Experimental Research on the TrkA Gene Inhibition of Angiogenesis in Human Neuroblastoma

Objective This study was designed to investigate the feasibility of gene therapy for human neuroblastoma (NB) with the TrkA gene inhibition of tumor angiogenesis, growth and metastasis. Methods Three groups of cells including SY5Y, SY5Y-TrkA and SY5Y-Vec NB cells, were cultured by routine methods. Comparison of oncogenicity was performed among the three groups of cells. Tumor volume and angiogenesis in nude mice were also compared with VEGFmRNA expression (by RT-PCR analysis), immunohistochemistry and microvessel counting. Results The TrkA-transfected SY5Y NB cells showed significantly reduced oncogenicity and tumor angiogenesis. Tumor volumes were statistically different among the control, Empty-Vec and the experimental group, namely 1.736±0.485cm³, 1.803±0.751cm³ and 0.395±0.015cm³, respectively (P< 0.01). The difference of vascular endothelial growth factor (VEGF) expression between the experimental group and the control group was significant (P<0.01 ). Microvessel density (MVD) of the control, Empty-Vec and the experimental group were 27.21±14.58, 27.76±14.15 and 4.08±4.72 respectively, with statistical differences from the experimental group (P< 0.001). Conclusion The tumor angiogenesis and growth of NB were significantly inhibited by the TrkA gene. These studies provide a theoretical basis for application of NB antiangiogenesis gene therapy. This work was supported by National Nature Science Foundation (No. 39470739).     

靶向IGFIR的siRNA抑制人肝癌细胞SMMC7721裸鼠移植瘤的实验研究

Objective:To investigate the effect of insulin-like growth factor 1 receptor (IGF1R) small interfering RNA (siRNA)on the growth of human liver cancer SMMC7721 cell xenograft in nude mice.Methods:siRNAtargeting IGF1R was designed,and plasrnid SMMC7721-1GF1R-siRNA was constructed and transfected into SMMC7721 cells (SMMC7721-1GF1R-siRNAcells); the cells transfected with SMMC7721-1GF1 R-mutation (SMMC7721-1GF1 R-mutation cells) were used as negative control,and untransfected cells as empty control.Stable cell clones were screened by G418,and transplanted into nude mice to establish cancer xenograft.Tumor growth was monitored.Tumor morphology was observed with HE staining.The expression of IGF1R protein in tumor tissues was detected by Western blot.Microvessel density (MVD) in tumor tissues was detected by SP immunohistochemistry.Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay.Results:The tumor volume was significantly smaller in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups (P＜0.05).Necrosis and cell apoptosis were found in SMMC7721-1GF1R-siRNA group.The expression of tGF1R protein was significantly lower in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups (P＜0.05).MVD was significantly lower in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups (11.3±4.4 vs.36.7±7.6 and 28.4±6.5,P＜0.05).The apoptosis rate of tumor cells was significantly higher in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1 R-mutation and SMMC7721 groups [(50.2±6.4)% vs.(5.4±1.0)% or (6.0±2.1)%,P＜0.05].Conclusion:1GF1R siRNA can inhibit the growth of SMMC7721 cell xenograft in nude mice.     

放疗对胰腺癌细胞种植瘤血管生成的影响

Objective

The aim of this study was to observe the effect of radiation on micro-vessel density (MVD) and vascular endothelial growth factor (VEGF) expression in an transplanted tumor of human pancreatic cancer in nude mice and to explore the role of radiotherapy on pancreatic cancer angiogenesis.

Methods

After vaccinated pancreatic cancer PANC-1 cells, 20 nude mice were randomly divided into radiation group and control group. Radiation group received the radiation (dose of 20 Gy) and were killed 5 days later. The MVD and VEGF expression were determined by immunohistochemistry and then compared with the control group.

Results

The number of MVD in the radiation group was significantly lower than that of the control group (8.30 ± 4.55 vs. 13.60 ± 4.28, P < 0.01). The optical density (OD) of VEGF in the radiation group was higher (2.11 ± 0.54 vs. 1.32 ± 0.61, P < 0.05) compared with the control group.

Conclusion

The radiotherapy can reduce the number of vessels and increase the VEGF expression of human pancreatic cancer transplanted tumor in nude mice.     

Relationship belween Gene Expression of bFGF and Prolifercrtive Activity in Meningioma

Objective To study the relationship between bFGF gene expression and proliferative activity in meningiomas. Methods Thirty-seven samples of meningioma were examed using Northern hybridization for bFGF-gene expression and immunohistochemistry for bFGF protein expression and the Ki-67LI. Results bFGF mRNA was detected in 22 meningiomas of Grade I and in all samples of Grade II and III. Six of the Grade I tumors were negative, giving an overall positive rate of 83.8 % for bFGF mRNA. Autoradiography was conducted using a thin scaning apparatus. bFGF mRNA expression compared to that for β-actin was 0.34±0.06 for Grade I tumors compared to 0.82±0.12 for Grade II and III tumors. The difference was highly significant (P<0.001). The results of immunohistochemistry showed that the number of the positive cells for the bFGF protein was 29.7±7.6% in Grade I tumors and 63.2±11.7 % in Grade II plus III, resulting in a significant difference between them (P<0.001). The Ki-67LI was found to be 2.8+1.1 % in Grade I and 6.5±1.3 % in Grade II plus III. The former was significantly lower than the later (P<0.001). Conclusion The expression of bFGF was correlated well with the malignancy of the meningiomas.     

Discussion of modifying stage IV gastric cancer based on Borrmann classification

This study aims to investigate the prognostic significance of reclassification of stage IV gastric cancers in conjunction with Borrmann type. A total of 1,673 gastric cancer patients who received a gastrectomy between 1980 and 2003 were retrospectively evaluated. Of the patients, 244 (14.58 %), 344 (20.56 %), 589 (35.21 %), and 496 (29.65 %) had stage I, II, III, and IV cancers, respectively. After Cox regression analysis, Borrmann type was identified to be the independent prognostic factor in stage IV gastric cancer. The disease-specific postoperative survival of patients with Borrmann I, II, and III tumors was clearly distinguished by TNM classification system (P?<?0.05), while it failed to classify Borrmann IV tumors (P?=?0.147). Interestingly, the disease-specific postoperative survival of stage IV patients with Borrmann IV tumors (group 1) was significantly poor than the cases with stage IV but not Borrmann IV tumors (group 2), as well as the patients with Borrmann IV while not included in stage IV tumors (group 3) (P?=?0.022 and P?=?0.000, respectively). Meanwhile, the disease-specific postoperative survival was not observed as significantly different between group 2 and group 3 (P?=?0.063); furthermore, group 2?+?3 had a better prognosis than group 1 (introduced stage IVa vs. stage IVb; P?=?0.006). Reclassification of stage IV through combining the present TNM classification system with Borrmann type may more accurately predict the prognosis of patients.     

RETROVIRAL-MEDIATED SUICIDE GENE THERAPY OF EXPERIMENTAL GLIOMA

Ｍａｌｉｇｎａｎｔｇｌｉｏｍａ，ｔｈｅｍｏｓｔｃｏｍｍｏｎｉｎｔｒａｃｒａｎｉａｌｔｕｍｏｒ，ｉｓｏｎｅｏｆｔｈｅｍｏｓｔｌｅｔｈａｌｃａｎｃｅｒｓｉｎｈｕｍａｎ．Ｔｈｅｍｅｄｉａｎｓｕｒｖｉｖａｌｔｉｍｅａｆｔｅｒｄｉａｇｎｏｓｉｓｉｓｌｅｓｔｈａ...     

裸鼠肺癌移植瘤中NRP-1的表达及其意义

Objective:To establish angiogenesis model of xenografts of lung cancer cell in nude mouse and investigate the expression of the neuropilin-1 (NRP-1) protein in tumors and its role in progression and angiogenesis of lung cancer.Methods:Human lung adenocarcinoma cells A549 were analyzed for the expression of vascular endothelial growth factor165(VEGF165)mRNA using RT-PCR in vitro.TWo groups of nude mice were subcutaneously inoculated with A549 at different tumor-loading time.Two groups of xenografts were jdentified by hematoxylin and eosin (HE) staining.their microvessel density (MVD) were analyzed meanwhile.Two groups were analyzed for the expression of NRP-1 protein and their mean absorbency by using immunohistochemistry and automatic image analysis system respectively.Results:A549 expressed VEGF165 mRNA,and xenografts of A549 in nude mice were successfully established and confirmed by HE staining.The atypia of cancer cells and angiogenesis were occurred in two groups.Two groups of MVD were 13.06±1158.23.61±3.11(vessels/mm2)(P＜0.01).NRP-1 protein was expressed in cytoplasm of vascular endothelium cells and partial tumor cells.Two groups of mean absorbency of NRP-1 were 0.1095±O.0228,0.1784±0.0151 (P＜0.01).Conclusion:The angioqenesis models of xenografts in nude mice with lung cancer cell A549 expressing VEGF165 mRNA at different tumor-loading times were established successfully.The expression of NRP-1 protein and MVD were increased with the tumor progression.Our results demonstrate that NRP-1 protein in lung cancer is related to angiogenesis.     

Lentivirus-mediated CD/TK fusion gene transfection neural stem cell therapy for C6 glioblastoma

A suicide gene can convert nontoxic prodrugs into toxic products to kill tumor cells. In this study, our aim was to transfect lentivirus-mediated CD/TK fusion gene into Wistar rat’s neural stem cells (NSC) and then implant the NSC into a C6 glioma model to observe a C6 glioma growth inhibition effect. Primary NSC and stable transfection CD/TK fusion gene cell lines were established. To observe the tumor size and rat survival period in different groups, C6 glioma cell apoptosis and cell viability rate were applied to analyze the tumor inhibition effect of the neural stem cells’ transfected CD/TK fusion gene. C6 cell viability showed that CDglyTK-NSC + GCV/5-Fc (group 1) was lower than CDglyTK-NSC (group 2), NSC + GCV/5-Fc (group 3), and control (group 4) from day 2 (p?<?0.05), and the apoptosis rate was higher in group 1 compared with that of other groups (50.6 %, p?<?0.05) either in vitro or in vivo (35.47 %, p?<?0.05); both cell viability and apoptosis had no significance in the other three groups. In vivo, tumor size in group 1 was 7.76?±?1.37 mm³, which is smaller than the others (group2 27.28?±?4.11 mm³, group3 27.94?±?2.08 and 28.61?±?2.97 mm³; p?<?0.05). The other groups’ tumor size was not significant (p?>?0.05). Survival time of rats treated with CDglyTK-NSC + GCV/5-Fc (group 1) was significantly longer than that of the other groups (p?<?0.05; group 1 48.86?±?1.97, group 2 28.67?±?3.75, group 3 31.5?±?1.27, group 4 29.3?±?1.33). We also showed that the transfected C6 cells had a migratory capacity toward gliomas in vivo. Transfected CD/TK fusion gene neural stem cells combined with propyl–guanosine and 5-flucytosine double prodrug significantly inhibit the development of glioma.     

Effects of a selective cyclooxygenase-1 inhibitor in SKOV-3 ovarian carcinoma xenograft-bearing mice

To evaluate the effect of a cyclooxygenase-1 (COX-1) inhibitor, SC-560, on the growth inhibition of s.c. human ovarian SKOV-3 carcinoma and on angiogenesis. Human ovarian SKOV-3 carcinoma cells xenograft-bearing mice were treated with SC-560, a COX-1-selective inhibitor, 6 mg/kg alone i.g. daily, and i.p. injections of cisplatin 3 mg/kg every other day for 21 days. Prostaglandin E₂ (PGE₂) levels were determined by ELISA. Microvessel density (MVD) of ovarian carcinoma was determined with anti-CD₃₄ as the label by immunohistochemistry. In addition, the expression of COX-1 at protein levels in the control group was detected by immunohistochemistry. SC-560 reduced the growth of tumors when SKOV-3 cells were xenografted in nude female mice. The inhibitory rates in SC-560 group and cisplatin group were 47.1% and 51.7%, respectively, which is significant statistically compared to that of control group (all, P < 0.05). In treatment groups, SC-560 significantly reduced intratumor PGE₂ levels (P < 0.01). MVDs in SC-560 group were 35.73 ± 9.87, which are significant statistically compared to that of control group (74.33 ± 9.50) (P < 0.01). COX-1, not COX-2, protein levels are elevated in tumor tissues. These findings may implicate COX-1 as a suitable target for the treatment of ovarian cancer and that antiangiogenic therapy can be used to inhibit ovarian cancer growth.     

Prognostic significance of serum superoxide dismutase activity in patients with gastric cancer

Background: Although superoxide dismutase (SOD) may play an important role in helping to protect against carcinogenesis or tumor progression, little information is available regarding the clinical evaluation of antioxidant defense in patients with gastric cancer. Methods: Serum SOD activity in 34 patients with gastric cancer was estimated and the data compared with clinicopathological parameters. Results: The mean serum SOD activity in the patients was 15.9% ± 2.6%, which was higher than the value obtained in healthy donors. The serum SOD activity in patients over 70 years of age was 14.5% ± 3.0%, which was significantly lower than the value of 16.6% ± 7.3% in those under 70 years of age (P < 0.01). According to the stage of disease, the reduction in SOD activity in the patients aged over 70 years was significant in those with far advanced tumor, classified as stage IV (P < 0.01), but not in those with stage I–III disease. When the cutoff value for high- and low-SOD groups was determined as 18.0%, based on the median value for serum SOD activity in 15 patients with stage IV gastric cancer, the survival rate of patients with stage IV tumor was significantly higher in the high-SOD group than in the low-SOD group (P < 0.05). Conclusion: These findings suggested that the reduction in serum SOD activity in elderly patients may be due to weakness of the antioxidant defense of the host, thus resulting in a poor prognosis in those with far-advanced (stage IV) gastric cancer. Received: December 11, 2001 / Accepted: April 23, 2002 Acknowledgments This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (Frontier Research Grant and Grants for Science Research 12770677). Offprint requests to: M. Yasuda     

Radiofrequency ablation combined with liposomal quercetin to increase tumour destruction by modulation of heat shock protein production in a small animal model

Purpose: To investigate the effect of heat shock protein (HSP) modulation on tumour coagulation by combining radiofrequency (RF) ablation with adjuvant liposomal quercetin and/or doxorubicin in a rat tumour model.

Methods: Sixty R3230 breast adenocarcinoma tumours/animals were used in this IACUC-approved study. Initially, 60 tumours (n?=?6, each subgroup) were randomised into five groups: (1) RF alone, (2) intravenous (IV) liposomal quercetin alone (1?mg/kg), (3) IV liposomal quercetin followed 24?h later with RF, (4) RF followed 15?min later by IV liposomal doxorubicin (8?mg/kg), (5) IV liposomal quercetin 24?h before RF followed by IV liposomal doxorubicin 15?min post-ablation. Animals were sacrificed 4 or 24?h post-treatment and gross coagulation diameters were compared. Next, immunohistochemistry staining was performed for Hsp70 and cleaved caspase-3 expression. Comparisons were performed by using Student t-tests or ANOVA.

Results: Combination RF-quercetin significantly increased coagulation size compared with either RF or liposomal quercetin alone (13.1?±?0.7?mm vs. 8.8?±?1.2?mm or 2.3?±?1.3?mm, respectively, P?<?0.001 for all comparisons). Triple therapy (quercetin-RF-doxorubicin) showed larger coagulation diameter (14.5?±?1.0?mm) at 24?h than quercetin-RF (P?=?0.016) or RF-doxorubicin (13.2?±?1.3?mm, P?=?0.042). Combination quercetin-RF decreased Hsp70 expression compared with RF alone at both 4?h (percentage of stained cells/hpf 22.4?±?13.9% vs. 38.8?±?16.1%, P?<?0.03) and 24?h (45.2?±?10.5% vs. 81.1?±?3.6%, P?<?0.001). Quercetin-RF increased cleaved caspase-3 expression at both 4?h (percentage of stained cells/hpf 50.7?±?13.4% vs. 41.9?±?15.1%, P?<?0.03) and 24?h (37.4?±?7.8% vs. 33.2?±?6.5%, P?=?0.045); with, triple therapy (quercetin-RF-doxorubicin) resulting in the highest levels of apoptosis (45.1?±?10.7%) at 24?h. Similar trends were observed for rim thickness.

Conclusions: Suppression of HSP production using adjuvant liposomal quercetin can increase apoptosis and improve RF ablation-induced tumour destruction. Further increases in tumour coagulation can be seen including an additional anti-tumour adjuvant agent such as liposomal doxorubicin.     

NUCLEOLAR ORGANIZER REGIONS IN CUTANEOUS T CELL LYMPHOMAS

The present work studied the application of AgNOR count to differential diagnosis between cutaneous T cell lymphoma (CTCL) and cutaneous pseudolymphoma (CPL). Paraffin sections from 50 mycosis fungoides (22 MFI-Premycotic stage, 24 MF Ⅰ infiltrative stage and 4 MF Ⅲ - tumor stage), 2 nonepidermotropic cutaneous T cell lymphoma (NECTCL) and 9 CPL were investigated. In each case, 200 cells randomly selected were examined using a × 100 oil immersion lens. The mean number, standard deviation and standard error of the mean of AgNOR counts were as follows: MFⅠ 1.17±0.09, SEM = 0.01; MⅡ 1.17±0.01, SEM = 0.01; MF Ⅲ. 3.55±0.87, SEM = 0.43; NECTCL 4.5±0.28, SEM -0.199; CPL 1.17±0.1, SEM ± 0.03. The results revealed a highly significant difference between CTCL (MFⅢ NECTCL) and CPL (t = 4.75, P<0.001), tumor stage (MF Ⅲ) and pretumor stage (MFI, MF Ⅱ) of mycosis fungoides (t = 4.75, P<0.001). Thus. AgNOR count is valuable in differential diagnosis.     

MicroRNAs 182 and 375 Sera Expression as Prognostic Biochemical Markers in Breast Cancer

Introduction

Breast cancer (BC) is the most common malignancy among women; supporting the need for identification of novel prognostic biomarkers, circulating microRNAs (miRNAs) could serve as such in various cancers. The aim of this study was to explore the association between miRNAs 182 and 375 with BC stages and its receptors, based on their expression using real time PCR.

Relationship among COX-2 protein expression,PGs levels and biologic behavior in ovarian carcinoma tissues

Recent epidemiological studies shows thatlong-term use of aspirin or other NSAIDs (non-steroidanti-inflammatory drugs, NSAIDs) reduces the risk ofcolon cancer development by 40% — 60%[1,2].Although the exact mechanisms of NSAIDs on cancerprevention have not been clarified, one of them maybe via the inhibition of COX-2 enzyme. In the presentstudy, we investigated the COX-2 protein,prostaglandin (PG)E2, 6-keto-PGF1α and thromboxane(TX)B2 by Western blot analysis and radio-immunoass…     

Prognostic factors and long‐term outcomes of childhood nasopharyngeal carcinoma

BACKGROUND:

The authors studied the survival and long‐term morbidities of children with nasopharyngeal carcinoma (NPC).

METHODS:

This was a retrospective review of children with NPC who were treated at St. Jude Children's Research Hospital between 1961 and 2004. Prognostic factors and long term effects of therapy were analyzed.

RESULTS:

Fifty‐nine patients (median age, 14.1 years) were identified. Most were male (66.1%) and black (54.2%) and had lymphoepithelioma (93.2%). Thirty‐five patients had stage IV disease (59.3%), 20 patients had stage III disease (33.9%), and 4 patients had stage II disease (6.8%). All patients received radiotherapy (RT) to the primary tumor, and most received cervical RT (98.3%) and chemotherapy (88.1%). The 15‐year survival and event‐free survival (EFS) rates were 67.2% ± 7.5% and 63.5% ± 7.8%, respectively. Five patients (8.5%) developed subsequent malignancies 8.6 to 27 years after NPC diagnosis. EFS was improved in patients who were diagnosed after 1980 (74.8% ± 10% vs 45.5% ± 10.1%; P = .031), in patients who had stage III disease compared with patients who had stage IV disease (79.3% ± 9.6% vs 56.2% ± 11.8%; P = .049), in patients who received cisplatin (81% ± 10.7% vs 45.8% ± 9.7%; P = .013), and in patients who received ≥50 grays of RT (71.4% ± 9.3% vs 43.8% ± 11.6%; P = .048). White patients had higher distant failure rates than black patients (41.7% ± 10.4% vs 15.6 ± 6.5%; P = .045). The 15‐year cumulative incidence (CI) of any morbidity was 83.7% ± 5.4%, the CI of sensorineural hearing loss was 52.9% ± 6.7%, the CI of primary hypothyroidism was 42.7% ± 6.6%, and the CI of growth hormone deficiency (GHD) was 14.1% ± 4.7%. Dose‐response relations were observed between the RT dose and primary hypothyroidism and GHD.

CONCLUSIONS:

The outcome of children with NPC improved over the past 4 decades with the use of cisplatin‐based chemotherapy and higher RT doses. However, many survivors had long‐term treatment‐related morbidities. Cancer 2011. © 2010 American Cancer Society.     

Ki-ras mutation and cell proliferation of lung lesions induced by 1-nitropyrene in A/J mice

In this study, lung lesions were found in male A/J mice 24 wk after intraperitoneal injection of 1-nitropyrene (1-NP). The lesions were classified into three categories: alveolar/bronchiolar hyperplasia, adenoma, and adenocarcinoma. The proliferation kinetics of cells in the lesions were evaluated by assessing proliferating cell nuclear antigen (PCNA) expression and silver-staining nucleolar organizer regions (AgNORs). Furthermore, the role of the Ki-ras gene in tumorigenesis was studied by detecting point mutations in Ki-ras codons 12, 13, and 61 by polymerase chain reaction and sequence analysis. The PCNA-positive rates (± standard deviations) in various samples were as follows: 0% for specimens from six untreated animals and six uninvolved areas, 4.26 ± 3.94% for 19 hyperplasias (hyperplasias vs normal lung tissue, P < 0.01), 13.24 ± 6.35% for 25 adenomas (adenomas vs hyperplasias, P < 0.01), and 38.0 ± 9.63% for four adenocarcinomas (adenocarcinomas vs adenomas, P < 0.01). The corresponding mean AgNOR scores were as follows: 1.10 ± 0.05 for the untreated animals, 1.32 ± 0.09 for the uninvolved areas, 1.72 ± 0.59 for the hyperplasias (hyperplasias vs normal lung tissue, P < 0.05), 2.74 ± 0.70 for the adenomas (adenomas vs hyperplasias, P < 0.01), and 5.22 ± 0.62 for the adenocarcinomas (adenocarcinomas vs adenomas, P < 0.01). Ki-ras gene mutations were identified in three of four (75%) adenocarcinomas, six of 23 (26%) adenomas, and two of 17 (12%) hyperplasias. No mutations were found in normal lung tissue. The most frequent Ki-ras mutation was an arginine (CGA) AT → GC transition at codon 61 in exon 2. The PCNA-positive rates and AgNOR scores of cases with Ki-ras mutations were higher than those without an identified mutation (P < 0.05). Ki-ras mutations at codon 61 (Arg) may therefore influence the growth or development of 1-NP–induced lung lesions in A/J mice. Mol. Carcinog. 22:258–264, 1998. © 1998 Wiley-Liss, Inc.     

冬虫夏草雄激素样作用及对人前列腺癌VCaP细胞放射敏感性影响

目的 探讨冬虫夏草雄激素样作用及对人前列腺癌VCaP细胞放射敏感性影响。方法检测冬虫夏草灌胃后小鼠激素水平及性器官重量指数变化。克隆形成实验方法观察冬虫夏草对VCaP细胞放射敏感性影响。MTS、流式细胞术、裸鼠移植瘤检测冬虫夏草对VCaP细胞(雄激素受体AR阳性)及PC-3细胞(AR阴性)体外及体内增殖能力影响,比较裸鼠血清PSA水平。单因素方差分析或t检验差异。     

A tandemly repeated thyroglobulin core promoter has potential to enhance efficacy for tissue-specific gene therapy for thyroid carcinomas

Recombinant adenoviruses, carrying herpes simplex virus thymidine kinase (HSVtk) genes, were developed to evaluate the possibility of tissue-specific gene therapy for thyroid carcinomas. The HSVtk gene was driven by a minimal thyroglobulin (TG) promoter (AdTGtk) and a tandemly repeated minimal TG promoter (Ad2 x TGtk) to obtain thyroid-specific cell killing ability. The transduction of HSVtk genes by infection with Ad2 x TGtk followed by ganciclovir (GCV) treatment showed more powerful cytotoxicity for TG-producing FRTL5 cells, a rat normal thyroid cell line, and FTC-133 cells, a human follicular thyroid carcinoma cell line, than when infected with AdTGtk in vitro. The cell killing ability of Ad2 x TGtk was 10- to 30-fold higher than that of AdTGtk and similar to that of AdCMVtk, which carries HSVtk under the control of CMV promoter. Whereas after treatment with adenovirus/GCV to non-TG-producing cell lines (undifferentiated thyroid carcinoma cell lines and carcinoma cell lines from other tissues), Ad2 x TGtk and AdTGtk needed more than 100-fold concentrated GCV to reach IC(50) compared to AdCMVtk. We confirmed the enhanced efficacy of Ad2 x TGtk for tissue-specific cytotoxicity in vivo. After adenovirus/GCV treatment for FTC-133 tumor-bearing nude mice, Ad2 x TGtk enhanced tumor growth inhibition and survival rates compared to AdTGtk. Tumor growth inhibition and survival rates by Ad2 x TGtk were similar to that by AdCMVtk. Moreover, any toxic effect for rat normal tissues was not revealed after intravenous injections with Ad2 x TGtk and intraperitoneal administrations with GCV in vivo, whereas severe liver damages were observed after treatment with AdCMVtk/GCV. These data indicate a beneficial effect of Ad2 x TGtk for tissue-specific gene therapy for TG-producing thyroid carcinomas without toxicity for normal tissues.     

In vitro and in vivo potentiating the cytotoxic effect of radiation on human U251 gliomas by the c-Met antisense oligodeoxynucleotides

C-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, and invasion, and are known to be overexpressed in gliomas, which are related to the repair of damaged DNA. In this study, we investigated both in vitro and in vivo whether inhibition of the c-Met gene by antisense oligonucleotides (ODNs) enhances the cytotoxic effect of radiation on human U251 gliomas. A volume of 100 nM of c-Met antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70%. Treatment of human U251 glioma cells with 100 nM of c-Met antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with nonsense ODNs. When the glioma cells were implanted in the cisterna magna of nude mice followed by treatment with c-Met antisense ODNs, the survival time of the nude mice was markedly prolonged compared to that of the untreated group (P < 0.001, logrank test). In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing nude mice much longer than could be achieved with radiation alone (P < 0.0001, logrank test). These results suggest that inhibition of c-Met can be expected to serve as a novel potentiator for radiation therapy in human U251 gliomas.