RESUMO
Expression of the TUSC2/FUS1 tumor suppressor gene in TUSC2 deficient EGFR wildtype lung cancer cells increased sensitivity to erlotinib. Microarray mRNA expression analysis of TUSC2 inducible lung cancer cells treated with erlotinib uncovered defects in the response to oxidative stress suggesting that increasing reactive oxygen species (ROS) would enhance therapeutic efficacy. Addition of the thioredoxin reductase 1 inhibitor (TXNRD1) auranofin (AF) to NSCLC cells treated with combination of TUSC2 forced expression with erlotinib increased tumor cell apoptosis and inhibited colony formation. TXNRD1 overexpression rescued tumors from AF-TUSC2-erlotinib induced apoptosis. Neutralizing ROS with nordihydroguaiaretic acid (NDGA) abrogated cell death induced by AF-TUSC2-erlotinib, indicating a regulatory role for ROS in the efficacy of the three drug combination. Isobologram-based statistical analysis of this combination demonstrated superior synergism, compared with each individual treatment at lower concentrations. In NSCLC tumor xenografts, tumor growth was markedly inhibited and animal survival was prolonged over controls by AF-TUSC2-erlotinib. Microarray mRNA expression analysis uncovered oxidative stress and DNA damage gene signatures significantly upregulated by AF-TUSC2-erlotinib compared to TUSC2-erlotinib. Pathway analysis showed the highest positive z-score for the NRF2-mediated oxidative stress response. Taken together these findings show that the combination of TUSC2-erlotinib induces additional novel vulnerabilities that can be targeted with AF.
Assuntos
Auranofina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Receptores ErbB/biossíntese , Cloridrato de Erlotinib/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Objective Establish an experimental model for primary subchondral bone osteoblasts culture of neonatal SD rat's condylar process. Methods Under the condition of sterile,24-hour SD rat was executed and its condylar process was isolated. Removing cartilage layer, the subchondral bone was exposed obviously, then it was cultured with modified repeating enzymatic digestion-adherent explants method. The cellular morphology was identified with invert microscope and immunohistochemistry staining, the osteoblasts were identified by alkaline phosphorase (ALP) staining and calcified nodules staining, and the proliferation of the acquired cells was examined by methyl thiazolyl tetrazolium (MTT) assay. Results A variety of cell morphologies were observed, such as spindle-shaped, triangular and irregular-shape, and their cell processes were significant. The alkaline phosphatase staining and calcified nodules staining of cultured osteoblasts with mineralized nodules were positive. Cells grew slowly in 1-3 days, and the cells growth reached the highest level at the 8th day. The cells growth trend has gradually slowed down after 8 days. Conclusion The method is an efficient way to culture and obtain purified neonatal SD rat's subchondral bone osteoblasts with typical characteristics.
