Celecoxib pre-treatment in human colorectal adenocarcinoma patients.

Pharmacological inhibition of cyclooxygenase-2 (COX-2) is being explored as a chemotherapeutic option because COX-2 protein expression is often elevated in many cancers. Cancer cells treated with COX-2 inhibitors, such as the selective COX-2 inhibitor celecoxib, show growth inhibition and the induction of apoptosis, through alterations in inflammatory processes, angiogenesis, cell adhesion and transforming growth factor-β signaling. This study was conducted to determine if the same processes are relevant to celecoxib’s effects on human colorectal adenocarcinomas treated in vivo. A cohort of 23 patients with primary colorectal adenocarcinomas was randomized to receive a 7-day course of celecoxib (400 mg b.i.d.) or no drug prior to surgical resection. Gene expression profiling was performed on resected adenocarcinomas from patients with and without celecoxib pre-treatment. Using fold change (>1.5) and p-value (<0.05) cut-offs, 190 genes were differentially expressed between adenocarcinomas from patients receiving celecoxib and those that did not. Of the differentially expressed genes, multiple genes involved in cellular lipid and glutathione metabolism showed decreased expression levels in celecoxib pre-treated samples; changes associated with diminished cellular proliferation. Other observed gene expression changes consistent with reduced proliferation include: altered expression of genes involved in cell adhesion (including collagen, laminin, von Willebrand factor and tenascin C), increased expression of inflammatory modulators (including inerleukin-6, S100 calcium binding protein A8, and several chemokines) and decreased expression of the pro-angiogenic gene, angiogenin. Celecoxib pre-treatment for 7 days in vivo is associated with alterations in colorectal adenocarcinoma gene expression which are suggestive of diminished cellular proliferation. Keywords: treatment outcome Patients undergoing surgical resection of histologically proven primary colorectal adenocarcinomas were consented for participation in the study. The patients enrolled in this study were randomized to receive either 400 mg celecoxib two times per day (n=11) or no COX-2 inhibitor (n=12) for 7 days prior to surgical resection. Total RNA (5 ug) from each sample was converted to double stranded cDNA using a dT-T7 promoter primer. The double stranded cDNA was then used as a template to synthesize biotinylated RNA, which was fragmented and hybridized to the Affymetrix HG_U95av2 microarray chip using Affymetrix’s labeling and hybridization protocol. The array data was imported into GeneSpring GX 7.3 using the GC-RMA file preprocessor. The data was normalized by: (1) setting all expression measurements <0.01 to 0.01, (2) a per chip normalization to the 50th percentile, and (3) a per gene normalization to the median value across all chips.

环氧化酶-2（cyclooxygenase-2, COX-2）的药理学抑制作为化疗方案的潜力正被广泛探索，因COX-2蛋白在多种癌症中常呈高表达状态。使用选择性COX-2抑制剂塞来昔布（celecoxib）等COX-2抑制剂处理癌细胞，可通过调控炎症过程、血管生成、细胞黏附及转化生长因子-β（transforming growth factor-β, TGF-β）信号通路，实现生长抑制并诱导细胞凋亡。本研究旨在探究上述效应机制是否同样适用于塞来昔布对体内人结直肠腺癌的作用。

招募23例经组织学确诊的原发性结直肠腺癌患者，随机分为两组：分别在手术切除前接受7天塞来昔布治疗（400mg，每日两次）或不使用任何药物。对接受塞来昔布预处理与未预处理患者的手术切除腺癌组织进行基因表达谱分析。以倍数变化（fold change, FC）>1.5且p值<0.05作为筛选阈值，共筛选得到190个在塞来昔布处理组与对照组腺癌组织中差异表达的基因。

在差异表达基因中，多个参与细胞脂质代谢与谷胱甘肽代谢的基因在塞来昔布预处理样本中表达下调，该变化与细胞增殖减弱相关。其他与增殖降低相符的基因表达改变包括：细胞黏附相关基因（包括胶原蛋白、层粘连蛋白、血管性血友病因子及腱糖蛋白C）的表达异常；炎症调节因子（包括白细胞介素-6、S100钙结合蛋白A8及多种趋化因子）的表达上调；以及促血管生成基因血管生成素（angiogenin）的表达下调。

体内7天塞来昔布预处理可诱导结直肠腺癌基因表达谱发生改变，这些改变提示细胞增殖受到抑制。
关键词：治疗结局

本研究纳入拟接受手术切除的经组织学确诊的原发性结直肠腺癌患者，所有患者均签署知情同意书。入组患者被随机分为两组：塞来昔布组（n=11，400mg，每日两次）与对照组（n=12，未使用COX-2抑制剂），均在手术切除前接受为期7天的干预。

从每份样本中提取总RNA（5μg），使用寡聚dT-T7启动子引物反转录为双链cDNA。以双链cDNA为模板合成生物素标记的RNA，经片段化后按照Affymetrix公司的标记与杂交流程，与Affymetrix HG_U95av2基因芯片进行杂交。芯片数据通过GC-RMA文件预处理器导入GeneSpring GX 7.3软件进行分析。数据标准化步骤如下：(1) 将所有表达量<0.01的数值统一调整为0.01；(2) 以50百分位数对每张芯片进行标准化；(3) 以所有芯片的中位数对每个基因进行标准化。