple bars) and control A431 (red bars) cells. (d) Z13 phage binding to endometrial cell lines (blue bars) and control non-endometrial cell lines (pink bars). In c,d, each cloned phage was added to a monolayer of each cell line at 37 °C for 30 min. Internalized phage was counted by a plaque-forming assay. Experiments shown in panels b–d were repeated three times. Each error bar represents s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109024&req=5

f1: Identification of an endometriosis-targeting peptide by subtractive phage library screening.(a) Illustration of library screening steps. A peptide displaying phage library was selected by subtraction using female mouse peritoneum followed by adhesion to and internalization by Ishikawa cells. (b) Binding efficiency of phage pools obtained after each screening round assessed by plaque-forming assay. (c) Binding of cloned phage to Ishikawa (purple bars) and control A431 (red bars) cells. (d) Z13 phage binding to endometrial cell lines (blue bars) and control non-endometrial cell lines (pink bars). In c,d, each cloned phage was added to a monolayer of each cell line at 37 °C for 30 min. Internalized phage was counted by a plaque-forming assay. Experiments shown in panels b–d were repeated three times. Each error bar represents s.d.
Mentions: As yet, there is neither an in vitro model nor an in vivo animal model available to study endometriosis, other than non-human primates. To identify a peptide that specifically binds to epithelial cells in endometriosis, we hypothesized that some, if not all, human endometrial adenocarcinoma cell lines would express cell surface proteins expressed in glandular epithelial cells in endometriosis. To devise a probe that specifically binds to the endometriosis surface but not to the surface of other organs facing the peritoneum, we undertook subtractive phage library screening1819. A T7 phage-based library (109 clones, 1011 plaque-forming unit) of linear 9-mer peptides was injected into the peritoneal cavity of a female mouse to allow absorption of phage clones to the peritoneal surface in vivo for 1 h. The precleared library was recovered from peritoneal fluid and added to a monolayer of human endometrial adenocarcinoma Ishikawa cells cultured in vitro. We choose Ishikawa cells as this cell line shares characteristics with mature endometrial epithelial cells2021. We also wanted to identify a peptide internalized by endometrial glandular epithelial cells so that a drug conjugated with that peptide would penetrate target cells. We therefore incubated phage with live Ishikawa cells at 37 °C for 30 min to facilitate phage internalization. Selected phage clones were recovered after solubilizing cells with detergent and amplified in bacteria (Fig. 1a). After three rounds of subtractive library screening, the number of phage clones with Ishikawa cell-binding activity relative to the total number of added phage increased 10,000-fold (Fig. 1b).

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