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Identification of the sAPRIL binding peptide and its growth inhibition effects in the colorectal cancer cells.

He XQ, Guan J, Liu F, Li J, He MR - PLoS ONE (2015)

Bottom Line: The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively.The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner.In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Institute of Digestive Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China; Oncology Department, Wuzhou Red Cross Hospital, Wuzhou 543002, Guangxi Province, China.

ABSTRACT

Background: A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer.

Methods: A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP.

Results: Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells.

Conclusions: sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL.

No MeSH data available.


Related in: MedlinePlus

Selection of sAPRIL-BP.(A) The binding affinity of phage clones No.1–20 for sAPRIL were determined by ELISA. Clone 21 was used as a positive control. The fold change of the optical density was normalized to the positive control. Clones that had at least a 6-fold greater affinity than the positive control were considered ‘positive’ for sAPRIL binding. (B) Three binding peptides were synthesized and their binding affinity with sAPRIL (black bars) was determined and compared with the negative control (NC) using ELISA. Cross-reactivity was assessed by measuring the binding affinity to BAFF (grey bars). (C) Clone BP1 (sAPRIL-BP) was mixed with sAPRIL at different doses to compete for binding with fixed LOVO cells.
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pone.0120564.g001: Selection of sAPRIL-BP.(A) The binding affinity of phage clones No.1–20 for sAPRIL were determined by ELISA. Clone 21 was used as a positive control. The fold change of the optical density was normalized to the positive control. Clones that had at least a 6-fold greater affinity than the positive control were considered ‘positive’ for sAPRIL binding. (B) Three binding peptides were synthesized and their binding affinity with sAPRIL (black bars) was determined and compared with the negative control (NC) using ELISA. Cross-reactivity was assessed by measuring the binding affinity to BAFF (grey bars). (C) Clone BP1 (sAPRIL-BP) was mixed with sAPRIL at different doses to compete for binding with fixed LOVO cells.

Mentions: A phage library was used to identify 20 single clones expressing potential sAPRIL binding peptides. The clones were randomly selected after four rounds of panning. Eight single clones were identified as ‘positive’ indicating a high binding affinity (defined as an OD ≥6-fold the OD of the positive control) for sAPRIL by ELISA (Fig 1A). The eight clones represented three DNA sequences that correspond to the following binding polypeptides (BP): AAAPLAQPHMWA, SSTTTSDKYLSA, and SNLHDNNTEKNV (Table 1). The affinity for sAPRIL was measured for each polypeptide by ELISA and compared to an unrelated polypeptide (HWDPFSLSAYFP) as a negative control. The three peptides identified using the phage library had sAPRIL binding affinities that were 13.7, 10.8, and 9.3-fold higher than the control peptide, respectively (Fig 1B). The binding affinity of the sAPRIL-BPs was also tested against another TNF superfamily ligand BAFF (B-cell activation factor of the TNF family). The binding affinities for each sAPRIL-BP were 1.2, 1.1, and 0.9, respectively (Fig 1B). Taken together, these results indicated that the sAPRIL-BPs specifically binds to APRIL and do not cross-react with BAFF. sAPRIL-BP1 (AAAPLAQPHMWA) had the highest binding affinity and was subsequently used in vitro to assess whether it was able to inhibit sAPRIL binding in the fixed human colorectal cancer cell line LOVO cells (Fig 1C). sAPRIL-BP1 showed a dose-dependent inhibitory effect on sAPRIL binding to the LOVO cells. Therefore, sAPRIL-BP1 (hereafter sAPRIL-BP) was chosen for further functional characterization.


Identification of the sAPRIL binding peptide and its growth inhibition effects in the colorectal cancer cells.

He XQ, Guan J, Liu F, Li J, He MR - PLoS ONE (2015)

Selection of sAPRIL-BP.(A) The binding affinity of phage clones No.1–20 for sAPRIL were determined by ELISA. Clone 21 was used as a positive control. The fold change of the optical density was normalized to the positive control. Clones that had at least a 6-fold greater affinity than the positive control were considered ‘positive’ for sAPRIL binding. (B) Three binding peptides were synthesized and their binding affinity with sAPRIL (black bars) was determined and compared with the negative control (NC) using ELISA. Cross-reactivity was assessed by measuring the binding affinity to BAFF (grey bars). (C) Clone BP1 (sAPRIL-BP) was mixed with sAPRIL at different doses to compete for binding with fixed LOVO cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380366&req=5

pone.0120564.g001: Selection of sAPRIL-BP.(A) The binding affinity of phage clones No.1–20 for sAPRIL were determined by ELISA. Clone 21 was used as a positive control. The fold change of the optical density was normalized to the positive control. Clones that had at least a 6-fold greater affinity than the positive control were considered ‘positive’ for sAPRIL binding. (B) Three binding peptides were synthesized and their binding affinity with sAPRIL (black bars) was determined and compared with the negative control (NC) using ELISA. Cross-reactivity was assessed by measuring the binding affinity to BAFF (grey bars). (C) Clone BP1 (sAPRIL-BP) was mixed with sAPRIL at different doses to compete for binding with fixed LOVO cells.
Mentions: A phage library was used to identify 20 single clones expressing potential sAPRIL binding peptides. The clones were randomly selected after four rounds of panning. Eight single clones were identified as ‘positive’ indicating a high binding affinity (defined as an OD ≥6-fold the OD of the positive control) for sAPRIL by ELISA (Fig 1A). The eight clones represented three DNA sequences that correspond to the following binding polypeptides (BP): AAAPLAQPHMWA, SSTTTSDKYLSA, and SNLHDNNTEKNV (Table 1). The affinity for sAPRIL was measured for each polypeptide by ELISA and compared to an unrelated polypeptide (HWDPFSLSAYFP) as a negative control. The three peptides identified using the phage library had sAPRIL binding affinities that were 13.7, 10.8, and 9.3-fold higher than the control peptide, respectively (Fig 1B). The binding affinity of the sAPRIL-BPs was also tested against another TNF superfamily ligand BAFF (B-cell activation factor of the TNF family). The binding affinities for each sAPRIL-BP were 1.2, 1.1, and 0.9, respectively (Fig 1B). Taken together, these results indicated that the sAPRIL-BPs specifically binds to APRIL and do not cross-react with BAFF. sAPRIL-BP1 (AAAPLAQPHMWA) had the highest binding affinity and was subsequently used in vitro to assess whether it was able to inhibit sAPRIL binding in the fixed human colorectal cancer cell line LOVO cells (Fig 1C). sAPRIL-BP1 showed a dose-dependent inhibitory effect on sAPRIL binding to the LOVO cells. Therefore, sAPRIL-BP1 (hereafter sAPRIL-BP) was chosen for further functional characterization.

Bottom Line: The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively.The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner.In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Institute of Digestive Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China; Oncology Department, Wuzhou Red Cross Hospital, Wuzhou 543002, Guangxi Province, China.

ABSTRACT

Background: A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer.

Methods: A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP.

Results: Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells.

Conclusions: sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL.

No MeSH data available.


Related in: MedlinePlus