Marine Lectins DlFBL and HddSBL Fused with Soluble Coxsackie-Adenovirus Receptor Facilitate Adenovirus Infection in Cancer Cells BUT Have Different Effects on Cell Survival

Abstract

Cancer development and progression are usually associated with glycosylation change, providing prognostic and diagnostic biomarkers, as well as therapeutic targets, for various cancers. In this work, Dicentrarchus labrax fucose binding lectin (DlFBL) and Haliotis discus discus sialic acid binding lectin (HddSBL) were genetically fused with soluble coxsackie-adenovirus receptor (sCAR), and produced through a bacterial expression system. Results showed that recombinant sCAR-DlFBL not only facilitated adenovirus Ad-EGFP infection in K562/ADR and U87MG cells, but also enhanced the cytotoxicity of adenovirus harboring gene encoding Pinellia pedatisecta agglutinin (PPA) or DlFBL (Ad-PPA or Ad-DlFBL) on U87MG cells through inducing apoptosis. Recombinant sCAR-HddSBL facilitated Ad-EGFP infection, but dramatically counteracted the cytotoxicity of both Ad-PPA and Ad-DlFBL in U87MG cells. Further analysis revealed that sCAR-HddSBL, but not sCAR-DlFBL, significantly upregulated transcription factor E2F1 levels in U87MG cells, which might be responsible for the adverse effect of sCAR-HddSBL on Ad-PPA and Ad-DlFBL. Taken together, our data suggested that sCAR-DlFBL could be further developed to redirect therapeutic adenoviruses to infect cancer cells such as U87MG, and the sCAR-lectin fusion proteins for adenoviral retargeting should be carefully examined for possible survival signaling induced by lectins, such as HddSBL.

Keywords: DlFBL; E2F1; HddSBL; adenovirus.

Figure 1

The structure and production of recombinant sCAR-lectin proteins. (a) Schematic structure of the sCAR-lectins fusion proteins. The recombinant proteins consist of a 6his-tag, an extracellular domain of CAR with 239 amino acids, a flexible linker (SASASASAPGS), and a lectin region; (b) the production of recombinant sCAR-lectin proteins. The pQE30-sCAR-lectin plasmids were transformed to Escherichia coli strain M15 and induced by IPTG. The expression of sCAR-DlFBL and sCAR-HddSBL proteins were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. The sCAR-lectin proteins were purified through a Ni-NTA-Sepharose column, and subjected to SDS-PAGE, followed by Coomassie brilliant blue staining (c), and subjected to Western blotting analysis with a goat anti-CAR antibody (d).

Figure 2

Recombinant sCAR-DlFBL and sCAR-HddSBL proteins enhanced adenoviral infection in K562/ADR leukemia cells and U87MG glioblastoma cells. (a) K562/ADR cells were treated with 30 MOI Ad-EGFP combined with 10 μg/mL of sCAR-DlFBL or sCAR-HddSBL for 48 h. Cells treated with Ad-EGFP alone served as the control. The portion of EGFP positive cells was analyzed through fluorescence microscopy and flow cytometry. Shown is a representative from three separate experiments; (b) U87MG cells were treated with 5 MOI Ad-EGFP combined with 42 μg/mL sCAR-DlFBL or 31.8 μg/mL sCAR-HddSBL for 48h. Cells treated with Ad-EGFP alone served as the control. EGFP positive cells was analyzed through fluorescence microscope and flow cytometry. Shown is a representative from three separate experiments.

Figure 3

Recombinant sCAR-DlFBL and sCAR-HddSBL had different effects on the cytotoxicity of Ad-PPA and Ad-DlFBL in U87MG cells. U87MG cells were treated with 42 μg/mL sCAR-DlFBL or 31.8 μg/mL sCAR-HddSBL in combination with 6.8 MOI of Ad-PPA or 8.2 MOI of Ad-DlFBL for 48 h. Cells were also treated with PBS, sCAR-DlFBL, sCAR-HddSBL, Ad-PPA, Ad-DlFBL alone as a control. Cell morphology was observed under a microscope.

Figure 4

Recombinant sCAR-DlFBL enhanced the cytotoxicity of Ad-PPA and Ad-DlFBL. U87MG cells were treated with (a) 8.2 MOI of Ad-DlFBL or (b) 6.8 MOI of Ad-PPA in combination with sCAR-DlFBL at concentrations indicated for 96 h. Cells were also treated with PBS, sCAR-DlFBL, Ad-PPA, or Ad-DlFBL alone as a control. Cell viability was analyzed through MTT assay. *: p < 0.05

Figure 5

Recombinant sCAR-HddSBL counteracted the cytotoxicity of Ad-PPA and Ad-DlFBL. U87MG cells were treated with (a) 8.2 MOI of Ad-DlFBL or (b) 6.8 MOI of Ad-PPA in combination with sCAR-HddSBL at concentrations indicated for 96 h. Cells were also treated with PBS, sCAR-HddSBL, Ad-PPA, or Ad-DlFBL alone as a control. Cell viability was analyzed through MTT assay. *: p < 0.05

Figure 6

Recombinant sCAR-DlFBL but not sCAR-HddSBL induced apoptosis in combination with Ad-PPA or Ad-DlFBL in U87MG cells. U87MG cells were treated with 42 μg/mL sCAR-DlFBL or 31.8 μg/mL sCAR-HddSBL in combination with 6.8MOI of Ad-PPA or 8.2MOI of Ad-DlFBL for 48 h. Cells were also treated with PBS, sCAR-DlFBL, sCAR-HddSBL, Ad-PPA, and Ad-DlFBL alone as a control. Cells were then collected and stained with by Annexin V-FITC Apoptosis Detection Kit, followed by flow cytometry analysis.

Figure 7

Recombinant sCAR-HddSBL upregulated E2F1 levels in U87MG cells. U87MG cells were treated with (a) 42 μg/mL sCAR-DlFBL or (b) 31.8 μg/mL sCAR-HddSBL in combination with 6.8MOI of Ad-PPA or 8.2MOI of Ad-DlFBL for 48 h. Cells were also treated with PBS, sCAR-DlFBL, sCAR-HddSBL, Ad-PPA, and Ad-DlFBL alone as a control. Cells were lysed and analyzed for phosphor-ERK, ERK, and E2F1 through Western blot. Actin served as the loading control; and (c) the effects of recombinant of sCAR-DlFBL and sCAR-HddSBL on the NF-κB activation in U87MG cells were analyzed through a NF-κB reporter assay.