The incidence of colorectal cancer is increasing and is associated with the fourth highest cancer associated mortality rate worldwide [
]. Despite advances in chemotherapy, radiotherapy and the development of new drugs, the prognosis for patients remains poor. Therefore, new targets and therapeutic substances are desperately needed [
One promising strategy may be targeted suicidal cancer gene therapy, including an approach by which foreign toxic molecules are specifically delivered to tumor cells [
]. Attractive candidates include bacterial toxins, which have demonstrated efficient cell-killing capacity in several in vitro and in vivo studies [
]. Pore-forming bacterial toxins, such as streptolysin O (
enterotoxin (CPE), are of particular interest [
an anaerobic gram-positive bacterium, produces the CPE protein, associated mainly with food poisoning [
]. The protein binds claudin-4 and claudin-3 on targeted cells [
]. The claudin family consists of at least 27 proteins that are essential for tight-junction formation in epithelial and endothelial cells and are important in controlling paracellular transport and the maintenance of cell polarity [
]. The binding of CPE to claudins triggers the formation of a multi-protein membrane pore complex, leading to a loss of cellular osmotic equilibrium and rapid cell lysis [
]. Cells that lack claudin-3 or -4 expression are unaffected by the toxin [
]. Numerous studies have shown that colon carcinoma and other epithelial tumors exhibit increased claudin-3 and/or -4 expression, suggesting that CPE might selectively target such tumors [
]. In our previous study we reported the successful tumor targeted in vitro and in vivo suicide gene therapy [
]. Based on this, the present approach is employing CPE gene therapy to selectively eradicate claudin-3 and -4 expressing colon carcinomas as a new strategy for this tumor entity.
Here, we use in vitro and in vivo approaches to demonstrate that claudin-3 and -4 expressing human colon cancers can be successfully treated by CPE gene transfer. CPE expression in these cells permits a rapid and selective eradication of colon cancer, further enhanced by a toxin-mediated bystander effect. We show that CPE specifically binds to the claudins in these cells and provide data on the kinetics of cytotoxicity as well as intracellular distribution of CPE after gene transfer. Our study reveals that CPE gene therapy can be used for the successful treatment of colon cancer.
Human SW480, SW620, HCT116 colon carcinoma and isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines were grown in RPMI medium (Gibco, Life technologies, Darmstadt, Germany), 10% FCS (Biochrom, Berlin,Germany). The colon carcinoma lines CaCo-2 and HT-29 were grown in DMEM (Gibco), 10% FCS (Biochrom). All lines were kept at 37 °C, 5% CO
. Claudin-3-YFP stably transfected cells were selected with 0.5–1.5 mgml
G418 (Gibco). The expression vector for Cldn-3-YFP is based on pEYFP-N1 [
]. The identity of all cell lines was confirmed by STR-genotyping (DSMZ, Braunschweig, Germany).
Quantitative real-time RT-PCR
Cell lysis and isolation of total RNA was done using GeneMatrix Universal RNA Purification Kit EURx (Roboklon, Berlin, Germany). 50 ng of RNA was reverse transcribed and real-time RT-PCR (qRT-PCR) was performed with SYBR GREEN. Each real-time PCR (qPCR) was done using the LightCycler 480 (Roche Diagnostics, Mannheim, Germany). Following primers were used for claudin-3: forward 5’-CTGCTCTGCTGCTCGTGTCC-3’; reverse 5’-TTAGACGTAGTCCTTGCGGTCGTAG-3’; for claudin-4: forward 5’-CCTCTGCCAGACCCATATAA-3’; reverse 5’-CACCGTGAGTCAGGAGATAA-3’. The cycle conditions were as follows: 90 °C for 30 s, 95 °C for 5 s, 57 °C for 5 s and 72 °C for 10 s for 45 cycles. Normalization was done with the human housekeeping gene glucose-6-phosphate dehydrogenase (hG6PDH) using the hG6PDH Roche Kit (Roche Diagnostics).
For protein analysis, cells or tissue cryosections were lysed in RIPA buffer (50 mM TRIS, 150 mM, NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, protease inhibitor, ddH
2O) and 25 μg of protein was electrophorezed in 10% precast NuPAGE gels (Invitrogen), 1 h at 180 V. The proteins were transferred to nitrocellulose membranes (Hybond-C Extra, Amersham, Freiburg, Germany) by semidry blotting (Turbo Blot BioRad, Munich, Germany) at 20 V, 25 min. Membranes were blocked for 1 h at room temperature (RT) in TBS (50 mM Tris, 150 mM NaCl, pH 7.5, 5% fat-free dry milk and 2.5% casein) and washed in TBST (0.05% Tween 20 in PBS), 2 x 5 min at RT. As primary antibody rabbit anti-claudin-3 antibody (1:3000, Acris, Herford, Germany), rabbit anti-claudin-4 antibody (1:3000, Acris), rabbit anti-CPE (1:4000, Acris), mouse monoclonal anti-β-tubulin (1:1000, BD Bioscience, MD, USA) or mouse monoclonal anti-β-actin antibody (1:10000, Sigma-Aldrich, MI, USA) was added respectively over night at 4 °C and washed in TBST. As secondary HRP-labeled goat anti-rabbit-IgG antibody (1:10000, Promega, Madison, WI, USA) or goat anti-mouse IgM/IgG (1:10000, Sigma-Aldrich) was added for 1 h, RT. Membranes were washed in TBST. Detection was done using ECL solution (Amersham) and exposure to Kodak X-Omat AR film (Kodak, Stuttgart, Germany).
Clostridium perfringens enterotoxin (CPE) expressing plasmids
For transfection experiments the pCpG-optCPE (optCPE) plasmid was used [
]. For construction of plasmid encoding optCPE-GFP fusion protein, cDNA of CPE was amplified by PCR from optCPE and cloned into the Pst I site of pcDNA3.1/CT-GFP (GFP Fusion TOPO TA Expression Kit, Invitrogen Life Technology) resulting in pcDNA3-optCPE-GFP (optCPE-GFP). Preparation of plasmid-DNA was done using the Jetstar Plasmid Purification Maxi Kit (Genomed, Löhre, Germany).
Transfection of human tumor cell lines
Cells were seeded into 6-well plates and transfected with 1.75–3.5 μg DNA (pCpG-optCPE, pCpG-mcsG2/pcDNA3.1 as empty vector or pcDNA3-optCPE-GFP fusion protein vector) using the transfection reagents Fugene X-treme (Roche), Fugene HD (Roche) or Metafectene (Biontex) as recommended by the manufacturer. To ensure comparable transfection rates, transfection efficiency for each cell line was determined by transfection of green fluorescent protein expressing plasmid pEGFP-N1 (Clontech, Mountain View, CA, USA) and analyzed using FACS Calibur (Becton Dickinson, San Jose, CA, USA). The number of green fluorescence protein expressing cells was quantified 48 h after transfection and given as a percentage of green fluorescent protein positive cells.
For knock-down experiments cells were seeded in 6-well plates and transfected with 50 nM siRNA control, siRNA Claudin-3 (iBONi siRNA box Cldn3 gene ID1363, Riboxx, Germany) or siRNA Claudin-4 (iBONi siRNA box Cldn4 gene ID1364, Riboxx, Germany) using Lipofectamine RNAiMax Reagent (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s instructions. Cells were seeded for recCPE treatment or optCPE gene transfer 48 h after siRNA transfection.
MTT cytotoxicity assay
MTT assay was performed to test cytotoxicity of recombinant CPE or after optCPE transfection and biological activity of released CPE from transfected cells. For sensitivity testing of the cell lines towards recombinant CPE 6 × 10
3- 4 × 10
5 cells were seeded into 96-well plates and 24 h later the toxin was added at different concentrations (0, 50, 100, 150 ng ml
-1) and incubated for 72 h. For determination of the biological activity of CPE in supernatants of transfected cells, 6 × 10
3 non-transfected cells were seeded into 96-well plates. After 24 h, 100 μl of supernatants from optCPE transfected cells were added to the respective non-transfected cells and incubated for 72 h. For all cytotoxicity assays MTT (3-(4,5-dimethylthiazyol-2yl)-2,5-diphenyltetrazolium bromide (5 mg ml-1, Sigma) was added after 72 h of CPE incubation and absorbance was measured in triplicates at 560 nm in a microplate reader (Tecan, Groedig, Austria). Values are expressed as percentage of untreated controls.
Clostridium perfringens Enterotoxin ELISA (R-Biopharm, Darmstadt, Germany) was performed to quantify CPE in supernatants 24 h or 48 h after transfection. For this, 4 × 10
5 cells were seeded into 6-well plates and transfected with pCpG-optCPE or pCpG-mscG2 (e.v.) plasmid-DNA. Supernatants were used for the detection as recommended by the manufacturer. For analyzing potential shedding of CPE into the blood of animals, which received gene transfer, blood was collected and CPE was quantified in serum samples. Recombinant CPE was used as standard at serial dilutions of 0.4 ng to 25 ng CPE ml
-1. Measurements were done in duplicates at 450 nm in the microplate reader (Tecan). Values are expressed as percentage of untreated controls.
Immunofluorescence and immunohistochemistry
For immunofluorescence, 2 × 10
5 cells were seeded onto cover slips (Steiner GmbH, Siegen Eiserfeld, Germany). After 24 h cells were treated with Hoechst 33342 (5 μM) or recombinant CPE (250 ng ml
-1, R-Biopharm) or were transfected with pcDNA3.1 as empty vector, pCpG-optCPE (optCPE) or pcDNA3-optCPE-GFP (optCPE-GFP) as described. Cells were washed with PBS, fixed 15 min in 3.7% (v/v) formaldehyde in PBS, quenched 20 min with 0.1 M glycin in PBS and blocked 1 h with 1% (w/v) bovine serum albumin and 0.05% Tween 20 in PBS at RT. As primary antibody, rabbit anti-human claudin-3 or rabbit anti- human claudin-4 antibody (1:100, Acris) or rabbit anti-CPE (1:1000, Acris) was added for 2 h at RT. Cells were washed with TBST. Alexa 488 labeled goat anti-rabbit-IgG (1:1000, Invitrogen) or Alexa 555 labeled goat anti-rabbit-IgG (1:1000, Invitrogen) was added as secondary antibody for 1 h at RT. For staining of nuclei DAPI (Sigma-Aldrich) and for staining of the cytoplasm Alexa 555 phalloidin (Thermo Fisher Scientific) was used. Cells were evaluated in a fluorescence microscope (Zeiss, Jena, Germany).
For immunohistochemistry of the patient-derived xenotransplant (PDX, EPO GmbH, Berlin, Germany) tumor samples 3–5 μm paraffin embedded tumor sections were deparaffinized, fixed with 0.04% glutaraldehyde for 15 min at RT, quenched 20 min with 0.1 M glycin, incubated 10 min with 3% H
2, washed with PBS, permeabilized by 0.2% Triton X-100 in PBS for 10 min, RT and blocked 1 h with 1% (w/v) bovine serum albumin and 0.05% Tween 20 in PBS at RT. Primary antibody rabbit anti-human claudin-3/-4 (1:200, Acris) was added for 2 h, RT. Sections were washed with PBS and as secondary antibody HRP-labeled goat anti-rabbit antibody (1:200, Promega) was added for 1 h, RT, then washed in PBS and incubated with diamino-benzidine (DAB, DAKO, Hamburg, Germany) 1 min, at RT, washed in PBS, counterstained for 30 s with hemalum (Roth, Karlsruhe, Germany), rinsed in water, covered with glycergel (DAKO) and evaluated in a light microscope (Zeiss).
in vivo optCPE gene transfer
For establishment of subcutaneous tumors, pieces of app. 3 × 3 mm in size of patient derived colorectal cancer xenograft tissue (PDX, Co7515*, lung metastasis of colon cancer, see Additional file
) were inoclulated into the left flank of female NMRI: nu/nu mice (
= 5 animals per group). When tumors reached a mean volume of 0.3 cm
, animals were randomized into treatment groups. Intratumoral non-viral optCPE gene transfer was performed in anesthetized animals by jet-injection. Therefore, 50 μg plasmid DNA of respective vector construct was applied by 5 injections (jet injector, EMS Medical Systems SA, Nyon, Switzerland) of 10 μl injection volume (1 μg DNA μl
PBS). The in vivo optCPE gene transfer gene transfer was performed once at day 46 post tumor inoculation.
Tumor volumes (TV) were measured at indicated time points and calculated using the formula: TV = (width
2 x length)/2. As toxicity parameters body weight, clinical signs and behavior were recorded for all mice twice a week. Animals were sacrificed and tumors were harvested for further analysis.
For statistical analyses of the in vitro experiments the Student’s t-test, 1way-ANOVA test and 2way-ANOVA test was used. For the analyses of in vivo optCPE gene transfer experiments the non-parametric Mann-Whitney test was used. Error values for the in vitro experiments are S.D. and for in vivo optCPE gene transfer experiment S.E.M.
We report for the first time the successful tumor-targeted CPE gene therapy for colon carcinoma in vitro and in vivo optCPE gene transfer. This approach demonstrates that CPE gene transfer could be a promising and efficient option for a targeted suicide gene therapy of claudin-3 and/or -4 expressing colon carcinoma.
The CPE gene therapy is of particular interest if applied locally to treat either unresectable residual cancer tissue or to treat unresectable or refractory liver or lung metastasis of colorectal cancers. In fact, we did use in our in vivo optCPE gene transfer experiment the Co7515 PDX model, which highly expresses claudin-3 and -4 and is derived from lung metastasis of colon cancer. This provides some hint that such approach might be of value for the local control of the disease. Therefore, this strategy could be of particular value for treatment of therapy-refractory tumors or metastases thereof.
We gratefully thank Iduna Fichtner, EPO GmbH Berlin, Germany, for supporting the in vivo optCPE gene transfer experiments and Jörg Piontek, Charité Campus Benjamin Franklin, Berlin, Germany, for providing the Cldn3-YFP expression vector for in vitro studies. We thank Renate Fischer, R-Biopharm, Darmstadt, Germany for providing the recombinant CPE for the in vitro studies. We thank for the support of Matthias Richter and Anje Sporbert, core facility of the Max-Delbrück-Center in performing all confocal microscopy analyses.
The concept of the study and the experimental work was funded by the Deutsche Krebshilfe (DKH110838), Germany and the Berliner Krebsgesellschaft, Berlin, Germany.
Availability of data and materials
All datasets are available from the corresponding author on reasonable request and IRB approval.
JP carried out experiments, analyzed data and prepared the manuscript. LM and NN carried out experiments and analyzed data. MR established the PDX models, was involved in in vivo optCPE gene transfer study design and data collection. JA supported in vitro experiments and DK generated expression vectors, was involved in study design and data interpretation. WW conceived experiments, designed study and was involved in data interpretation and manuscript writing and editing. All authors reviewed and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
All animal experimental procedures are performed in accordance with the in-house guidelines of the Institutional Animal Care and in accordance with the United Kingdom Coordinating Committee on Cancer Research regulations for the Welfare of Animals (Workman P. et al. Br. J. Cancer 2010; 102: 1555-77) and of the German Animal Protection Law and approved by the local responsible and official authorities, State Office of Health and Social Affairs (LaGeSo Berlin, Germany; Approval No. A0452/08). The animal care, housing and the experiments are performed in accordance to the ethical guidelines.