RESUMO
Cytochrome P450 enzymes (CYP450s) exert mighty catalytic actions in cellular metabolism and detoxication, which play pivotal roles in cell fate determination. Preliminary data shows differential expression levels of CYP27C1, one of the "orphan P450s" in human lung cancer cell lines. Here, we study the functions of CYP27C1 in lung cancer progression and drug endurance, and explore its potential to be a diagnostic and therapeutic target for lung cancer management. Quantitative real-time PCR and immunoblot assays were conducted to estimate the transcription and protein expression level of CYP27C1 in human lung cancer cell lines, which was relatively higher in A549 and H1975 cells, but was lower in H460 cells. Stable CYP27C1-knockdown A549 and H1975 cell lines were established, in which these cells showed enhancement in cell proliferation, colony formation, and migration. In addition, aberrant IGF-1R/Akt/p53 signal transduction was also detected in stable CYP27C1-knockdown human lung cancer cells, which exhibited greater tolerance towards the treatments of anticancer agents (including vinorelbine, picropodophyllin, pacritinib, and SKLB610). This work, for the first time, reveals that CYP27C1 impacts lung cancer cell development by participating in the regulation of the IGF-1R/Akt/p53 signaling pathway, and the level of CYP27C1 plays indispensable roles in dictating the cellular sensitivity towards multiple anticancer agents.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cell lines which stably express reporter proteins through CYP3A4 gene activation have been developed for use in predicting CYP3A4 induction. Twelve clones showing distinct profiles on chemical-induced response were isolated. Among them, two clones showing high response for CYP3A4 inducers, namely clone 3-1-10 and 3-1-20, were further evaluated for their sensitivities, reproducibilities and applicabilities to predict CYP3A4 induction in human. Clone 3-1-10 showed higher response to rifampicin than to clotrimazole, whereas clone 3-1-20 had rather higher response to clotrimazole. Optimal plating density and highly reproducible response were observed at the range of 1.65-5.0 x 10(4) cell/cm2. Clear induction responses of more than ten chemicals were observed in both cell lines. The reporter activity was further dramatically increased after an introduction of human PXR. Induction with rifampicin was, however, not much altered between the absence and presence of hPXR. The luciferase activity remained unaltered and showed little fluctuation during the culture for more than 6 months. Due to the strikingly high sensitivity and reproducibility of this system, as compared to previously published systems, these HepG2-derived cell lines showing distinct response profiles as developed in the present study will offer high advantages for chemical screening of CYP3A4 inducibility.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Adenoviridae/genética , Linhagem Celular , Células Cultivadas , Células Clonais , Citocromo P-450 CYP3A , DNA/biossíntese , DNA/genética , Indução Enzimática/efeitos dos fármacos , Genes Reporter/genética , Vetores Genéticos , Humanos , Luciferases/biossíntese , Luciferases/genética , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genéticaRESUMO
Assessment of foreign chemical inducibility on CYP3A4 is necessary to optimize drug therapies. The properties of chemicals such as pesticides, however, are not well investigated. In the present study, properties of various pesticides on human CYP3A4 induction have been tested using HepG2-derived cells stably expressing the CYP3A4 promoter/enhancer (3-1-10 cells) and the human pregnane X receptor (hPXR)-small interfering RNA (siRNA) system. Among the examined pesticides, 13 pesticides were observed to activate the CYP3A4 gene. Surprisingly, pyributicarb was found to increase the CYP3A4 reporter activity at 0.1 to 1 microM more strongly than typical CYP3A4 inducer rifampicin. Expression of hPXR-siRNA clearly diminished the pyributicarb-stimulated CYP3A4 reporter activity in 3-1-10 cells and decreased the endogenous CYP3A4 mRNA levels in HepG2 cells. Pyributicarb caused enhancement of CYP3A4-derived reporter activity in mouse livers introduced with hPXR by adenovirus. These results indicate pyributicarb as a potent activator of CYP3A4 gene, suggesting the existence of pesticides leading to CYP3A4 induction in our environment.