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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 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.Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells. 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.

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

Effect of sAPRIL-BP on the expression of cell cycle-related proteins.LOVO cells were treated with the indicated doses of sAPRIL-BP for 48 h. (A) The expression levels of the indicated cell cycle proteins were assessed by Western Blotting analysis. GAPDH was used as the internal control. The protein size of Cyclin D1 is 34 kDa, Cyclin A 49 kDa, Cyclin E 50 kDa, Cyclin B1 55 kDa, CDK4 34 kDa, CDK6 37 kDa, p53 53 kDa, p27 27 kDa, p16 40 kDa, and GAPDH 36 kDa. The optical densities of the cyclin D1 (B) and CDK4 (C) protein bands were analyzed and normalized to the internal control as fold change. *P<0.05 compared to Vehicle group; #P<0.05 compared to 10 μM group, †P<0.05 compared to 20 μM group.
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pone.0120564.g005: Effect of sAPRIL-BP on the expression of cell cycle-related proteins.LOVO cells were treated with the indicated doses of sAPRIL-BP for 48 h. (A) The expression levels of the indicated cell cycle proteins were assessed by Western Blotting analysis. GAPDH was used as the internal control. The protein size of Cyclin D1 is 34 kDa, Cyclin A 49 kDa, Cyclin E 50 kDa, Cyclin B1 55 kDa, CDK4 34 kDa, CDK6 37 kDa, p53 53 kDa, p27 27 kDa, p16 40 kDa, and GAPDH 36 kDa. The optical densities of the cyclin D1 (B) and CDK4 (C) protein bands were analyzed and normalized to the internal control as fold change. *P<0.05 compared to Vehicle group; #P<0.05 compared to 10 μM group, †P<0.05 compared to 20 μM group.

Mentions: Since the proliferation of cancer cells is primarily regulated by the cell cycle, we next determined which phase of the cell cycle was affected by sAPRIL treatment. Based on the proliferation results (Fig 3A), we chose to test 20 μM and 40 μM of sAPRIL-BP in LOVO cells to determine how inhibiting sAPRIL altered the cell cycle and apoptosis. By flow cytometry, sAPRIL-BP significantly increased the percentage of cells in the G0/G1 phase at the low dose (20 μM sAPRIL-BP: 73.1 ± 0.6%; p<0.001) and the high dose (40 μM sAPRIL-BP: 76.2 ± 0.1%; p<0.001) when compared to the Vehicle control (65.2 ± 0.8%). sAPRIL-BP also significantly reduced the percentage of cells in the G2/M stage compared to the Vehicle control (24.3 ± 0.8%) at the low dose (20 μM sAPRIL-BP: 15.1 ± 0.5%; p<0.05) and the high dose (40 μM sAPRIL-BP: 13.7 ± 0.5%; p<0.001; Fig 4A and 4B). This suggests that the anti-proliferative effects of sAPRIL-BP in LOVO cells are due to an accumulation of cells in the G0/G1 stage, which blocks cell cycle progression. Regarding the apoptotic effects of sAPRIL-BP, flow cytometry analysis showed that sAPRIL-BP had dose-dependent effects on the percentage of LOVO cells in early apoptosis (PI- Annexin V+ cells). Compared to the Vehicle control (1.76 ± 0.12%) the low dose (20 μM sAPRIL-BP: 2.49 ± 0.23%; p<0.05) and high dose (40 μM sAPRIL-BP: 3.82 ± 0.36%; p<0.05) significantly increased early apoptotic cells (Fig 4C and 4D). We further investigated the possible molecular mechanism underlying the effect of sAPRIL-BP on cell cycle progression. Treatment with sAPRIL-BP had no effect on cyclin A, B, E1, CDK6, p53, p27, and p16 expression (Fig 5A). But the expression of the G1/S-specific protein cyclin D1 (Fig 5A and 5B) and cell division kinase cyclin-dependent kinase 4 (CDK4) (Fig 5A and 5C) were downregulated in a dose-dependent manner by treatment with sAPRIL-BP.


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)

Effect of sAPRIL-BP on the expression of cell cycle-related proteins.LOVO cells were treated with the indicated doses of sAPRIL-BP for 48 h. (A) The expression levels of the indicated cell cycle proteins were assessed by Western Blotting analysis. GAPDH was used as the internal control. The protein size of Cyclin D1 is 34 kDa, Cyclin A 49 kDa, Cyclin E 50 kDa, Cyclin B1 55 kDa, CDK4 34 kDa, CDK6 37 kDa, p53 53 kDa, p27 27 kDa, p16 40 kDa, and GAPDH 36 kDa. The optical densities of the cyclin D1 (B) and CDK4 (C) protein bands were analyzed and normalized to the internal control as fold change. *P<0.05 compared to Vehicle group; #P<0.05 compared to 10 μM group, †P<0.05 compared to 20 μM group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4380366&req=5

pone.0120564.g005: Effect of sAPRIL-BP on the expression of cell cycle-related proteins.LOVO cells were treated with the indicated doses of sAPRIL-BP for 48 h. (A) The expression levels of the indicated cell cycle proteins were assessed by Western Blotting analysis. GAPDH was used as the internal control. The protein size of Cyclin D1 is 34 kDa, Cyclin A 49 kDa, Cyclin E 50 kDa, Cyclin B1 55 kDa, CDK4 34 kDa, CDK6 37 kDa, p53 53 kDa, p27 27 kDa, p16 40 kDa, and GAPDH 36 kDa. The optical densities of the cyclin D1 (B) and CDK4 (C) protein bands were analyzed and normalized to the internal control as fold change. *P<0.05 compared to Vehicle group; #P<0.05 compared to 10 μM group, †P<0.05 compared to 20 μM group.
Mentions: Since the proliferation of cancer cells is primarily regulated by the cell cycle, we next determined which phase of the cell cycle was affected by sAPRIL treatment. Based on the proliferation results (Fig 3A), we chose to test 20 μM and 40 μM of sAPRIL-BP in LOVO cells to determine how inhibiting sAPRIL altered the cell cycle and apoptosis. By flow cytometry, sAPRIL-BP significantly increased the percentage of cells in the G0/G1 phase at the low dose (20 μM sAPRIL-BP: 73.1 ± 0.6%; p<0.001) and the high dose (40 μM sAPRIL-BP: 76.2 ± 0.1%; p<0.001) when compared to the Vehicle control (65.2 ± 0.8%). sAPRIL-BP also significantly reduced the percentage of cells in the G2/M stage compared to the Vehicle control (24.3 ± 0.8%) at the low dose (20 μM sAPRIL-BP: 15.1 ± 0.5%; p<0.05) and the high dose (40 μM sAPRIL-BP: 13.7 ± 0.5%; p<0.001; Fig 4A and 4B). This suggests that the anti-proliferative effects of sAPRIL-BP in LOVO cells are due to an accumulation of cells in the G0/G1 stage, which blocks cell cycle progression. Regarding the apoptotic effects of sAPRIL-BP, flow cytometry analysis showed that sAPRIL-BP had dose-dependent effects on the percentage of LOVO cells in early apoptosis (PI- Annexin V+ cells). Compared to the Vehicle control (1.76 ± 0.12%) the low dose (20 μM sAPRIL-BP: 2.49 ± 0.23%; p<0.05) and high dose (40 μM sAPRIL-BP: 3.82 ± 0.36%; p<0.05) significantly increased early apoptotic cells (Fig 4C and 4D). We further investigated the possible molecular mechanism underlying the effect of sAPRIL-BP on cell cycle progression. Treatment with sAPRIL-BP had no effect on cyclin A, B, E1, CDK6, p53, p27, and p16 expression (Fig 5A). But the expression of the G1/S-specific protein cyclin D1 (Fig 5A and 5B) and cell division kinase cyclin-dependent kinase 4 (CDK4) (Fig 5A and 5C) were downregulated in a dose-dependent manner by treatment with sAPRIL-BP.

Bottom Line: 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.Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells. 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.

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