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. 2011 Sep 7:10:109.
doi: 10.1186/1476-4598-10-109.

Tumor cell-selective apoptosis induction through targeting of K(V)10.1 via bifunctional TRAIL antibody

Franziska Hartung  1 , Walter Stühmer, Luis A Pardo
Affiliations

Tumor cell-selective apoptosis induction through targeting of K(V)10.1 via bifunctional TRAIL antibody

Franziska Hartung et al. Mol Cancer. .

Abstract

Background: The search for strategies to target ion channels for therapeutic applications has become of increasing interest. Especially, the potassium channel K(V)10.1 (Ether-á-go-go) is attractive as target since this surface protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in approximately 70% of tumors from different origins.

Methods: We designed a single-chain antibody against an extracellular region of K(V)10.1 (scFv62) and fused it to the human soluble TRAIL. The K(V)10.1-specific scFv62 antibody -TRAIL fusion protein was expressed in CHO-K1 cells, purified by chromatography and tested for biological activity.

Results: Prostate cancer cells, either positive or negative for K(V)10.1 were treated with the purified construct. After sensitization with cytotoxic drugs, scFv62-TRAIL induced apoptosis only in K(V)10.1-positive cancer cells, but not in non-tumor cells, nor in tumor cells lacking K(V)10.1 expression. In co-cultures with K(V)10.1-positive cancer cells the fusion protein also induced apoptosis in bystander K(V)10.1-negative cancer cells, while normal prostate epithelial cells were not affected when present as bystander.

Conclusions: K(V)10.1 represents a novel therapeutic target for cancer. We could design a strategy that selectively kills tumor cells based on a K(V)10.1-specific antibody.

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Figures

Figure 1
Figure 1
Construction, expression and characterization of scFv62-TRAIL. A) Schematic structure of the recombinant fusion construct scFv62-TRAIL, scFv62 (30 kDa) is genetically linked through a flexible Ser-Ser-Gly-Ser-Gly linker to soluble TRAIL (21 kDa). The monomeric scFv62-TRAIL fusion has a molecular weight of 51 kDa and the active trimer a molecular weight of ~150 kDa. B) Stable transfected CHO-K1 cells were used to produce the scFv62-TRAIL fusion protein. Cells were seeded on cell culture flasks, after allowing cell attachment normal medium was change into serum- and protein-free medium and cells were incubated at 37°C or 30°C. The scFv62-TRAIL yield in the medium was analyzed after 5 days by immunoblot using an anti-TRAIL antibody. Under non-reducing conditions scFv62-TRAIL was detected as trimers (150 kDa). C) The scFv62-TRAIL preparation was analyzed using size exclusion chromatography; the received protein peaks were analyzed by immunoblot using an anti-TRAIL antibody. D) Stability of scFv62-TRAIL in mouse serum. The scFv62-TRAIL preparation was incubated in mouse serum at 37°C up to 72 h and analyzed for its ability to induce apoptosis on DU145 cells in the presence of 5 μg/ml CHX (n = 3).
Figure 2
Figure 2
KV10.1 expression analysis and apoptosis induction by scFv62-TRAIL. A) Kv10.1 mRNA levels determined by quantitative real-time PCR. B) Apoptosis analysis using Annexin assay and flow cytometry of different cell lines treated with 50 U/ml scFv62-TRAIL or medium for 20 h (n = 2). C) Immunoblot analysis using anti-caspase-3 antibody: 1) medium supernatant of CHO-K1, scFv62 preparation, 2) scFv62-TRAIL preparation, 3) scFv62-TRAIL dialyzed through a 100 kDa cut-off membrane.
Figure 3
Figure 3
Effect of CHX on cell cycle and in combination with scFv62-TRAIL. A) Histograms of cell cycle analysis with flow cytometer of DU145 cells treated or non-treated with 5 μg/ml CHX and B) quantitation of cell cycle analysis. (n = 3) C) DU145 cells were treated for 18 h with different amounts of scFv62-TRAIL in presence of 5 μg/ml CHX, and subsequently analyzed for apoptosis with Annexin-FITC/PI staining in a flow cytometer (n = 3). D) and E) flow cytometer measurements and analysis: DU145 were treated with 50 U/ml scFv62-TRAIL in the presence of 5 μg/ml CHX and the progression of apoptosis was monitored at different time points with Annexin-FITC/PI staining (live cell: negative for both staining, early apoptotic: Annexin-positive, late apoptotic: Annexin and PI positive).
Figure 4
Figure 4
KV10.1-specific apoptosis induction. A) Annexin/PI staining and flow cytometry analysis of DU145 cells treated with scFv62-TRAIL in combination with different chemotherapeutic agents: cisplatin (10 μM), 17-AAG (5 μM), actinomycin D (800 nM), doxorubicin (1.8 μM), etoposide (50 μM), roscovitine (10 μM), 5-fluorouracil (100 μM), CHX (5 μg/ml) (n = 2). B) Cell lines were treated for 18 h with 50 U/ml scFv62-TRAIL in combination with 5 μg/ml CHX and analyzed for apoptosis with Annexin/PI staining and flow cytometry (n = 2). C) Blocking assay: DU145 were treated with scFv62-TRAIL, scFv62-TRAIL pre-incubated with anti-TRAIL antibody or scFv62-TRAIL pre-incubated with antigen, in all cases in the presence of CHX. Alternatively, cells were pre-incubated with scFv62 for 1 h and then treated with scFv62-TRAIL and CHX. As a control, DU145 cells were treated with scFv62 preparation. Apoptosis induction was analyzed with Annexin/PI staining and flow cytometry (n = 3). D) Proliferation assay: DU145 cells were treated with 50 U/ml scFv62-TRAIL, astemizole (4 μM), CHX (5 μg/ml) or etoposide (5 μM) and proliferation was measured after 24, 48 and 72 h (n = 3). E) Quantitative real-time PCR analysis of the four TRAIL receptors.
Figure 5
Figure 5
Influence of TRAIL receptors and KV10.1. A) DU145 cells were pre-incubated with anti-TRAIL-R1 antibody, anti-TRAIL-R2 antibody or a mixture of both for 2 h. Cells were then treated with scFv62-TRAIL in the presence of CHX (n = 2). B) DU145 cells were transfected with siRNA against TRAIL-R1 and/or TRAIL-R2 for 24 h and then treated with 50 U/ml scFv62-TRAIL in presence of 5 μg/ml CHX; apoptosis was measured by flow cytometry (n = 3). C) Quantitative real-time PCR analysis: DU145 cells were transfected with siRNA against TRAIL-R1 and/or TRAIL-R2 and analyzed for mRNA expression of death receptors. Quantitative real-time PCR analysis of D) TRAIL-R1, E) TRAIL-R2 and F) KV10.1 expression after chemotherapeutic treatment: doxorubicin (1.8 μM), etoposide (50 μM), roscovitine (10 μM), 5-fluorouracil (100 μM).
Figure 6
Figure 6
Bystander effect. Illustration of the scFv62-TRAIL bystander effect. Binding of scFv62-TRAIL to KV10.1 results in the membrane-bound form of TRAIL. Thereby, it is possible to induce apoptosis in the same cell (autocrine) or in a neighboring cell (paracrine) independently of KV10.1 expression. Targeting of KV10.1-negative cells is defined as bystander effect.
Figure 7
Figure 7
Determination of bystander effect. A) Upper panel. DU145 cells stably transfected with a vector expressing the fluorescent protein Venus to identify DU145 cells in mixed cultures. Two populations are clearly distinguishable by Venus fluorescence in mixed cultures. Lower panels. PC3 and PNT2 cells were co-cultured with DU145-venus cells and treated with 50 U/ml scFv62-TRAIL in the presence of 5 μg/ml CHX for 18 h. Apoptosis induction was analyzed with Annexin/PI staining and flow cytometry. Venus-positive (DU145 cells) are depicted in orange; green represents PC3/PNT2 cells (bystander cells). B) Quantification of the change in apoptosis of bystander cells was calculated.
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