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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2010, Vol. 4 Issue (3) : 323-328    https://doi.org/10.1007/s11684-010-0026-5
RESEARCH ARTICLE
Inhibition of NF-kappa B can enhance Fas-mediated apoptosis in leukemia cell line HL-60
Li WANG1, Shi ZHAO1,2(), Hong-Xiang WANG1, Ping ZOU2
1. Department of Hematology, The Central Hospital of Wuhan, Wuhan 430014, China; 2. Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
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Abstract

This study explored the effects of nuclear factor-kappa B (NF-κB) inhibitor Bay 11-7082 on Fas/FasL system and Fas-mediated apoptosis in cell line HL-60 cells. The mRNA and protein levels of Fas, FasL, and X-linked inhibitor of apoptosis protein (XIAP) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM); the level of sFasL was evaluated by enzyme-linked immunosorbent assay (ELISA); and apoptosis was determined by FCM. After treatment with Bay 11-7082, the mRNA and protein levels of FasL and XIAP in HL-60 cells were significantly lower than in the controls (P<0.05), but the mRNA and protein levels of Fas and sFasL did not change significantly (P>0.05). Apoptotic rate of HL-60 cells treated with Bay 11-7082 was significantly higher than in the controls (P<0.05). Therefore, we conclude that Bay 11-7082 can enhance Fas-mediated apoptosis in HL-60 cells by downregulating FasL and XIAP levels.

Keywords nuclear factor-kappa B      Fas/FasL system      HL-60      Bay 11-7082     
Corresponding Author(s): ZHAO Shi,Email:wlmarx96@yahoo.com.cn   
Issue Date: 05 September 2010
 Cite this article:   
Li WANG,Shi ZHAO,Hong-Xiang WANG, et al. Inhibition of NF-kappa B can enhance Fas-mediated apoptosis in leukemia cell line HL-60[J]. Front Med Chin, 2010, 4(3): 323-328.
 URL:  
https://academic.hep.com.cn/fmd/EN/10.1007/s11684-010-0026-5
https://academic.hep.com.cn/fmd/EN/Y2010/V4/I3/323
geneprimer sequenceslength of products/bp
Fasforward: 5'-TGGCATCAACTTCATGGAAA-3'347
reverse: 5'-AAACATCCTTGGAGGCAGAA-3'
FasLforward: 5'-CCCTGAGCCACAAGGTCTAC-3'284
reverse: 5'-TTCTCGGTGCCTGTAACAAA-3'
XIAPforward: 5'-GAAGACCCTTGGGAACAACA-3'389
reverse: 5'-CGCCTTAGCTGCTCTTCAGT-3'
β-actinforward: 5'- TGAGACCTTCAACACCCCAG-3'312
reverse: 5'-GCCATCTCTTGCTCGAAGTC-3'
Tab.1  Primer sequences and length of products
Fig.1  The survival rate of HL-60 cells detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after treated by Bay 11-7082, and 5 μmol/L was the suitable concentration for the treatment.
Fig.2  The expression of Fas, FasL in HL-60 cells after treatment with Bay 11-7082. The mRNA level of FasL was downregulated after treatment with Bay 11-7082, while Fas did not change significantly. M: DNA marker;2, 4, 5, 7: β-actin; 1, 3: Fas; 6, 8: FasL; 1, 2, 7, 8: Bay 11-7082, 5 μmol/L; 3, 4, 5, 6: Bay 11-7082, 0 μmol/L.
Fig.3  The expression of XIAP in HL-60 cells after treatment with Bay 11-7082. The mRNA level of XIAP was downregulated after treatment with Bay 11-7082. M: DNA marker; 1, 2: Bay 11-7082, 0 μmol/L; 3, 4: Bay 11-7082, 2.5 μmol/L; 5, 6: Bay 11-7082, 5 μmol/L; 1, 3, 5: β-actin; 2, 4, 6: XIAP.
Fig.4  FCM analysis of Fas (a) and FasL (b) protein in HL-60 cells treated with Bay 11-7082. In accordance with the results detected by RT-PCR, the protein level of FasL was downregulated after treatment with Bay 11-7082, while Fas did not change significantly. Blue: control; Red: Bay 11-7082, 0 μmol/L; Green: Bay 11-7082, 5 μmol/L.
groupsmean fluorescence intensity
FasFasL
1: control6.80±1.305.41±1.41
2: Bay 11-7082 (0 μmol/L)9.98±2.7861.62±2.00
3: Bay 11-7082 (5 μmol/L)9.92±1.98Δ51.51±2.11?
Tab.2  Expression of mFas and mFasL in HL-60 cells in control and experimental groups detected by flow cytometry
Fig.5  Examination of the apoptosis rate by flow cytometry. The apoptosis rate of combined group is obviously higher than Bay 11-7082 or CH-11 individually.
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