RESUMEN

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.

Asunto(s)

Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Farmacogenética/métodos , Biología de Sistemas/métodos , Biomarcadores , Línea Celular Tumoral , Biología Computacional/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Ontología de Genes , Genómica/métodos , Humanos , Transducción de Señal , Flujo de Trabajo

RESUMEN

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.

Asunto(s)

Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células A549 , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

Asunto(s)

Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/uso terapéutico , Células A549 , Línea Celular Tumoral , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genética

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/fisiología , Neoplasias Pulmonares/tratamiento farmacológico , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Humanos

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Transición Epitelial-Mesenquimal , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , MicroARNs/fisiología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Regulación hacia Abajo , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología