Scholarly article on topic '194. Induction of Apoptosis in a Human Testicular Cancer Cell Line by Overexpression of REIC/Dkk-3 In Vitro and In Vivo'

194. Induction of Apoptosis in a Human Testicular Cancer Cell Line by Overexpression of REIC/Dkk-3 In Vitro and In Vivo Academic research paper on "Biological sciences"

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Academic research paper on topic "194. Induction of Apoptosis in a Human Testicular Cancer Cell Line by Overexpression of REIC/Dkk-3 In Vitro and In Vivo"

effects of Ad-INFp therapy and an Ad-vaccine, and should therefore be considered for use in clinical immunotherapy trials.

Cancer: Apoptosis & Suicide

192. Anti-Tumor Activity of Mesenchymal Stem Cells Producing IFN-a in a Mouse Melanoma Lung Metastasis Model

Changchun Ren,1 Sanjay Kumar,1 Diptiman Chanda,1 Selvaran-gan Ponnazhagan.1

'Pathology, The University of Alabama at Birmingham, Birmingham, AL.

Tumor metastasis is the leading cause of mortality in melanoma as it is in most malignant tumors. However, melanoma shows preferential metastasis to the brain, lung, liver, and skin. Type 1 interferons (ot/p) have significant antitumor activity but their clinical utilities have been limited by their short half-life and systemic side effects. Gene therapy is an alternative way to overcome these barriers. Cell-based gene therapy approach using mesenchymal stem cells (MSC) have been shown to be useful for the delivery of transgenes specifically to the target cells or tissues. In addition to thcirstem cell property for tissue regeneration, MSC represent cellular vehicles for delivery of therapeutic agents to tumor cells. In the present study, we evaluated the anti-tumor activity of mouse MSC, transduced with a recombinant adeno-associaied virus 6 vector (rAAV) expressing the murine interferon (IFN) a in a mouse melanoma lung metastases model, rAAV expressing mouse IFNa was constructed under the control of CMV/chicken fl-actin promoter. High-level expression and bioactivity of transgenic protein were confirmed by western blot and cell proliferation assays respectively. Lung metastasis model of melanoma was developed by intravenous injection ofSxlO'B I6FI0 cells in 6-8 week-old C57BL/6 mice. Ten days after the implantation of tumor cells, IxlO4 MSC, transduced with rAAV-IFNa or GFP or untransduced MSC were injected intravenously. The MSC injections were given two times. Cohorts of mice were sacrificed at different time points to determine the effects of the therapy. Results indicated that systcmic application of MSC-AAV-IFNa but not control MSC or that transduccd with rAAV-GFP strongly rcduccd the growth of B16F10 melanoma cells and significantly prolonged survival (P<0.05). The data demonstrate that rAAV-IFNa transduced MSC therapy can be used to reduce the growth of lung metastasis in melanoma.

193. Novel Adenoviral Vector Two-Step Transcription Amplification Expression System (TSTA) for Expressing CDK2-Associated Cell Cycle Regulators for Cancer Gene Therapy

Marxa L. Figueiredo,' May N. Chen,1 Makoto Sato,1 Russell Powell,1 Ewa Jaruga,2 Walter Rayford,2 Lily Wu.1 'Urology, UCLA, Los Angeles, CA;2Urology, LSU, New Orleans, LA,

Expression ofp27Lipl is commonly downregulated during prostate cancer progression by its increased targeting for ubiquitin protea-some degradation. Since p27 is a cyclin-dependent kinase 2 (CDK2) inhibitor that regulates G1/S cell cycle progression, we hypothesized that p27 can be used as a therapeutic gene strategy for inhibiting prostate canccr growth. In particular, a degradation-resistant mutant (TI87M/P188I; p27mt) may be more effective in promoting cell cycle arrest or apoptosis. We previously have utilized the TSTA system to efficiently amplify the expression of tissue-specific promoters to CMV levels, while maintaining prostate specificity. The TSTA system typically utilizes two separate Ads. one containing a prostate-specific promoter (PSE) driving the expression of an activa-

tor (GAL4-VP 16 or VP2), and another Ad containing the target gene downstream of 5 GAL4-binding sites (G5). We now have generated an all-in-one bidirectional TSTA vector containing the p27-G5-FL cassette in the ДЕ1 and the PSE-VP2 in the ДЕЗ region. This Ad directs more efficient, amplified, and specific prostate-targeted expression of p27/p27mt and FL reporter in a highly inducible manner following androgen treatment as assayed by p27 Western blot and luc activity assays in 22Rvl and LNCaP ccl Is. Analyses of protein stability over time following cycloheximide treatment suggested p27mt is more stable within transduced cells. Cell growth rate analyses by the CCK8 colorimelric assay in 22Rv] and LNCaP cells showed a higher growth inhibition for the AdTSTA-p27mt-FL virus (26-58%) compared to FL control, and although both p27 vectors significantly inhibited 22Rvl colony formation by 25-72% of control FL by day 16 in a colony formation assay, the p27mt Ad was more efficient. FACS analyses showed significant induction of a subGl fraction by 24h post-infection (pi) by both p27 Ads (-4.5%) compared to controls, and increasing over time in both LNCaP and 22Rv 1 (-16% by 96h pi). AnnexinV/PI analyses confirmed an increased percentage of early and late apoptotic cells following treatment with p27 Ads starting at 48h pi. The p27mt Ad induced a stronger G1 arrest, and further characterization will include BrdU/7AAD assays to confirm G l/S arrest by FACS. hi vivo studies are in progress using renilla luc marked prostate tumors to facilitate monitoring of tumor establishment. growth rate, magnitude and effectiveness of gene therapy by optical imaging. AdTSTA-p27FL-mediated gene expression will be monitored by optical FL imaging. Coupling prostate-targeted gene therapy with non-invasive imaging affords us a sensitive, real-time assessment of therapeutic activity in vivo, and possibly the ability to track or target metastasis. If succcssful, this promising prc-ciinical approach may be adapted for PET imaging and translated to clinical settings. OtherCDK2 regulators under exam ¡nation include p 12°ilfcil'pl and strategies are being developed and adapted for the treatment of head and neck cancers using the Ad vector systems described. Supported by R25CA098010: 1K01CA117921 (MLF) and IROl CA101904 (LW)

194. Induction of Apoptosis in a Human Testicular Cancer Cell Line by Overexpression of REIC/Dkk-3 In Vitro and In Vivo

Ryuta Tanimoto,1-2 Abarzua Fernando,1-2 Masakiyo Sakaguchi,2 Yasutomo Nasu,1 Hiromi Kumon,1 Nam-ho Huh.2 'Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistiy and Pharmaceutical Sciences, Okayama, Japan.

(Introduction and Object) REIC/Dkk-3 was originally isolated as a gene whose expression was reduced in immortalized human fibroblasts compared with that in normal counterparts, and subsequent analyses revealed that it is a candidate of tumor suppressor gene. Expression of REIC/Dkk-3 gene was reduced in many human cancer cells and tissues including prostate cancer (F, Abarzua et al. Cancer Res 65 (2005) 9617-9622), renal clear cell carcinoma(K. Kurosc el al. J Urol 171 (2004) 1314-1318). Forced expression of REIC/Dkk-3 using an adenovirus vector selectively induced apoptotic cell death in human prostate canccr ccll lines with marginal effect in normal epithelial and stromacells of prostate. In the present study, we examined potential utility of REIC/Dkk-3-based gene therapy against human testicular cancer. (Materials and Methods) A testicular carcinoma tissue microarray for seminoma and surgical resectcd tissues for non-scminoma were used for immunostaining for REIC/Dkk-3. For further studies on relevance of REIC/Dkk-3 to human testicular cancers, we chose a cell line derived from non-seminomatous germ cell tumor, NCCIT. REIC/Dkk-3 was

Molecular Therapy Volume 15, Supplement I. Мяу 2007 Copyright © ThL1 amlncui Society of Gl-пс Theripy

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.

| 1600 E

? 1200

Weeks after vector injection

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