63ª Reunião Anual da SBPC

C. Ciências Biológicas - 3. Bioquímica - 1. Biologia Molecular

EXPRESSION OF MULTIDRUG RESISTANCE GENE MDR1(ABCB1) IN HUMAN LUNG ALVEOLAR CARCINOMA EPITHELIAL CELLS (A549)

Cesar Augusto Sam Tiago Vilanova-Costa 1 Flávia de Castro Pereira 2 Aliny Pereira de Lima 3 Lucas Carlos Gomes Pereira 4 Bruno Pereira Carvalho 5 Elisângela de Paula Silveira-Lacerda 6

1. Laboratório de Genética Molecular e Citogenética – ICB – UFG 2. Laboratório de Genética Molecular e Citogenética – ICB – UFG 3. Laboratório de Genética Molecular e Citogenética – ICB – UFG 4. Laboratório de Genética Molecular e Citogenética – ICB – UFG 5. Laboratório de Genética Molecular e Citogenética – ICB – UFG 6. Profa. Dra./Orientadora – Laboratório de Genética Molecular e Citogenética – ICB – UFG

INTRODUÇÃO:

Multidrug resistance is a frequent cause of treatment failure in cancer patients. One mechanism of MDR is over-expression of ATP-binding cassette (ABC) transporter proteins that function as a drug efflux pump. These ABC transporter proteins include P-glycoprotein (P-gp), which is a member of the multidrug resistance-associated protein (MRP) family. Lung cancer is one of the leading cause of cancer mortality in industrialized countries, with non-small cell lung cancer (NSCLC) accounting for nearly 80%. First-line treatment for patients with advanced NSCLC includes platinum compounds such cisplatin, carboplatin and oxaliplatin. Nevertheless, the clinical utility of these drugs has been proven limited due to the relatively narrow range of tumors affected and the development of acquired drug resistance. Ruthenium complexes have shown potential utility in chemotherapy with lower toxicities compared to cisplatin attributed to their specific accumulation in cancer tissues. To our knowledge, the mechanism of multidrug resistance to chemotherapy remained largely unknown in A549 tumor cells. The present study aimed to investigate the relationship between the expression of MDR1 in human lung alveolar carcinoma epithelial cells (A549) and treatment with two Ruthenium(III) compounds.

METODOLOGIA:

Human human lung alveolar carcinoma epithelial cells A549 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cell line was maintained at 37°C under 5% CO2 in DMEM medium (pH 7.2-7.4) supplemented with 100 U mL-1 penicillin G, 100 g mL-1 streptomycin, 2 mM L-glutamine, 1.5 g L-1 sodium bicarbonate, 4.5 g L-1 glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10% fetal calf serum 1% (w/v) (all reagents were obtained from Gibco, Grand Island, NY). The tumor cells were cultured in the presence or absence of two Ruthenium(III) Complexes CRu(III) and DRu(III). Total RNA was isolated from A549 cells with Trizol reagent (Invitrogen, USA). cDNA was prepared according to standard methods . For PCR, 30 cycles of denaturation (94°C for 45s), annealing (60°C for 45s), and elongation (72°C for 1 min) was performed using the following primer pairs for ABCB1 gene forward: 5'-CTATGCTGGATGTTTCCGGT-3', reverse: 5'-GCTTTGGCATAGTCAGGAGC-3', which yielded a 147-bp product. The GAPDH control PCR was performed using the following primer pairs: forward: 5'-ACAGTCAGCCGCATCTTCTT-3', reverse: 5'-GTTAAAAGCAGCCCTGGTGA-3', which yielded a 127-bp product. PCR reactions were performed using a LineGene K fluorescence quantitative PCR detection system (BIOER Tech Co. LTD).

RESULTADOS:

In this study, we described the expression of a protein associated with multidrug resistance in human lung carcinoma cell A549. The tested marker exhibited some changes in their expression pattern when tumor cells were treated with different concentrations of Ruthenium(III) compounds CRu(III) and DRu(III) and Carboplatin when compared with untreated A549 cells. Expression of MDR1 in A549 cells was detected by RT-PCR analysis and results showed a the the relative quantities of ABCB1 mRNA expression in A549 treated with DRu(III), CRu(III), and Carboplatin were 0.2850±0.0777, 5.4250±0.4313 and 8.9023±2.6162, respectively, when compared to untreated A549 cells (P = 0.011). The current results showed that MDR1(ABCB1) was weakly expressed in the membranous and intracellular regions of A549 cells treated with Ruthenium(III) complexes, but highly expressed when tumor cells were treated with the antitumor agent Carboplatin.

CONCLUSÃO:

The reduced induction of MDR1 in A549 by CRu(III) and DRu(III) are particularly attractive attributes of these complexes and indicates that they are worthy of further studies as potential anti-tumor agents. The identification of new chemotherapeutics agents is critical for further progress in the treatment of NSCLC.

Palavras-chave: A549, MDR1, Ruthenium(III) complexes.