Caracterización del perfil quimiosensibilidad y del estado de ampliación génica de un panel de líneas celulares de origen tumoral pulmonar / Characterization of chemosensitivity profile gene amplification status of a panel of lung cancer cell lines

En este trabajo se presentan los resultados de un análisis de amplificación génica y de sensibilidad a fármacos antineoplásicos, realizado para un panel de líneas celulares de origen tumoral pulmonar. Para los ensayos de quimiosensibilidad las células fueron tratadas durante 48 h con concentraciones variables de taxol, cisplatino, doxorrubicina y 5-fluoracilo. La citotoxicidad de los fármacos se cuantificó usando el ensayo de reducción de resazurina y se reportó en valores de concentración inhibitoria 50 (CI50). Para los análisis de amplificación génica se emplearon sondas TaqMan® dirigidas contra los genes AKT2, PIK3CA, ERBB2, EGFR, c-REL y genes de la familia MYC. El número de copias para cada gen fue calculado usando el método de doble delta Ct, empleando ACTB como gen de referencia y la línea MRC-5 como muestra control. Los resultados mostraron que la viabilidad de todas las líneas celulares se afectó por el tratamiento con taxol, cisplatino y doxorrubicina, pero no con el tratamiento con 5-fluoracilo. Las CI50 calculadas se ubicaron entre 0,38 ± 0,03 µM y 111,3 ± 3,58 µM, siendo el taxol y la doxorrubicina los fármacos más potentes. Del panel evaluado las células NCI-H292 resultaron ser las más sensibles y las células LSPG8G las más resistentes a los fármacos. Interesantemente en las células NCI-H292 ningún gen se encontró amplificado; por el contrario en las células LSPG8G los genes cMYC, MYCN, MYCL y AKT2 mostraron un aumento en el número de copias con respecto al de las células control. Estos resultados sugieren que eventos de amplificación génica podrían contribuir con el fenómeno de quimioresistencia en líneas celulares de cáncer de pulmón, sin embargo otros estudios deben realizarse para confirmar esta hipótesis.

In this paper, we show results of anticancer drug sensitivity assays and studies of gen amplification performed for a panel of lung cancer cell lines. For the chemosensitivity assays the cells were treated for 48 h with different concentrations of taxol, cisplatin, doxorubicin and 5-fluorouracil. The cytotoxic effect of each drug was determined using the resazurin reduction assay and reported in terms of inhibitory concentration 50 (IC50). For the analysis of gene amplification we used TaqMan® probes designed against AKT2, PIK3CA, ERBB2, EGFR, REL and MYC family members. Copy number for each gene was calculated using the delta-delta-CT method, employing ACTB as reference gen and MRC-5 cell line as control sample. In the chemosensitivity assays, we observed a clear decrease in cell viability in the cells treated with taxol, cisplatin and doxorubicin but not in the cells treated with 5-fluorouracil. IC50 values ranging between 0,38± 0,03 µM and 111,3 ±3,58 µM, being the taxol and doxorubicin the most potent drugs. NCI-H292 cell line was the most sensitivity and LSPG8G cell line was the most resistant. Interestingly, NCI-H292 cells did not show increase in the copy numbers for the gene evaluated, in contrast, we observed changes in the gene dosage for cMYC, MYCN, MYCL and AKT2 in LSPG8G cells. These results suggest that gene amplification could contribute to drug resistance in lung cancer cell lines; however, more studies are needed to confirm this hypothesis.

The Effect of Doxycycline on PMA-Induced MUC5B Expression via MMP-9 and p38 in NCI-H292 Cells

OBJECTIVES: Doxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated. METHODS: In human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis. RESULTS: PMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9. CONCLUSION: The results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.

Regulation of IL-1beta-Mediated MUC2 Gene and Mucin in Human Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Mucin secretion is regulated by the mucin genes (MUC) in the respiratory, gastrointestinal and reproductive system. Inflammation induces mucin hypersecretion in the human body. This study demonstrates the effects of IL-1beta on the regulation of mucin protein expression as well as the MUC2 gene in cultured airway epithelial cells. MATERIALS AND METHOD: Analysis of MUC2 gene was done by RT-PCR and the protein analysis was done by a flow cytometric analysis and an immunoassay method using cultured human airway epithelial cells, and NCI-H292 cells. RESULTS: The expression of MUC2 mRNA and protein induced by IL-1beta increased in a dose-and time-dependent manner. The maximum mRNA level of the MUC2 gene was approximately 3-fold, compared to that of the control cell. The IL-1beta-mediated MUC2 protein started at 6 hours of exposure to IL-1beta (20 ng/ml) and the maximum level was 12 hours. The MUC2 protein data of flow cytometric analysis corresponded to that of immunoassay analysis. The expression of MUC2 gene was suppressed by actinomycin D, but not attenuated by cycloheximide. CONCLUSION: These results suggest that the IL-1beta-mediated MUC2 gene and protein expression were increased in a dose- and time-dependent pattern and regulated by transcriptional step.

IL-1beta Mediated COX-2 Expression in Human Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Prostaglandin is one of the important inflammatory mediator in inflammatory diseases. Cyclooxygenase-2 (COX-2) plays a key role in biosynthesis of prostaglandins. In this study, we aimed to investigate COX-2 expression and prostaglandin E2 (PGE2) production by interleukin-1beta (IL-1beta) in cultured human airway epithelial cells. MATERIALS AND METHOD: COX-2 gene expression, and COX-2 protein, PGE2 production by IL-1beta were analyzed by RT-PCR, Western blot, and enzymeimmunoassay (EIA) in cultured human airway NCI-H292 epithelial cells. RESULTS: The COX-2 protein production was increased when the cells were exposed to IL-1beta in a dose dependent manner. The maximum level of COX-2 protein was detected at 20 ng/ml of IL-1beta. After 4 hours, the production of COX-2 protein was detected by IL-1beta(20 ng/ml) and this was held up to 12 hour. The maximum level of COX-2 protein production reached at 8 hour of exposure to IL-1beta and this was held up to 12 hour. The release of PGE2 occurred in the same pattern as the IL-1beta-mediated COX-2 protein production. The COX-2 gene expression was induced by IL-1beta (20 ng/ml). The IL-1beta-mediated COX-2 expression was suppressed by actinomycin D, but was not affected by cycloheximide. CONCLUSION: These results suggest that the IL-1beta-mediated COX-2 expression and the PGE2 production were increased in dose and time dependent manner and regulated in the transcriptional step.

Expression of IL-8 induced by Pyocyanin in NCI-H292 cells by PKC-NF-?B signal pathway / 中国药理学通报

Aim To study IL-8 expression of human airway epithelium-like NCI-H292 cells directly induced by Pyocyanin and its mechanism through protein kinase C(PKC) and nuclear factor-kappa B(NF-??).Methods ELISA methods were performed to test the expression of IL-8 in NCI-H292 cells infected by Pyocyanin.Western blot was employed to examine the expression of NF-?B protein.In addition,NCI-H292 cells were cultured together with PKC inhibitor,calphostin C or NF-?? inhibitor,pyrrolidine dithiocarbamate(PDTC),respectively before Pyocyanin infection,then the expression of IL-8 and NF-?? was assayed using the above methods.Results Pyocyanin was able to induce IL-8 protein secretion in NCI-H292 cells and had marked concentration-dependent relations.Pyocyanin could also significantly promote the activation of NF-?B,which peaked at 60~90 min.PDTC and calphostin C could significantly decrease the activation of NF-?? and the expression of IL-8.Conclutions Pyocyanin can induce IL-8 production.The direct induction of Pyocyanin can promote the activation of NF-?B by PKC signal pathway,then cause the expression and secretion of IL-8.