Inhibition of tissue factor by siRNA enhances doxorubicin-induced apoptosis in human neuroblastoma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the regulation of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblastoma.</p><p><b>METHOD</b>The expression of TF was examined by Western blotting. TF siRNA-pSUPER plasmid was constructed by inserting a specific 19-nt silencing sequence targeting TF gene into pSUPER vector. Transfection of TF siRNA-pSUPER was performed using lipofectamine 2000. The activation of caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest 33342 and counted under fluorescence inverted microscope.</p><p><b>RESULTS</b>(1) Human neuroblastoma cell line SK-N-MC expressed high level of TF. (2) Downregulation of TF expression was achieved by transfection of TF siRNA-pSUPER into SK-N-MC cells in a dose-dependent manner. (3) Cleavage of caspase-3 and PARP was increased in transfected SK-N-MC cell with down-regulation of TF. (4) TF siRNA treatment at 1 microg/ml for 8 h significantly increased apoptotic cell number in transfected SK-N-MC cells compared to that in non-transfected cells (P < 0.05) while exposing to 1 microg/ml doxorubicin for 8 h.</p><p><b>CONCLUSIONS</b>Downregulation of TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human neuroblastoma cells.</p>

Regulation of NF-kappaB/P65 by MDM2 in acute lymphoblastic leukemia in childhood / 中华儿科杂志

<p><b>OBJECTIVE</b>MDM2 is considered a proto-oncogene due to its ability to inhibit P53 tumor-suppressor function. But, evidence showed that MDM2 might have a P53-independent role in tumorigenesis. MDM2 is over-expressed in human sarcoma and carcinoma. Recent studies showed that MDM2 might act as a transcriptional factor to modulate expressions of other genes involved in cell cycle regulation and transformation. In the present study, the investigators hypothesized that MDM2 directly affected NF-kappaB expression and function in a P53-independent manner.</p><p><b>METHODS</b>MDM2 was transfected to acute lymphoblastic leukemia (ALL) line EU-4 cells lacking P53 expression and expressing very low levels of MDM2. MDM2 and P65 expression in mRNA level and protein level were detected by Western blot and Northern blot after transfection. Since the expression of E-selectin is P65 dependent, E-selectin promoter-CAT construct and P65 and MDM2 expression plasmids were co-transfected to EU-4 cells. CAT activation was determined with ELISA. The effect of adriamycin (ADM) at the concentrations of 15 micro g/ml, 7.5 micro g/ml, 5 micro g/ml and 1 micro g/ml on MDM2-transfected EU-4 cells and the parent cells was detected by MTT assay.</p><p><b>RESULTS</b>The results showed that MDM2 up-regulated P65 expression at both mRNA and protein levels, and MDM2 increased P65-mediated transactivation of E-selectin promoter. Without P65, MDM2 had no effect on the transactivation of E-selectin. Moreover, MDM2 antisense could not change the transactivation of E-selectin. MTT results showed that the survival rate of MDM2 transfected EU-4 cells was higher than that of parental cells. The results suggested that MDM2 transfection increase drug resistance of EU-4 cells to ADM compared with parent cells.</p><p><b>CONCLUSION</b>(1) MDM2 up-regulated transcriptionally P65 expression. (2) MDM2 increased drug resistance of leukemia cells to ADM. (3) MDM2 elevated NF-kappaB activity in a P53-independent manner in childhood lymphoblastic leukemia cell line.</p>