Scholarly article on topic '196. Adenoviral Delivery of Secretable Trimeric TRAIL in Combination with Conventional Chemotherapy for Brain Tumors Induces Synergistic Tumor Suppression and Improves Survival in an Intracranial Human Malignant Glioma Xenograft Mouse Model'

196. Adenoviral Delivery of Secretable Trimeric TRAIL in Combination with Conventional Chemotherapy for Brain Tumors Induces Synergistic Tumor Suppression and Improves Survival in an Intracranial Human Malignant Glioma Xenograft Mouse Model Academic research paper on "Biological sciences"

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Academic research paper on topic "196. Adenoviral Delivery of Secretable Trimeric TRAIL in Combination with Conventional Chemotherapy for Brain Tumors Induces Synergistic Tumor Suppression and Improves Survival in an Intracranial Human Malignant Glioma Xenograft Mouse Model"

overexpressed by infecting cells with an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC). A vector cariying LacZ (Ad-LacZ) was used as a negative control. Seventy two hours after infection of the virus vcctors at 20 MOI, apoptotic cclls were monitored by TUNEL method. To see whether activation of c-Jun terminal kinase (JNK) is causally linked to the induction of apoptosis, we examined effect of SP600126 (JNK inhibitor) on Ad-REIC-induced apoptosis. We determined the protein levels and phosphorylation state of JNK and other apoptosis-related proteins by Western blot analysis. NCCIT cells were subcutaneously injected into the right flank of nude mice. Three weeks after injection of the cells. Ad-REIC or Ad-LacZ in a 100 [il buffer was infected intratumorally. The size of tumors was measured every 3 or 4 days over 30 days after the infection. (Results) Expression of RElC/Dkk-3 was reduced in all the human seminoma and non-seminomatous germ eel I tumor tissues exam ined. Overexpression of REIC/Dkk-3 using Ad-REIC induced apoptosis in a testicular germ cell cancer cell line NCCIT but not in normal human fibroblasts. JNK was activated by Ad-REIC and the induction of apoptosis was abrogated by a JNK inhibitor. A single intrntu moral injection of Ad-REIC markedly inhibited tumorigenic growth of NC-C1T cells in nude mice (Fig. I), (Conclusion)These results indicate that Ad-REIC may lead to developing less insulting non-genotoxic therapeutic measures against human testicular cancer.

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195. Conditionally-Replicative Adenovaral Vectors with Bioluminescence-Based Replication Monitoring Capability for Lung Cancer

Julia Davydova. Eric J. Brown, Masato Yamamoto. 'Surgery, University of Minnesota, Minneapolis, MN.

The employment of Conditionally-replicative adenoviruses (CRAds) constitutes promising tools for cancer gene therapy. However, to maintain sarety and efficacy in clinical application, a noninvasive detection system of viral replication is required. In this study, we developed a novel CycIooxygenase-2 (Cox-2) promoter-controlled rcplicative agent with biolumincscencc-bascd replication monitoring capability and applied it to treat lung cancer. With exception of adenoviral death protein (ADP). most £3 genes in our reporter vectors were deleted or mutated and replaced with the firefly luciferase gene. We combined the E3-modified vector with the Cox-2 promoter-controlled EI expression cassette and designed six different structures such as configurations with and without ADP and vcctors equipped with different fibers to optimize therapeutic and monitoring vector features. The presence or absence of a poly-adenylation signal following the reporter gene has been examined for effect on stability of transgene expression. Cox-2 CRAds with the imaging cassette demonstrated increasing reporter expression and subsequent oncolytic effect in A549 lung cancer cells but not in Cox-2-negative A43I lung cancer cells in vitro, supporting the principle that the oncolytic effect of these CRAds depends on the Cox-2 promoter activity. The detected gene expression closely correlated with the viral DNA quantity resulting from viral replication. No expression or cytocidal effect was observed in mouse hepatoma BNL-ING-A.2 cells in which human adenoviruses do not productively replicate, indicating the dependence of reporter expression on productive replication. In vivo therapeutic data revealed that the Cox2CRAd_AE3_ADP_Luc vector significantly suppressed the tumor growth of established A549 Cox-2-positive xenografts and its therapeutic effect was as high as that of the fully replicative wild type virus. In contrast, the Cox-2-negative xenografts (A431) the same Cox2-bascd vector showed no antitumor effect while its signal was barely dctcctablc and comparable to the negative-contra I group treated with a non-replicativc lucifcrasc vector. Notably, live imaging allowed dynamic visualization of viral replication and viral spread in tumors, showing close correlation to therapeutic effect. These data demonstrate that our noninvasive CRAd replication monitoring system is applicable Tor further development of oncolytic therapeutic agents for lung cancer,

196. Adenoviral Delivery of Secretable Trimeric TRAIL in Combination with Conventional Chemotherapy for Brain Tumors induces Synergistic Tumor Suppression and Improves Survival in an Intracranial Human Malignant Glioma Xenograft Mouse Model

Moonsup Jeong,1 Yong-Sam Kvvon,1 Soon-Hye Park,' Min-Tae Park,1 Chae-Young Kim,' Kang-Won Son,3 Yong Ko,2 Paul D. Robbins,3 Dai-Wu Seol,4 Byong-Moon Kim.1 'Research Laboratories, Dong-A Pltarm. Co., Ltd, Yongin, Kyunggi, Republic of Korea; 'Neurosurgery, Hanyang University Medical Center, Sttngdong, Seoul, Republic of Korea;3Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; ^Surgery, University of Pittsburgh, Pittsburgh, PA.

Treatment of malignant gliomas is still a significant clinical challenge. The conventional therapies for malignant gliomas yield a median survival of only 9 months. TRAIL is a novel therapeutic candidate for treating a wide variety of cancers. In particular, a secreted, soluble trimeric TRAIL (stTRAIL) easily disseminates to nearby cancer cells and induces more extensive induction of apop-

Molecular Therapy Volume 15, Supplement I, May 2A07 Copyright © The American jwiciciy of Gene Therapy

tosis than [hat of full length transmembrane TRAIL. In (his study, we compared tumor suppressive activity of adenovirus mediated delivery of stTRAlL(Ad-stTRAIL) with l,3-bis(2-chloroethyl)-l-nifrosourea (BCNU) treatment, the conventional adjuvant therapy for malignant glioma and evaluated the anti-tumor efficacy of combining Ad-stTRAIL and BCNU. Although Ad-stTRAIL or BCNU alone induccd tumor-killing effects in vitro, some gliomas were resistant to each treatment. However, the combination of Ad-stTRAIL and BCNU reversed their resistance and enhanced tumor-killing effect induced in other sensitive gliomas. To examine the effects of stTRAI L and BCN U in vivo, a disease animal model was used where U87-MG ccils were injected into the brain of athymic mouse. The median survival ofxenografled mice was 38.0 ± 1.7 days. The in-tratumoral treatment with Ad-stTRAIL alone produced an increased survival (46.0 ± 3.4 day) as compared with systemically delivered BCNU (43.0 ± 3.2 day), and the combination of both treatments caused a longer growth delay and a significant increased survival (54.0 ± 5.0 day). MR imaging allowed the precise localization of tumors in brain and showed how the established tumors were suppressed. Through MR Imaging, Ad-stTRAIL alone or combination with BCNU induced significant suppression of tumor mass as compared with BCNU alone. In situ immunohistochcmical staining for TRAIL and Cyclin A and TUNEL staining showed the established tumors were derived from human malignant gliomas and the regression of Ad-stTRAIL treated tumor mass was due to the apoptotic cell death induced by expressed stTRAlL from Ad-stTRAIL. Our results suggest that Ad-stTRAIL treatment together with chemotherapy may achieve maximal tumor control and is a promising alternative or cooperative adjuvant therapy for malignant brain tumor.

197. mda-7 (INGN-241) Kills Chemoresistant Cancer Cells and Restores Chemosensitivity to Cisplatin

Began Gopalan,1 Carlos Rachcd,1 Daniel Pearson,1 Manish Shanker,1 Sunil Chada,3 Rajagopal Ramesh.1 'Thoracic and Cardiovascular Surgery, M. D. Anderson Cancer Center, Houston, TX; 2Research and Clinical Development, Introgen Therapeutics, Inc., Houston, TX,

Cisplatin (CDDP) is the mainstay chemodrug for cancer treatment that include cancer of the lung, breast and colon. However, development of chemotherapy-resistant tumor cells during treatment is a major problem in cancer therapy. Therefore, novel therapeutic strategies that can restore chemosensitivity and/or effectively kill drug resistant tumor cells are warranted. In the present study we have utilized a gene therapy strategy and investigated if the tumor supprcssor/eytokinc melanoma differentiation associated gcne-7 (mda-7) can kill chemoresistant tumor cells. Treatment ofCisplatin (CDDP)-sensitive (2008: IC50 = 30 |iM; CDDP-S) and resistant (2008/C13; IC50 = 125 ¡iM: CDDP-R) ovarian tumor cells with an adenovirus vector carrying the mda-7 gene (Ad-mda7; INGN-241) resulted in effective growth inhibition of both cell lines. However, CDDP-R cells were found to be at least two times more sensitive to Ad-mda7 than CDDP-S cells and demonstrated increased activation of proapototic markers. Molecular analysis for the enhanced sensitivity of CDDP-R cells to Ad-mda7 revealed no significant increase in the transduction efficiency or adenovirus receptor (CAR, aV[J3 oraVp5) expression compared to CDDP-S cells. A similar enhanced sensitivity to Ad-mda7 was observed in CDDP-R lung tumor (I I1437R) cells compared to CDDP-S (I I1437) cells. These results showed Ad-mda7 can effectively kill CDDP-R cells of both ovarian and lung origin. We next determined ifAd-mda7 can restore chemosensitivity to CDDP-R cells. Treatment of CDDP-R cells with Ad-mda7 (3000 vp/cell) plus CDDP (25 pM: IC50 = 125 pM for 2008/C13) resulted in a significant growth inhibition compared to cells that were treated with Ad-mda7 alone or CDDP alone (P

< 0.05: 87% inhibition). Additionally, the growth inhibitory effcct in Ad-mda7 plus CDDP-treated CDDP-R cells was synergistic compared to cells that were untreated or treated with CDDP (< 5%), Ad-luciferase (Ad-luc; <5%)), Ad-mda7 (12%), or Ad-luc plus CDDP (20%). The underlying mechanism by which Ad-mda7 (INGN-241) restores chemoscnsitivty is not known and is currently under investigation in the laboratory. Finally, our studies show that Ad-mda7 (INGN-241) effectively kills chcmorcsistant tumor cells when used as monotherapy and in combination with CDDP restores chemosensitivity to CDDP. Our findings are of clinical importance and warrant further investigation for clinical translation.

198. Engineering Cytosine Deaminase/Uracil Phosphoribosyltransferase Fusion for Improved Cancer Gene Therapy

Andressa Ardiani,1 Michi Fuchita,' Margaret E, Black.11 'School of Molecular Bioscience, Washington State University, Pullman, IVA;1Department of Pharmaceutical Sciences. Washington State University, Pullman, WA.

Suicide gene therapy is a particularly attractive cancer therapy because of its ability to localize toxicity to tumor cells. We are interested in the bacterial uracil phosphoribosyltransferase (UPRT) and its application in conjunction with the cytosine deaminase (CD)/5-fluorocytosinc (5FC) system to enhance tumor ablation. Cytosine deaminase (CD) is an enzyme responsible for deaminat-ing cytosine to form uracil. It also recognizes 5FC, an anti-fungal drug, and is able to convert 5FC into 5-Huorouracil (5FU), a highly toxic anti-cancer drug. UPRT is an important enzyme involved in the pyrim ¡dine salvage pathway, catalyzing the phosphorylation of uracil to uracil-monophosphate. The enzyme also phosphotylates 5FU to form 5FUMP which is then further catalyzed to antimetabolites by endogenous enzymes. Because the conversion of 5FC to 5FU by bCD has been shown to be rate limiting, our lab performed regio-specific random mutagenesis within the substrate binding site of bCD. From these series of experiments, we have successfully identified 3 bCD variants. Kinetic analysesofthe 3 variants suggest that substrate preference for 5FC is shifted by a decrease of normal substrate (cytosine) specificity of 100-fold to an increased specificity for 5FC by approximately 19-fold. In vitro cytotoxicity and bystander effect assays were performed in rat C6 glioma, HCT116 colorectal and DUI45 prostate cancer eel Is. fo viVro data revealed that cells stably transfected with these mutants have 3- to 18-fold lower ICfn values than wild type transfected cells. Experiments also showed that bCD mutants have significant increase in bystander activities compared to wild type bCD. It has been previously demonstrated that the fusion of CD with UPRT (CD/UPRT) along with 5FC imparts a greater tumor killing activity than CD alone. Therefore, to further enhance the production of cytotoxic compounds, we sought to incorporate improvements previously identified in bCD into new fusion constructs containing both bCD and UPRT activities. We hypothesize that such fusion enzymes will offer more efficient prodrug activation, confer significantly improved sensitivity toward 5FC and therefore, improved tumor ablation. The fusion constructs were evaluated for their cell killing effect and bystander effect in vitro. Mutants with exceptional prodrug converting properties will be beneficial because they allow administration of lower doses of 5FC, thereby minimizing side effects without the loss of potency. The use of such novel mutant fusion constructs will advance suicide gene therapy treatment for cancer and improve the likelihood of complete tumor ablation.

Molecular Tlierapj" Volume 15, Supptcmcw 1. May 2007 Copyriglii © The Aimncin Soeieiy of (itne Therapy