Abstract
OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells, and to further determine whether the proteasomal inhibitor, MG132, can potentiate apoptosis in triptolide-treated K562 cells.
METHODS Apoptosis was assessed via annexin V/PI doublelabeled cytometry. The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blotting.
RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner, and treatment with triptolide plus MG132 potentiated the apoptotic rate. Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α, and MG132 potentiated the effect of triptolide. Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.
CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.
keywords
Introduction
Triptolide was first isolated and structurally characterized in 1972 when it was extracted from a Chinese medicinal herb, a member of the Celastraceae family. We now know that triptolide exerts its anti-inflammatory and immunosuppressant effects by inhibition of cytokine production. The isolation of triptolide has also led to studies supporting its potential development as an antineoplastic agent[1]. The NF-κB/Rel family of inducible transcription factors is involved in the expression of numerous genes involved in processes such as growth, development, apoptosis, and inflammatory and immune responses. One important function of NF-κB is its ability to protect cells from apoptosis[2]. A key step in controlling NF-κB activity is the regulation of the NF-κB subcellular localization through its interaction with the IκB protein in both pre-induced and post-induced cellular states. The proteasomal inhibitor, Z-LLL-CHO (MG132), can inhibit the NF-κB activation through degradation of the IκBα protein[3].
To explore the anticancer mechanism of triptolide in human leukemic K562 cells, and to further determine whether MG132 can potentiate the apoptotic rate in K562 cells treated by triptolide, we invested the affects of triptolide plus MG132 on the IκBα and NF-κB/p65 proteins in K562 cells.
Materials and Methods
Drugs and reagents
Triptolide (molecular weight, 360, from Tripterygium wilfordii 98% [HPLC] solid) was used. Cbz-Leu-Leu-leucinal (MG-132) was obtained from Sigma Chemicals. In studies exploring the use of MG-132, the compound was dissolved in dimethyl sulfoxide (DMSO) on the day of use, and was added to the cells at a concentration of 5 μM. In all studies, the concentration of DMSO was always less than 0.1%. TNF-α was from (PEPROTECH-EC, USA, ≥ 2 × 107 U/mg).An Annexin V-FITC Detection KitII was purchased from Biosciences Pharmingen (San Jose, CA, USA), and anti-IκBα (SC-371), anti-p65 (SC-372) and HRP-conjugated secondary antibodies (goat IgG-HRP, SC-2020) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescence (ECL) reagent kits were purchased from Pierce Biotechnology (Rockford, USA).The K562 cell line was obtained from the China Center for Typical Culture Collection (Wuhan, China). All cells were grown in RPMI-1640 culture medium containing 10% FCS and L-glutamine (2 mmol/l), penicillin (100 U/ml), and streptomycin (100 U/ml) at 37°C in a humidified 5 % CO2 atmosphere.
MTT assay
The antiproliferative effect of triptolide in different groups was determined by using the MTT assay. Briefly, the final concentrations of triptolide were 0, 6.25, 12.5, 25, 50 and 100 nmol/L, and the cells collected following different times of incubation at 24, 48 and 72 h. Each concentration was added to 6 wells, respectively. Thereafter, 20 μl MTT solution (5 mg/mL in PBS) was added to each well. After incubation for 4 h at 37°C, the supernatant was re moved and 150 μl DMSO was added. When the blue crystals were dissolved, the optical density (OD) was detected in a microplate reader at a wavelength of 570 nm using a 96-well multiscanner autoreader (Biotech Instruments, USA). The following formula was used: The cell inhibitory rate (%) = [1 - (OD of the experimental samples/OD of the control)] × 100% (n = 6, mean ± SD).
Western blotting
The cells were harvested and lysed in 100 μl of lysis buffer by incubation on ice for 30 min, after which the extracts were centrifuged at 18,000 × g for 15 min to remove cell debris. Protein concentrations were determined by using the Bio-Rad protein assay. After the addition of 5 × loading buffer, the samples were incubated at 95°C for 5 min and then resolved using SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and probed with anti-IκBα and anti-p65 antibody. The antigen-antibody complexes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies at a final dilution of 1:1500. After the mixture was washed 3 times with Tris-buffered saline, antibody binding was visualized using ECL and autoradiography. Quantification of the bands was conducted using the quantity one densitometric analysis software (Bio-Rad).
Preparation of nuclear extracts for NF-κB/p65
The nuclear extracts were prepared according to the method described by Schreiber et al.[4] Briefly, 5 × 106 cells were washed with PBS and suspended in 4 ml hypotonic lysis buffer containing protease inhibitors for 30 min. The cells were then lysed with 12.5 μl 10% Non-idet P-40. The homogenate was centrifuged, and supernatant containing the cytoplasmic extracts was stored at -80°C. The nuclear pellet was resuspended in 25 μl ice-cold nuclear extraction buffer. After 30 min of intermitten mixing, the extract was centrifuged, and supernatants containing nuclear extracts were obtained. The protein contents was measured by using the Bradford method. If the nuclear extracts were not used immediately, they were stored at -80°C.
Annexin V/PI double-labeled cytometry
The expression of annexin-V-FITC and exclusion of PI were simultaneously detected by using two color flow cytometry. The cells were washed with PBS and resuspended in binding buffer containing 5 μl of FITC-labeled anti-annexin-V antibody and 10 μl of 20 μg/ml PI. After incubation for 10 min at room temperature in a light-protected area, all specimens were quantified on the FACScan.
Statistical analysis
All data were expressed as the mean ± SD, and were analyzed using SPSS 10.0. The linear t-test was used for statistical analysis, and P < 0.05 was considered to be statistically significant.
Results
K562 cell inhibitory rates following triptolide treatment
The cellular inhibitory rates following triptolide treatment at different times were measured by the MTT assay. The results are shown in Fig.1. The inhibitory rates gradually increased in a dose- and time-dependent manner. At the different concentrations of triptolide (0, 6.25, 12.5, 25, 50 and 100 nmol/L) treatment, at 48 h the inhibitory rates were 0.86%, 15.78%, 24.67%, 59.87% and 76.54% respectively. The IC50 value was 25 nmol/l.
The cells were incubated with triptolide for 24, 48 and 72 h followed by analysis of cell viability. The values are the means of 3 experiments ± SD.
Proteasomal inhibitor MG132 potentiated the apoptosis of K562 cells treated with triptolide
Fig.2. shows the cell viability following treatment with triptolide plus MG132 measured by the MTT assay. Apoptosis was tested by the annexin V-FITC assay, as shown in Fig.3 (A, B, C, D). There was little binding of annexin V-FITC in untreated K562 cells (0.07%). The binding of annexin V-FITC was increased following cell treatment with 25 nmol/l triptolide and 5 μmol/ml MG132 (25.12% and 9.18%, respectively). The binding of annexin V-FITC with combined treatment with 25 nmol/triptolide plus MG132 was 75.34% higher than treatment with triptolide alone (P = 0.003).
The cells were treated with trip-tolide (25 nmol/l) alone or with triptolide plus MG132 (5 μmol/ml) for 24 h. The cell viability was tested by the MTT assay. The values are means of 3 experiments ± SD, P < 0.01 vs. control.
The cells were treated with triptolide alone or with triptolide plus MG132 for 24 h fellowed by an annexin V-FITC assay. The K562 cells were treated with triptolide (25 nmol/l), MG132 (5 μmol/ml), or triptolide plus MG132 for 24 h. The cells were resuspended in 0.5 ml of binding buffer, reacted with 2 μl annexin V-FITC for 30 min at room temperature, and the binding analyzed by flow cytometry. Values are means of 3 experiments ± SD. A, Control; B, triptolide; C, MG132; D, triptolide plus MG132.
The cellular level of the IκBα protein and nuclear translocation of NF-κB/p65
In Western blot experiments, we found that the expression of the IκBα was decreased after treatment by TNF-α, and the NF-Bκ/p65 protein was released and translocated into the nucleus. MG132 and triptolide potently inhibited the degradation of the IκBα protein and the translocation of p65 protein induced by TNF-α. Triptolide plus MG132 almost completely blocked TNF-α-induced NF-κB activation (Fig.4A, B).
A, IκBα degradation in the control, or treatment with triptolide (25 nmol/L), MG132 (5 μmol/ml), and/or TNF-α (10 ng/ml) for 30 min. Cytoplasmic proteins were prepared, and 30 μg was subjected to 10% SDS-PAGE. IκBα was identified as a 37 kDa protein. B, the p65 level in the control, or treatment with TNF-α, and/or triptolide, MG132 for 30 min. The cell nuclear extracts for NF-κB/p65 were prepared and 30-50 μg was subjected to 10% SDS-PAGE. Immunoblotting revealed p65 to be a 65 kDa protein. Representative blots are shown here.
Discussion
In this study, we found that the inhibitory rates following triptolide treatment of K562 cells were gradually increased in a dose- and time-dependent manner. Triptolide induced apoptosis in the K562 cells and the proteasomal inhibitor, MG132, potentiated apoptosis induced by triptolide. Triptolide inhibited the degradation of IκBα and the nuclear localization of NF-κB/p65 proteins induced by TNF-α, and MG132 potentiated the effect. Triptolide plus MG132 almost completely blocked TNF-α-induced NF-κB activation. It was shown previously that triptolide and chemotherapy can cooperate to induce tumor cell apoptosis[5]. Shamon et al.[6] showed that trip-tolide inhibited growth of several human cancer-derived cell lines (including breast, prostate, and lung) grown in culture. Triptolide was also shown to induce apoptosis in human promyelocytic leukemic cells, T-cell lymphoma, and hepatocellular carcinoma cell lines grown in culture[7]. Triptolide potently inhibited TNF-α-induced activation of NF-κB and also blocked TNF-α-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of a family of inhibitors of apoptosis (IAP). Interestingly, triptolide did not block DNA binding of NF-κB, but it blocked transactivation of NF-κB. Our identification of a compound that blocks TNF-α-induced activation of NFκ-B may enhance the cytotoxicity of TNF-α on tumors in vivo and limit its proinflammatory effects[8].
Proteasome inhibitors, the well-known inhibitors of NF-kappaB, have recently been considered as therapeutic agents for inflammation. Nakayama et al.[9] found that the MG132 induced expression of MCP-1 in a dose-dependent manner at the transcriptional level. These data revealed that proteasome inhibition triggered the expression of MCP-1 and other genes via a multistep induction of the JNK-c-Jun/AP-1 pathway. Incubation of cultured rat mesangial cells (MCs) with tumor necrosis factor (TNF-α), interleukin (IL)-1beta, platelet-derived growth factor (PDGF)-AB or basic fibroblast growth factor (bFGF) significantly up-regulated CX3CL1/fractalkine mRNA and protein expression. This cytokine- and growth factor-stimulated CX3CL1/fractalkine expression could be abolished by the nuclear factor-kappaB inhibitors, curcumin and MG132. The ubiquitin-dependent protein degradation pathway is involved in the regulation of many basic cellular processes such as the cell cycle and division, differentiation and development, signal transduction, and apoptosis[10,11]. Ubiquitin-dependent mechanisms are also responsible for the selective degradation of transcriptional activators[12].
Overall, our results indicated that triptolide induced apoptosis in K562 cells and that the proteasomal inhibitor, MG132, potentiated apoptosis. Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α, and MG132 can potentiate the effect. MG132 potentiated the apoptosis of triptolide-treated K562 cells by regulating the NF-κB-signal pathway.
Acknowledgements
We thank Dr. Lishen Wang of the Institute of Radiation Medicine, Academy of Military Medical Sciences for critical review of the manuscript.
Footnotes
This work was supported by a grant from the National Natural Science Foundation of China (No.30570776).
- Received March 14, 2008.
- Accepted July 10, 2008.
- Copyright © 2008 by Tianjin Medical University Cancer Institute & Hospital and Springer
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