Fig 1.
(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.
Table 1.
DNA and amino acid sequence of positive clones.
Fig 2.
APRIL mRNA and protein expression in human colorectal cell lines.
APRIL expression was assessed in five human colorectal cell lines (indicated) using RT-PCR (A) and Western Blotting (C). Representative gel images are shown. GAPDH was used as the internal control. The optical densities of the APRIL mRNA (B) and protein (D) bands were analyzed and normalized to the internal control. *P<0.05 compared to SW620, HT-29, or SW480.
Fig 3.
Effect of sAPRIL-BP on the proliferation of LOVO and SW620 cells.
(A) APRILhigh LOVO and HCT116 cells and (B) APRILlow SW620 and HT-29 cells were treated with the indicated doses of sAPRIL binding peptides for 24, 48, and 72 h, and proliferation was determined using the CCK-8 kit. The rate of proliferation inhibition was calculated as: (%) = [(mean of ODcontrol—mean of ODexperimental) / mean of ODcontrol]×100%.
Fig 4.
Effect of sAPRIL-BP on cell cycle and apoptosis of LOVO cells.
LOVO cells were treated with the indicated doses of sAPRIL-BP for 48 h. Cells were stained with PI for cell cycle analysis (A and B) and PI + Annexin V for apoptosis analysis (C and D) by flow cytometry. *p <0.05 compared to Vehicle control; #p <0.05 compared to the low dose group.
Fig 5.
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.
Fig 6.
In vivo effects of sAPRIL-BP on tumor development.
LOVO cells were injected subcutaneously into nude mice and allowed to grow for 3 weeks. Once the tumor was establishes, the mice were divided into 3 groups (N = 5) and treated with PBS (control), low (20 mg/kg), or high (40 mg/kg) dose of sAPRIL-BP every other day. (A) Representative examples of the tumors from each group are shown. Top panel: Scale bar, 8 mm. Bottom panel: Scale bar, 7 mm. (B) Mice were sacrificed after two weeks of treatment with sAPRIL-BP and the tumor weights were recorded. *p <0.05 compared to control. #p <0.05 compared to the low dose group. (C) The tumor volume was recorded every two days during treatment. *p <0.05 compared to control.
Fig 7.
In vivo effect of sAPRIL-BP on proliferation and apoptosis in xenograft tumors.
Paraffin embedded tumor tissues were used for morphological analysis with H&E staining (A), proliferation analysis with Ki67 staining (B), and apoptosis analysis with cleaved caspase-3 (cle-casp-3) staining (C). Representative pictures of the tumors from each group are shown (magnification 200×). The proliferation index (D) and apoptosis index (E) were calculated and normalized to the control (Con) group. *p <0.05 compared to control. #p <0.05 compared to 20 mg/kg group.
Fig 8.
In vivo effect of sAPRIL-BP on liver metastasis.
LOVO cells were injected into the spleens of nude mice to observe experimental liver metastasis. Three weeks after injection, the mice were divided into 3 groups (N = 5) and treated with PBS (control), low (20 mg/kg), or high (40 mg/kg) doses of sAPRIL-BP every other day. Mice were sacrificed after two weeks of treatment with sAPRIL-BP. (A) Representative pictures of the metastatic liver tumors from each group are shown. (B) Numbers of metastatic nodules per mouse were recorded. *P <0.05 compared to control. #P <0.05 compared to the low dose group. (C) Numbers of metastatic nodules with the indicated size were recorded. Total numbers of metastatic nodules: n = 197 (Con), n = 130 (20 mg/kg), and n = 84 (40 mg/kg).