Alterations in tumour necrosis factor signaling pathways associated with cytotoxicity and resistance to taxanes in tumour cells

The taxanes are widely used in the treatment of breast and other cancers. While their cytotoxicity has been attributed to the induction of cell cycle arrest in mitosis through the stabilization of microtubules, we found that docetaxel promotes soluble tumor necrosis factor alpha (TNF-alpha production in MCF-7 breast tumor cells. TNF-alpha induces apoptotic cell death in a variety of cell types by binding to one of its receptors (TNFR1) which promotes death-inducing signaling complex (DISC) formation. Consistent with this view, we also report that selection of MCF-7 cells for survival in increasing concentrations of paclitaxel or docetaxel results in selection of drug-resistant variants that are resistant to TNF-alpha cytotoxicity. MCF-7 cells selected to 3-5 nM docetaxel produced >30-fold more TNF-alpha than control MCF-7CC cells but had strongly reduced levels of TNFR1. In contrast, expression of TNFR2 was unchanged, resulting in enhanced cell survival through the activation of the NF-kappaB p50 and p65 subunits. Gene expression profiling of docetaxel resistant MCF-7 cells compared to parental MCF-7 cells was performed for the changes of TNF related genes, and also confirmed in ovarian cell line A2780. Two docetaxel resistant cell lines of breast MCF-7 and ovarian A2780 were generated for gene expression profilling. Two colour microarray of Agilent whole human genome nucleotide arrays was conducted with two replicate of both forward and reverse labellings for MCF-7. Four arrays were used for this experiments. And it was four replicate of both forward and reverse labellings for A2780 cells. Eight arrays were used for this experiments

紫杉烷类（taxanes）被广泛应用于乳腺癌及其他癌症的治疗。既往研究认为，其细胞毒性源于通过稳定微管诱导有丝分裂期细胞周期阻滞。本研究发现，多西他赛（docetaxel）可诱导MCF-7乳腺癌细胞分泌可溶性肿瘤坏死因子α（tumor necrosis factor alpha, TNF-alpha）。

肿瘤坏死因子α可通过结合其受体之一（tumor necrosis factor receptor 1, TNFR1）激活死亡诱导信号复合物（death-inducing signaling complex, DISC）的形成，进而在多种细胞类型中诱导凋亡性细胞死亡。与此观点一致，本研究同时证实：在逐步升高浓度的紫杉醇（paclitaxel）或多西他赛环境中筛选MCF-7细胞，可获得对肿瘤坏死因子α细胞毒性产生耐药性的耐药变异株。

经3~5 nM多西他赛持续筛选得到的MCF-7细胞，其肿瘤坏死因子α分泌量较对照MCF-7CC细胞高出30倍以上，但肿瘤坏死因子受体1的表达水平显著降低。与之相反，肿瘤坏死因子受体2（TNFR2）的表达未发生明显变化，该变化通过激活核因子κB（NF-kappaB）p50与p65亚基，增强了细胞的存活能力。

本研究针对多西他赛耐药MCF-7细胞与亲本MCF-7细胞，开展了肿瘤坏死因子相关基因的基因表达谱（gene expression profiling）分析，并在卵巢癌细胞系A2780中验证了该结果。我们构建了乳腺癌MCF-7与卵巢癌A2780的两株多西他赛耐药细胞系，用于基因表达谱分析。

针对MCF-7细胞，采用安捷伦全人类基因组核苷酸芯片（Agilent whole human genome nucleotide arrays）进行双色微阵列（two colour microarray）检测，设置正向标记与反向标记各2次重复，该实验共使用4张芯片；针对A2780细胞，设置正向标记与反向标记各4次重复，该实验共使用8张芯片。