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Objective: To investigate the expression of inhibitor of nuclear factor κBα (IκBα) in bladder cancer cell lines and its correlation with epithelial-mesenchymal transition (EMT) and invasive potential in vitro., Methods: The expressions of IκBα, epithelia cadherin (E-cadherin), neuronal cadherin (N-cadherin) and vimentin in human bladder cancer RT4, 5637, 235J, J82 and T24 cell lines were detected by Western blotting. Invasive potential of these cell lines was evaluated by Transwell(TM) assay. In addition, morphological characteristics of those cells were carefully observed under a microscope., Results: RT4 and 5637 cells exhibited epithelial cell morphological features, but 253J, J82 and T24 cells had mesenchymal cell morphological features. E-cadherin was over-expressed in RT4 and 5637 cell lines, but the expressions of vimentin and N-cadherin could not been detected in the two cells. E-cadherin could be detected in 253J cells with a lower expression level; J82 and T24 cells had no expression of E-cadherin; vimentin and N-cadherin were highly expressed in 253J, J82 and T24 cells. Transwell(TM) assay showed higher invasive potential of 253J, J82 and T24 cells than RT4 and 5637 cells in vitro. In addition, RT4 and 5637 cells expressed higher levels of IκBα than 253J, J82 and T24 cells., Conclusion: Expression of IκBα in human bladder cancer cells is negatively correlated with EMT and tumor invasion in vitro.

Background & Objective: Recent studies have shown that activation of nuclear factor-kappaB(NF-kappaB) can regulate the invasion and metastasis of cancer cells. The present study was to investigate inhibition of NF-kappaB activity on invasion of human lung cancer cell line A549 and the possible mechanism., Methods: The recombinant plasmid pcDNA3.1(+)/IkappaBalpha, expressing the alpha isoform (IkappaBalpha) of the NF-kappaB inhibitor, was constructed. A549 cells were cultured in vitro and divided into non-transfection group, pcDNA3.1(+) transfected group and pcDNA3.1(+)/IkappaBalpha transfected group. The expression of IkappaBalpha was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The activity of NF-kappaB was determined by electrophoretic mobility shift assay (EMSA). Invasion of A549 cells was assessed by transwell chamber assay. The expression of MMP-2 and MMP-9 was detected by RT-PCR and gelatin zymography., Results: Plasmid pcDNA3.1(+)/IkappaBalpha was successfully constructed and expressed in A549 cells. The activity of NF-kappaB, the number of invasive cells, the activity of MMP-2 and MMP-9 of A549 cells in pcDNA3.1(+)/IkappaBalpha transfected group were significantly lower than those in non-transfection group and pcDNA3.1(+) transfected group (all P<0.05)., Conclusion: Transfection of IkappaBalpha can inhibit NF-kappaB activity, thus inhibit cell invasion of A549, which may be through the down-regulation of MMP-2 and MMP-9 expressions.

Objective: To investigate the efficacy of transfection with a mutant IkappaBalpha gene (mIkappaBalpha) in enhancing the radiosensitivity of hepatocellular carcinoma cells., Methods: The hepatocellular carcinoma Hep G2 cells were divided into 3 group and transfected with the adenovirus containing mIkappaBalpha vector (Ad-mIkappaBalpha group), Ad vector (Adv group), or without treatment (parental control cells). Before and after irradiation of the cells with 6 Gy high-energy X ray, Western blotting was performed to measure the expression level of IkappaBalpha in the cytoplasm, and electrophoresis mobility shift assay (EMSA) carried out to evaluate the nuclear factor-kappaB (NF- kappa;B) activity in the cell nuclei, with the cell apoptosis detected using TUNEL assay. The radiosensitivity of the HepG2 cells were determined by comparison between the 3 groups in term of the surviving cell fractions (SF2) after 2-Gy X-ray exposure, and of the Do and Dq values obtained using the single-hit multi target model., Results: Before the X-ray exposure, the Hep G2 cells in Adv group and the control group showed low levels of IkappaBalpha absorbance in the cytoplasm, which were further decreased after the exposure. The NF-kappaB activity in the nuclei of the cells in the two groups was positive (+) before irradiation, and substantially enhanced (++) after the exposure and maintained the stably activated state. The apoptotic index of the cells in the two groups was 1.4 and 1.6 before irradiation, and increased to 8.9 and 11.7 after the irradiation, respectively. The cells in the Ad-mIkappaBalpha group, however, exhibited high levels of IkappaBalpha absorbance either before or after the irradiation, which were approximately 3 times that of the Adv group, but the NF-kappaB activity remained negative irrespective of the irradiation. The apoptotic index of the cells in the Ad-mIkappaBalpha group was 18.2 before irradiation, was increased to 88.3 after the irradiation. Among the 3 groups, Ad-mIkappaBalpha group had the smallest SF(2) value of 0.301 but the highest sensitivity enhancement ratio (SER) of2.99, with the lowest Do and Dq values (1.468 and 0.709, respectively)., Conclusion: mIkappaBalpha gene transfection into HepG2 cells inhibits the anti-apoptotic activity of NF-kappaB to enhance the radiosensitivity of the cells.

Objective: To study the activation of nuclear factor-kappaB (NF-kappaB) in gastric carcinoma SGC-7901 cells after treating with As(2)O(3) and the enhancement of the therapeutic effect of As(2)O(3) with recombinant adenovirus IkappaBalphaM., Methods: Culture of gastric carcinoma SGC-7901 cells was carried out. The cells both uninfected and infected with Ad-IkappaBalpha but not treated with As(2)O(3) were used as control. Electrophoretic mobility shift assay (EMSA) and immunohistochemistry were used to detect the activation of NF-kappaB in the cells after treatment of As(2)O(3) and the combination with Ad-IkappaBalphaM. MTT, Hoechst staining and TUNEL were used to assay the change of apoptosis induced by As(2)O(3) after infection with Ad-IkappaBalphaM., Results: The results of EMSA and immunohistochemical method showed that after the treatment of As(2)O(3) the cells showed high activity of NF-kappaB. Simultaneous infection with Ad-IkappaBalphaM can inhibit the activation of NF-kappaB; MTT method indicated that after the treatment of As(2)O(3) infected with Ad-IkappaBalphaM apoptosis rate of the cells (59.2 +/- 2.5)% was higher than that of the cells treated with As(2)O(3) and infected with Ad-IkappaBalpha but not treated with As(2)O(3) (47.5 +/- 2.3)% and these neither infected nor treated (40.0 +/- 1.2%), P < 0.01. The result of Hoechst staining method indicated that, in the group of cells treated with As(2)O(3) and infected with Ad-IkappaBalphaM, apoptosis rate is (27.7 +/- 2.6)%, which was higher than the that of the cells infected with Ad-IkappaBalpha (18.3 +/- 1.5)% but not treated with As(2)O(3) and these neither infected nor treated (11.0 +/- 1.7%), P < 0.05. Hoechst staining method was in accordance with TUNEL technique; it was shown that in the group of cells treated with As(2)O(3) and infected with Ad-IkappaBalphaM, apoptosis rate was (31.1 +/- 2.5)%, being still higher than that of cells infected with Ad-IkappaBalpha but not treated with As(2)O(3) (20.7 +/- 2.1)% and these neither infected nor treated (13.0 +/- 1.7)%, P < 0.01. Therefore, infection with Ad-IkappaBalphaM can significantly increase the apoptosis induced by As(2)O(3)., Conclusions: Gastric carcinoma cells treated with As(2)O(3) show activity of NF-kappaB. It is indicated that the activity of NF-kappaB may be the mechanism of the antagonism of gastric carcinoma cells against the apoptosis induced by As(2)O(3). Infection with Ad-IkappaBalphaM can effectively inhibit the activation of NF-kappaB in gastric carcinoma cells and increase the cell apoptosis induced by As(2)O(3).

Objective: To investigate the interaction between p38 mitogen-activated protein kinase signal transduction pathway and nuclear factor (NF)-kappaB/IkappaB system on the proinflammatory cytokines release after burn trauma., Methods: Human monocyte line THP-1 were incubated with serum from eight healthy controls, burn sera, burn sera pretreatment with SB203580, and burn sera pretreatment with pyrrolidine dithiocarbamate (PDTC). After 24 hours incubation with serum, tumor necrosis factor (TNF)-alpha and interleukin-1beta (IL-1beta) levels in THP-1 culture supernatants were measured by ELISA. The activities of p38 MAPK and expressions of IkappaBalpha in THP-1 were measured by Western blot analysis. The EMSA method was used to characterize the binding activities of NF-kappaB and activating protein (AP)-1 in THP-1., Results: In comparison with normal controls, burn sera resulted in a significant higher level release of TNF-alpha and IL-1beta in THP-1 [(7.30 +/- 0.84) ng/ml vs (2.20 +/- 0.28) ng/ml, P < 0.05; (2.88 +/- 0.38) ng/ml vs (0.81 +/- 0.14) ng/ml, P < 0.05], which were significantly inhibited by pretreatment with SB203580 or PDTC. Burn sera showed increased activities of p38 MAPK and AP-1 in THP-1 (4728 +/- 582 vs 1291 +/- 163, P < 0.05; 946 +/- 137 vs 361 +/- 40, P < 0.05), which were abolished by pretreatment with SB203580 but not PDTC. The expression of IkappaBalpha in THP-1 incubated with burn sera was significantly decreased than those incubated with control sera (1211 +/- 115 vs 2658 +/- 318, P < 0.05), which were abolished by pretreatment with PDTC but not SB203580. Burn sera also leaded to an increased activity of NF-kappaB in THP-1 (1636 +/- 170 vs 317 +/- 32, P < 0.05), which were abolished by pretreatment with PDTC but not SB203580., Conclusions: There are no direct interaction between p38 MAPK signal transduction pathway and NF-kappaB/IkappaB pathway. These two pathways, which regulate the production of TNF-alpha and IL-1beta in monocyte following burn trauma, are parallel and independent.

Objective: To investigate the inhibition consequence of NF-kappaB activity and cell viability by transfecting mutated inhibitor kappa B alpha (mI(kappa)B(alpha)) into liver cancer cell line of SMMC-7721 cells., Methods: The nucleic proteins of SMMC-7721 cells transfected with mI(kappa)B(alpha) plasmid and cells with empty pcDNA3 vector were used to determine not only the binding of the 32P-labelled kappaB probes by EMSA, but also the expression of NF-kappaB by western blot. Cell viability was also analyzed., Results: NF-kappaB nuclear translocation was inhibited remarkably in SMMC-7721 cells transfected with mI(kappa)B(alpha) at 0, 24, 48 and 96 hours. Furthermore, NF-kappaB was not detected in the nucleic protein of mI(kappa)B(alpha) -transfected cells at the same intended time by western blot. Compared with that of control cells, the growth of SMMC-7721 cells transfected with mI(kappa)B(alpha) was suppressed evidently, especially on the second day, the cpm values of mI(kappa)B(alpha) -transfected cells, pcDNA3-transfected cells, and control cells were 5,092.63+/-541.41, 7,851.87+/-72.76, and 8,240.8+/-603.26 respectively (t = 14.29, P<0.01; t = 10.99, P<0.01)., Conclusion: Stable expression of mI(kappa)B(alpha) in SMMC-7721 cells transfected with mI(kappa)B(alpha) plasmid inhibits NF-kappaB nuclear translocation, then suppresses the cell growth.