Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCa on resistance to methotrexate in human HT29 colon cancer cells. Homo sapiens

A summary of the work associated to these microarrays is the following: Methotrexate (MTX) is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we have performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the DHFR gene or as a result of a transcriptome screening using microarrays. Genes adjacent to dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCa genes in treated cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. We provide functional evidences indicating that caveolin 1 and E-cadherin may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy. Keywords: DHFR, Methotrexate, drug resistance Overall design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M MTX. Six samples are provided which correspond to triplicated of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX v7.3.1.

本研究针对这批微阵列（microarray）相关工作的概述如下：甲氨蝶呤（Methotrexate, MTX）是最早应用于癌症治疗的细胞毒性药物之一，尽管后续已分离出多种其他叶酸拮抗剂，甲氨蝶呤仍在多种癌症及其他疾病的治疗中占据重要地位。然而甲氨蝶呤的临床应用受限于耐药性的产生，而耐药性可通过多种途径获得。为深入解析甲氨蝶呤耐药与增敏的相关机制，本研究针对甲氨蝶呤耐药细胞中失调的基因开展了功能分析——这些基因的失调或源于与二氢叶酸还原酶（Dihydrofolate Reductase, DHFR）基因的共扩增，或源于采用微阵列进行的转录组筛选结果。位于DHFR基因座旁、且包含于5q14扩增子中的基因，在HT29甲氨蝶呤耐药细胞中呈现过表达。针对上述基因的小干扰RNA（small interfering RNA, siRNA）处理，可在HT29敏感细胞与耐药细胞中均轻微降低细胞存活率。另一方面，通过对HT29甲氨蝶呤敏感细胞与HT29甲氨蝶呤耐药细胞的微阵列分析，本研究发现耐药细胞中窖蛋白1（caveolin 1）、烯醇化酶2（enolase 2）与蛋白激酶Cα（PKCa）基因呈现过表达，且该现象未伴随拷贝数的增加。针对这三个基因的siRNA处理可有效降低细胞存活率，并削弱细胞的甲氨蝶呤耐药能力。此外，在甲氨蝶呤耐药细胞中表达下调的上皮钙黏蛋白（E-cadherin），其过表达也可使细胞对化疗药物增敏。本研究提供的功能实验证据表明，窖蛋白1与上皮钙黏蛋白可能在细胞存活中发挥关键作用，或可成为辅助治疗的潜在靶点。关键词：二氢叶酸还原酶（DHFR）、甲氨蝶呤（Methotrexate）、耐药性。整体实验设计：本研究比较两种细胞系：对甲氨蝶呤敏感的HT29结肠癌细胞，以及对10^-5 M甲氨蝶呤产生耐药的HT29细胞。本次提供的6份样本分别对应两种细胞系的生物学重复各3次。后续对样本进行归一化处理，并采用GeneSpring GX v7.3.1专用软件进行分析。