Molecular Mode of Action and Role of TP53 in the Sensitivity to the Novel Epothilone Sagopilone (ZK-EPO) in A549 Non-Small Cell Lung Cancer Cells

Sagopilone, an optimized fully synthetic epothilone, is a microtubule-stabilizing compound that has shown high in vitro and in vivo activity against a broad range of human tumor models. We analyzed the differential mechanism of action of sagopilone in non-small cell lung cancer cell lines in vitro. Sagopilone inhibited proliferation of non-small cell lung cancer cell lines at lower nanomolar concentration. The treatment with sagopilone caused strong disturbances of cellular cytoskeletal organization. Two concentration-dependent phenotypes were observed. At 2.5 nM sagopilone or 4 nM paclitaxel an aneuploid phenotype occur whereas a mitotic arrest phenotype was induced by 40 nM sagopilone or paclitaxel. Interestingly, treatment with 2.5 nM of sagopilone effectively inhibited cell proliferation, but - compared to high concentrations (40 nM) - only marginally induced apoptosis. Treatment with a high versus a low concentration of sagopilone or paclitaxel regulates a non-overlapping set of genes, indicating that both phenotypes substantially differ from each other. Genes involved in G2/M phase transition and the spindle assembly checkpoint, like Cyclin B1 and BUBR1 were upregulated by treatment with 40 nM sagopilone. Unexpectedly, also genes involved in DNA damage response were upregulated under that treatment. In contrast, treatment of A549 cells with a low concentration of sagopilone revealed an upregulation of direct transcriptional target genes of TP53, like CDKN1A, MDM2, GADD45A, FAS. Knockdown of TP53, which inhibited the transcriptional induction of TP53 target genes, led to a significant increase in apoptosis induction in A549 cells when treated with a low concentration of sagopilone. The results indicate that activation of TP53 and its downstream effectors like CDKN1A by low concentrations of sagopilone is responsible for the relative apoptosis resistance of A549 cells and might represent a mechanism of resistance to sagopilone.

沙戈匹隆（Sagopilone）是一种经优化的全合成埃博霉素（epothilone）类微管稳定化合物，在体外及体内实验中对多种人类肿瘤模型均展现出广谱强效活性。我们在体外实验中分析了沙戈匹隆在非小细胞肺癌（non-small cell lung cancer）细胞系中的差异化作用机制。沙戈匹隆可在低纳摩尔浓度下抑制非小细胞肺癌细胞系的增殖，该药物处理会显著干扰细胞骨架的组织结构。研究观察到两种浓度依赖型表型：当沙戈匹隆浓度为2.5 nM或紫杉醇（paclitaxel）浓度为4 nM时，会触发非整倍体（aneuploid）表型；而当沙戈匹隆浓度为40 nM或紫杉醇达到对应浓度时，则诱导产生有丝分裂阻滞（mitotic arrest）表型。值得注意的是，2.5 nM浓度的沙戈匹隆即可有效抑制细胞增殖，但与高浓度（40 nM）处理相比，仅能极轻微地诱导细胞凋亡。相较于低浓度处理，高浓度的沙戈匹隆或紫杉醇可调控一组非重叠的基因表达谱，这表明两种表型存在显著差异。经40 nM沙戈匹隆处理后，参与G2/M期转换（G2/M phase transition）及纺锤体组装检验点（spindle assembly checkpoint）的基因（如细胞周期蛋白B1（Cyclin B1）与BUBR1）均出现上调。出乎意料的是，该处理下参与DNA损伤应答的基因也被上调。与之相反，用低浓度沙戈匹隆处理A549细胞时，TP53的直接转录靶基因（如CDKN1A、MDM2、GADD45A、FAS）出现上调。敲低TP53可抑制其靶基因的转录诱导，此时用低浓度沙戈匹隆处理A549细胞，会显著增强细胞凋亡的诱导效果。上述结果表明，低浓度沙戈匹隆通过激活TP53及其下游效应因子（如CDKN1A），是A549细胞产生相对凋亡抗性的原因所在，这或许也代表了肿瘤对沙戈匹隆产生耐药性的一种机制。