Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN 47907, USA; Purdue Oncological Sciences Center, Purdue University, West Lafayette, IN 47907, USA.
Urol Oncol. 2013 Nov;31(8):1761-9. doi: 10.1016/j.urolonc.2012.03.015. Epub 2012 May 19.
More than 14,000 people die from invasive urothelial carcinoma (iUC) of the urinary bladder each year in the USA, and more effective therapies are needed. Naturally occurring canine iUC very closely resembles the disease in humans and serves as a highly relevant translational model for novel therapy of human iUC. Work was undertaken to identify new targets for anticancer therapy in dogs with the goal of translating successful therapeutic strategies into humans with iUC.
Microarray expression analyses were conducted on mRNA extracted from canine normal bladder (n = 4) and iUC tissues (n = 4) using Genome Array 1.0 and analyzed by GeneSpring GX 11, with the stringency of P < 0.02 and a ≥ 2-fold change. The genes thus identified were further analyzed for functional and pathway analysis using Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System. In selecting genes for further study, consideration was given for evidence of a role of the gene in human iUC. From these analyses, DNA methyltransferase 1 (DNMT1) was selected for further study. Immunohistochemistry (IHC) of canine normal bladder and iUC tissues was performed to confirm the microarray expression analyses. The effects of targeting DNMT1 in vitro was assessed through MTT assay and Western blot of canine iUC cells treated with 5-azacitidine (5-azaC) and trichostatin A (TSA).
DNMT1 was expressed in 0 of 6 normal canine bladder samples and in 10 of 22 (45%) canine iUC samples. The proliferation of canine iUC cells was inhibited by 5-azaC (at concentrations ≥ 5 μm) and by TSA (at concentrations ≥ 0.1 μm). Western blot results were supportive of DNMT1-related effects having a role in the antiproliferative activity.
Microarray expression analyses on canine tissues identified DNMT1 as a potentially "targetable" gene. Expression of DNMT1 in canine iUC was confirmed by IHC, and in vitro studies confirmed that drugs that inhibit DNMT1 have antiproliferative effects. These findings are similar to those recently reported in human iUC and are also in line with results of a preclinical (prehuman) trial of 5-azaC in dogs with naturally occurring iUC. DNMT1 has excellent potential as a target for iUC therapy in humans.
在美国，每年有超过 14000 人死于侵袭性尿路上皮癌（iUC），因此需要更有效的治疗方法。犬类自然发生的 iUC 与人类疾病非常相似，是研究人类 iUC 新型治疗方法的高度相关转化模型。本研究旨在鉴定犬类癌症治疗的新靶点，目的是将成功的治疗策略转化为患有 iUC 的人类。
使用基因组阵列 1.0 对来自 4 只正常犬膀胱（n=4）和 4 只 iUC 组织的 mRNA 进行微阵列表达分析，并使用 GeneSpring GX 11 进行分析，严格标准为 P<0.02 和 ≥2 倍变化。通过蛋白质分析通过进化关系（PANTHER）分类系统对由此鉴定的基因进行功能和途径分析。在选择进一步研究的基因时，考虑了该基因在人类 iUC 中的作用证据。从这些分析中，选择 DNA 甲基转移酶 1（DNMT1）进行进一步研究。通过免疫组织化学（IHC）对犬类正常膀胱和 iUC 组织进行检测，以确认微阵列表达分析的结果。通过 5-氮杂胞苷（5-azaC）和曲古抑菌素 A（TSA）处理犬类 iUC 细胞的 MTT 测定和 Western blot 评估了靶向 DNMT1 的作用。
在 6 只正常犬膀胱样本中，DNMT1 未表达，在 22 只犬 iUC 样本中，DNMT1 表达 10 只（45%）。5-azaC（浓度≥5μm）和 TSA（浓度≥0.1μm）抑制犬类 iUC 细胞的增殖。Western blot 结果支持 DNMT1 相关作用在抗增殖活性中起作用。
犬类组织的微阵列表达分析鉴定出 DNMT1 是一种潜在的“可靶向”基因。通过免疫组织化学证实了 DNMT1 在犬类 iUC 中的表达，并且体外研究证实抑制 DNMT1 的药物具有抗增殖作用。这些发现与最近在人类 iUC 中报告的发现相似，也与自然发生的 iUC 犬类的 5-azaC 临床前（非人类）试验结果一致。DNMT1 作为人类 iUC 治疗的靶标具有巨大的潜力。
