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Combinatorial Treatment with Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Gene and a Chemotherapeutic Agent Suppressed Hepatic Metastasis of Colon Carcinoma
CANCER TUMOR SUPPRESSOR GENE AND APOPTOSIS 942. Combinatorial Treatment with Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Gene and a Chemotherapeutic Agent Suppressed Hepatic Metastasis of Colon Carcinoma 1,2
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943. Loss of Neoplastic Phenotype by Specific Inhibition of RET Oncogene Signalling in a Transgenic Model of Primary Medullary Thyroid Carcinoma 2
Michiaki Ishii, Masaki Iwai, Yoshinori Harada, Jiro Imanishi, Osam Mazda,2 Takeshi Okanoue.1 1 Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Kyoto, Japan; 2 Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Kyoto, Japan.
As a therapeutic approach to eradicate highly malignant neoplasms including metastatic liver tumors, a powerful, specific and systemically applicable tool that directly destroys tumor cells is required. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various transformed cells, while most normal cells are unsusceptible to the TRAIL-mediated apoptosis induction. In the present study, we evaluated therapeutic efficacy of intravascular TRAIL gene therapy against multiple hepatic metastasis model in mice. A chemotherapeutic agent, actinomycin D, was also administered to assess whether the tumoricidal activity of the TRAIL is further improved by the combination therapy. First, recombinant human TRAIL (rTRAIL) was added to the CT26 (murine colon carcinoma) in culture in the presence or absence of 10 ng/ml actinomycin D, and cell proliferation and apoptosis were assessed in vitro by means of the tetrazolium and flow cytometric analyses. The treatment with rTRAIL alone failed to affect viability of CT-26 cells, while rTRAIL plus actinomycin D treatment effectively induced apoptosis in a dose dependent fashion. Antitumor effect in vivo of the TRAIL plus actinomycin D treatments was then investigated. pGEG.TRAIL was constructed so that the plasmid encoding the 3’ extracellular domain of human TRAIL fused with a secretory signal sequence. When the vector construct was intravenously transfected into mice, a considerable concentration of TRAIL molecule was detected in the sera of the animals, whereas delivery of a wildtype TRAIL construct did not result in a significant elevation of the genetic product in the murine sera. To establish liver metastasis, CT-26 cells were injected into the spleen of syngenic BALB/c mice (day 0). On days 1 and 8, mice received intravascular transfection with pGEG.TRAIL or pGEG.4 (a control plasmid), while actinomycin D was intraperitoneally injected into some groups of mice. The mice were sacrificed on day 14, when the numbers of metastatic nodules were revealed to be 63.8±31.5 (untreated), 49.4±21.0 (pGEG.4 plus actinomycin D), 52.3±32.7 (pGEG.TRAIL alone) and 14.8±17.3 (pGEG.TRAIL plus actinomycin D). The statistical analyses demonstrated that combination treatment significantly suppressed the liver metastasis in comparison with the untreated and pGEG.4 plus actinomycin D groups (p=0.015 and 0.021, respectively). All of the mice given injections with either the pGEG.sTRAIL or actinomycin D died within 44 days, which was not significantly longer than the longevity of control animals. In drastic contrast, 18% of the mice treated with the both agents survived for more than 90 days without any evidence of tumor progression. These results strongly suggest that the soluble TRAIL gene transfer and the administration with a chemotherapeutic agent synergistically elicit anti-tumor activities in vivo, and the combinatorial chemogene therapy may become an efficient and feasible means to suppress metastatic liver tumors.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
Matthias Drosten,1 Gero Hilken,2 Florian Rodicker,1 Miriam Bockmann,1 Carmen C. Theseling,1 Nikica Mise,1 Aaron N. Cranston,3 Andreja Frilling,4 Bruce A. Ponder,3 Brigitte M. Putzer.1 1 Department of Vectorology and Experimental Gene Therapy, University of Rostock Medical School, Rostock, Germany; 2 Central Animal Facility, University of Essen Medical School, Essen, Germany; 3Cancer Research UK Department of Oncology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; 4Department of General and Transplantation Surgery, University of Essen Medical School, Essen, Germany. Background: Dominant activating mutations in the RET protooncogene trigger the development of medullary thyroid carcinoma (MTC). These mutations render the RET receptor independent of ligand-induced stimulation and turn it into a dominant acting oncoprotein by inducing constitutive trans-autophosphorylation. Methods: To study effects related to endogenous RET autophosphorylation in MTC cells and orthotopic MTCs in RET transgenic mice, we constructed adenoviral vectors expressing a dominant-negative truncated form of the receptor (RETDTK). Results: Expression of RET DTK in MTC cells impaired the autophosphorylation activity leading to decreased cell survival. This effect was specific to oncogenic RET expressing cells as RETnegative cells did not exhibit any treatment related cytoxicity. To delineate mechanisms responsible for the observed effect, we analyzed downstream targets potentially implicated in RET mediated transformation. We show that phosphorylation of Akt and ERK, as the major contributors to RET triggered transformation, is abolished after RETDTK expression. RETDTK mediated inactivation of Akt and ERK signaling is further accompanied by reduced cell cycle progression (through downregulation of cyclin D1 and upregulation of p21CIP1/WAF1and p27KIP1) and stimulation of apoptosis (through downregulation of Bcl-2) in MTC derived cells. Furthermore, we demonstrate that tumor growth and maintenance of orthotopic MTCs in transgenic mice expressing oncogenic RET in their germline is significantly affected after inhibition of oncogenic RET through RETDTK expression (p<0,0001; two-sided unpaired Student’s t test). Conclusion: Our results indicate that targeted therapy by selective disruption of oncogenic RET signaling responsible for cellular transformation can be an effective new treatment for primary MTC in vivo. This work was supported by grant PU188/3-1/3-2/3-3 from the Deutsche Forschungsgemeinschaft.
944. The Histone Deacetylase Inhibitor Depsipeptide (FR901228) Enhances the Cytotoxic Activity of Ad5-TRAIL Against Human Prostate Tumor Cells Thomas S. Griffith,1 Jill Moore,1 Rebecca Van Oosten.1 1 Urology, University of Iowa, Iowa City, IA. The administration of genes into tumor sites in situ through the use of various gene delivery systems, such as non-replicative viral vectors, is becoming a viable alternative therapy for treating cancer. Most gene therapy protocols currently being investigated for cancer are designed to eradicate tumor cells directly with toxic genes or indirectly by using genes that elicit antitumor immune responses. Prostate cancer is a prime target for gene transfer therapy because, at present, local treatment protocols are effectively used. There are increasingly more clinical gene therapy trials underway and although S361
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943. Loss of Neoplastic Phenotype by Specific Inhibition of RET Oncogene Signalling in a Transgenic Model of Primary Medullary Thyroid Carcinoma 2
Michiaki Ishii, Masaki Iwai, Yoshinori Harada, Jiro Imanishi, Osam Mazda,2 Takeshi Okanoue.1 1 Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Kyoto, Japan; 2 Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Kyoto, Japan.
As a therapeutic approach to eradicate highly malignant neoplasms including metastatic liver tumors, a powerful, specific and systemically applicable tool that directly destroys tumor cells is required. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various transformed cells, while most normal cells are unsusceptible to the TRAIL-mediated apoptosis induction. In the present study, we evaluated therapeutic efficacy of intravascular TRAIL gene therapy against multiple hepatic metastasis model in mice. A chemotherapeutic agent, actinomycin D, was also administered to assess whether the tumoricidal activity of the TRAIL is further improved by the combination therapy. First, recombinant human TRAIL (rTRAIL) was added to the CT26 (murine colon carcinoma) in culture in the presence or absence of 10 ng/ml actinomycin D, and cell proliferation and apoptosis were assessed in vitro by means of the tetrazolium and flow cytometric analyses. The treatment with rTRAIL alone failed to affect viability of CT-26 cells, while rTRAIL plus actinomycin D treatment effectively induced apoptosis in a dose dependent fashion. Antitumor effect in vivo of the TRAIL plus actinomycin D treatments was then investigated. pGEG.TRAIL was constructed so that the plasmid encoding the 3’ extracellular domain of human TRAIL fused with a secretory signal sequence. When the vector construct was intravenously transfected into mice, a considerable concentration of TRAIL molecule was detected in the sera of the animals, whereas delivery of a wildtype TRAIL construct did not result in a significant elevation of the genetic product in the murine sera. To establish liver metastasis, CT-26 cells were injected into the spleen of syngenic BALB/c mice (day 0). On days 1 and 8, mice received intravascular transfection with pGEG.TRAIL or pGEG.4 (a control plasmid), while actinomycin D was intraperitoneally injected into some groups of mice. The mice were sacrificed on day 14, when the numbers of metastatic nodules were revealed to be 63.8±31.5 (untreated), 49.4±21.0 (pGEG.4 plus actinomycin D), 52.3±32.7 (pGEG.TRAIL alone) and 14.8±17.3 (pGEG.TRAIL plus actinomycin D). The statistical analyses demonstrated that combination treatment significantly suppressed the liver metastasis in comparison with the untreated and pGEG.4 plus actinomycin D groups (p=0.015 and 0.021, respectively). All of the mice given injections with either the pGEG.sTRAIL or actinomycin D died within 44 days, which was not significantly longer than the longevity of control animals. In drastic contrast, 18% of the mice treated with the both agents survived for more than 90 days without any evidence of tumor progression. These results strongly suggest that the soluble TRAIL gene transfer and the administration with a chemotherapeutic agent synergistically elicit anti-tumor activities in vivo, and the combinatorial chemogene therapy may become an efficient and feasible means to suppress metastatic liver tumors.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
Matthias Drosten,1 Gero Hilken,2 Florian Rodicker,1 Miriam Bockmann,1 Carmen C. Theseling,1 Nikica Mise,1 Aaron N. Cranston,3 Andreja Frilling,4 Bruce A. Ponder,3 Brigitte M. Putzer.1 1 Department of Vectorology and Experimental Gene Therapy, University of Rostock Medical School, Rostock, Germany; 2 Central Animal Facility, University of Essen Medical School, Essen, Germany; 3Cancer Research UK Department of Oncology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; 4Department of General and Transplantation Surgery, University of Essen Medical School, Essen, Germany. Background: Dominant activating mutations in the RET protooncogene trigger the development of medullary thyroid carcinoma (MTC). These mutations render the RET receptor independent of ligand-induced stimulation and turn it into a dominant acting oncoprotein by inducing constitutive trans-autophosphorylation. Methods: To study effects related to endogenous RET autophosphorylation in MTC cells and orthotopic MTCs in RET transgenic mice, we constructed adenoviral vectors expressing a dominant-negative truncated form of the receptor (RETDTK). Results: Expression of RET DTK in MTC cells impaired the autophosphorylation activity leading to decreased cell survival. This effect was specific to oncogenic RET expressing cells as RETnegative cells did not exhibit any treatment related cytoxicity. To delineate mechanisms responsible for the observed effect, we analyzed downstream targets potentially implicated in RET mediated transformation. We show that phosphorylation of Akt and ERK, as the major contributors to RET triggered transformation, is abolished after RETDTK expression. RETDTK mediated inactivation of Akt and ERK signaling is further accompanied by reduced cell cycle progression (through downregulation of cyclin D1 and upregulation of p21CIP1/WAF1and p27KIP1) and stimulation of apoptosis (through downregulation of Bcl-2) in MTC derived cells. Furthermore, we demonstrate that tumor growth and maintenance of orthotopic MTCs in transgenic mice expressing oncogenic RET in their germline is significantly affected after inhibition of oncogenic RET through RETDTK expression (p<0,0001; two-sided unpaired Student’s t test). Conclusion: Our results indicate that targeted therapy by selective disruption of oncogenic RET signaling responsible for cellular transformation can be an effective new treatment for primary MTC in vivo. This work was supported by grant PU188/3-1/3-2/3-3 from the Deutsche Forschungsgemeinschaft.
944. The Histone Deacetylase Inhibitor Depsipeptide (FR901228) Enhances the Cytotoxic Activity of Ad5-TRAIL Against Human Prostate Tumor Cells Thomas S. Griffith,1 Jill Moore,1 Rebecca Van Oosten.1 1 Urology, University of Iowa, Iowa City, IA. The administration of genes into tumor sites in situ through the use of various gene delivery systems, such as non-replicative viral vectors, is becoming a viable alternative therapy for treating cancer. Most gene therapy protocols currently being investigated for cancer are designed to eradicate tumor cells directly with toxic genes or indirectly by using genes that elicit antitumor immune responses. Prostate cancer is a prime target for gene transfer therapy because, at present, local treatment protocols are effectively used. There are increasingly more clinical gene therapy trials underway and although S361