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

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.


The Production of sCAR-DlFBL and sCAR-HddSBL Fusion Proteins
The sCAR-lectin fusion proteins presented in this work contain a 6his-tag, a human soluble coxsackie-adenovirus receptor (sCAR), a short flexible linker, and a lectin region (Figure 1a). A bacterial expression system was used to produce sCAR-lectin proteins. The production and purification of sCAR-DlFBL and sCAR-HddSBL proteins were examined through SDS-PAGE followed by Coomassie brilliant blue staining (Figure 1b,c). The production of sCAR-DlFBL and sCAR-HddSBL proteins was verified by Western blotting analysis for CAR (Figure 1d). Results indicated that purified sCAR-DlFBL and sCAR-HddSBL with expected molecular weights were successfully obtained through the bacterial expression system. , 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).

Recombinant sCAR-DlFBL and sCAR-HddSBL Proteins Facilitated Adenovirus Infection
The sCAR-DlFBL and sCAR-HddSBL proteins were then tested for the activity of facilitating adenovirus infection in cancer cells. Leukemic K562/ADR cells were treated with Ad-EGFP alone, as well as Ad-EGFP combined with sCAR-DlFBL or sCAR-HddSBL proteins, followed by fluorescent microscope observation and flow cytometry analysis. As observed under fluorescent microscope, cells treated with Ad-EGFP combined with sCAR-DlFBL or sCAR-HddSBL showed significantly higher portion of EGFP positive population, compared to cells treated with Ad-EGFP alone. Flow cytometry analysis verified the elevated Ad-EGFP infection in K562/ADR cells by sCAR-DlFBL and sCAR-HddSBL proteins (Figure 2a). To further confirm the activity of sCAR-DlFBL and sCAR-HddSBL proteins, the glioblastoma cell line U87MG was infected with Ad-EGFP alone, as well as Ad-EGFP combined with sCAR-DlFBL or sCAR-HddSBL proteins. As shown in Figure 2b, both fluorescent microscope observation and flow cytometry analysis confirmed that sCAR-DlFBL and sCAR-HddSBL proteins were capable of facilitating adenovirus infection. , 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).

Recombinant sCAR-DlFBL and sCAR-HddSBL Proteins Facilitated Adenovirus Infection
The sCAR-DlFBL and sCAR-HddSBL proteins were then tested for the activity of facilitating adenovirus infection in cancer cells. Leukemic K562/ADR cells were treated with Ad-EGFP alone, as well as Ad-EGFP combined with sCAR-DlFBL or sCAR-HddSBL proteins, followed by fluorescent microscope observation and flow cytometry analysis. As observed under fluorescent microscope, cells treated with Ad-EGFP combined with sCAR-DlFBL or sCAR-HddSBL showed significantly higher portion of EGFP positive population, compared to cells treated with Ad-EGFP alone. Flow cytometry analysis verified the elevated Ad-EGFP infection in K562/ADR cells by sCAR-DlFBL and sCAR-HddSBL proteins (Figure 2a). To further confirm the activity of sCAR-DlFBL and sCAR-HddSBL proteins, the glioblastoma cell line U87MG was infected with Ad-EGFP alone, as well as Ad-EGFP combined with sCAR-DlFBL or sCAR-HddSBL proteins. As shown in Figure 2b, both fluorescent microscope observation and flow cytometry analysis confirmed that sCAR-DlFBL and sCAR-HddSBL proteins were capable of facilitating adenovirus infection.

Recombinant sCAR-DlFBL and sCAR-HddSBL Had Different Effects on the Cytotoxicity of Ad-PPA and Ad-DlFBL in U87MG Cells
Previously, non-replicating adenovirus harboring gene encoding exogenous lectins, such as PPA and DlFBL have shown significant cytotoxicity to a variety of cancer cells [8,9]. In this work, we tested whether the cytotoxicity of Ad-PPA and Ad-DlFBL could be further enhanced by sCAR-DLFBL or sCAR-HddSBL. As observed under microscope (Figure 3), sCAR-DLFBL in combination with Ad-PPA or Ad-DlFBL dramatically induced higher level of cytotoxicity to U87MG cells than Ad-PPA or Ad-DlFBL alone. Differently, sCAR-HddSBL combined with Ad-PPA or Ad-DlFBL did not induce obvious toxicity to U87MG cells ( Figure 3). The higher cytotoxicity of sCAR-DLFBL in combination with Ad-PPA or Ad-DlFBL was confirmed by MTT assay (Figure 4), in which the effect of sCAR-DlFBL showed a dose-dependent tendency. Interestingly, sCAR-HddSBL significantly counteracted the cytotoxicity of Ad-PPA and Ad-DlFBL at a dose-dependent manner ( Figure 5). Recombinant sCAR-HddSBL at 31.8 µg/mL almost completely suppressed Ad-DlFBL induced anti-proliferative effect on U87MG cells, suggesting that sCAR-HddSBL possibly activated survival signaling pathways which counteracted the cytotoxicity of Ad-DlFBL and Ad-PPA. Further staining with Annexin V-FITC and PI followed by flow cytometry analysis showed that sCAR-DLFBL but not sCAR-HddSBL in combination of Ad-PPA or Ad-DlFBL enhanced apoptosis in U87MG cells ( Figure 6). Our results indicated that sCAR-DlFBL but not sCAR-HddSBL is capable of enhancing the cytotoxicity of Ad-PPA and Ad-DlFBL in U87MG cells through inducing apoptosis.

Recombinant sCAR-DlFBL and sCAR-HddSBL Had Different Effects on the Cytotoxicity of Ad-PPA and Ad-DlFBL in U87MG Cells
Previously, non-replicating adenovirus harboring gene encoding exogenous lectins, such as PPA and DlFBL have shown significant cytotoxicity to a variety of cancer cells [8,9]. In this work, we tested whether the cytotoxicity of Ad-PPA and Ad-DlFBL could be further enhanced by sCAR-DLFBL or sCAR-HddSBL. As observed under microscope (Figure 3), sCAR-DLFBL in combination with Ad-PPA or Ad-DlFBL dramatically induced higher level of cytotoxicity to U87MG cells than Ad-PPA or Ad-DlFBL alone. Differently, sCAR-HddSBL combined with Ad-PPA or Ad-DlFBL did not induce obvious toxicity to U87MG cells ( Figure 3). The higher cytotoxicity of sCAR-DLFBL in combination with Ad-PPA or Ad-DlFBL was confirmed by MTT assay (Figure 4), in which the effect of sCAR-DlFBL showed a dose-dependent tendency. Interestingly, sCAR-HddSBL significantly counteracted the cytotoxicity of Ad-PPA and Ad-DlFBL at a dose-dependent manner ( Figure 5). Recombinant sCAR-HddSBL at 31.8 μg/mL almost completely suppressed Ad-DlFBL induced anti-proliferative effect on U87MG cells, suggesting that sCAR-HddSBL possibly activated survival signaling pathways which counteracted the cytotoxicity of Ad-DlFBL and Ad-PPA. Further staining with Annexin V-FITC and PI followed by flow cytometry analysis showed that sCAR-DLFBL but not sCAR-HddSBL in combination of Ad-PPA or Ad-DlFBL enhanced apoptosis in U87MG cells ( Figure 6). Our results indicated that sCAR-DlFBL but not sCAR-HddSBL is capable of enhancing the cytotoxicity of Ad-PPA and Ad-DlFBL in U87MG cells through inducing apoptosis.       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.

Recombinant sCAR-HddSBL Upregulated E2F1 Levels in U87MG Cells
We then investigated the underlying mechanism of the suppressive effect of sCAR-HddSBL on the cytotoxicity of Ad-DlFBL and Ad-PPA. U87MG cells were treated with sCAR-DlFBL or sCAR-HddSBL in combination with Ad-PPA or Ad-DlFBL as shown in Figure 7a,b. Both sCAR-DlFBL and sCAR-HddSBL combined with Ad-PPA or Ad-DlFBL upregulated the phosphorylation of extracellular regulated protein kinases (ERK), suggesting that ERK was not involved in the sCAR-HddSBL induced suppression effect on the cytotoxicity of Ad-PPA and Ad-DlFBL. Furthermore, NF-κB reporter assay showed that sCAR-DlFBL and sCAR-HddSBL did not significantly alter NF-κB activation (Figure 7c). Interestingly, different from sCAR-DlFBL which had no obvious effect on E2F1 levels (Figure 7a), sCAR-HddSBL alone, or sCAR-HddSBL in combination with Ad-PPA or Ad-DlFBL, significantly upregulated E2F1 levels in U87MG cells as compared to the PBS control, Ad-PPA, or Ad-DlFBL treatment (Figure 7b). Our data indicated that transcription factor E2F1 was significantly upregulated by sCAR-HddSBL.

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.

Recombinant sCAR-HddSBL Upregulated E2F1 Levels in U87MG Cells
We then investigated the underlying mechanism of the suppressive effect of sCAR-HddSBL on the cytotoxicity of Ad-DlFBL and Ad-PPA. U87MG cells were treated with sCAR-DlFBL or sCAR-HddSBL in combination with Ad-PPA or Ad-DlFBL as shown in Figure 7a,b. Both sCAR-DlFBL and sCAR-HddSBL combined with Ad-PPA or Ad-DlFBL upregulated the phosphorylation of extracellular regulated protein kinases (ERK), suggesting that ERK was not involved in the sCAR-HddSBL induced suppression effect on the cytotoxicity of Ad-PPA and Ad-DlFBL. Furthermore, NF-κB reporter assay showed that sCAR-DlFBL and sCAR-HddSBL did not significantly alter NF-κB activation (Figure 7c). Interestingly, different from sCAR-DlFBL which had no obvious effect on E2F1 levels (Figure 7a), sCAR-HddSBL alone, or sCAR-HddSBL in combination with Ad-PPA or Ad-DlFBL, significantly upregulated E2F1 levels in U87MG cells as compared to the PBS control, Ad-PPA, or Ad-DlFBL treatment (Figure 7b). Our data indicated that transcription factor E2F1 was significantly upregulated by sCAR-HddSBL.

Discussion
Due to lytic replication, efficient gene transfer, and low pathogenicity, oncolytic adenovirus, or conditionally replicating adenoviruses, has become a promising strategy for cancer therapy [30][31][32][33]. CAR acts as the primary receptor for the infection of serotype 5 (Ad5) adenoviruses, the most commonly used adenoviral vector in cancer gene therapy [34,35]. However, many tumors only express low levels of CAR, resulting in resistance to Ad5 infection [36]. Previously, we designed a novel strategy to redirect oncolytic adenoviruses to leukemia cell membrane receptors though carrying a sCAR-ligand expression cassette in the viral genome [12,37]. To retarget oncolytic adenoviruses to

Discussion
Due to lytic replication, efficient gene transfer, and low pathogenicity, oncolytic adenovirus, or conditionally replicating adenoviruses, has become a promising strategy for cancer therapy [30][31][32][33]. CAR acts as the primary receptor for the infection of serotype 5 (Ad5) adenoviruses, the most commonly used adenoviral vector in cancer gene therapy [34,35]. However, many tumors only express low levels of CAR, resulting in resistance to Ad5 infection [36]. Previously, we designed a novel strategy to redirect oncolytic adenoviruses to leukemia cell membrane receptors though carrying a sCAR-ligand expression cassette in the viral genome [12,37]. To retarget oncolytic adenoviruses to interleukin-3 receptor α subunit (CD123) or CD47, a sCAR-IL-3, or sCAR-4N1 expression cassettes were genetically inserted into the viral genome. During viral packaging, the sCAR-ligand fusion protein would be expressed in packaging cells and non-covalently installed on viral surface, bridging oncolytic adenoviruses to CD123+ or CD47+ leukemia cells. After infection and replication in leukemia cells, the sCAR-ligand expression would help newly-produced oncolytic adenoviruses to be further modified and infect more leukemia cells. Therefore, harboring a sCAR-ligand expression cassette in the viral genome may become a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resistant to Ad5 adenovirus infection. In this study, we showed that recombinant sCAR-DlFBL not only facilitated Ad-EGFP infection in K562/ADR leukemia cells and U87MG glioblastoma cells, but also enhanced cytotoxicity of Ad-PPA and Ad-DlFBL in U87MG cells. However, recombinant sCAR-HddSBL enhanced Ad-EGFP infection in U87MG cells, but dramatically counteracted the cytotoxicity of Ad-PPA and Ad-DlFBL. Therefore, our data strongly suggested that sCAR-DlFBL could be further developed to redirect oncolytic adenoviruses to infect cancer cells, such as U87MG. Our data also suggested that sCAR-lectins for adenoviral retargeting should also be carefully examined for possible survival signaling induced by lectins, such as HddSBL in cancer cells.
Transcription factor E2F-1 has been identified as both activator of cell cycle progression and apoptosis inducer [38]. Overexpression of E2F1 was shown to promote leukemia cell proliferation in a cytokine independent manner, and a variety of cell cycle dependant cyclins were maintained by E2F1 without cytokine stimulation [39]. On the other hand, E2F1 induces cell apoptosis through cooperating with either p53 [40] or p73 [41]. In response to DNA damage, Chk2 activates E2F1, which subsequently induces apoptosis [42]. E2F1 was determined to be a direct substrate for PRMT1 and PRMT5 [43,44]. Interestingly, E2F1 methylated by PRMT1 augmented cell apoptosis, whereas E2F1 methylated by PRMT5 favored cell proliferation [44], suggesting a key role of differed arginine methylation in determining E2F1 biological activities. Our previous studies have identified several exogenous lectins such as DlFBL, Anguilla japonica lectin 1, as well as Strongylocentrotus purpuratus rhamnose binding lectin interacted with PRMT5 and induced downregulation of E2F1 in cancer cells [9,11]. In this work, we further showed that recombinant sCAR-HddSBL upregulated E2F1 in U87MG cells, suggesting that E2F1 may play as a target for various lectins. However, the underlying mechanism is still not clear, pending further investigations.

Production of sCAR-DlFBL and sCAR-HddSBL Fusion Proteins
Plasmid pQE30-sCAR has been constructed previously. In this work, gene encoding Dicentrarchus labrax fucose-binding lectin (DIFBL, GenBank accession number: EU877448) or Haliotis discus discus sialic acid-binding lectin (HddSBL, GenBank accession No. EF103404) was inserted to form pQE30-sCAR-DlFBL or pQE30-sCAR-HddSBL. The pQE30-sCAR-DlFBL or pQE30-sCAR-HddSBL plasmid was then transformed to Escherichia coli strain M15 and the expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). Inclusion bodies were suspended in a buffer containing 8 M urea, 0.1 M sodium phosphate buffer, and 0.01 M Tris-HCl (pH 8.0) at 5 mL per gram of wet weight, and centrifuged at 12,000 rpm for 30 min. The supernatant was diluted by PBS at 1 mL per 20 mL PBS and dialyzed against PBS over night at 4 • C, followed by mixture with Ni-NTA slurry (Merck Biosciences, Darmstadt, Germany). The lysate-Ni-NTA mixture was loaded into a column and washed twice with a washing buffer containing 300 mM NaCl, 50 mM sodium phosphate buffer, and 20 mM imidazole (pH 8.0). The column was eluted with an elution buffer containing 300 mM NaCl, 50 mM sodium phosphate buffer, and 250 mM imidazole (pH 8.0). The eluted protein was dialyzed against PBS at 4 • C overnight to remove imidazole.

Adenoviral Infection
The recombinant serotype 5 adenovirus carrying an enhanced green fluorescent protein gene (Ad-EGFP) was generated in our laboratory previously. K562/ADR cells were treated with 30MOI of Ad-EGFP in combination with PBS or 10 µg/mL of sCAR-lectin proteins. U87MG/Ctr and U87MG/SLMAP cells were treated with 5MOI of Ad-EGFP in combination with PBS or 10 µg/mL of sCAR-lectin proteins. After one day, EGFP positive cells were examined under a fluorescent microscope (Olympus Corporation, Tokyo, Japan) or a BD FACSAria flow cytometry (BD Biosciences, San Jose, CA, USA).

Cytotoxicity Detection
Cells were plated on 96-well plates at 5 × 10 3 per well one day before infected with adenoviruses. Then cells were treated with adenoviruses in combination with sCAR-lectins as indicated for 96 h. The cytotoxicity detection assay was carried out as the procedure of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Meanwhile, cells treated with adenoviruses in combination with sCAR-lectins as indicated for 48 h were collected and stained with Annexin V-FITC Apoptosis Detection Kit (KeyGEN Biotech Co., Ltd., Nanjing, China) following the manufacturer's instruction, followed by analyzing under a BD FACSAria flow cytometry (BD Biosciences, San Jose, CA, USA).

Western Blot Analysis
The cell extracts were subjected to SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were then blocked with Tris-buffered saline and Tween 20 contaning 5% of bovine serum albumin at room temperature for 2 h and incubated with corresponding antibodies overnight at 4 • C. The membranes were washed and incubated with appropriate dilution of secondary antibodies for 1 h at room temperature. After washing with Tris-buffered saline, the bands were detected under a Tanon 5500 chemiluminescence image system (Tanon Inc., Shanghai, China). Prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA) and sCAR-lectins bands were detected separately and incorporated together through the software provided by the manufacturer (Tanon Inc.).

Reporter Assay
NF-κB firefly luciferase reporter plasmid was constructed previously. Reporter assay was performed using a duo-luciferase assay kit (GeneCopoeia, Inc., Rockville, MD, USA) following the manufacturer's instructions. Briefly, U87MG cells were co-transfected with NF-κB luciferase reporter plasmid and Renilla luciferase control plasmid, followed by treatment with PBS, 42 µg/mL of sCAR-DlFBL or sCAR-HddSBL for 24 h. Cells were then lysed and NF-κB firefly luciferase activity was normalized to Renilla luciferase activity.

Statistical Analysis
Differences among the treatment groups were assessed by Student's t-test. p < 0.05 was considered significant.

Conclusions
In this work, recombinant sCAR-DlFBL and sCAR-HddSBL produced in a bacterial expression system successfully facilitated Ad-EGFP infection in K562/ADR and U87MG cells. Recombinant sCAR-DlFBL was further shown to enhance the cytotoxicity of Ad-PPA and Ad-DlFBL on U87MG cells through inducing apoptosis. However, recombinant sCAR-HddSBL dramatically counteracted the cytotoxicity of Ad-PPA and Ad-DlFBL. Further analysis revealed that sCAR-HddSBL 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 oncolytic adenoviruses to infect cancer cells, such as U87MG, and the sCAR-lectin strategy for adenoviral retargeting should be carefully examined for possible survival signaling induced by lectins, such as HddSBL.