Tumor Biol. (2015) 36:8137–8145 DOI 10.1007/s13277-015-3555-3
RESEARCH ARTICLE
A combinatory use of adenoviruses expressing melanoma differentiation-associated gene-7 and replication-competent adenoviruses produces synergistic effects on pancreatic carcinoma cells Guangyu Ma 1 & Boya Zhong 1,2,4 & Shinya Okamoto 2,3 & Yuanyuan Jiang 2,4 & Kiyoko Kawamura 2 & Hongdan Liu 2 & Quanhai Li 5,6 & Masato Shingyoji 7 & Ikuo Sekine 7 & Yuji Tada 3 & Koichiro Tatsumi 3 & Hideaki Shimada 8 & Kenzo Hiroshima 9 & Masatoshi Tagawa 2,4
Received: 16 March 2015 / Accepted: 11 May 2015 / Published online: 20 May 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Type 5 adenoviruses expressing mda-7 gene (Admda-7) induced cell death in various kinds of human tumors, but pancreatic carcinoma cells were relatively resistant to Admda-7-mediated cytotoxicity. We then examined whether infection of Ad-mda-7 together with replication-competent Ad produced combinatory cytotoxic effects. We prepared replication-competent Ad, defective of the E1B55kDa gene or activated by a transcriptional regulatory region of the midkine or the survivin gene of which the expression was up-regulated in human tumors. Type 5 Ad bearing the exogenous regulatory region were further modified by replacing the fiber-knob region with that of type 35 Ad. Pancreatic carcinoma cells were infected with replication-incompetent Ad-mda7 and the replication-competent Ad. Combinatory effects were examined with the CalcuSyn software and cell cycle analyses. Ad-mda-7 and the replication-competent Ad achieved cytotoxicity to pancreatic carcinoma. A combinatory use of Admda-7 and either Ad defective of the E1B55kDa gene or Ad
activated by the regulatory region produced synergistic cytotoxic effects. Cell cycle analyses demonstrated that the combination increased sub-G1 populations. These data collectively suggest that expression of MDA-7 augments cytotoxicity of replication-competent Ad and achieves adjuvant effects on Ad-mediated cell death.
Keywords Replication-competent adenovirus . MDA-7 . Pancreatic carcinoma . Cytotoxicity
Abbreviations Ad Ad-delE1B55 Ad-mda-7 Ad-LacZ
Adenoviruses Ad defective of E1B55kDa molecules Ad expressing the mda-7 gene Ad expressing the β-galactosidase gene
Guangyu Ma and Boya Zhong contributed equally to this work. * Masatoshi Tagawa
[email protected] 1
Department of Hematology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China
2
Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan
3
Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan
4
Department of Molecular Biology and Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan
5
Department of Immunology, Hebei Medical University, Shijiazhuang, China
6
Cell Therapy Center, The First Hospital of Hebei Medical University, Shijiazhuang, China
7
Department of Thoracic Diseases, Chiba Cancer Center, Chiba, Japan
8
Department of Surgery, School of Medicine, Toho University, Tokyo, Japan
9
Department of Pathology, Tokyo Women’s Medical University Yachiyo Medical Center, Yachiyo, Japan
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AdF AdF-MK AdF-Sur AdF-LacZ CAR CI Fa IL MDA-7 MK ROS Sur
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Type 5 Ad bearing type 35-derived fiberknob region AdF in which E1 gene is activated by MK regulatory region AdF in which E1 gene is activated by Sur regulatory region AdF expressing the β-galactosidase gene Coxsakie adenovirus receptor Combination index Fractions affected Interleukin Melanoma differentiation-associated gene-7 Midkine Reactive oxygen species Survivin
Introduction Pancreatic carcinoma remains one of the intractable diseases partly because of difficulty in the detection at an early stage and the invasiveness into adjacent vital organs [1]. Chemotherapy with gemcitabine and 5-fluorouracil derivatives is effective; however, a novel therapeutic strategy is further required to improve the prognosis. A gene medicine is one of the candidate agents and replication-competent adenoviruses (Ad), which produce the progenies in infected cells, achieved cytotoxicity in a manner different from conventional chemotherapeutic agents. Human interleukin (IL)-24 belongs to the IL-10 family and is regarded as one of the pro-inflammatory cytokines [2]. The cytokine is also known as a melanoma differentiationassociated gene-7 (MDA-7), and the expression was linked inversely with the pathological progression of melanocytes to melanoma [3]. Subsequent studies demonstrated that recombinant Ad expressing the mda-7 gene (Ad-mda-7) had a potent apoptosis-inducing activity in various types of human tumors but not in normal tissues. The anti-tumor effects were achieved through a number of non-immunological mechanisms including endoplasmic reticulum stress responses and induction of autophagy and/or apoptosis [3]. Moreover, reactive oxygen species (ROS) have an important role in the MDA-7-mediated cell signaling and cell death. Previous studies showed that pancreatic carcinoma was less susceptible to MDA-7/IL-24-induced apoptosis than other tumor types, but the cytotoxicity to pancreatic carcinoma was enhanced by different kinds of therapeutics such as pharmacologic and genetic agents targeting the K-ras mutations [3, 4]. Replication of Ad is primarily regulated by expression of immediately early E1 genes, which facilitate cell cycle progression of the infected cells and production of viral proteins necessary for producing viral progenies [5]. A regulatory region of the genes which are expressed in human tumor cells
but scarcely in normal tissues can activate a target gene preferentially in tumors. We demonstrated that the regulatory region of the survivin (Sur) gene and the midkine (MK) gene transactivated an exogenous gene in tumors [6, 7]. Replication-competent Ad in tumors are thereby produced by replacing the authentic Ad E1 promoter region with such a regulatory region. Previous studies demonstrated that both Sur and MK genes were expressed in human pancreatic carcinoma [8, 9] and indicated that replication-competent Ad activated by the Sur or the MK regulatory region produced cytotoxic effects in tumors. Another type of replication-competent Ad is one being defective of a region that interacts with cellular proteins. Ad defective of E1B55kDa molecules (AddelE1B55) is one of the agents which achieved cytotoxicity in human tumors [10]. The Ad-delE1B55 induced apoptotic cell death and produced synergistic effects with chemotherapeutic agents [11]. A number of subtypes are known in Ad, and repertoires of receptors are different among the subtypes. Type 5 and type 35 Ad use the coxsakie adenovirus receptor (CAR) and CD46 molecules as the major receptor for infection, respectively. Replacement of the Ad fiber-knob region, which is responsible for the binding to CAR or CD46 molecules, can change the Ad tropism between type 5 and type 35 species, and we can create type 5 Ad bearing type 35-derived fiber-knob region (AdF) [12]. These fiber-knob modifications can improve Ad infectivity when CD46 is expressed better than CAR molecules in target cells. Moreover, the fiber-knob-modified AdF have another advantage in infectivity. Ad infection downregulates the receptor expression on target cells and suppresses subsequent infection by Ad of the same type. Combination of different Ad types thereby does not inhibit the reciprocal infectivity, and in fact, type 5 Ad and AdF can transduce target cells without such interference caused by downregulated receptor expression [13]. In this study, we examined possible combinatory effects of Ad-mda-7 and replication-competent Ad on pancreatic carcinoma cells and influence of MDA-7 expression on Admediated cytotoxicity.
Materials and methods Cells Human pancreatic carcinoma cells, PANC-1, AsPC-1, MIAPaCa-2, and BxPC-3 (Cell Resource Center for Biomedical Research, Sendai, Japan), were cultured with RPMI 1640 supplemented with 10 % fetal calf serum. All of them are either p53-mutated (PANC-1, BxPC-3, and MIA-PaCa-2) or p53null (AsPC-1) in the genotype. Human embryonic kidney HEK 293 cells (American Type Culture Collection, Manassas,
Tumor Biol. (2015) 36:8137–8145
VA, USA) were cultured with in Dulbecco’s modified Eagle’s medium containing 10 % fetal calf serum. Ad preparation Replication-incompetent type 5 Ad-mda-7 or type 5 Ad expressing the β-galactosidase gene (Ad-LacZ) were prepared with an Adeno-X expression system (Takara, Shiga, Japan). Replication-competent type 5 Ad defective of the 55-kDaencoding E1B region (Ad-delE55) were also produced as described [11]. AdF, type 5 Ad modified with the receptor binding sites, were produced by replacing the fiber-knob region with that of type 35 Ad (Avior Therapeutic, Seattle, WA, USA). Replication-competent AdF in which E1 gene was activated by Sur (AdF-Sur) or MK regulatory region (AdF-MK) were constructed by replacing authentic E1 promoter region with 5′ regulatory sequences of the 0.5-kb human Sur [6] or the 0.6-kb human MK gene [7]. AdF expressing βgalactosidase gene (AdF-LacZ) were also produced as a control. These Ad were purified with an Adeno-X virus purification kit (BD Biosciences, San Jose, CA, USA), and the numbers of virus particles (vp) per milliliter were estimated with the formula, absorbance at 260 nm of purified Ad in the presence of 0.1 % sodium dodecyl sulfate×1.1×1012. In vitro cytotoxicity Cells (5×103/well) were seeded in 96-well plates and were cultured for 5 days with different amounts of Ad (vp/cell). Cell viability was determined with a cell-counting water soluble tetrazolium (WST) kit (Wako, Osaka, Japan). The amount of formazan produced was determined with the absorbance at 450 nm, and the relative viability was calculated based on the absorbance without any treatments. Combinatory effects were examined with CalcuSyn software (Biosoft, Cambridge, UK). Combination index (CI) values at respective fractions affected (Fa) points, which showed relative suppression levels of cell viability, were calculated based on the WST assay. CI1 indicate synergistic, additive, and antagonistic actions, respectively.
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Transcriptional activation Genomic fragments of a 5′ regulatory region of the Sur (0.5 kb) or the MK (0.6 kb) gene, which were used for Ad construction, were cloned into pGL-2 basic vector (Promega, Madison, WI, USA) that contained the firefly luciferase gene. Plasmid DNA containing the genomic fragments, pGLcontrol vector (Promega) harboring the SV40 T antigen promoter-linked firefly luciferase gene, or pGL-basic vector without any transcriptional regulatory regions, and the renilla luciferase gene fused with the herpes simplex virus-thymidine kinase gene promoter (pRL-TK, Promega), at a molar ratio of 10:1, was transfected into tumors with a Lipofectin reagent (Life Technologies, Gaithersburg, MD, USA). Cell lysate on day 2 was assayed for the luciferase activity with the dual luciferase reporter assay (Promega). The firefly luciferase activity was standardized by the amounts of luminescence produced by renilla luciferase, and the relative activity was expressed as a percentage of the SV40 T antigen promotermediated activity. Expression of Ad receptors Cells were stained with fluorescein isothiocyanate (FITC)conjugated anti-CD46 Ab (BD Bioscience, San Jose, CA) or were reacted with anti-CAR (Upstate, Lake Placid, NY, USA) followed by FITC-conjugated goat anti-mouse IgG Ab (Kirkegaard & Perry, Gaithersburg, MD, USA). They were then analyzed for the fluorescence intensity with FACS Calibur (BD Bioscience) and CellQuest software (BD Bioscience). Mean fluorescence intensity of the staining profile was expressed as an arbitrary FL1 unit, and the intensity was expressed as a percentage of that of HEK293 cells as a control. Cell cycle analysis Cells were treated with Ad were fixed in ice-cold 70 % ethanol, incubated with RNase (50 μg/ml), and stained with propidium iodide (50 μg/ml). The staining profiles were analyzed with FACSCalibur and CellQuest software (BD Biosciences).
Western blot analysis
Results
Cells were transduced with Ad-LacZ or Ad-mda-7 (6,000 vp/ cell) and the cell lysate were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was transferred to a nylon filter and was hybridized with antibody (Ab) against MDA-7 (Santa Cruz Biotech, Dallas, TX, USA) and actin (Sigma-Aldrich, St Louis, MO, USA). The membranes were developed with the ECL system (GE Healthcare, Buckinghamshire, UK).
Cytotoxicity of Ad-mda-7 and Ad-delE1B55 We examined cytotoxicity of replication-incompetent Admda-7 and replication-competent Ad-delE1B55 to four kinds of pancreatic cell lines, PANC-1, AsPC-1, BxPC-3, and MIAPaCa-2 (Fig. 1). The cytotoxicity of both Ad types was different among the cells tested. PANC-1 cells were more sensitive to Ad-delE1B55 than Ad-mda-7, but the other cells showed
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Fig. 1 Cytotoxicity of Ad-mda-7 and Ad-delE1B55 on pancreatic carcinoma. a Cells were infected with various doses of Ad-mda-7, AddelE1B55, or Ad-LacZ as a control, and the viability was assayed with the WST assay. The relative viability was calculated based on uninfected cells. Averages and the SE bars are shown (n=3). *P
