Selga Elisabet, Morales Cristina, Noé Véronique, Peinado Miguel A, Ciudad Carlos J
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.
BMC Med Genomics. 2008 Aug 11;1:35. doi: 10.1186/1755-8794-1-35.
Methotrexate 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 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.
Gene expression levels were compared between triplicate samples from either HT29 sensitive cells and resistant to 10-5 M MTX by hybridization to the GeneChip(R) HG U133 PLUS 2.0 from Affymetrix. After normalization, a list of 3-fold differentially expressed genes with a p-value < 0.05 including multiple testing correction (Benjamini and Hochberg false discovery rate) was generated. RT-Real-time PCR was used to validate the expression levels of selected genes and copy-number was determined by qPCR. Functional validations were performed either by siRNAs or by transfection of an expression plasmid.
Genes adjacent to the 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 PKCalpha genes in resistant 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. Combined treatments targeting siRNA inhibition of caveolin 1 and overexpression of E-cadherin markedly reduced cell viability in both sensitive and MTX-resistant HT29 cells.
We provide functional evidences indicating that caveolin 1 and E-cadherin, deregulated in MTX resistant cells, may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy.
甲氨蝶呤是最早用于癌症治疗的细胞毒性药物之一，尽管已分离出多种其他叶酸拮抗剂，但甲氨蝶呤在不同类型癌症及其他疾病的治疗中仍发挥着重要作用。甲氨蝶呤治疗的有效性受到耐药性发展的限制，耐药性可能通过不同方式获得。为深入了解与耐药性和致敏相关的机制，我们对甲氨蝶呤耐药细胞中失调的基因进行了功能分析，这些细胞的耐药性要么是由于其与二氢叶酸还原酶（dhfr）基因共扩增，要么是通过使用微阵列进行转录组筛选的结果。
通过与Affymetrix公司的GeneChip(R) HG U133 PLUS 2.0杂交，比较来自HT29敏感细胞和对10 - 5 M甲氨蝶呤耐药的细胞的一式三份样本之间的基因表达水平。标准化后，生成一份3倍差异表达基因列表，其p值<0.05，包括多重检验校正（Benjamini和Hochberg错误发现率）。采用RT - 实时PCR验证所选基因的表达水平，并通过qPCR确定拷贝数。通过小干扰RNA（siRNAs）或转染表达质粒进行功能验证。
位于dhfr基因座附近且包含在5q14扩增子中的基因在HT29甲氨蝶呤耐药细胞中过表达。用针对这些基因的siRNAs处理导致HT29敏感细胞和耐药细胞的细胞活力略有降低。另一方面，对HT29和HT29甲氨蝶呤耐药细胞的微阵列分析揭示，耐药细胞中小窝蛋白1、烯醇化酶2和蛋白激酶Cα（PKCalpha）基因过表达，且无伴随的拷贝数增加。针对这三个基因的siRNAs有效降低了细胞活力，并导致甲氨蝶呤耐药能力下降。此外，在甲氨蝶呤耐药细胞中发现表达下调的E - 钙黏蛋白的过表达也使细胞对化疗药物敏感。针对小窝蛋白1的siRNA抑制和E - 钙黏蛋白过表达的联合治疗显著降低了敏感和甲氨蝶呤耐药的HT29细胞的细胞活力。
我们提供了功能证据，表明在甲氨蝶呤耐药细胞中失调的小窝蛋白1和E - 钙黏蛋白可能在细胞存活中起关键作用，并可能构成辅助治疗的潜在靶点。
