Survivin启动子驱动腺病毒介导LRIG1基因治疗前列腺癌的体外实验

目的 由于去势抵抗性前列腺癌（CRPC）缺乏有效治疗方法,因此催生了探索新治疗模式的需求,其中靶向基因治疗可能是治疗CRPC的较理想模式;但是CRPC的靶向性基因治疗尚没有受到应有的关注.本课题拟研究Survivin启动子驱动的重组腺病毒Ad-Surp-LRIGl对前列腺癌细胞株的体外治疗效果.方法 用Ad-Surp-LRIGl和Ad-LRIGl分别感染人前列腺癌细胞PC-3M及人前列腺上皮细胞CRL-11609 RWPE-1,根据报告基因EGFR表达的不同计算病毒的细胞转染率.MTT法评估Ad-Surp-LRIGl和Ad-LRIGl对PC-3M细胞生长的抑制作用.结果 当MOI=25时,Ad-Surp-LRIGl在PC-3M细胞中的转染效率为81.24%,在CRL-11609 RWPE-1细胞中转染率为0,在两种细胞内的转染率比较差异有显著性意义（χ2=65.18,P=0.000）;Ad-LRIGl在PC-3M细胞中的转染效率为77.22%,在CRL-11609 RWPE-1细胞中转染率为71.68%,转染率比较差异无显著性意义（χ2=0.051,P=0.802）.Ad-Surp-LRIGl和Ad-LRIGl在PC-3M细胞中的转染率相比,差异无显著性意义（χ2=0.013,P=0.796）.在常规培养1d 后PBS组的细胞生长速度即开始超过Ad-Surp-LRIGl组和Ad-LRIGl组细胞,4 d 后细胞生长速度差异显著（χ2=15.37,P=0.001）,而Ad-Surp-LRIGl组和Ad-LRIGl组细胞生长速度无明显差异.结论 Ad-Surp-LRIGl能选择性转染人前列腺癌细胞PC-3M,能明显抑制体外前列腺癌细胞的生长.     

Progress report on phase I/II clinical trial of Ad-OC-TK plus VAL therapy for metastatic or locally recurrent prostate cancer: Initial experience at Kobe University

There is no effective therapy for hormone-refractory prostate cancer and a novel therapeutic modality, such as a gene therapy, should be actively pursued. Previously, Gardner and Chung conducted a phase I clinical trial of Ad-OC-TK (recombinant adenoviral vector containing osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) for the treatment of hormone-refractory prostate cancer at the University of Virginia. We report on our ongoing phase I/II clinical trial of Ad-OC-TK plus VAL for the treatment of advanced prostate cancer at the Kobe University Hospital, Japan.     

GM-CSF gene therapy using adenoviral vector in hamster pancreatic cancer

The aim of this study was to examine the antitumor effect of irradiated granulocyte macrophage-colony-stimulating factor (GM-CSF)-gene-transduced hamster pancreatic cancer cells and its relationship to the amount of GM-CSF produced by transduced tumor cells. Hamster pancreatic adenocarcinoma cells, HPD1NR, which spontaneously secrete 15.0 ± 0.4 pg/10⁶ cells per 24 h of GM-CSF, and HPD2NR cells, which do not secrete GM-CSF, were used. When these cells were infected with recombinant adenovirus harboring the GM-CSF gene, HPD1NR and HPD2NR secreted 624.2 ± 9.9 and 157.8 ± 5.7 pg/10⁶ cells per 24 h, respectively. Vaccination with irradiated GM-CSF-secreting HPD2NR completely protected syngeneic hamsters challenged with live parental cells. On the other hand, vaccination with irradiated HPD1NR protected 60% of hamsters from tumor development after challenge with parental cells. None of the tumor-free hamsters initially vaccinated with irradiated GM-CSF-producing HPD2NR cells developed tumor upon repeated challenge with parental cells during the entire observation period. Irradiated GM-CSF-gene-transduced hamster pancreatic cells are promising as a novel adjuvant cancer therapy after surgery for primary and metastatic pancreatic cancer. The results indicate the necessity for a therapeutic strategy for cancer based on the cytokine status of tumors. Received for publication on Dec. 16, 1999; accepted on April 6, 2000     

腺病毒介导HSV-TK／GCV自杀基因系统靶向性治疗前列腺癌实验研究

目的观察腺病毒介导单纯疱疹病毒胸苷激酶基因／丙氧鸟苷（HSV-TK／GCV）自杀基因系统在人端粒酶逆转录酶（hTERT）启动子调控下对人前列腺癌细胞的靶向性体外杀伤效应。方珐利用不同感染复数（MOI）的重组腺病毒携带增强型绿色荧光蛋白（EGFP）基因感染前列腺癌细胞LNCaP和人成纤维细胞MRC-5,荧光显微镜下观察其感染效率;利用携带不同启动子的重组腺病毒Ad-hTERT-HSV／TK以及Ad-CMV-HSV／TK感染LNCaP和MRC-5细胞,加入不同浓度GCV,MTT法观察受转染细胞的存活率。结杲重组腺病毒Ad-hTERT-EGFP能特异地转染LNCaP,其转染效率随重组病毒的MOI增加而升高（P〈0.01）,MOI为1时转染率为8.3％,MOI为1000时转染率达100％;应用GCV处理后,Ad-CMV-HSV／TK对LNCaP和MRC-5细胞均有杀伤作用,而Ad-hTERTp-HsV／TK只杀伤LNcaP（P〈0.001）,随着MOI和GCV浓度的增加,LNCaP细胞存活率明显降低（P〈0.01）,MOI为1和GCV浓度为1μmol／L时存活率为95.4％,MOI为100和GCV浓度为1000μmol／L时存活率仅为6.1％,并有旁观者效应。结论重组腺病毒携带EGFP报告基因可准确、简便地确定转染效率;hTERT启动子调控的重组腺病毒介导的HSV-TK／GCV自杀基因系统对人前列腺癌细胞有靶向杀伤作用。     

Novel small molecule inhibitors for prostate-specific antigen

BACKGROUND: Prostate-specific antigen (PSA or KLK3) has been shown to inhibit angiogenesis, but it might also have tumor promoting activities. Thus, it may be possible to modulate prostate cancer growth by stimulating or inhibiting the activity of PSA. To this end we have previously identified peptides that stimulate the activity of PSA. As peptides have several limitations as drug molecules, we screened a chemical library to find drug-like compounds that could be used to modulate the function(s) of PSA. METHODS: Almost 50,000 compounds were analyzed for their ability to modulate PSA activity towards a fluorescent PSA-substrate. The ability of the most active compounds to affect the anti-angiogenic activity of PSA was analyzed by human umbilical vein endothelial cell (HUVEC) tube formation assay. RESULTS: In the initial screening we identified two compounds that inhibited PSA activity. Based on these, similar compounds were selected and tested for activity to define structure-activity relationships. Several compounds with micromolar IC50-values were found, but they were not entirely specific towards PSA, e.g., they inhibited chymotrypsin, which has similar substrate specificity as PSA. However, it was possibly to improve the selectivity of the compounds towards PSA by small structural changes. These compounds inhibited the anti-angiogenic activity of PSA in the HUVEC model, proving that the proteolytic activity of PSA is essential for inhibition of angiogenesis. CONCLUSIONS: We found several PSA inhibitors that could be useful tools for studying the role of PSA in cancer models and in normal physiology as showed in angiogenesis model.     

Adenovirus-mediated expression of a soluble fibroblast growth factor receptor inhibits in vitro growth of prostate DU145 cells

BACKGROUND: Fibroblast growth factor (FGF) family plays a key role in prostate cancer. The soluble FGF receptor (sFGFR) has been studied with regards to inhibiting cancer growth and was shown to have a dominant negative effect on cellular signaling and function. Using replication deficient adenovirus-mediated gene transfer, we tested if sFGFR expression may have a suppressive effect on in vitro growth of prostate cancer cells. METHODS: Western analysis was used to verify expression of sFGFR1 and to examine the effect of sFGFR1 on MAP kinase phosphorylation. The effect on proliferation and invasiveness of DU145 cells was examined using the WST-1 and Matrigel Invasion assay, respectively. RESULTS: Activation of MAP kinase (pERK1 and 2) by exogenous FGF1, 2, and 7 was suppressed to baseline levels by sFGFR, which was not seen with EGF. Proliferation and invasion of DU145 cells were significantly suppressed by sFGFR. CONCLUSIONS: A replication deficient adenoviral vector system reproducibly expresses sFGFR in prostate cells. Suppression of in vitro growth in DU145 cells by sFGFR provides the basis of a novel therapeutic approach.     

Exploration of target molecules for prostate cancer gene therapy

BACKGROUND: Focusing on Adv-FZ33, a modified adenovirus in which a synthetic 33-amino-acid immunoglobulin G-binding domain was inserted into the adenoviral fiber protein, we tried to identify suitable target molecules for prostate cancer-specific gene therapy. METHODS: Hybridomas were established from mice immunized with prostate cancer cell lines. The hybridomas were screened using Adv-FZ33 to create monoclonal antibodies (mAbs) that induced high gene transfer efficiency for PC-3 cells. Furthermore, we identified target antigens of the mAbs by immunoprecipitation and mass spectrometry, and investigated the expression of target molecules by flow cytometry and immunocytochemistry. RESULTS: Using Adv-FZ33, we established four different mouse mAbs that increased transduction efficiency for PC-3. The target antigens identified were Ep-CAM, CD155, HAI-1, and Na,K-ATPase beta1. These antigens were expressed in several cancer cell lines, including prostate cancer. Human prostatic myofibroblast cells lacked expression of Ep-CAM and HAI-1. CONCLUSIONS: We established anti-Ep-CAM mAb and anti- HAI-1 mAbs. Gene transduction via Ep-CAM and HAI-1 may be a novel strategy for treatment of prostate cancer.     

Human coxsackie adenovirus receptor (CAR) expression in transgenic mouse prostate tumors enhances adenoviral delivery of genes

BACKGROUND: Transgenic mice that recapitulate the progression of human diseases are potentially useful models for testing the effectiveness of new therapeutic strategies. Their use in pre-clinical testing of adenovirally-delivered gene therapies, however, is limited because of restricted cell surface expression of Coxsackie adenovirus receptor (CAR) in mice. METHODS: To develop a more suitable transgenic mouse model for testing adenoviral-based gene therapies for prostate cancer, we generated prostate specific antigen/human CAR (PSA/hCAR) transgenic mice in which a chimeric enhancer/promoter sequence of the human PSA gene drives expression of a functional hCAR coding sequence. RESULTS: Expression of an adenovirally-delivered luciferase reporter gene in prostate tumor cells in bigenic mice (PSA/hCAR + TRAMP) was enhanced compared to the level in tumor cells lacking the PSA/hCAR transgene. CONCLUSIONS: Breeding PSA/hCAR mice to existing transgenic mouse models for prostate cancer (e.g., TRAMP) results in improved mouse models for testing adenovirally-delivered therapeutic genes.     

A comparative performance analysis of total prostate-specific antigen, percentage free prostate-specific antigen, prostate-specific antigen velocity and urinary prostate cancer gene 3 in the first, second and third repeat prostate biopsy

Study Type – Diagnostic (cohort) Level of Evidence 2b What's known on the subject? and What does the study add? Although non‐recommended PSA testing has been reported in men younger than 40 years of age, there are few recognized data on PSA in younger American men, particularly younger African‐American men, to provide age‐ and race‐specific references. Using data from an existing large study of young, male members of the US military, aged 28–36 years, the present study provides PSA reference distributions for young Caucasian‐American men (median = 0.56, 95th percentile = 1.42, range: <0.01–3.34 ng/mL) and African‐American men (median = 0.64, 95th percentile = 1.89, range: 0.12–6.45 ng/mL). Previous estimates from the literature are also summarized.

OBJECTIVE

• ? To provide race‐specific prostate‐specific antigen (PSA) reference distributions for young men less than 40 years of age who might have undergone non‐recommended PSA testing because of their family history of prostate cancer or inadvertently as part of a standard panel of tests.

MATERIALS AND METHODS

• ? We used data from a large existing study of young, male Caucasian‐ and African‐American members of the US military with stored serum in the Department of Defense serum repository.
• ? As part of this previous study, we selected a random sample of 373 Caucasian‐ and 366 African‐American men aged 28–36 years with an archived serum specimen collected for standard military purposes from 2004 to 2006.
• ? We measured serum total PSA concentration in this specimen using the Beckman Coulter Access Hybritech PSA assay.

RESULTS

• ? The PSA level ranged from <0.01 to 3.34 ng/mL among Caucasian‐American men, with a median of 0.56 ng/mL and a 95th percentile of 1.42 ng/mL.
• ? The PSA level ranged from 0.12 to 6.45 ng/mL among African‐American men, with a median of 0.64 ng/mL and 95th percentile of 1.89 ng/mL.
• ? The PSA level was significantly higher in African‐ than in Caucasian‐American men (P= 0.001).

CONCLUSION

• ? The PSA estimates, together with those summarized from the literature, provide age‐ and race‐specific PSA reference distributions for young men who might have undergone non‐recommended PSA testing.
• ? Comparisons by race could also begin to inform the timing of divergence of prostate cancer risk by race.

     

前列腺特异性膜抗原为靶标的前列腺癌靶向疗法研究进展

前列腺特异性膜抗原(PSMA)是一种Ⅱ型跨膜糖蛋白,特异性表达于前列腺癌(PCa)腺上皮和其他非前列腺恶性实体肿瘤的新生毛细血管内皮细胞。这些发现促进了以PSMA为靶向的PCa靶向疗法研究的快速发展,目前第一代产品已进入临床试验阶段,但限于相关临床研究的缺乏,要在临床上推广应用PSMA靶向疗法,还需要进行大量的研究以验证其临床安全性、稳定性和有效性等。本文就PSMA的免疫疗法、基因疗法、放射免疫疗法和化学疗法作一综述。     

Prostate-tumor targeting of gene expression by lentiviral vectors containing elements of the probasin promoter

BACKGROUND: Lentiviruses are retroviruses that can infect and stably integrate into the chromatin of non-dividing cells. The purpose of this study was to determine whether lentiviral vectors containing the probasin (PB) promoter displayed prostate-specific, androgen-regulated, and persistent gene expression. METHODS: Three lentiviral-PB promoter/enhanced green fluorescent protein (EGFP)-reporter vectors together with a control lentiviral-CMV-EGFP, were tested by microscopy and flowcytometry for expression of EGFP after infection of human prostate cancer cells (LNCaP, PC-3, PC-3(hAR), and Du145 cells) and non-prostate cells (COS-1, HeLa, HeLa(hAR), and MCF-7 cells). RESULTS: All cells infected in vitro with lentiviral-CMV vectors expressed EGFP, whereas with lentiviral-PB vectors (the most potent being Lv-ARR(2)PB), reporter expression was only observed in LNCaP cells with a small amount seen in androgen-independent PC-3 cells. Stable or transient transfection of androgen receptor only raised EGFP expression in prostate-derived cell lines, but did not change tumor specificity. With Lv-ARR(2)PB infected LNCaP cells, androgens regulated EGFP both in vitro and in vivo. After intra-tumor injection of this vector, EGFP expression was observed in LNCaP tumors, but not in A-549 lung or CaKi-2 kidney tumors. CONCLUSIONS: Lv-ARR(2)PB may be an ideal vector for prostate-tumor targeting and for persistent, hormone-enhanced expression of a therapeutic gene to treat slow growing prostate tumors.     

Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets

There is considerable interest in the viral modification of insulin-producing islets, including porcine islets, in the context of islet xenotransplantation to treat type 1 diabetes. Adenovirus (Adv) gene delivery offers the potential to modify pre-transplant islets for enhanced survival. Modifications include transfer of cytoprotective molecules to ensure islet survival immediately post-transplant, and molecules to dampen the immune system and prevent chronic islet graft rejection. In this study, we compared different promoters (three promiscuous and two tissue-specific promoters) for their efficiency in driving gene expression in neonatal pig islet tissue after Adv delivery. We also compared the efficiency of these promoters in adult islets from mouse and human pancreata. We observed that the promiscuous cytomegalovirus promoter was the most potent, eliciting high luciferase expression in neonatal pig islets, as well as in human and mouse islets. In contrast, the mammalian EF1-alpha promoter educed comparatively intermediate gene expression. The mouse major histocompatibility complex class I promoter H-2K(b) and the pancreatic-specific promoters insulin and human pdx-1 (area II) performed poorly in islets from all three species. This has important implications for the generation of modified neonatal pig islets for transplantation into humans.     

腺病毒-VEGF基因重组体促进预构皮瓣成活的实验研究

目的：应用大鼠预构皮瓣模型,探讨基因治疗技术产生的血管内皮生长因子促进预构皮瓣血管新生和皮瓣存活的可能性,为临床上寻找加速预构皮瓣成熟的新方法提供实验依据。方法：20只SD大鼠每只腹部两侧各构建一个预构皮瓣,共构建40个皮瓣,每只大鼠两侧皮瓣按随机原则进行不同的处理,分别归于实验组或对照组,每组各20个皮瓣。于大鼠腹部两侧各标记3cm×2cm矩形预构区,短边平行于腹股沟韧带,自尾侧短边中点向后纵向切开后肢皮肤,剥离出长约2cm的股动静血管束,远端结扎切断。在两侧预构区域的中轴线上,于真皮与肉膜层间各制作一皮下隧道,实验组的隧道壁皮下组织内注射携带有VEGF基因的腺病毒,同法向所有对照组的隧道壁软组织内注射等量生理盐水。将已剥离好的血管束向颅侧翻转置入相应皮下隧道内。所有已植入股血管的预购区域2周后均被制成以植入血管束为蒂的岛状皮瓣,从两组中各取一个皮瓣进行免疫组化染色,观察有无VEGF生成,其余岛状皮瓣均缝回原处。形成岛状皮瓣后第七天观察皮瓣存活及血管新生情况。结果：实验组与对照组的皮瓣平均存活率分别为（90．48±1．89）％、（69．75±2．36）％,其差异有统计学意义（P〈0．01）;血管放射显影图上,实验组植入血管周围见广泛白色显影,尤以血管两端明显,而对照组新生血管显影仅局限于植入血管周围;组织学切片显示实验组植入血管周围新生血管丰富,以毛细血管为主,并见肉芽成份,对照组新生血管相对较少,两组间新生小血管管腔大小则无明显差异;免疫组化检测显示仅实验组皮瓣中有VEGF表达。结论：腺病毒-VEGF基因重组体能通过促进预构皮瓣的血管新生,增加预构皮瓣的存活率。