|Year : 2016 | Volume
| Issue : 1 | Page : 155-160
Synergism between NF-kappa B inhibitor, celastrol, and XIAP inhibitor, embelin, in an acute myeloid leukemia cell line, HL-60
Yaghub Pazhang1, Hossein Zarei Jaliani2, Mehdi Imani3, Hassan Dariushnejad4
1 Department of Biology, Faculty of Science, Urmia University, Urmia, Iran
2 Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
3 Department of Biochemistry, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
4 Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
|Date of Web Publication||13-Apr-2016|
Hossein Zarei Jaliani
Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Shohadaye Gomnam Blvd, Yazd - 8915816653
Source of Support: None, Conflict of Interest: None
Aim of Study: Embelin and celastrol, inhibitors of XIAP and NF-κB proteins respectively, have been derived from natural sources and shown anti-tumor activities against different cancer cell lines. Some interactions have recently been discovered between XIAP and NF-κB pathways, but the effects of these inhibitors in combination have not been investigated yet. We have studied possible synergistic effects of embelin in combination with celastrol, in an acute myeloid leukemia model, HL-60 cell line.
Materials and Methods: Cytotoxicity of embelin and celastrol, separately and in combination, was determined by MTT assay and flow cytometry. Chou-Talalay's method was used to assess the synergistic effect of two components. Immunocytochemistry and western blot analysis of the two tumor marker proteins. (survivin and COX-2) was also performed to investigate downstream effects of two components.
Results: Analysis of MTT assay and flow cytometry showed that there is a substantial synergistic effect in some affected fractions of drug-treated HL-60. cells, while in other affected fractions a mild synergism or additive effect was observed. Immunocytochemistry and western blot analysis revealed that the expression of survivin and COX-2 proteins was reduced in treated cells.
Conclusion: Embelin and celastrol showed potent antitumor activity and synergistic effects in combination. Therefore targeting XIAP and NF-κB pathways simultaneously can be investigated in more detail to make use of embelin and celastrol as a combination therapy of cancer.
Keywords: Acute myeloid leukemia, HL-60 cell line, NF-kB, synergism, XIAP
|How to cite this article:|
Pazhang Y, Jaliani HZ, Imani M, Dariushnejad H. Synergism between NF-kappa B inhibitor, celastrol, and XIAP inhibitor, embelin, in an acute myeloid leukemia cell line, HL-60. J Can Res Ther 2016;12:155-60
|How to cite this URL:|
Pazhang Y, Jaliani HZ, Imani M, Dariushnejad H. Synergism between NF-kappa B inhibitor, celastrol, and XIAP inhibitor, embelin, in an acute myeloid leukemia cell line, HL-60. J Can Res Ther [serial online] 2016 [cited 2019 Dec 6];12:155-60. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/155/150407
| > Introduction|| |
Inhibitors of apoptosis proteins (IAPs) inhibit apoptosis and promote cell proliferation in many eukaryotic cells. X-linked inhibitor of apoptosis protein (XIAP) is a member of anti-apoptotic proteins and its overexpression has been observed in several cancer cell lines. XIAP inhibits apoptotic pathway of cell death by binding and inhibiting essential molecules of apoptosis, caspases. XIAP plays its role by dual actions; direct sequestration of caspases by binding to a cleft in their surfaces, and targeting caspases and other apoptotic proteins to proteasome by ubiquitinating them., Regarding its roles in apoptosis inhibition and cell proliferation, it is not so surprising that XIAP overexpression has been shown in various malignancies. Therefore a couple of small molecule inhibitors or peptide drugs have been tried to suppress XIAP activity and let caspases to start apoptosis where needed. This strategy has been specially followed in leukemia and lymphoma.,,
Embelin, a small molecule inhibitor of XIAP protein, has been discovered in 2004 in a structure-based computational screening of a database of chemicals against the XIAP structure. The source of this XIAP inhibitor was from the Japanese Ardisia herb. It inhibits growth of prostate cancer cells and induces apoptosis, with marginal effects on the normal prostate epithelial and fibroblast cells. Embelin has shown anti-tumor effect in myeloma cell line (HL-60) through down-regulation of XIAP protein. The mechanism of this effect seems to be apoptosis.,
Several types of tumors can survive by developing resistance to chemotherapy. NF-κB is one of the main cell survival pathways attenuating efficacy of therapeutics. This nuclear transcription factor makes tumor cells more resistant by several mechanisms, largely by increasing the number of membrane transporter molecules pumping drugs outside of the cell., This transcription factor plays important roles in the regulation of tumor proteins such as cyclooxygenase-2 (COX-2),, survivin , and many other cancer-related proteins. COX-2,, and survivin , upregulation has been shown in different malignancies.
Celastrol, a quinone methide triterpene, is a pharmacologically active compound present in Thunder of God vine root extracts which has multiple effects including anti-inflammatory and antitumor effects., Its anti-inflammatory effect is mediated by inhibition of NF-κB protein. There have recently been some evidences concerning the relationship between NF-κB pathway and XIAP anti-apoptotic protein (BIR1 domain). It seems that XIAP protein modulates NF-κB pathway of signal transduction. In acute myeloid leukemia (AML), one of the main causes of cancer in adults, chemotherapy resistance is occurred through action of the two above-mentioned proteins, XIAP and NF-κB.,
Therefore we have evaluated the inhibitory effect of XIAP and NF-κB inhibitors in combination in this study (Embelin and Celastrol respectively), in a model of acute myeloid leukemia cell line, HL-60, and as the probable consequence of this combination study, the alteration of COX-2 and survivin expression has been evaluated in drug-treated HL-60 cells.
| > Materials and Methods|| |
Human acute myeloid leukemia cell line (HL-60) was purchased from Pasteur Institute of Iran (Tehran, Iran). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS plus glutamine, streptomycin and penicillin, pH 7.4, in a humidified atmosphere of 95% air plus 5% CO2 at 37°C.
The anti-proliferative activity of celastrol and embelin (purchased from Sigma) independently and in combination was measured by MTT assay. The inhibition rate of each well was calculated as [(A570 control cells–A570 treated cells)/A570 control cells] ×100%.
Analysis of apoptosis by Annexin V/PI Apoptosis Detection Kit
Apoptosis was measured using the Annexin V/PI Apoptosis Detection Kit (Invitrogen Inc.), according to the manufacturer's instructions. Firstly, cells treated with each component or combination of them were collected and incubated with 5 µL Annexin V for 15 minutes in the dark at room temperature. Before analysis, 1 µL of propidium iodide (PI) working solution (100 µg.ml -1) was added to the samples. For each sample, 1 × 105 cells were collected and analyzed using a Fluorescence-activated cell sorting (FACS) cytometer.
Synergistic effect analysis
Synergism, if any, was assessed using the Chou-Talalay method  and Compusyn software. Dose-effect relationship for celastrol and embelin (each component alone) and their combination were determined using the median-effect principle. Then combination index (CI) for experimental combination (with the ratio of 0.6:0.4 celastrol and embelin, respectively) was calculated by Compusyn software. Combination index values less than 1 indicates synergism, (with the exception of 0.9–1 which approximately indicates additive effect.
Cells were first cultured on poly-l-lysine coated coverslips and the coverslip, bearing cells, were immersed in - 20 methanol for 10 minutes, then rehydrated with PBS and washed thrice with Ab buffer (PBS, 3% BSA, 0.1% Triton X-100 and 0.02% azide). Anti survivin (purchased from Sigma) and anti COX-2 antibodies (purchased from cell signaling Co) were diluted in Ab buffer (by 1/100), added to the coverslips and incubated for 90 min at room temperature. Samples were washed thrice with antibody buffer and incubated with secondary antibody, Fluorescein isothiocyanate (FITC) -conjugated anti-rabbit antibody (Sigma) for 45 minutes. Serial dilutions were tested for all antibodies and the best dilution was finally chosen for further experiments. Cells were washed once with antibody buffer, and then incubated in Hoechst 333492 (Sigma) at a concentration of 1 µg/ml for 30 minutes to stain DNA. Then cells were washed and the coverslips were mounted on the slides using glycerol containing mounting buffer (1 volume 0.2 M Na2 HPO4 (pH 9.0, 9 volumes of glycerol). Samples were observed via a Zeiss fluorescence microscope and the micrographs were recorded using a CCD camera. Cells were visualized with DAPI (Excitation = 330, Emission = 480) and FITC filters. Apoptosis was detected in individual cells on the basis of characteristic changes in nuclear morphology.
Whole-cell extracts were prepared by washing the treated and control cells with PBS buffer. Then cells were sonicated for 20 seconds in the presence of protease inhibitor cocktail. Protein concentrations were quantified using Bradford (Coomassie blue G250 dye) assay and equivalent quantities of cell lysates were loaded on a 10% SDS-PAGE. Separated samples were transferred to Polyvinylidene Fluoride (PVDF) membrane at 75 V for 1.5 h. Membrane was blocked for 1 h with PBS containing 2.5% nonfat dried milk and 0.05% Tween-20. After washing with PBS buffer (with 0.05% Tween-20), membrane was subjected to primary antibody against survivin and Cox-2 proteins separately at 4°C overnight. Horse-radish-peroxidase (HRP) -conjugated secondary antibody was applied after another three rounds of washing followed by diaminobenzidine (DAB) staining.
| > Results|| |
Celastrol and embelin reduce cell viability
HL-60 cells were treated with increasing doses of celastrol and embelin for 24 hours. By analyzing data, it was clear that components have dose-dependently affected HL-60 cell growth and viability. As shown in [Figure 1], IC50 of celastrol and embelin after 24 hours of treatment were determined to be 21.3 and 28.2 µM, respectively (analyzed by Compusyn software).
|Figure 1: Dose-response curve analysis of celastrol and embelin and their combination. Data were produced from MTT assay in a 24 hour treatment experiment|
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Synergistic effect of celastrol and embelin
When combined at the ratio of 0.6:0.4 (for celastrol and embelin, respectively), there is an additive effect of the combination therapy below 0.2 affected fractions, that becomes a mild synergism between 0.2 and 0.6 affected fractions (because the combination index falls below the 0.9). Stronger synergism can be seen above 0.6 of affected fractions which have CI (Combination Index) of lower than 0.7 [Figure 2].
|Figure 2: Combination Index (CI) analysis of the celastrol and embelin treatment. The analysis has been performed by Compusyn software and data were depicted as a curve in Graphpad prism version 6|
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Apoptosis induced by celastrol and embelin
As shown in [Figure 3], apoptotic portions of HL-60 cells treated with celastrol (5 µM), embelin (7 µM) and their combination (total concentration of 7.5µM for the 6:4 ratio) were 19.3, 17.1, and 31.8% respectively. This portion for untreated cells (control) was 5.2%. Results showed that the combination effect of two components in apoptosis induction is higher than each component individually. Not very surprisingly, the number of apoptotic cells was higher at 48 hour than 24 hour treatments [summarized in [Table 1].
|Figure 3: Apoptosis inducing effect of celastrol and embelin. Celastrol and embelin have apoptosis inducing effect on HL-60 cell line, (a) untreated cells, (b) celastrol-treated cells, (c) embelin-treated cells, (d) celastrol and embelin-treated cells in combination. Left) Annexin-PI results, right) Hoechst staining|
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|Table 1: Apoptosis induced by embelin and celastrol and their combination|
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Decreased survivin protein level after treatments
HL-60 cells were treated with IC50 concentration of celastrol and embelin and their combination. The results showed that survivin protein level decreased after treatment. As shown in [Figure 4], survivin staining remarkably decreased after treatment with both celastrol and embelin and decreased further when two components administered simultaneously. The results of apoptotic cells were also in accordance when combination of two components applied.
|Figure 4: Survivin protein staining in HL-60 cell line. IC50concentrations of celastrol and embelin and their combination caused reduction in survivin protein staining. C) untreated cells, Ce) celastrol-treated cells, Em) embelin treated cells, Ce + Em) celastrol and embelin combination treated cells. Left) survivin staining, right) nucleus staining by Hoechst|
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Decreased COX-2 protein level after treatments
For further illustration of celastrol and embelin effect, HL-60 cells were treated with IC50 concentrations of the two drug and their combination. As shown in [Figure 5], celastrol and embelin decreased COX-2 protein staining. Diminished staining in celastrol -treated HL-60 cells is stronger than in embelin-treated cells. Also COX-2 staining in combination therapy of two components was less than when each component was applied individually.
|Figure 5: COX-2 protein staining. IC50concentrations of celastrol and embelin and their combination caused reduction in COX-2 protein staining. C) untreated cells, Ce) celastrol-treated cells, Em) embelin-treated cells, Ce + Em) celastrol and embelin combination treated cells. Left) COX-2 staining, right) nucleus staining by Hoechst|
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In order to verify the immunocytochemistry results, we examined survivin and Cox-2 protein expression level by western blotting. As shown in [Figure 6], HL-60 cells naturally express survivin and Cox-2 proteins while treatment of cells with combination of the two drugs, decreased the staining indicating that the combination drug treatment resulted in decreased survivin and Cox-2 protein levels.
|Figure 6: Western blot analysis of survivin and Cox-2 protein levels. Analysis of survivin and Cox-2 protein levels after combination treatment of celastrol and embelin have been shown. C; control, Ce + Em; combination of the two drugs|
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| > Discussion and Conclusion|| |
Embelin is a small-molecule inhibitor of X-linked inhibitor of apoptosis (XIAP) with a potent anti-tumor activity against different cancer cells. Although, it can be a promising agent in treatment of a variety of cancers, but a challenging feature of using any anti-tumor agent addressed by many researchers is the resistance of tumor cells.,,
One of the major causes of chemotherapy resistance agents is NF-κB which has just recently been recognized to have cross-talk with XIAP pathway of signal transduction. If overexpression of XIAP anti-apoptotic protein or any over-activity of it can lead to an increase in the activity of NF-κB, then tumor cells might overcome chemotherapy of embelin treatment by enhancing this cross-talk leading to increase in NF-κB activity and finally rising tumor cell resistance against treatment.
The relationships between NF-κB and XIAP may not be as simple as noted above. In glioma cell lines which embelin does not have any activity on XIAP, it seems that embelin itself have inhibitory activity on the NF-κB transcription factor. Also it was shown that over-expression of NF-κB factor decreased embelin induced apoptosis in gliomas. It seems that inhibition of NF-κB is essential for the apoptosis induced by embelin in glioma cell lines.
Our results showed that there is an obvious synergistic apoptosis induction effect between embelin and celastrol as two anti-tumor agents in HL-60 cell line. Immunocytochemistry results showed that two components down-regulate the expression of the anti-apoptotic proteins, leading to higher sensitivity of HL-60 cancer cells to anti-tumor components. COX-2 and survivin proteins, two targets of the transcription factor NF-κB, paly essential roles in survival of cancer cell lines. Their overexpression may promote various malignancies, so down-regulation of these targets of NF-κB protein might have anti-tumor indications., Based upon our findings, celastrol and embelin synergizes to down-regulate Survivin and COX-2 probably by acting on the various activating pathways upstream of NF-κB transcription factor, leading to higher sensitivity of HL-60 cancer cells.
However these two components have diverse roles in different cell types, with various targets inside them. Therefore illustrating the main pathway of this synergistic effect requires additional investigations.
Moreover, the results of our study showed that celastrol and embelin had cytotoxic effect on HL-60 cell line, especially when they were applied as a combination. Their combination effectively reduced the survivin and COX-2 protein levels even more than when they used separately. Reduction in the COX-2 and survivin protein levels was probably mediated by components effects on NF-κB pathway activity. The synergistic effects of celastrol and embelin on cell growth and apoptosis can lead us to the potential use of these components as antitumor agents especially in a combination therapy. However these two components have diverse roles in different cell types, with various targets inside cells. Therefore illustrating the main pathway of this synergistic effect requires additional investigations.
| > Acknowledgment|| |
Financial supports from the Iran National Science Foundation (INSF) are gratefully acknowledged.
| > References|| |
Morizane Y, Honda R, Fukami K, Yasuda H. X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO. J Biochem 2005;137:125-32.
Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 2002;9:459-70.
Carter BZ, Milella M, Tsao T, McQueen T, Schober W D, Hu W, et al.
Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia. Leukemia 2003;17:2081-9.
Lima RT, Martins LM, Guimaraes JE, Sambade C, Vasconcelos MH. Chemosensitization effects of XIAP downregulation in K562 leukemia cells. J Chemother 2006;18:98-102.
Schimmer AD. Novel therapies targeting the apoptosis pathway for the treatment of acute myeloid leukemia. Curr Treat Options Oncol 2007;8:277-86.
Nikolovska-Coleska Z, Xu L, Hu Z, Tomita Y, Li P, Roller PP, et al.
Discovery of embelin as a cell-permeable, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database. J Med Chem 2004;47:2430-40.
Mannhold R, Fulda S, Carosati E. IAP antagonists: Promising candidates for cancer therapy. Drug Discov Today 2010;15:210-9.
Hu R, Wu B, Zhang GJ, Wang HT, Zhu K, Yang W, et al.
Effect of Embelin on proliferation, differentiation and aopotosis of HL-60 cells. Zhonghua Xue Ye Xue Za Zhi 2010;31:442-5.
Hu R, Zhu K, Li Y, Yao K, Zhang R, Wang H, et al.
Embelin induces apoptosis through down-regulation of XIAP in human leukemia cells. Med Oncol 2011;28:1584-8.
Bours V, Bentires-Alj M, Hellin AC, Viatour P, Robe P, Delhalle S, et al.
Nuclear factor-kappa B, cancer, and apoptosis. Biochem Pharmacol 2000;60:1085-9.
Choi BH, Kim CG, Lim Y, Shin SY, Lee YH. Curcumin down-regulates the multidrug-resistance mdr1b gene by inhibiting the PI3K/Akt/NF kappa B pathway. Cancer Lett 2008;259:111-8.
Inoue H, Tanabe T. Transcriptional role of the nuclear factor kappa B site in the induction by lipopolysaccharide and suppression by dexamethasone of cyclooxygenase-2 in U937 cells. Biochem Biophys Res Commun 1998;244:143-8.
Yu LL, Yu HG, Yu JP, Luo HS. Nuclear factor-kappa B regulates cyclooxygenase-2 expression and cell proliferation in human colorectal carcinoma tissue. Eksp Onkol 2004;26:40-7.
Takizawa BT, Uchio EM, Cohen JJ, Wheeler MA, Weiss RM. Downregulation of survivin is associated with reductions in TNF receptors' mRNA and protein and alterations in nuclear factor kappa B signaling in urothelial cancer cells. Cancer Invest 2007;25:678-84.
Tracey L, Perez-Rosado A, Artiga MJ, Camacho FI, Rodriguez A, Martinez N, et al.
Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas, respectively. J Pathol 2005;206:123-34.
Bernard MP, Bancos S, Sime PJ, Phipps RP. Targeting cyclooxygenase-2 in hematological malignancies: Rationale and promise. Curr Pharm Des 2008;14:2051-60.
Sobolewski C, Cerella C, Dicato M, Ghibelli L, Diederich M. The role of cyclooxygenase-2 in cell proliferation and cell death in human malignancies. Int J Cell Biol 2010;2010:215158.
Trifan OC, Hla T. Cyclooxygenase-2 modulates cellular growth and promotes tumorigenesis. J Cell Mol Med 2003;7:207-22.
Nestal de Moraes G, Silva KL, Vasconcelos FC, Maia RC. Survivin overexpression correlates with an apoptosis-resistant phenotype in chronic myeloid leukemia cells. Oncol Rep 2011;25:1613-9.
Small S, Keerthivasan G, Huang Z, Gurbuxani S, Crispino JD. Overexpression of survivin initiates hematologic malignancies in vivo
. Leukemia 2010;24:1920-6.
Kannaiyan R, Shanmugam MK, Sethi G. Molecular targets of celastrol derived from Thunder of God Vine: Potential role in the treatment of inflammatory disorders and cancer. Cancer Lett 2011;303:9-20.
Liu Z, Ma L, Zhou GB. The main anticancer bullets of the Chinese medicinal herb, thunder god vine. Molecules 2011;16:5283-97.
Lee JH, Koo TH, Yoon H, Jung HS, Jin HZ, Lee K, et al.
Inhibition of NF-kappa B activation through targeting I kappa B kinase by celastrol, a quinone methide triterpenoid. Biochem Pharmacol 2006;72:1311-21.
Lu M, Lin SC, Huang Y, Kang YJ, Rich R, Lo YC, et al.
XIAP induces NF-kappaB activation via the BIR1/TAB1 interaction and BIR1 dimerization. Mol Cell 2007;26:689-702.
Bueso-Ramos CE, Rocha FC, Shishodia S, Medeiros LJ, Kantarjian HM, Vadhan-Raj S, et al.
Expression of constitutively active nuclear-kappa B RelA transcription factor in blasts of acute myeloid leukemia. Hum Pathol 2004;35:246-53.
Rushworth SA, Zaitseva L, Murray MY, Shah NM, Bowles KM, MacEwan DJ. The high Nrf2 expression in human acute myeloid leukemia is driven by NF-kB and underlies its chemo-resistance. Blood 2012;120:5188-98.
Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 2010;70:440-6.
Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006; 58:621-81.
Baud V, Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov 2009;8:33-40.
Bentires-Alj M, Barbu V, Fillet M, Chariot A, Relic B, Jacobs N, et al.
NF-kappaB transcription factor induces drug resistance through MDR1 expression in cancer cells. Oncogene 2003;22:90-7.
Gottesman MM. Mechanisms of cancer drug resistance. Annu Rev Med 2002;53:615-27.
Park SY, Lim SL, Jang HJ, Lee JH, Um JY, Kim SH, et al.
Embelin induces apoptosis in human glioma cells through inactivating NF-kB. J Pharmacol Sci 2013;121:192-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]