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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

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.
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f5: 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.

Mentions: We next assessed cytotoxic activity of dKLAK-z13 in the presence of HLAH-z13 using a cell viability assay (Fig. 5a,b). When CNGB3–MYC-transfected A431 cells were cultured in a medium containing z13, dKLAK-z13 or HLAH-z13 at 37 °C for 6 h, cell viability was not affected, except at high concentrations of dKLAK-z13 (>200 μg ml−1) or HLAH-z13 (>50 μg ml−1). By contrast, a mixture of dKLAK-z13 and HLAH-z13 each at a low concentration showed clear cytotoxicity, but had no toxic effect on control CNGB3-negative A431 cells. Cytotoxicity of KLAK-z13 plus HLA-z13 was blocked by excess z13 (Fig. 5c).


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)

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.
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Related In: Results  -  Collection

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f5: 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.
Mentions: We next assessed cytotoxic activity of dKLAK-z13 in the presence of HLAH-z13 using a cell viability assay (Fig. 5a,b). When CNGB3–MYC-transfected A431 cells were cultured in a medium containing z13, dKLAK-z13 or HLAH-z13 at 37 °C for 6 h, cell viability was not affected, except at high concentrations of dKLAK-z13 (>200 μg ml−1) or HLAH-z13 (>50 μg ml−1). By contrast, a mixture of dKLAK-z13 and HLAH-z13 each at a low concentration showed clear cytotoxicity, but had no toxic effect on control CNGB3-negative A431 cells. Cytotoxicity of KLAK-z13 plus HLA-z13 was blocked by excess z13 (Fig. 5c).

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