Inhibition of NF-kappa B can enhance Fas-mediated apoptosis in leukemia cell line HL-60

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

Introduction

Apoptosis plays an important role in the development and progress of tumor and tumor resistance to antitumor drugs. Chemotherapeutics may activate Fas-mediated apoptosis to treat tumor. However, there is resistance to the apoptosis in most leukemia cells [1,2], so it provides a novel way to treat leukemia by reducing or even reversing the resistance. Studies in recent years indicate that continuous abnormal activation of NF-κB exists in various types of leukemia cells, while it has a relationship with Fas-mediated apoptosis resistance in leukemia cells [3-5]. NF-κB is considered to be a predictor of poor treatment outcome in childhood acute lymphoblastic leukemia, and inhibitors for the activity of NF-κB and stimulants to Fas could have a potential therapeutic benefit [6]. Many researches indicate that the sensitivity of tumor cells, such as the lymphoma cell line HuT78 and T cell line H9, to apoptosis can be enhanced by inhibiting NF-κB, but the mechanism is not clear yet [7-9]. This study intended to clarify the potential mechanism by observational study.

Materials and methods

Major reagents

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was the product of Sigma (USA), Bay 11-7082 was procured from ALEXIS (USA), and FITC series were the products of the Biolegend (USA). CH-11 (activating anti-Fas) and sFasL enzyme-linked immunosorbent assay (ELISA kit), cDNA synthesis kit, and the Annexin V-FITC were separately produced by the Upstate, Bender, TOYOBO (Japan) and the Shen Zhen Jing Mei Biologic Engineering Limited Company (China).

Cell culture

The human myeloid leukemia cell line (HL-60), introduced by the Wuhan Typical Culture Reserve Center (China), was cultured in the RPMI 1640 cultivating liquid with 10% serum of embryo calf (Tianjing Three Benefits Biology Engineer Institute, China) at 37°C and in 5% CO₂, and it was passaged once daily. The doubling time of HL-60 was 24±1 h. The cells in logarithmic growing phase were harvested.

Measurement of cell growth and viability

The HL-60 cells in logarithmic growing phase were adjusted to 1 × 10⁵/mL with the RPMI 1640 liquid containing 10% serum and inoculated in 96-well cultivation plates, and then, Bay 11-7082 was added at different concentrations. As a result, the final concentration of Bay 11-7082 was 0, 2.5, 5, and 10 μmol/L, separately. The group without Bay 11-7082 (0 μmol/L) was considered as the control group. Four parallel wells were established in each group. A culture medium without cells was added into the blank control well. After the cells were cultured for 24 h, 20 μL MTT working solution (5 mg/mL) was added to each well, and the cells were incubated for 5 h at 37°C; the supernatant was discarded after centrifugation. Dimethyl sulphoxide (DMSO) (0.15 mL) was added to each well and shaken for 10 min to dissolve the crystal thoroughly. The absorbance (A) value at 570 nm was measured, and the active cell percentage was calculated according to the A value.

Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of Fas, FasL, and XIAP gene expression

The cells of the control group and the HL-60 cells treated with Bay 11-7082 (5 μmol/L) for 6 h were harvested, and total RNA was extracted using TRIzol reagent. Then, 2 μg of total RNA was reversely transcribed by M-MuLV reverse transcriptase according to the manufacturer’s instructions; PCR was then performed as described by the manufacturer with specific primers. The primer sequences and length of products are presented in Table 1. The amplification products were electrophoresed on a 2% agarose gel and scanned, and the ratios of Fas/β-actin, FasL/β-actin and XIAP/β-actin were considered as the relative expression levels of Fas, FasL, and XIAP.

Flow cytometry analysis of mFas and mFasL

The cells of the control group and the HL-60 cells treated with Bay 11-7082 (5 μmol/L) for 6 h were collected with tubes. After centrifugation, the supernatant was discarded, and the cells were suspended in 100 μL of ice-cold PBS. The cell number was adjusted to 10⁶/tube. Twenty μL FITC-anti-Fas and 20 μL PE-anti-FasL were added into the tubes, and then, the mixture was incubated on ice for 30 min in darkness. After centrifugation, the supernatant was removed. Four mL PBS was added to remove the unbinding antibody. The mixture was then suspended in 300 μL PBS for subsequent flow cytometry detection. The relative intensity of mFas or mFasL was presented as the average cell fluorescent intensity.

ELISA analysis of sFasL

The supernatant of the cells before and after being treated with Bay 11-7082 (5 μmol/L) for 6 h was collected. sFasL content was detected by ELISA method. The detailed steps were as follows: The microtiter plate was coated with anti-FasL monoclonal antibody overnight at 4°C. After the plates were washed in assay buffer, 100 µL of standards or samples were added into each well and incubated at room temperature for 2 h. The diluted HRP-IgG was then added to each well, followed by incubation at 37°C for 1 h. Intermittent washings of the microplate with buffer were carried out before each addition. The chromogenic enzyme reaction was initiated by adding the substrate solution. The reaction was terminated by 100 µL of H₂SO₄(2 mmol/L) per well 20 min later. The A value was read at 490 nm. A standard curve was constructed for calculation of the level of sFasL.

Flow cytometry analysis of apoptosis cells

The flow cytometry analysis included four groups: negative control group, Bay treated group, CH-11 treated group, and CH-11 combined with Bay group. The last group was obtained as follows: after HL-60 was treated with Bay 11-7082 (5 μmol/L) for 1 h, CH-11 (20 μmol/L) was added. The cells of each group were collected after treatment for 24 h, and then, cold PBS was added and centrifuged at 4°C. After the cells were washed twice, resuspended in 200 μL binding buffer, 10 μL Annexin V-FITC and 5 μL PI were added, respectively. The mixture was then incubated at 37°C for 30 min in the dark; 300 μL binding buffer was added, and the apoptosis rates were detected within 1 h.

Statistical analysis

The data were expressed as \overline{x}\pm s. T test was applied to analyze the average ratio of two samples; while if the number of samples was above two, it was analyzed by one-way ANOVA. SPSS 12.0 for Windows statistic software was employed for analysis.

Results

Inhibiting effect of Bay 11-7082 on the proliferation of HL-60

As it is shown in Fig. 1, the inhibiting effect of the Bay 11-7082 on the proliferation of HL-60 depended on the dosage. After being treated with 0, 2.5, 5, 7.5, and 10 μmol/L Bay 11-7082 for 24 h, the survival rates were 97.76%, 94.05%, 76.96%, 55.88%, and 21.87%, respectively, and 5 μmol/L was chosen as the dealing concentration of Bay 11-7082.

Influence of Bay 11-7082 on Fas, FasL, and XIAP gene expression

After treatment with 5 μmol/L Bay 11-7082 for 6 h, the level of the FasL and XIAP reduced obviously, while it had little effect on the intensity of Fas. The ratios of the Fas/β-actin, FasL/β-actin, and XIAP/β-actin were 0.41±0.10, 0.41±0.06, and 0.33±0.06 in the control group, respectively. The ratios of Fas/β-actin, FasL/β-actin and XIAP/β-actin were 0.39±0.09, 0.20±0.04, and 0.13±0.03 in the treatment group. More information is provided in Figs. 2 and 3.

Influence of Bay 11-7082 on mFas and mFasL protein

Figure 4 shows the average fluorescent intensities of Fas and FasL by using FCM, which is in accordance with the change of the transcription level. The average fluorescent intensity of mFasL declined after intervention, and the difference was significant. However, there was no significant change in mFas. Table 2 shows the average fluorescent intensities of Fas and FasL in the control and experimental groups, respectively.

Influence of Bay 11-7082 on serum sFasL level

A standard curve was drawn according to the instruction of ELISA kit, and then, sFasL in the supernatant of the control and experimental groups was detected. There was no difference in the concentration of sFasL between the two groups, indicating that Bay11-7082 had little effect on the level of sFasL in the HL-60.

Bay 11-7082 enhanced the sensitivity of HL-60 cells to Fas-mediated apoptosis

The apoptosis cells were positive for Annexin V-FITC and negative for PI in the early period, and double positive for Annexin V-FITC and PI in the late period. The apoptosis rate of the cells treated with single Bay 11-7082 was (10.20±0.82)%. The apoptosis rate of the cells treated with single CH-11 was (11.09±1.11)%. The apoptosis rate of the cells treated with both Bay 11-7082 and CH-11 was (31.25±1.28)%. The apoptosis rate of combined group was obviously higher, suggesting that Bay 11-7082 and CH-11 had a synergistic relationship.

Discussion

There is a relationship between the development, progression of human leukemia, and the inhibition of differentiation and apoptosis. Fas/FasL system represents one of the main apoptotic pathways and has close relationship with the onset and development of leukemia and also the immune escape of tumor cells [10,11]. AML14.3D10 is an AML14 subline resistant to Fas-mediated apoptosis. Qin et al. [4] launched the study of AML14.3D10 and figured out that the combination of FasL and Fas could activate NF-κB and induce intracellular NF-κB heterodimer p65/p50 to transfer into nucleus in time- and dose-dependent manners. It could obviously augment CH-11-induced cell apoptosis by inhibiting NF-κB. Furthermore, while Fas-stimulation of resistant control 3D10 cells led to increases in the anti-apoptotic proteins cellular inhibitor of apoptosis protein-1 (CIAP), X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 family members, inhibition of NF-κB could lead to the downmodulation of these proteins. The above data suggested that the resistance of these leukemic eosinophils to Fas-mediated killing was due to induced NF-κB activation. Barnhart et al. [8] did the research on the T cell line H9 and B lymphoblastoid cell line SKW and found that the NF-κB inhibitor CAPE or Bay 11-7082 could make both of the two cell lines more sensitive to sFasL-induced apoptosis. Meli et al. [9] found that the NF-κB inhibitors MG132 and PDTC could significantly enhance CH-11-induced apoptosis of lymphoma cells

The result of our study demonstrated that the Fas-mediated apoptosis of HL-60 could be enhanced by inhibiting NF-κB. We also observed its effect on Fas/FasL system and XIAP and that Bay 11-7082 obviously decreased the protein and mRNA level of FasL, probably by inhibiting the transcription of FasL. Previous research indicated that NF-κB was a key positive transcriptional regulator of high expression of FasL in the active T cells and many solid tumor cells [12,13]. In addition, we found that Bay 11-7082 had little effect on the protein and mRNA level of Fas, which is in accordance with the result that NF-κB inhibitor MG132 had no effect on Fas [2]. In this study, Bay 11-7082 had little effect on sFasL level in HL-60 cell line. Therefore, we conclude that sFasL level directly relates with MMP-7 rather than NF-κB, because MMP-7 can specifically cleave mFasL to sFasL.

Inhibitor of apoptosis proteins (IAPs) is a family of inhibitors of apoptosis protein that is homologous in structure and located inside the cell. The main members of the human IAPs are X-linked IAP (XIAP), cIAP1, cIAP2, survivin, and so on. XIAP is the most effective caspase inhibitor among all of them. Its biological functions are various, having impact on the occurrence, development, and prognosis of tumor. Kater et al. [14] found that it could increase the sensitivity of the CLL cells to Fas-mediated apoptosis by inhibiting XIAP, which can activate caspases 3 and then cut poly (ADP-ribose) polymerase (PARP) and activate caspase 8 as well to form the waterfall magnifying effect, which leads to apoptosis. The study indicated that XIAP played an important role in the resistance of Fas-mediated apoptosis in CD40-activated chronic lymphocytic leukemia cells. Loeder et al. [15] found small molecule inhibitors that target XIAP sensitized ALL cells to Fas-induced apoptosis. Doyle et al. [16] figured out that IAP expression decreased during HL-60 cell differentiation. Ohashi et al. [17] found that the apoptosis sensitivity of the differentiated HL-60 increased. The current study confirmed that Bay 11-7082 could increase the sensitivity of the Fas-mediated apoptosis in HL-60 cell line, and at the meantime, mRNA level of XIAP decreased obviously. Therefore, we could expect that the decrease of the XIAP level had a relation with the increase of the sensitivity. There are several new studies which proved the important role of XIAP in the pathways of Fas-mediated apoptosis [18,19].

Reduced Fas susceptibility is usually accompanied with resistance to TNF-related apoptosis-inducing ligand (TRAIL) stimulation in U937 and other tumor cells because they partially share the intracellular apoptotic signaling mechanism [20]. TNF-α dramatically decreased the CH-11-induced cell death in eosinophilic acute myeloid leukemia cells. The process involved NF-κB transactivation and XIAP up-regulation. NF-κB inhibitor Bay 11-7085 reversed the suppression of Fas-mediated apoptosis by TNF-α [21]. XIAP inhibitors could sensitize childhood acute leukemia cells and activate T cells to be predisposed to TRAIL-induced apoptosis [22,23]. Triptodlide was proven to be a sensitizer to TRAIL-induced apoptosis in AML cells in part by inhibition of XIAP expression and NF-κB [24], so it was probably a sensitizer to Fas-mediated apoptosis in AML. NF-κB inhibitor Bay was toxic to cells, and the general caspase inhibitor Z-VAD-FMK could inhibit the cytotoxic effect. Thus, discovery of effective drugs like Z-VAD may be helpful in bringing NF-κB inhibitor into clinical practice [23].

In summary, Bay 11-7082 can reduce the expression of FasL and XIAP to increase Fas-mediated apoptosis in HL-60 cell line. It provides a new method to induce apoptosis of leukemia cells by inhibiting NF-κB or/and XIAP, and this finding may translate to a new way of treating leukemia.

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