. 2014 Jul 22:5:4478.
doi: 10.1038/ncomms5478.
Y Kobayashi 2 , A Suzuki 3 , N Tamura 2 , K Motamedchaboki 2 , C-T Huang 2 , T O Akama 2 , J Pecotte 4 , P Frost 4 , C Bauer 4 , J B Jimenez Jr 4 , J Nakayama 5 , D Aoki 3 , M N Fukuda 2
Affiliations
PMID: 25047118 PMCID: PMC4109024 DOI: 10.1038/ncomms5478
Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosis
K Sugihara et al. Nat Commun. 2014.
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.
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Conflict of interest statement
K.S. and M.N.F. are inventors of provisional US patent filed. The remaining authors declare no competing financial interests.
Figures Figure 1. 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. Figure 2. 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. Figure 3. Immunohistochemistry of paraffin tissue sections using mouse monoclonal anti-CNGB3 antibody.
(a) Endometriosis lesions from an endometriosis patient. Scale bar, 500 μm. (b) An endometrial gland in endometriosis tissue. Scale bar, 200 μm. (c) Peritoneal tissue from a cycling woman without endometriosis. Scale bar, 100 μm. (d) Peritoneal tissue from an endometriosis patient. Scale bar, 100 μm. (e) Uterine endometrium at secretory phase. Scale bar, 200 μm. (f) Uterine endometrium at proliferative phase. Scale bar, 200 μm. Figure 4. Design and activity of endosome-escaping peptide.
(a) Helical wheels of alpha helix (left), and alignment of KLAKLAKKLAKLAKKLAK (center) and HLAHLAHHLAHLAHHLAH (right). Note that polar or hydrophilic amino acids are concentrated on one side of helix and hydrophobic amino acids on the other. (b) Endosome-escaping activity of HLAH-z13. Biotin-z13 was bound to CNGB3–MYC-expressing HEK293T cells at 37 °C for 30 min with (b) or without (a) HLAH-z13. Cells were stained by Alexa 488-conjugated avidin (green) followed by 4',6-diamidino-2-phenylindole (blue) nuclear staining. Scale bar, 50 μm. Figure 5. Z13-targeted apoptosis of CNGB3-expressing cells in vitro.
(a) Cell viability assays of CNGB3-positive A431 cells (left) and control CNGB3-negative A431 cells (right) after incubating cells with z13, dKLAK-z13, HLAH-z13 or dKLAK-z13 plus HLAH-z13. Cells were cultured in a medium containing each peptide at the indicated concentrations at 37 °C for 6 h. ATP levels were measured by CellTiter Glo (Promega). dimethylsulphoxide alone at 37 °C for 6 h at a final concentration of 0.01% had no effect by this assay (data not shown). (b) Number of living CNGB3-positive and -negative A431 cells following treatment with the indicated reagents for 20 h. (c) Inhibition of the activity by KLA-z13 plus HLA-z13 by palmitoyl-z13 peptide. CNGB3-expressing A431 cells were treated with C16-z13 alone (blue) or with a mixture of KLAK-z13 (100 μg ml−1) and HLA-z13 (25 μg ml−1; red) at 37 °C for 6 h. Each data point represents average of triplicate measurements. This analysis was repeated three times. (d) TUNEL assay of CNGB3–MYC-expressing HEK293T cells cultured in a medium containing z13 peptide with or without an apoptosis-inducing peptide and/or endosome-escaping peptide. Cells were cultured at 37 °C for 20 h in a medium containing: no peptide (a); z13, 100 μg ml−1 (b); dKLAK, 100 μg ml−1 (c); dKLAK-z13, 100 μg ml−1 (d); HLAH-z13, 25 μg ml−1 (e); and dKLAK-z13, 100 μg ml−1, plus HLAH-z13, 25 μg ml−1 (f). Scale bar, 100 μm. (e) TUNEL assay of CNGB3–MYC-transfected HEK293T cells treated with a mixture of dKLAK-z13 (100 μg ml−1) and HLAH-z13 (25 μg ml−1) for the periods indicated. Scale bar, 100 μm. Figure 6. Induction of apoptosis in baboon endometriosis tissue in vivo.
(a) Quantitative TUNEL analysis. Shown is the percentage of TUNEL-positive endometrial glands in tissues from three control untreated baboons compared with three animals treated with a mixture of dKLAK-z13 and HLAH-z13 peptides (see Methods). The number of glands counted and the number of TUNEL-positive glands (indicated in parentheses) were 5 (0), 8 (0) and 18 (0) for untreated and 8 (1), 18 (2) and 26 (3) for treated. Percentages of TUNEL-positive glands from untreated and treated animals were 0.00%±0.00 (n=3) and 13.53%±1.56 (n=3), respectively. (b) TUNEL assay of baboon endometriosis. Upper panels: hematoxylin and eosin-stained images. Lower panels: TUNEL assay of corresponding areas. Left: an endometrial gland in the ovary. Scale bar, 100 μm. Right: TUNEL-positive cell debris in an endometrial gland in the omentum. Scale bar, 200 μm.
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