Gene therapy with TRAIL against renal cell carcinoma

2165 Gene therapy with TRAIL against renal cell carcinoma Hiroki Matsubara,1 Yoichi Mizutani,1 suggest that TRAIL gene therapy and/or 5-FU may be Fumiya Hongo,1 Hiroyuki Nakanishi,1 effective against renal cell carcinoma without harmful Yasunori Kimura,1 So Ushijima,1 toxic effects. [Mol Cancer Ther 2006;5(9):2165 – 71] Akihiro Kawauchi,1 Takahiro Tamura,2 Tsuneaki Sakata,2 and Tsuneharu Miki1 Introduction 1 Department of Urology, Kyoto Prefectural University of There are only a few effective therapies against metastatic Medicine, Kyoto, Japan and 2Shionogi Institute for renal cell carcinoma. Immunotherapy including IFNs Medical Science, Osaka, Japan against metastatic renal cell carcinoma is relatively effective (1 – 4). Many investigators have tried to enhance effective- ness of IFNs by combination treatment with other Abstract Downloaded from http://aacrjournals.org/mct/article-pdf/5/9/2165/1873952/2165.pdf by guest on 10 June 2022 biological agents or chemotherapeutic agents (3, 4). Tumor necrosis factor – related apoptosis-inducing ligand However, these response rates were f15% to 20% (1 – 4). (TRAIL) induces apoptosis in cancer cells. However, TRAIL Furthermore, systemic side effects and high cost have is not toxic against most normal cells. We have accord- limited the clinical validity of immunotherapy using ingly examined by in vivo electroporation whether TRAIL recombinant cytokine proteins. The development of new induces apoptosis in renal cell carcinoma. In addition, strategies for patients with metastatic renal cell carcinoma combination treatment with TRAIL and 5-fluorouracil is therefore much to be desired. (5-FU) against renal cell carcinoma was also investigated. Tumor necrosis factor (TNF) – related apoptosis-inducing The NC65 renal cell carcinoma line was used as a target. ligand (TRAIL) belongs to the TNF family (5), and the pCAGGS TRAIL was injected into the NC65 tumors in the corresponding cellular receptors have been identified (6). right flanks of severe combined immunodeficient mice. TNF-a is the first molecule in the TNF family to be tested Tumors were pulsed with the CUY21 electroporator. for its anticancer activity, followed by Fas ligand. These Electroporation was done once on day 0 or thrice on days two molecules are efficient in killing a variety of cancer 0, 2, and 4. Apoptosis was determined by terminal cells. However, they cause significant damage to normal deoxyribonucleotide transferase – mediated nick-end label- tissues, resulting in life-threatening toxicities (7, 8). TRAIL ing assay. When TRAIL gene therapy using in vivo has been shown to selectively induce apoptosis in cancer i.t. electroporation was done once only, the growth of cells and has minimal or no toxicity against normal tissues, NC65 tumors was not inhibited. However, when TRAIL as examined both in vitro and in vivo in mice (9, 10). gene therapy was done thrice, growth suppression of the Therefore, TRAIL may be effective in vivo as an anticancer NC65 tumors was observed. Transfection of the TRAIL agent, provided the cancer cells are sensitive to it. gene by in vivo electroporation induced apoptosis in Unfortunately, most cancer cells, including renal cell NC65 tumors. When NC65 cells were treated with TRAIL carcinoma, are not sensitive to TRAIL-mediated apoptosis. gene therapy in combination with 5-FU, stronger growth Electroporation was originally developed for taking suppression was obtained. TRAIL gene therapy did not anticancer agents into tumor cells. It is also a well- induce liver dysfunction in severe combined immunodefi- established laboratory technique for the transfection of cient mice. This study shows that TRAIL gene therapy genes in vitro for floating and adherent cells (11). In vivo induced growth suppression and apoptosis in NC65 electroporation is also an efficient method of gene transfer tumors without severe side effects, and that combination (12). Thus far, this technique has been widely applied to treatment of NC65 cells with TRAIL gene therapy and various tissues. For example, the cytotoxic effects on renal 5-FU resulted in higher antitumor activity. These findings cell carcinoma of the combinations of anticancer drugs with electrical impulses were investigated both in vitro and in vivo (13). The efficacy of electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma Received 12/19/05; revised 6/19/06; accepted 7/11/06. was studied (14). Grant support: Grant-in-aids from the Japanese Ministry of Education, Several anticancer chemotherapeutic drugs, as well as Culture, Sports, Science and Technology nos. 18390439 and 18659479. TRAIL, mediate apoptosis and may share common intra- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked cellular signaling pathway leading to apoptosis. We have advertisement in accordance with 18 U.S.C. Section 1734 solely to reasoned that cancer cells that are resistant to TRAIL/drugs indicate this fact. can be sensitized by combined treatment with TRAIL and Requests for reprints: Yoichi Mizutani, Department of Urology, Kyoto anticancer drugs. Indeed, several studies tested the Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Phone: 81-75-251-5595; Fax: 81-75-251-5598; hypothesis and corroborated the drug-mediated sensitiza- E-mail: [email protected]. tion of resistant cancer cells to TRAIL (15, 16). This study Copyright C 2006 American Association for Cancer Research. has investigated in vivo whether renal cell carcinoma is doi:10.1158/1535-7163.MCT-05-0522 sensitive to TRAIL gene therapy, and whether the Mol Cancer Ther 2006;5(9). September 2006 2166 Gene Therapy with TRAIL resistance of renal cell carcinoma to TRAIL gene therapy/ was removed from the wells and replaced with 100 AL 5-fluorouracil (5-FU) can be overcome by a combination isopropanol (Sigma Chemical) supplemented with 0.05 N of TRAIL gene therapy and 5-FU. HCl. The absorbance of each well was measured with a microculture plate reader (Immunoreader, Japan Intermed Co., Ltd., Tokyo, Japan) at 540 nm. The percentage Materials and Methods cytotoxicity was calculated by following formula: percent- Tumor Cells age cytotoxicity = [1  (absorbance of experimental wells / The NC65 human renal cell carcinoma line was used as a absorbance of control wells)]
100. target. The cell line was maintained in RPMI 1640 (Life In vivo Study Technologies, Inc., Gaithersburg, MD) supplemented with Into the right flanks of the SCID mice, 6 million NC65 100 units/mL penicillin, 100 Ag/mL streptomycin (Life cells were s.c. injected with a mixture of 50 AL RPMI 1640 Technologies), and 10% fetal bovine serum (Life Technol- without antibiotics and serum, and 50 AL Matrigel (Becton ogies, Inc., Bio-cult, Glasgow, Scotland, United Kingdom). Dickinson, Franklin Lakes, NJ). At 7 days following Reagents injection of the tumor cells, the maximum lengths of the 5-FU was supplied by Kyowa Hakkou Co., Ltd. (Tokyo, Downloaded from http://aacrjournals.org/mct/article-pdf/5/9/2165/1873952/2165.pdf by guest on 10 June 2022 NC65 tumors had developed to 5 to 7 mm in diameter. Japan). Tumor sizes were measured at the indicated days after Plasmid initial treatment. Tumor diameters were scaled with a Human TRAIL cDNA was obtained by PCR with the use digital scalper. Tumor volume was calculated as follows: of a spleen cDNA library (Clontech, Palo Alto, CA). volume = a
b 2 / 2, where a is the long diameter and b is Amplified products were cloned into expression plasmid the short diameter. pCAGGS NEO containing the CAG promoter, forming I.t. or I.m. DNA Injection and Electroporation pCAGGS NEO-TRAIL, and subjected to analysis to verify Mice were anesthetized with pentobarbital sodium. its sequence. The plasmids used in this study were purified Established s.c. NC65 tumors were injected with 50 AL with a Qiagen (Valencia, CA) plasmid Maxi kit. The saline, pCAGGS NEO (50 AL), pCAGGS TRAIL (50 AL), amounts of endotoxin in purified plasmids were assayed and/or 10 Amol/L/50 AL of 5-FU. Electrode needles were by the Limulus test (Wako Pure Chemical Industries, Osaka, injected around the tumor, and electric pulses were Japan). The plasmids contained endotoxin at <3.0 EU/Ag. delivered with an electric pulse generator (CUY-21; Bex). Animal Care The shape of the pulse was a square wave. The electrodes Female severe combined immunodeficient (SCID) mice, consisted of tungsten needles, 10 mm in length and 0.4 mm ages 8 to 9 weeks, were purchased from CLEA Japan in diameter. The conditions for electroporation were as (Osaka, Japan) and housed in a specific pathogen-free follows: 33 V, 50 ms on, 950 ms off, eight times. Injec- animal facility. The animals were fed irradiated mouse tions and electroporation were done once (day 0) or thrice chow and autoclaved reverse osmosis – treated water. The (days 0, 2, and 4). In another experiment, 50 AL saline, Committee for Animal Research in Kyoto Prefectural pCAGGS NEO, and pCAGGS TRAIL was injected into University of Medicine permitted the experimental thigh muscle six times (days 0, 2, 4, 6, 8, and 10). procedure. Luciferase Assay In vitro Electroporation Protocol and Cytotoxicity Tumors were homogenized in 200 AL reporter lysis buffer Assay using a sonicator. After freezing and thawing twice, the The target was the NC65 cell line. Electric pulses were extract was centrifuged at 10,000 rpm for 10 minutes. delivered with an electric pulse generator (CUY-21; Bex, Luciferase activity in the supernatant was measured using Tokyo, Japan). The electrodes consisted of tungsten a Luciferase assay kit (Promega, Madison, WI) according to needles, 4 mm in gap and 32 mm in diameter (CUY-510; the protocol of the manufacturer. Photoemission was Bex). The conditions for electroporation were as follows: measured during a 10-second period using a luminometer. 100 V, 10 ms on, 90 ms off, 10 times. The 3-(4, 5- Protein concentration in the supernatant was measured as dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide described (19). (MTT) assay was used to determine tumor cell lysis as Evaluation of Liver Function previously described (17, 18). Briefly, 100 AL of the target To evaluate the hepatotoxicity of TRAIL gene therapy in cell suspension (2
104) were added to each well of 96-well this mouse model, serum alanine aminotransferase (ALT) flat-bottomed microtiter plates (Corning Glass Works, and serum aspartate aminotransferase (AST) were mea- Corning, NY), and each plate was incubated for 24 hours sured on day 7. at 37jC in a humidified 5% CO2 atmosphere. After Apoptosis Detection by Terminal Deoxyribonucleo- incubation, 100 AL of drug solution or complete medium tideTransferase ^ Mediated Nick-End Labeling Staining for control were distributed in the 96-well plates and each For terminal deoxyribonucleotide transferase – mediated plate was incubated for 72 hours at 37jC. Following nick-end labeling (TUNEL) staining, the NC65 tumors were incubation, 20 AL MTT working solution (5 mg/mL; Sigma fixed in 4% paraformaldehyde at 4jC for 24 hours, Chemical Co., St. Louis, MO) were added to each culture embedded in optimum cutting temperature compound, well and the cultures were incubated for 4 hours at 37jC in and frozen at 80jC. Serial 10-Am sections were made, and a humidified 5% CO2 atmosphere. The culture medium TUNEL staining was done using the in situ apoptosis Mol Cancer Ther 2006;5(9). September 2006  Molecular Cancer Therapeutics 2167 detection kit (Takara, Kyoto, Japan) according to the pro- Antitumor Effect of TRAIL Gene Therapy by In vivo tocol of the supplier. For fluorescent microscopic observa- I.t. Electroporation on NC65 Tumors tion, tumors were fixed in 2% glutaraldehyde at 4jC for Because in vivo electroporation increased the gene 2 hours, followed by washing with 0.1 mol/L phosphate transfection efficiency, the growth-inhibitory effect on buffer and immersion in 1% osmium tetroxide for 2 hours. NC65 cells was investigated using TRAIL plasmid. We Dehydrated in a graded series of ethanol, the specimen did in vitro TRAIL gene therapy by electroporation for was embedded in Spurr resin before staining of the sections NC65 cells and confirmed inhibition of cell proliferation. with uranyl acetate and lead citrate. Observation was made When TRAIL gene therapy using in vivo i.t. electroporation with a Hitachi 7000 fluorescent microscope set at 75 kV. was done once (day 0), the growth of the NC65 tumors was Statistical Analysis not inhibited significantly. However, when TRAIL gene For tumor size in vivo, results were expressed as mean F therapy was done thrice (days 0, 2, and 4), growth SE, and the Mann-Whitney U test was used for statistical suppression of NC65 tumors was observed (Fig. 2A). The analysis. For luciferase assay and differences in ALT and combination of the control plasmid and electroporation AST, the Fisher’s protected least significant difference test increased the tumor growth compared with the control, but was used to determine statistical significance. P V 0.05 was Downloaded from http://aacrjournals.org/mct/article-pdf/5/9/2165/1873952/2165.pdf by guest on 10 June 2022 the difference was not significant. considered significant in all statistical evaluations. Antitumor Effect of TRAIL Gene Therapy and 5-FU by In vivo Electroporation on NC65 Tumors Results In a previous study, we showed that treatment of established and freshly isolated renal cell carcinomas with Luciferase Assay a combination of TRAIL and 5-FU resulted in the At first, to confirm whether electroporation increases the potentiation of cytotoxicity and apoptosis and reversed gene transfection efficiency, luciferase assay was used in the their resistance (16). To obtain a more remarkable in vivo experiment. Electroporation was set up at 33 V, 50 ms on, therapeutic outcome, we combined TRAIL gene transfer 950 ms off, eight times. The plasmid was directly injected with an anticancer agent, 5-FU. When NC65 cells were into the NC65 tumors. Luciferase activity of the pCAGGS treated with TRAIL gene therapy in combination with Luc with in vivo electroporation was 10 times higher than 5-FU, the antitumor effect of the combination therapy was the pCAGGS Luc injection alone (Fig. 1). We also confirmed more evident than either gene therapy alone or chemo- the effect in Luciferase assay in vitro (data not shown). therapy alone (Fig. 2B). Cytotoxicity of TRAIL Gene Transfection against TUNEL Staining of TRAIL Gene ^ Transfected NC65 NC65 Cells by Electroporation In vitro Tumors We investigated the cytotoxicity of TRAIL gene transfec- To confirm the induction of apoptosis by TRAIL gene tion using electroporation against NC65 cells in vitro, and transfection, TUNEL staining was done. TUNEL staining the cytotoxicity was measured by MTT assay. The revealed that the TRAIL gene – injected tumors had cytotoxicity of pCAGGS TRAIL + electroporation was abundant apoptotic nuclei with double-strand DNA breaks 59.2 F 10.1% (mean F SD). The viability of the cells that (Fig. 3B), whereas apoptosis was not evident in the NC65 get electroporated with a control plasmid was slightly tumors treated with saline (Fig. 3A). reduced. Thus, a significant cytotoxicity of pCAGGS Effect of In vivo Electroporation of TRAIL Gene on TRAIL + electroporation was observed. These findings Serum ALTand AST Levels of SCID Mice Bearing NC65 suggested that electroporation is useful for TRAIL gene Tumors transfection in vitro. The hepatic dysfunction due to TRAIL has been detected in some studies (20, 21). Effects of TRAIL gene transfection into NC65 tumors on serum ALT and AST levels were examined. TRAIL gene therapy had no effect on the serum levels of ALT and AST in SCID mice (Fig. 4). Antitumor Effect of TRAIL Gene Therapy by In vivo I.m. Electroporation on NC65 Tumors Aiming at clinical application, the TRAIL gene was intro- duced into thigh muscle, and the cell growth-inhibitory effect was investigated. It is clinically easier to do in vivo i.m. electroporation, compared with i.t. electroporation. When TRAIL gene therapy using in vivo electroporation was done six times (days 0, 2, 4, 6, 8, 10, and 12), growth suppression of the NC65 tumors was observed. On day 28, residual tumors receiving electroporation with the TRAIL gene were significantly smaller than those of tumors Figure 1. Luciferase assay. Luciferase activity of pCAGGS Luc with receiving PBS, pCAGGS NEO, or pCAGGS TRAIL only in vivo electroporation was 10 times higher than pCAGGS Luc only. *, P < 0.05, significant difference between the pCAGGS Luc and all of (Fig. 5). No muscular cell death, such as muscular atrophy the other groups. and necrosis, was noted. Mol Cancer Ther 2006;5(9). September 2006 2168 Gene Therapy with TRAIL suggest the potential clinical application of TRAIL gene therapy and/or 5-FU in the treatment of renal cell carcinoma. Electroporation was used to introduce the TRAIL gene into NC65 cells in the present study. Reports on the use of this method of introducing genes into melanoma and hepatocellular carcinoma cells have been published (25, 26). In this method, cell membrane permeability is increased by applying electrical pulses to more efficiently incorporate drugs and plasmids into cells. It has been reported that the application of electrical pulses after the injection of plasmids into cells is more efficient than the reverse process. Therefore, we used this method in the present study. With this method, we investigated the inhibitory Downloaded from http://aacrjournals.org/mct/article-pdf/5/9/2165/1873952/2165.pdf by guest on 10 June 2022 effect of TRAIL on the proliferation of NC65 cells trans- planted into SCID mice. A more potent inhibitory effect was observed in the groups with TRAIL gene therapy than in the control group, and the effect was greater when TRAIL gene therapy was done thrice. These findings suggested that TRAIL gene therapy may be clinically applicable by electroporation. The combination of TRAIL and chemotherapeutic agents, such as 5-FU, cis-diamminedichloroplatinum, doxorubicin, and CPT-11, was shown to augment TRAIL-induced apoptosis in some human cancer cells, including renal cell carcinoma (27 – 30). This synergy was also observed in multidrug-resistant cell lines (31) or TRAIL-resistant cell lines (26, 31, 32). It has been proposed that chemothera- Figure 2. A, antitumor effect of TRAIL gene therapy by in vivo electroporation on NC65 tumors. Y axis, tumor volume, which was peutic agents augment TRAIL-induced apoptosis by up- calculated as length
width2 / 2; X axis, time of treatment. Measurement regulating DR5 (29, 30). Ionizing radiation may also was done every 7 d and the results were compared between the following enhance TRAIL-induced apoptosis by up-regulation of five groups: mice with tumors that received injection of PBS on day 0 (control; n = 6; n); mice with tumors that received injection of pCAGGS DR5 (33, 34). We then did combination therapy with TRAIL TRAIL alone on day 0 (n = 6; .); mice with tumors that received pCAGGS gene therapy and 5-FU, which has been used for renal cell TRAIL injection plus electroporation on day 0 (n = 6; o); mice with tumors carcinoma, to obtain a more potent antitumor effect. 5-FU that received injection of pCAGGS NEO, a control for the plasmid, plus enhanced the antitumor activity of TRAIL gene therapy. electroporation on days 0, 2, and 4 (n = 6; E); and mice with tumors that were injected with pCAGGS TRAIL plus electroporation on days 0, 2, and These findings suggest that the combination of TRAIL gene 4 (n = 6; 4). *, P < 0.05, significant difference in tumor volume at day therapy and 5-FU is effective against renal cell carcinoma. 35 was observed between the pCAGGS TRAIL + electroporation (three Previously, we have investigated the mechanisms of times) and all of the other groups. B, antitumor effect of TRAIL gene therapy and 5-FU by in vivo electroporation on NC65 tumors. Electro- synergistic cytotoxicity of TRAIL and 5-FU in vitro. When poration and injections were done thrice on days 0, 2, and 4 in all groups, Caki-1 renal cell carcinomas were treated with a combina- including the control (n = 6; PBS; 50 AL; E), pCAGGS NEO (50 AL; tion of 5-FU and TRAIL, 5-FU and 5-fluoro-2¶-deoxyuridine n = 6; n), pCAGGS TRAIL (50 AL; n = 6; 5), 5-FU (10 Amol/L/50 AL; n = 6; .), and 5-FU (10 Amol/L/50 AL) + pCAGGS TRAIL (n = 6; y) 5¶-monophosphate accumulation inside the cells increased groups. *, P < 0.05. Significant difference in tumor volume at day 28 was modestly. Because increased i.m. accumulation of 5-FU and observed between the pCAGGS TRAIL group and the control, pCAGGS 5-fluoro-2¶-deoxyuridine 5¶-monophosphate by TRAIL was NEO, and 5-FU groups. #, P < 0.05. The 5-FU + pCAGGS TRAIL group observed, we examined the effect of TRAIL on the showed more potent inhibition of cell proliferation than the pCAGGS TRAIL group and there was a significant difference in tumor volume at day expression of enzymes involved in the metabolism of 28 compared with all the other groups. 5-FU. When Caki-1 cells were treated with TRAIL, thymidylate synthesis and dihydropyrimidine dehydro- genase expression was slightly reduced, whereas the Discussion expression of oratate phosphoribosyltransferase was up- Reports on gene therapy using TRAIL have been published regulated. However, the treatment had no effect on (22 – 25), suggesting the great potential of this cytokine in thymidine phosphorylase expression. Although immuno- the development of more effective anticancer drugs with cytochemical analysis failed to detect p53 in the Caki-1 few adverse reactions. This study showed that TRAIL gene cells, p53 expression was observed after treatment with therapy was effective against renal cell carcinoma. It also 5-FU. Treatment of Caki-1 cells with 5-FU did not change showed that combination treatment with TRAIL gene the expression of bcl-2. However, bax expression in the therapy and 5-FU was more effective against renal cell Caki-1 cells was up-regulated following their treat- carcinoma and did not harm normal cells. These findings ment with 5-FU. These findings suggest that synergistic Mol Cancer Ther 2006;5(9). September 2006  Molecular Cancer Therapeutics 2169 Downloaded from http://aacrjournals.org/mct/article-pdf/5/9/2165/1873952/2165.pdf by guest on 10 June 2022 Figure 3. TUNEL staining of TRAIL gene transfected NC65 tumors. A, TUNEL-positive cells are not observed in the PBS-control group. B, TUNEL- positive cells can be observed in the pCAGGS TRAIL + electroporation (three times) group, which indicates that apoptosis has been induced. Arrows, TUNEL-positive cells. cytotoxicity of TRAIL and 5-FU might be due, in part, to the the tumors, but electroporation was done six times. There modified expression of at least some of these enzymes by was a significant difference in the inhibitory effect on TRAIL and the enhanced expression of the proapoptotic tumor growth between the thigh muscle gene incorporation p53 and bax proteins (16). The mechanisms responsible for group and the control group, indicating that this method enhanced cytotoxicity of TRAIL and 5-FU, however, remain also seems to be clinically applicable. We assume that unclear. Further studies are necessary to analyze the TRAIL protein was produced by plasmids introduced into mechanisms. the muscle cells and then induced apoptosis by binding to We introduced the TRAIL gene directly into the tumors the receptors on the tumor cells after being transported in the present study. However, this method might be via the blood, thus inhibiting tumor proliferation. When difficult to be applied in clinical practice. Therefore, we also TRAIL is administered to mice in vivo, TRAIL protein introduced the TRAIL gene into the thigh muscles of mice requires frequent administration (20). However, in gene and investigated the outcome. The method of introducing therapy with plasmids, treatment effects were observed genes into the thigh muscle was the same as that used for after direct injection into the tumor thrice or after i.m. Figure 5. Antitumor effect of TRAIL gene transfer by in vivo i.m. electroporation on NC65 tumors. As would be done in clinical practice, we introduced genes into the thigh muscles. Y axis, tumor volume, which was Figure 4. Effect of in vivo electroporation of TRAIL gene on serum AST calculated as length
width2 / 2; X axis, time of treatment. Measurement and ALT levels of SCID mice bearing NC65 tumors. Hepatic dysfunction was done every 4 d. Mice were treated with PBS + electroporation due to TRAIL has been reported; thus, we measured serum ALT and AST (n = 12; 4), pCAGGS NEO + electroporation (n = 12; y), pCAGGS levels in mice (n = 6) with transplanted tumors treated with PBS (control), TRAIL (n = 12; o), or pCAGGS TRAIL + electroporation (n = 12; n) on pCAGGS NEO + electroporation (ELP ), PBS + electroporation, pCAGGS days 0, 2, 4, 6, 8, 10, and 12, and the antitumor effect was evaluated. TRAIL, and pCAGGS TRAIL + electroporation to investigate hepatic *, P < 0.05. The pCAGGS TRAIL + electroporation group showed a dysfunction. As a result, no evidence of hepatic dysfunction was significant difference in tumor volume at day 28 compared with all of the observed. other groups. Mol Cancer Ther 2006;5(9). 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