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Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library.

Shi LF, Wu Y, Li CY - J Gynecol Oncol (2015)

Bottom Line: Sequencing and translation identified three different peptides.In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed.These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, No.117 Center Military Hospital, Hangzhou, China. lifeng_shi@163.com.

ABSTRACT

Objective: Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy.

Methods: The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides.

Results: After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with K(aff) (affinity constant) of 16.4±8.6 μg/mL (n=3), 9.2±2.1 μg/mL (n=3), and 174.8±31.1 μg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed.

Conclusion: These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.

No MeSH data available.


Related in: MedlinePlus

Localization of peptide III to ovarian tumors in vivo. (A) Technetium-99 (99Tc)-labeled peptide (phage-WHWLPNLRHYAS) interacted with the ovarian tumor in vivo (arrow). (B) Free probe (99Tc) accumulated in the liver, kidney, and bladder, but not at the tumor (arrow).
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Figure 6: Localization of peptide III to ovarian tumors in vivo. (A) Technetium-99 (99Tc)-labeled peptide (phage-WHWLPNLRHYAS) interacted with the ovarian tumor in vivo (arrow). (B) Free probe (99Tc) accumulated in the liver, kidney, and bladder, but not at the tumor (arrow).

Mentions: After an in vivo ovarian tumor model was established, tumor-bearing mice received a tail vein injection of either 99mTc-labeled peptide or 9mTcO4 as a control. As shown in Fig. 6, 99Tc labeled peptide was observed ovarian tumor (Fig. 6A); however, no such labeling was observed in mice injected with the free probe, which accumulated in the liver, kidney, and bladder (Fig. 6B).


Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library.

Shi LF, Wu Y, Li CY - J Gynecol Oncol (2015)

Localization of peptide III to ovarian tumors in vivo. (A) Technetium-99 (99Tc)-labeled peptide (phage-WHWLPNLRHYAS) interacted with the ovarian tumor in vivo (arrow). (B) Free probe (99Tc) accumulated in the liver, kidney, and bladder, but not at the tumor (arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4620370&req=5

Figure 6: Localization of peptide III to ovarian tumors in vivo. (A) Technetium-99 (99Tc)-labeled peptide (phage-WHWLPNLRHYAS) interacted with the ovarian tumor in vivo (arrow). (B) Free probe (99Tc) accumulated in the liver, kidney, and bladder, but not at the tumor (arrow).
Mentions: After an in vivo ovarian tumor model was established, tumor-bearing mice received a tail vein injection of either 99mTc-labeled peptide or 9mTcO4 as a control. As shown in Fig. 6, 99Tc labeled peptide was observed ovarian tumor (Fig. 6A); however, no such labeling was observed in mice injected with the free probe, which accumulated in the liver, kidney, and bladder (Fig. 6B).

Bottom Line: Sequencing and translation identified three different peptides.In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed.These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, No.117 Center Military Hospital, Hangzhou, China. lifeng_shi@163.com.

ABSTRACT

Objective: Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy.

Methods: The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides.

Results: After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with K(aff) (affinity constant) of 16.4±8.6 μg/mL (n=3), 9.2±2.1 μg/mL (n=3), and 174.8±31.1 μg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed.

Conclusion: These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.

No MeSH data available.


Related in: MedlinePlus