Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor(NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated.The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed.The in vitro cultured A549 cells were transfected with pcDNA3.1( )/IκBα alone,or pcDNA3.1( )/IκBα combined with cisplatin.The mito-chondrial membrane potential(?ψm) was determined by rhodamine 123,the activity of caspase-3 was tested by colorimetric assay,and cell apoptosis was detected by flow cytometry with the annexin Ⅴ/propidium iodide assay.The results showed that the activity of NF-κΒ in A549 cells was inhibited by transfecting pcDNA3.1( )/IκΒα.Transfection of pcDNA3.1( )/IκΒα alone did not promote apoptosis.Treatment of cisplatin alone had a little effect on cell apoptosis.Transfection of pcDNA3.1( )/IκΒα combined with cisplatin treatment significantly induced apoptosis of A549 cells.It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Inhibitory activity of nuclear factor-κB potentiates cisplatin-induced apoptosis in A549 cells

Whether inhibiting the activity of nuclear factor （NF）-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1（＋）/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 （＋）/IκBα alone, or pcDNA3.1（＋）/IκBα combined with cisplatin. The mitochondrial membrane potential （△ψm） was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1（＋）/IκBα. Transfection of pcDNA3.1（＋）/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1（＋）/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 ceils. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

rmhTNF-αCombined with Cisplatin Inhibits Proliferation of A549 Cell Line In Vitro

Objective To explore the inhibitory effect of recombinant mutant human tumor necrosis factor-α （rmhTNF-α in combination with cisplatin on human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma cell line A549 was treated with varying concentrations of rmhTNF- （0.38, 0.75, 1.50, 6.00 and 12.00 IU/ml） or cisplatin （3.91, 7.81, 15.63, 31.25 and 62.50 μg/ml） for 24 hours. Viable cell number was analyzed by using crystal violet staining. The inhibitory rates of A549 cells growth by the two drugs were calculated. For analyzing whether there was a synergistic effect of rmhTNF-α with cisplatin, A549 cells were treated with 0.75 IU/ml rmhTNF-α and increased concentrations of cisplatin. Results rmhTNF-α or cisplatin inhibited the growth of A549 cell lines in a dose-dependent manner. The inhibitory effect of rmhTNF-α combined with cisplatin was significantly greater than cisplatin alone at the same concentration （all P〈0.01）. Conclusion rmhTNF-α combined with cisplatin might have synergistic inhibitory effect on human lung adenocarcinoma cell line A549.     

Hypoxia-Inducible Factor-lalpha Suppressing Apoptosis and Increasing Tolerance of Lung Cancer Cells to Chemotherapy

In order to construct plasmid of hypoxia-inducible factor-lalpha （HIF-1α）, and transfect into human lung cancer cells A549, the change in sensitivity of lung cancer cells A549 to chemotherapy was observed. HIF-1α mRNA structure region was amplified by RT-PCR and inserted into plasmid pcDNA3. The expression plasmid pcDNA3/HIF-1α was transfected into A549 with LipofectAMINE^TM2000. The expression of HIF-1α protein was detected by Western blot. After A549 cells were transfected with HIF-1α prior to addition of 5-Fu, the growth activity was measured by growth curve, apoptosis was detected by flow cytometry at 48 h, and the levels of caspase3 and MDR-1 were determined by Western blot. The results showed that the constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two DNA lanes at 2.55 kb and 5.4 kb respectively were found, which were consistent with that expected. The growth rate in 5-Fu group was significantly inhibited, and the apoptosis index and caspase3 activity were increased significantly as compared with control group. After HIF-1α being transfected into A549, the activity of MDR-1 was increased and the effect of 5-Fu was weakened. In conclusion, HIF-1α can promote chemoresistance by increasing the activation of MDR1 and suppressing apoptosis during lung cancer cells A549 in- duced with 5-Fu.     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

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

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

Effects of Banxia Xiexin Decoction (半夏泻心汤) on Cisplatin-Induced Apoptosis of Human A549 Lung Cancer Cells

Objective: To examinie the synergistic effects of Banxia Xiexin Decoction(半夏泻心汤,Known as Banhasasim-tang in Korean) extract(BXDE) on cisplatin-induced cytotoxicity in the A549 human lung cancer cell lines. Methods: A549 cells were treated with varying concentrations(50–200 μg/m L) of cisplatin and BXDE alone or in combination for 96 h. We used 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay and flow cytometry to analyze cell viability and apoptosis, respectively. Results: The exposure of cells to cisplatin and BXDE alone or in combination decreased cell viability dose-and time-dependently(P0.05), which was found to be mediated by the apoptotic pathway as confirmed by the increase in the annexin V+/propidium iodide-stained cell population and a ladder pattern of discontinuous DNA fragments. Furthermore, the apoptosis was inhibited by the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethylketone(z-VAD-FMK). Conclusions: BXDE significantly potentiated apoptotic effects of cisplatin in A549 cells. Moreover, apoptosis induced by BXDE might be the pivotal mechanism mediating its chemopreventative action against cancer.     

The Role of IκBα in TNF-α-induced Apoptosis in Hepatic Stellate Cell Line HSC-T6

To investigate the role of NF-κB in TNF-α induced apoptosis in HSC-T6, a mutant IκBα was transfected into HSC-T6 cells by lipofectin transfection technique and its transient effect was examined 48 h after the transfection. The activation of NF-κB was detected by immune fluorescence cytochemistry and Western blotting with anti-p65 antibody. The apoptosis and the rate of inhibition by TNF-α in both transfected and untransfected HSC-T6 cells were measured respectively by FAC-Scan side scatter analysis and MTF methods. Our results showed that TNF-α could activate NF-κB in untransfected cells but not in transfected HSC-T6 cells. The percentage of apoptosis in transfected cells were significantly higher than that in the untransfected ones （P〈0.01） and it was also true of the inhibition rate （P〈0.01）. It is concluded that the resistance of HSC-T6 towards apoptosis induced by TNF-α can be mediated by NF-κB activation. The inhibition of NF-κB activation by mutant IκBα can attenuate the resistance of HSC-T6 cells and increase its sensitivity to TNF-α.     

Hypoxia-Inducible Factor-1alpha Suppressing Apoptosis and Increasing Tolerance of Lung Cancer Cells to Chemotherapy

In order to construct plasmid of hypoxia-inducible factor-lalpha (HIF-1α), and transfect into human lung cancer cells A549, the change in sensitivity of lung cancer cells A549 to chemotherapy was observed. HIF-1αmRNA structure region was amplified by RT-PCR and inserted into plasmid pcDNA3. The expression plasmid pcDNA3/HIF-1αwas transfected into A549 with Lipofec-tAMINE?000. The expression of HIF-1αprotein was detected by Western blot. After A549 cells were transfected with HIF-1αprior to addition of 5-Fu, the growth activity was measured by growth curve, apoptosis was detected by flow cytometry at 48 h, and the levels of caspase3 and MDR-1 were determined by Western blot. The results showed that the constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two DNA lanes at 2.55 kb and 5.4 kb respectively were found, which were consistent with that expected. The growth rate in 5-Fu group was significantly inhibited, and the apoptosis index and caspase3 activity were increased significantly as compared with control group. After HIF-1αbeing transfected into A549, the activity of MDR-1 was increased and the effect of 5-Fu was weakened. In conclusion, HIF-1αcan promote chemoresistance by increasing the activation of MDR1 and suppressing apoptosis during lung cancer cells A549 induced with 5-Fu.     

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma <Emphasis Type="Italic">in vitro</Emphasis>

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B(NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.     

The role of NF-κB in hepatocellular carcinoma cell

Objective To evaluate the role of nuclear factor-kappaB (NF-κB) and IκBα in hepatocellular cacinoma (HCC) SMMC7721 cells, the consequence of NF-κB inhibition in SMMC7721 cells transfected with mutated IκBα (mIκBα) plasmid and the effect of stable inhibition of NF-κB activity in combination with Doxorubicin.Methods Western blot was used to determine the expression of NF-κB and IκBα in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the 32P-labeled tandem κB sequence using electrophoretic mobility shift assay and the expression of NF-κB using Western blot between SMMC7721 cells transfected with mIκBα plasmid (SMMC7721-MT) and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of κB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated.Results Western blot analysis for nuclear extract showed more P50 (NF-κB1) and P65 (RelA) expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IκBα protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-κB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-κB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-κB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin (P&lt;0.01）.Conclusions The present study demonstrates that upregulation of NF-κB and downregulation of inhibitory kappaB (IκBα) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIκBα in SMMC7721-MT cells can inhibit NF-κB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-κB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-κB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation.     

Inhibitory Effects of Parthenolide on the Activity of NF-κB in Multiple Myeloma via Targeting TRAF6

This study examined the mechanism of the inhibitory effect of parthenolide(PTL) on the activity of NF-κB in multiple myeloma(MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6(TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.     

Pyrrolidine dithiocarbamate Inhibits NF- κB Activation and Enhance TNFα- Induced Apoptosis in Human Breast Cancer Cells

Objective: Tumor necrosis factor α(TNFα) induced apoptosis is limited by its coactivation of nuclear factor kappa B(NF- κB) -dependent antiapoptosis genes. We examined whether pyrrolidine dithiocarbamate (PDTC) enhance TNFα - induced apoptosis in cultured breast cancer cells and explored the role of NF - κB in TNFα - induced apoptosis. Methods: Human breast cancer cell lines MCF-7 and MDA - MB -435s were treated with TNFα、 PDTC and combination therapy . Induction of apoptosis was detected by TUNEL staining and flow cytometry. NF- κB DNA binding activity was detected using electrophoresis mobility shift assay(EMSA) . Western blots of cytoplasmic lysates were performed to demonstrate IκBα (Inhibitor protein of nuclear factor κB) phosphorylation and degradation. Results:TNFα-induced IκBo phosphorylation and degradation, which was inhibited by PDTC in both cell lines. TNFα-induced apoptosis (TUNEL) increased significantly when both cells were pretreated with PDTC. Flow cytometry also confirmed this. EMSA showed that PDTC continuously inhibited TNFo-induced NF- κB DNA binding activity . Conclusions:PDTC enhances TNFo-induced apoptosis whileinhibiting IκBα phosphorylation and degradation in human breast cancer cells. NF - κB has a protective role on TNFα-induced apoptosis.     

ROLE OF ERK1／2 KINASE IN CISPLATIN-INDUCED APOPTOSIS IN HUMAN OVARIAN CARCINOMA CELLS

Objective To investigate the role of extracellular regulated kinase (ERK1/2) pathway in cisplatin-induced apoptosis in human ovarian carcinoma cells.Methods Cisplatin-induced apoptosis were stained with DAPI and was assessed microscopically in human epithelial adenocarcinoma ovarian cell line SKOV3 cells. ERK activation was determined by Western blotting using an anti-phosphoERK antibody to detect ERK activity. The effect of PD98059 on ERK activity induced by cisplatin was detected by MTT assay.Results Marked apoptosis of SKOV3 cells resulted from 48 hours treatment with 20 μg/mL cisplatin. Strong activation of ERK was led to by 15 μg/mL cisplatin. Dose response and time course of cisplatin induced apoptosis in SKOV3 cells.Cisplatin-induced ERK activation occurred at 12 hours and increased to highest induction at 24 hours by Western blotting.The effect of PD 98059 on ERK activity induced by cisplatin at the concentration of 100 μmol/L PD 98059. Statistically significant decreased in cell survival were observed with 100 μmol/L PD 98059 at 15 and 20 μg/mL cisplatin (P＜ 0.05).Conclusions Cisplatin activates the ERK signaling pathway in ovarian cancer cell line SKOV3. Inhibition of ERK activity enhances sensitivity to cisplatin cytotoxity in ovarian cancer cell line SKOV3. Evaluation of ERK activity could be useful in predicting which ovarian cancer will response most favorably to cisplatin therapy.     

Increased expression and activity of MMP-9 in C-reactive protein- induced human THP-1 mononuclear cells is related to activation of nuclear factor kappa-B

The relation between the expression and activity of MMP-9 in C-reactive protein （CRP）-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B （NF-κB） was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 （control group）, 25, 50 and 100 μg/mL （CRP groups） for 24 h. In PDTC （a specific NF-κB inhibitor） group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media （CM） and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α （IκB-α） and NF-κB p65 was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IiB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB p65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB p65 expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.     

MDR1 and MDR3 Genes and Drug Resistance to Cisplatin of Ovarian Cancer Cells

To investigate the relationship between MDR1 and MDR3 gene and drug resistance to cisplatin of ovarian cancer cells. Two siRNAs (MDR1, MDR3) which specifically targeted MDR1 and MDR3 genes were transfered into A2780/DDP cells. Then double staining with Annexin- V-FITC/PI was used to detect cell apoptosis by the flow cytometry (FCM). A2780/DDP cell viability was determined by MTT. MDR1 and MDR3 mRNA were assessed by RT-PCR. Caspase-3 protein was detected by Western blotting. Transfection of MDR1 and MDR3 siRNA into A2780/DDP cells failed to reverse the drug-resistance of A2780/DDP cells to cisplatin (P>0.05). No significant differ- ence in the apoptosis efficiency was observed between the MDR1 and MDR3 siRNA, pSuppressor- Neo vector transfection cells and untreated cells (P>0.05). In the presence of cisplatin of different concentrations, the viability of A2780/DDP cells was not significantly decreased after the transfection. No changes in MDR1 and MDR3 mRNA were found in MDR1 and MDR3 siRNA-transfected A2780/DDP cells. As compared with pSuppressorNeo and untreated groups, no significant difference existed in the expression of MDR1 and MDR3 mRNA (P>0.05). The expression of caspase-3 protein in MDR1 and MDR3 siRNA transfected A2780/DDP cells was not significantly increased. It is con- cluded that multidrug resistance induced by cisplatin in ovarian carcinoma cell lines is not due to overexpression of MDR1 and MDR3 gene. The drug resistance of ovarian carcinoma cells to cisplatin is not mediated by P-glycoprotein.