Abstract
BackgroundCisplatin plus pemetrexed combination therapy is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC). However, advanced NSCLC has a 5-year survival rate of below 10%, which is mainly due to therapy resistance. We previously showed that the NSCLC cell line A549 harbors different subpopulations including a mesenchymal-like subpopulation characterized by increased chemo- and radiotherapy resistance. Recently, therapy resistance in hematological and solid tumors has been associated with increased mitochondrial activity. Thus, the aim of this study was to investigate the role of the mitochondrial activity in NSCLC chemotherapy resistance.MethodsBased on MitoTracker staining, subpopulations characterized by the highest 10% (Mito-High) or lowest 10% (Mito-Low) mitochondrial mass content were sorted by FACS (Fluorescence-Activated Cell Sorting) from paraclonal cultures of the NSCLC A549 cell line . Mitochondrial DNA copy numbers were quantified by real-time PCR whereas basal cellular respiration was measured by high-resolution respirometry. Cisplatin and pemetrexed response were quantified by proliferation and colony formation assay.ResultsPemetrexed treatment of parental A549 cells increased mitochondrial mass over time. FACS-sorted paraclonal Mito-High cells featured increased mitochondrial mass and mitochondrial DNA copy number compared to the Mito-Low cells. Paraclonal Mito-High cells featured an increased proliferation rate and were significantly more resistant to cisplatin treatment than Mito-Low cells. Interestingly, cisplatin-resistant, paraclonal Mito-High cells were significantly more sensitive to pemetrexed treatment than Mito-Low cells. We provide a working model explaining the molecular mechanism underlying the increased cisplatin- and decreased pemetrexed resistance of a distinct subpopulation characterized by high mitochondrial mass.ConclusionsThis study revealed that cisplatin resistant A549 lung cancer cells can be identified by their increased levels of mitochondrial mass. However, Mito-High cells feature an increased sensitivity to pemetrexed treatment. Thus, pemetrexed and cisplatin target reciprocal lung cancer subpopulations, which could explain the increased efficacy of the combination therapy in the clinical setting.
Highlights
Cisplatin plus pemetrexed combination therapy is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC)
MitoTracker Deep Red dye localizes to the mitochondria and confers toxicity at higher doses We established an experimental approach to investigate whether mitochondrial activity is associated with chemotherapy resistance
We previously showed that distinct subpopulations co-exist in the parental NSCLC cell line A549, which are characterized by significant differences in epithelial-mesenchymal transition (EMT) status [15]
Summary
Cisplatin plus pemetrexed combination therapy is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC). Lung cancer is the most common cause of cancer-related mortality worldwide This is mainly due to the difficulty of early detection and lack of effective treatment methods, more effective treatment options are desperately needed. The major target of MTA, is mainly localized in the mitochondria, which contain the complete pathway to conduct de novo thymidylate biosynthesis [2]. In this context, the expression of genes involved in pyrimidine metabolism, which are targeted by antifolates such as MTA, is highly upregulated in NSCLC stem cells [3]. Current strategies of MTA-based therapeutic regimens are challenged by relapse of the disease [1]
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