RESUMEN
BACKGROUND: Increased apolipoprotein B100 (apo B) and decreased apolipoprotein A-I (apo A-I) production are important risk factors in atherosclerosis. Urotensin II (UII), as the most potent vasoconstrictor in human, is related with hypertension and probably atherosclerosis. Because of the relationship between the hypertension and lipoprotein metabolism in atherosclerosis, the aim of this study was to test the effect of urotensin II on apo B and apo A-I expression in hepatic (HepG2) cell line. MATERIALS AND METHODS: HepG2 cells were treated with 10, 50, 100, and 200 nmol/L of urotensin II (n = 6). Relative apo B and apo A-I messenger RNA (mRNA) levels in conditioned media, normalized to glyceraldehyde-3-phosphate dehydrogenase, were measured with quantitative real-time polymerase chain reaction method. In addition, apo B and apo A-I levels were also estimated and compared with the controls using the western blotting method. Data were analyzed statistically by ANOVA and non-parametric tests. RESULTS: The apo B mRNA levels were not increased significantly following the treatment with UII. However, apo B protein levels were increased significantly after the treatment with urotensin II, especially at 100 and 200 nmol/L. The apo A-I mRNA and protein levels in conditioned media also were not significantly changed. However, there was a significant decrease in apo A-I mRNA and protein levels at 200 nM UII. CONCLUSIONS: UII might increase apo B at protein level probably through participating factors in its synthesis and/ or stability/degradation. In addition, UII may have decreasing effect at more than 200 nM concentrations on apo A-I.
RESUMEN
Epidermal growth factor receptor (EGFR) is over-expressed in several human cancers. This would suggest that inhibition of EGFR is a reasonable approach for cancer treatment. In this study we investigated EGFR blocking and its effects on the mediated signaling such as MAPK and STATb in HT29 cells. For this aim we used FITC-labeled EGFR antisense oligonucleotides encapsulated with PAMAM nanoparticles to inhibit EGFR expression. Cellular uptake of antisense was investigated by fluorescence microscopy and flow cytometry analysis. The effect of EGFR antisense on the expression of EGFR in HT29 cells was examined by real time PCR and Western blots, which showed that antisense encapsulated with PAMAM decreased the level of EGFR mRNA and protein. In addition, real time PCR results confirmed that EGFR inhibition had an effective role in the reduction of EGFR dependent downstream genes. In conclusion, EGFR antisense encapsulated with PAMAM nanoparticles down regulated EGFR and EGFR-mediated genes.
Asunto(s)
Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Oligonucleótidos Antisentido/farmacología , Factor de Transcripción STAT5/genética , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Nanopartículas , ARN Mensajero/metabolismoRESUMEN
Colon cancer continues to be one of the most common cancers, and the importance and necessity of new therapies needs to be stressed. The most important proto-oncogen factors for colon cancer appear to be epidermal growth factor receptor, EGFR, and c-Src with high expression and activity leading to tumor growth and ultimately to colon cancer progression. Application of c-Src and EGFR antisense agents simultaneously should theoretically therefore have major benefit. In the present study, anti-EGFR and c-Src specific antisense oligodeoxynucleotides were combined in a formulation using PAMAM dendrimers as a carrier. Nano drug entry into cells was confirmed by flow cytometry and fluorescence microscopy imaging and real time PCR showed gene expression of c-Src and EGFR, as well as downstream STAT5 and MAPK-1 with the tumor suppressor gene P53 to all be downregulated. EGFR and c-Src protein expression was also reduced when assessed by western blotting techniques. The effect of the antisense oligonucleotide on HT29 cell proliferation was determined by MTT assay, reduction beijng observed after 48 hours. In summary, nano-drug, anti-EGFR and c-Src specific antisense oligodeoxynucleotides were effectively transferred into HT-29 cells and inhibited gene expression in target cells. Based on the results of this study it appears that the use of antisense EGFR and c-Src simultaneously might have a significant effect on colon cancer growth by down regulation of EGFR and its downstream genes.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Familia-src Quinasas/genética , Análisis de Varianza , Dendrímeros , Regulación hacia Abajo/efectos de los fármacos , Receptores ErbB/metabolismo , Células HT29 , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Oligonucleótidos Antisentido/genética , Factor de Transcripción STAT5/genética , Proteína p53 Supresora de Tumor/genética , Familia-src Quinasas/metabolismoRESUMEN
c-Src is one member of non-receptor tyrosine kinase protein family that has over expression and activation in many human cancer cells. It has been shown that c-Src is implicated in various downstream signaling pathways associated with EGFR-dependent signaling such as MAPK and STAT5 pathways. Transactivation of EGFR by c-Src is more effective than EGFR ligands. To inhibit the c-Src expression, we used c-Src antisense oligonucleotide complexed with PAMAM Denderimes. The effect of c-Src antisense oligonucleotide on HT29 cell proliferation was determined by MTT assay. Then, the expression of c-Src, EGFR and the genes related to EGFR-depended signaling with P53 was applied by real time PCR. We used western blot analysis to elucidate the effect of antisense on the level of c-Src protein expression. The results showed, c-Src antisense complexed with PAMAM denderimers has an effective role in decrease of c-Src expression and EGFR-dependent downstream genes.