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Background: Our previous studies demonstrated that mutant IkappaBalpha (IkappaBalphaM) inhibited the occurrence, growth and angiogenesis of human glioblastoma multiform (GBM). However, the specific mechanism by which IkappaBalphaM regulates protein-degrading enzymes secreted from GBM to inhibit invasion and metastasis has remained unclear. The aim of the present study was to investigate the regulatory role and significance of IkappaBalphaM genes in the expression of tissue inhibitor of metalloproteinase (TIMP)-2 and matrix metalloproteinase (MMP)-9 in human GBM., Methods: We established the following four GBM cell lines stably expressing IkappaBalphaM by plasmid construction, gene transfection and screening for IkappaBalphaM protein expression: mutant IkappaBalpha-transfected cells (G36Delta-M), wild-type IkappaBalpha-transfected cells (G36Delta-W), empty plasmid transfected cells (G36Delta-P) and untransfected cells (G36Delta). The TIMP-2 and MMP-9 expression was detected by RT-PCR and Western blotting. Tumor cells were then implanted subcutaneously into nude mice to establish an animal model of ectopic tumor growth, and TIMP-2 and MMP-9 expression was determined by immunohistochemical methods., Results: The results showed that there was a significant increase in TIMP-2 expression and a significant decrease in MMP-9 expression in the G36Delta-M group at both the RNA and protein levels compared with the G36Delta-W group, G36Delta-P group and G36Delta group. Similar results were observed in the immunohistochemical staining analysis of tumor tissues. In the G36Delta-M group, TIMP-2 expression was significantly higher while MMP-9 expression was significantly lower than in the other three groups., Conclusions: Our findings indicate that IkappaBalphaM inhibits the activation of NF-kappaB. It significantly up-regulates TIMP-2 expression in human malignant glioma cells and down-regulates the expression of MMP-9. Thus, IkappaBalphaM maintains the integrity of the extracellular matrix and further inhibits the growth and metastasis of tumor tissues.

Background: Previous studies have showed that photooxidative stress can lead to down-modulation of nuclear factor-kappa B (NF-kappaB) activity causing apoptosis of cultured photoreceptor cells. This study aimed at investigating whether NF-kappaB was involved in photoreceptor cells apoptosis induced by N-methyl-N-nitrosourea (MNU) in rats., Methods: A single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats. At different intervals after MNU treatment, the animals were sacrificed. Retinal damage was examined by a light microscope. The apoptotic index of the photoreceptor cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). NF-kappaB was analysed by Western blot and Transcriptin Factor Assay Kits., Results: The pyknosis of the photoreceptor nuclei and the disorientation of the outer segment of the photoreceptor layer was seen after MNU treatment for 24 hours. The outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. Photoreceptor cells apoptosis reached the peaked value at 24 hours. In apoptotic cascade, the protein levels of NF-kappaB p65 were only detected after MNU treatment for 12 and 24 hours in the nucleus. Conversely, the amounts of IkappaBalpha were markedly increased in the cytoplasm as well as in the nucleus. The activity of NF-kappaB p65 in the nucleus was down-modulated in the end., Conclusions: MNU-induced photoreceptor cell destruction was attributed to the apoptotic process by down-regulating the activation of NF-kappaB p65.

Background: The aim of this study was to explore whether the inhibition of nuclear factor-kappaB (NF-kappaB) activation by mutant IkappaBalpha (S32, 36-->A) can enhance TNF-alpha-induced apoptosis of leukemia cells and to investigate the possible mechanism., Methods: The mutant IkappaBalpha gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IkappaBalpha were obtained by the limiting dilution method. TNF-alpha-induced NF-kappaB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-alpha. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM)., Results: Mutant IkappaBalpha protein was confirmed to exist by Western blot. The results of EMSA showed that NF-kappaB activation by TNF-alpha in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IkappaBalpha repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-alpha, but changed very little in transfected HL-60 cells. The inhibition of NF-kappaB activation by mutant IkappaBalpha enhanced TNF-alpha-induced apoptosis. The cytotoxic effects of TNF-alpha were amplified in a time- and dose-dependent manner., Conclusions: NF-kappaB activation plays an important role in the resistance to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB by mutant IkappaBalpha could provide a new approach that may enhance the anti-leukemia effects of TNF-alpha or even of other cytotoxic agents.