Pharmacogenomic Approach for the Identification of Novel Determinants of Resistance to Oxaliplatin in Colon Cancer. Homo sapiens

Oxaliplatin is a member of the family of Pt-containing chemotherapeutic agents that also include cisplatin (CDDP) and carboplatin. OXA is distinguished from these two older drugs by its different spectrum of activity both in preclinical models and in clinical trials. It is the only platinum analogue to have activity in colon cancer, a disease for which this drug has now become a mainstay of therapy. It mainly forms intrastrand adducts between two adjacent guanine residues or guanine and adenine, disrupting DNA replication and transcription. OXA has been reported to be involved in the Nucleotide Excision Repair Pathway (NER), p38 kinase activation, PI3K/AKT pathway and caspases cascade activation through apoptotic intrinsic pathway. However, the downstream molecular events underlying the cytotoxic effects of this chemotherapeutic agent have not been well characterized. This study was developed in order to clarify the multifactoriality of the resistance acquisition process and to identify genes and pathways that could play a role as markers in OXA sensitivity. Keywords: Drug resistance Overall design: The goal of our experiment was to determine a gene expression profile that discriminates between the OXA-sensitive colorectal cancer cell lines HT29, LoVo, DLD1 and LS513 group and the group of OXA-resistant derived cell lines HTOXAR3, LoVOXAR3, DLDOXAR3 and LSOXAR3 in order to clarify the multifactoriality of the resistance acquisition process and to identify genes and pathways that could play a role as markers in OXA sensitivity. The experimental design used was “RNA-Reference” and “Dye-Swap”. Each cell line was analyzed in duplicate, with RNA reference (Stratagene) as reference sample and labeled each biological condition once by Cy3 and once by Cy5. Taking the average of two arrays thus labeled, cancel the dye effect on any particular gene. In total we used 16 slides.

奥沙利铂（Oxaliplatin）属于含铂化疗药物家族，该家族成员还包括顺铂（cisplatin, CDDP）与卡铂。相较于这两款老牌药物，奥沙利铂在临床前模型与临床试验中展现出独特的活性谱，是目前唯一对结肠癌具有抗肿瘤活性的铂类类似物，现已成为该病临床治疗的核心药物之一。
奥沙利铂主要通过在相邻鸟嘌呤残基之间，或鸟嘌呤与腺嘌呤之间形成链内加合物，干扰DNA复制与转录过程。已有研究显示，奥沙利铂可通过内源性凋亡通路参与核苷酸切除修复通路（Nucleotide Excision Repair Pathway, NER）、p38激酶激活、PI3K/AKT通路以及半胱天冬酶级联激活过程。然而，该化疗药物细胞毒性效应背后的下游分子机制尚未得到充分阐释。
本研究旨在阐明耐药获得过程的多因素特征，并筛选可作为奥沙利铂敏感性标志物的基因与通路。
关键词：药物耐药
整体实验设计：本实验旨在构建可区分奥沙利铂敏感结直肠癌细胞系（HT29、LoVo、DLD1与LS513）与奥沙利铂耐药衍生细胞系（HTOXAR3、LoVOXAR3、DLDOXAR3与LSOXAR3）的基因表达谱，以阐明耐药获得过程的多因素特征，并筛选可作为奥沙利铂敏感性标志物的基因与通路。
本实验采用“RNA参照”与“染料互换”两种实验设计方案。每种细胞系均设置重复检测：以Stratagene公司的RNA作为参照样本，分别使用Cy3与Cy5对各生物学条件下的RNA进行荧光标记。取上述两张标记芯片的平均信号值，可消除荧光染料对特定基因表达检测的偏差影响。本研究共使用16张芯片玻片。