Cisplatin resistance is associated with altered signalling in NSCLC cells

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, the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 were compared with regard to cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA-damage response. In A549rCDDP2000 cells, the cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was significantly 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 (MDM2, XPC, SIP and GADD45a) as compared to resistant cells. This was underlined by significantly higher protein levels of pATM and p53 in A549 cells after cisplatin treatment compared to the respective untreated controls. Additionally, a data-driven method was used to identify further key candidates responsible for the different response of the two cell lines to the drug. The cellular transcriptome was screened for relevant gene candidates using a whole genome array. By combining statistical methods with available gene annotation without previously defined hypothesis, HRas, JNK3, p38, CCL2 and DOK1 were identified as genes relevant for cisplatin resistance. These genes were further analysed on transcriptome and proteome level to introduce a more systematic approach on different stages of cell signalling. Here, HRas showed lower protein levels and JNK3 a lower mRNA expression after treatment with cisplatin in A549rCDDP2000 cells. In addition to these short-term effects, SIP, JNK3 and p38 showed higher basal mRNA abundance in resistant control cells compared to the sensitive control cells. This circumstance was also observed with p53 on protein level and suggests a relevant long term effect caused during the development of resistance. All results were compiled in a preliminary model of resistance-associated signalling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is a consequence of altered signalling of the identified proteins leading to reduced G2/M cell cycle arrest and apoptosis

Key Players of Cisplatin Resistance: Towards a Systems Pharmacology Approach

The major obstacle in the clinical use of the antitumor drug cisplatin is inherent and acquired resistance. Typically, cisplatin resistance is not restricted to a single mechanism demanding for a systems pharmacology approach to understand a whole cell’s reaction to the drug. In this study, the cellular transcriptome of untreated and cisplatin-treated A549 non-small cell lung cancer cells and their cisplatin-resistant sub-line A549rCDDP2000 was screened with a whole genome array for relevant gene candidates. By combining statistical methods with available gene annotations and without a previously defined hypothesis HRas, MAPK14 (p38), CCL2, DOK1 and PTK2B were identified as genes possibly relevant for cisplatin resistance. These and related genes were further validated on transcriptome (qRT-PCR) and proteome (Western blot) level to select candidates contributing to resistance. HRas, p38, CCL2, DOK1, PTK2B and JNK3 were integrated into a model of resistance-associated signalling alterations describing differential gene and protein expression between cisplatin-sensitive and -resistant cells in reaction to cisplatin exposure

Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest

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

Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest

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

2017 CERN-Fermilab HCP Summer School

<p>Analysis of XPC in a) RT-PCR (n = 3) and b) Western Blot (n = 3) as fold change relative to untreated control in A549 and A549^rCDDP²⁰⁰⁰ cells, presented as mean ± SEM with c) one representative western blot showing A549 untreated, A549 treated with 11 μM cisplatin, A549^rCDDP²⁰⁰⁰ untreated, A549^rCDDP²⁰⁰⁰ treated with 11 μM cisplatin and A549^rCDDP²⁰⁰⁰ treated with 34 μM cisplatin.</p

p21 mRNA and protein analysis.

<p>Analysis of p21 in a) RT-PCR (n = 3) and b) Western Blot (n = 3) as fold change relative to untreated control in A549 and A549^rCDDP²⁰⁰⁰ cells, presented as mean ± SEM with c) one representative western blot showing A549 untreated, A549 treated with 11 μM cisplatin, A549^rCDDP²⁰⁰⁰ untreated, A549^rCDDP²⁰⁰⁰ treated with 11 μM cisplatin and A549^rCDDP²⁰⁰⁰ treated with 34 μM cisplatin.</p

SIP mRNA and protein analysis.

<p>Analysis of SIP in a) RT-PCR (n = 3) and b) Western Blot (n = 3) as fold change relative to untreated control in A549 and A549^rCDDP²⁰⁰⁰ cells, presented as mean ± SEM with c) one representative western blot showing A549 untreated, A549 treated with 11 μM cisplatin, A549^rCDDP²⁰⁰⁰ untreated, A549^rCDDP²⁰⁰⁰ treated with 11 μM cisplatin and A549^rCDDP²⁰⁰⁰ treated with 34 μM cisplatin.</p