Abstract
The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis.
Highlights
Non-small cell lung cancer (NSCLC) is associated with high incidence and mortality [1]
The human non-small cell lung carcinoma (NSCLC) cell line A549 was obtained from ATCC (Manassas, VA, USA) and its cisplatin resistant sub-line A549rCDDP2000 was derived from the Resistant Cancer Cell Line (RCCL) collection
Cisplatin cytotoxicity was markedly reduced in the A549rCDDP2000 cells compared to the A549 cell line after 24 h treatment
Summary
DrugsCis-diamminedichloroplatinum (II) (cisplatin) was obtained from Sigma–Aldrich, Steinheim, Germany and dissolved in 0.9% NaCl at a final concentration of 1.5 g/L. The sub-line had been established by adapting the growth of A549 cells in the presence of increasing concentrations of cisplatin until a final concentration of 2000 ng/mL cisplatin as described previously [9]. The medium of the A549rCDDP2000 cells contained 2 μg/mL cisplatin. Cells were allowed to attach overnight, experienced 4 h of serum starvation and were subsequently treated with cisplatin for 24 h in IMDM medium without any supplement. The cisplatin concentrations used were cell line dependent and based on the respective EC10. Both cell lines were treated with 11 μM cisplatin (EC10 of sensitive cell line). The resistant sub-line was besides treated with 34 μM cisplatin (the respective EC10). Equimolar treatment refers to treatment of sensitive and resistant cell line with 11 μM cisplatin and equitoxic treatment refers to treatment of sensitive cell line with 11 μM cisplatin and resistant cell line with 34 μM cisplatin
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