Melanoma cell acquired resistance to vinca-alkaloids

Malignant melanoma (MM) remains a therapeutic challenge on account of extreme primary resistance to apoptosis and fated acquired chemoresistance. In the current context of molecular classification of MM, the understanding of molecular mechanisms giving rise to these resistances should drive therapeutic choice trough personalized medicine. In order to study mechanisms of MM acquire resistance to VAs, we previously established three VA-resistant cell lines, CAL1R-VCR, CAL1R-VDS, and CAL1R-VRB, by long time exposure of the CAL1-wt MM cell line to IC50 (4nM) of VCR, VDS and VRB respectively. CAL1R-VAs cell lines consisted of polyclonal populations to respect biological diversity of tumour. Then, we searched for determine the resistance process impact on MM cells. We thus performed genome-wide analyses based on comparison of global transcriptomic profile of CAL1R-VAs cells to CAL1-wt. In this way, RNA extracted from each cell line was hybridized to Affimetrix HG-U133 Plus 2.00 GeneChips. After hybridization, microarray data were processed with Robust Multi-array Average (RMA) algorithm. We then performed bioinformatic analyses of microarray data and in vitro investigations in the aim to identify genes and pathways that might predict drug resistance. Melanoma model: CAL1 cell line. Treatments: vinorelbine (VRB), vincristine (VCR), vindesine (VDS). Etablishment of 3 resistant cell lines by long time exposure to vinka-alkaloids (4nM, 6-12 months): CAL1R-VRB, CAL1R-VCR, CAL1R-VDS. RNA extracted from a pooled population of each cell line were hybridized to Affimetrix HG-U133 Plus 2.00 GeneChips. Then, microarray data were processed with Robust Multi-array Average (RMA) algorithm.

恶性黑色素瘤（Malignant Melanoma, MM）由于对细胞凋亡（Apoptosis）存在极强的原发性耐药，且不可避免地会产生获得性化疗耐药，始终是临床治疗的棘手难题。在当前MM分子分型（Molecular Classification）的研究背景下，阐明此类耐药性产生的分子机制，可为通过个体化医疗（Personalized Medicine）制定治疗方案提供理论依据。为研究MM对长春花生物碱（Vinka Alkaloids, VAs）的获得性耐药机制，我们此前通过将CAL1野生型（CAL1-wt）黑色素瘤细胞系分别暴露于半数抑制浓度（Half Maximal Inhibitory Concentration, IC₅₀）为4nM的长春新碱（Vincristine, VCR）、长春地辛（Vindesine, VDS）和长春瑞滨（Vinorelbine, VRB）中进行长期培养，成功构建了三株VAs耐药细胞系CAL1R-VCR、CAL1R-VDS与CAL1R-VRB。CAL1R-VAs耐药细胞系均为多克隆群体（Polyclonal Population），以模拟肿瘤的生物学异质性。随后，我们旨在明确耐药进程对MM细胞的影响。因此，我们通过对比CAL1R-VAs细胞系与CAL1-wt细胞系的全转录组表达谱（Global Transcriptomic Profile），开展了全基因组水平分析。本次研究中，我们将各细胞系提取的核糖核酸（Ribonucleic Acid, RNA）与Affymetrix HG-U133 Plus 2.00基因芯片（Affymetrix HG-U133 Plus 2.00 GeneChips）进行杂交反应。杂交完成后，我们采用稳健多阵列平均算法（Robust Multi-array Average, RMA）对基因芯片（Microarray）数据进行预处理。随后，我们对芯片数据开展生物信息学分析（Bioinformatic Analysis），并辅以体外实验，以期筛选出可预测化疗耐药的关键基因与信号通路（Signaling Pathway）。黑色素瘤模型：CAL1细胞系。处理方式：长春瑞滨（VRB）、长春新碱（VCR）与长春地辛（VDS）。通过长期（6-12个月）暴露于4nM长春花生物碱构建三株耐药细胞系：CAL1R-VRB、CAL1R-VCR与CAL1R-VDS。将各细胞系混合群体提取的RNA与Affymetrix HG-U133 Plus 2.00基因芯片进行杂交，随后采用稳健多阵列平均算法（RMA）对芯片数据进行预处理。