This article has Open Peer Review reports available.
Curcumin induces the apoptosis of human monocytic leukemia THP-1 cells via the activation of JNK/ERK Pathways
- Chu-Wen Yang†1Email author,
- Chi-Lun Chang†1,
- Hsin-Chen Lee2,
- Chin-Wen Chi3,
- Jia-Ping Pan1 and
- Wen-Chin Yang2, 4
© Yang et al; licensee BioMed Central Ltd. 2012
Received: 23 October 2011
Accepted: 24 March 2012
Published: 24 March 2012
Curcumin is a principal compound of turmeric, commonly used to treat tumors and other diseases. However, its anti-cancer activity in human acute monocytic leukemia THP-1 cells is not clear. This study aimed to study the anti-cancer effect and action of curcumin on THP-1 cells.
THP-1 parental cells and PMA-treated THP-1 cells, were used as in vitro models to evaluate the anti-cancer effect and mechanism of curcumin. Apoptosis and its mechanism were evaluated by WST-1, flow cytometry and Western blotting. MAPK inhibitors were used to further confirm the molecular mechanism of curcumin-induced THP-1 cell apoptosis.
Curcumin induced cell apoptosis of THP-1 cells as shown by cell viability, cell cycle analysis and caspase activity. Curcumin significantly increased the phosphorylation of ERK, JNK and their downstream molecules (c-Jun and Jun B). Inhibitor of JNK and ERK reduced the pro-apoptotic effect of curcumin on THP-1 cells as evidenced by caspase activity and the activation of ERK/JNK/Jun cascades. On the contrary, the pro-apoptotic effect of curcumin was abolished in the differentiated THP-1 cells mediated by PMA.
This study demonstrates that curcumin can induce the THP-1 cell apoptosis through the activation of JNK/ERK/AP1 pathways. Besides, our data suggest its novel use as an anti-tumor agent in acute monocytic leukemia.
Acute myeloid leukemia (AML) is a hematopoietic cancer characterized by a disorder in differentiation of hematopoiesis; this disease results in the growth of a clonal population of neoplastic cells. Malignant hematopoietic cells lead to loss of normal hematopoietic functions, which results in death within weeks to months . AML is the most common type of leukemia in adults. It has the lowest survival rate of all leukemia . A better understanding of the molecular biology of AML will be helpful when developing new therapeutic strategies that specifically target molecular abnormalities.
Mitogen-activated protein kinases (MAPKs) such as ERK, JNK and p38 mediate the signaling transduction involved in cell proliferation, differentiation, transformation survival and death . Several publications showed the involvement of MAPKs in the apoptosis of HL-60 cells isolated from the patients with human promyelocytic leukemia, one type of acute myeloid leukemia. For instance, the activation of p38/ERK, JNK/ERK and p38/JNK by anti-cancer compounds, trifolin acetate , fucoidan  and 3,6-dihydroxyflavone , respectively, were observed during HL60 cell death. Accordingly, AP-1 transcription factor is associated with JNK mediated HL-60 cell apoptosis [7–10]. These data support the notion that the MAPKs and the downstream transcription factor AP-1 are the major mediators of HL-60 apoptosis.
Medicinal plants, used in complementary and alternative medicine, are an extraordinary source of chemopreventive and therapeutic agents for various human tumors [11, 12]. Turmeric has traditionally been used as a component to treat a variety of disorders in the Indian Ayurvedic medicine. Accumulating evidence shows that curcumin, the principal curcuminoid of turmeric, inhibits proliferation and induce apoptosis in various types of solid tumor and leukemia cell lines [13, 14]. Curcumin has been reported to possess inhibitory effects on MDR1 and WT1 gene expression in AML patient leukemic cells [15, 16]. Several studies have revealed that curcumin induces HL-60 cell line (a promyelocytic leukemia type of AML) apoptosis through several pathways, including the ornithine decarboxylase-dependent pathway , ER stress  and an inhibition of telomerase activity . However, little is known about the effects of curcumin on other types of AML.
In the present study, we investigated the effect and mode of action of curcumin on monocytic leukemia THP-1 cells. We first examined the effect of different concentrations of curcumin on THP-1 cell apoptosis. Next, interference of the inhibitor of ERK and JNK and PMA-treated THP-1 cells were used to study the likely mechanism of curcumin-mediated apoptosis.
Cell and reagents
The THP-1 cell line, derived from human acute monocytic leukemia, was purchased from American Type Culture Collection (TIB-202). Cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS (Gibco), 10 mM HEPES (GeneMark), 1% L-glutamine (Gibco), 1% non-essential amino acids (Gibco). Curcumin, dimethyl sulfoxide (DMSO), SP600125 (ERK inhibitor), U0126 (JNK inhibitor) and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma. Antibodies against caspase-3, cleaved caspase-8, Caspase-9, FoxO4, phospho-FoxO4 (Thr28), FoxO3a, FoxO1, phospho-FoxO1 (Ser256), phospho-FoxO3a (Ser253), p85, phospho-p85 (Tyr458), p110α, PDK1, Phospho-PDK1, JunB, c-Jun, phospho-c-Jun Ser63, AKT1, AKT2, AKT3, phospho-AKT (Ser473), phospho-AKT (Ser308), ATF2, phospho-ATF2 Thr71, phospho-JNK (Thr183/Tyr185), phospho-ERK (Thr202/Tyr2040), ERK, JNK, p38, phospho-p38 (Thr180/Tyr182), caspase-8 and histone H3 were purchased from Cell signaling laboratory and antibodies against PARP-1, caspase-3 and GAPDH were from Epitomics Inc. β-actin antibody and phospho-JunB (Ser259) were purchased from Sigma and Santa Cruz Biotechnology, respectively.
THP-1 cells, which had been treated with curcumin (30 μM, 40 μM and 50 μM), were harvested and fixed with 70% ethanol at 4°C overnight. After PBS washing, the cells were incubated with RNase A for 5 min. After incubation with propidium iodide (200 μg/mL), the cells underwent flow cytometry (Beckman, FC-500). For double staining, THP-1 cells were first treated with PhipPhiLux-G1D2/caspase-3 substrate (OncoImmuno, Inc) at 37°C for 45 min. After washing, the cells were stained with propidium iodide and analyzed using flow cytometry.
Protein extraction and immunoblotting
THP-1 cells were lyzed with RIPA lysis buffer (Sigma). Total cell lysates were extracted as described previously (Chen et al., 2009). The lysates were separated using polyacrylamide gel electrophoresis. After transfer, the membrane was blotted with antibody and developed with an enhanced chemiluminescent kit.
Caspase activity assay
THP-1 cells were treated with DMSO and curcumin (50 μM) in the presence of U0126 (20 μM) and SP600125 (30 μM) for 10 hours. The cells were subsequently incubated with Caspase-Glo® 3/7 reagent kit (Promega) and caspases-3/7 activity was detected and analyzed using a GloMax®-Multi Detection System (Promega) according to the manufacturer's instructions.
THP-1 cells and PMA-treated tHP-1 cells were seeded at the density of 50 000 cells/cm2 in 96-well plates. The cells were incubated with DMSO and 50 μM curcumin for 18 hr. After washing, the cells were incubated with WST-1 reagent at 37°C for 1 hr in accordance to the manufacturer's instructions (Roche). The quantity of formazan dye was determined with a photometer at 450 nm.
Data from three independent experiments are presented as mean ± standard deviation (SD). Student's t-test was used for statistical analysis between control and treatment groups. P less than 0.05 is considered statistically significant.
Curcumin induces THP-1 cell apoptosis
Apoptosis of THP-1 cells by curcumin is not mediated by PI3K/AKT pathway
Apoptosis of THP-1 cells by curcumin is mediated by the activation of JNK/ERK/Jun pathways
We turned to examine the involvement of MAPK pathways in the curcumin-mediated apoptosis in THP-1 cells. We found that curcumin increased the phosphorylation level of JNK and ERK to a greater extent than p38 in THP-1 cells (Figure 3B). Accordingly, curcumin augmented the phosphorylation of c-Jun and JunB, the downstream transcription factors of JNK and ERK, in THP-1 cells (Figure 3B).
PMA treatment reduces curcumin-induced THP-1 cell apoptosis by inhibiting ERK/JNK/Jun pathways
PMA is known to induce differentiation of THP-1 monocytic cells into macrophage-like cells (Auwerx et al., 1992). Next, we compared the effect of curcumin on PMA-treated THP-1 cells, differentiated/mature monocytic cells, and THP-1 cells using WST-1 assays. We found that cell viability of PMA-treated THP-1 cells and THP-1 cells after curcumin treatment was 25 ± 0.5% and 96 ± 3.7%, respectively. The data suggest that PMA treatment dramatically reversed curcumin-induced THP1 cell death.
In this work, we showed that curcumin induced the apoptosis of THP-1 cells, a human acute monocytic leukemia cell line. This cell death was associated with the MAKP and AP1 pathways. The study proves the concept that curcumin is therapeutically effective against human acute monocytic leukemia, one key type of acute myeloid leukemia.
PI3K, AKT and MAPKs are involved in regulation of life and death [3, 20]. Our results showed that curcumin increased apoptosis via the activation of ERK and JNK but not PI3K and AKT (Figure 3). Several anti-cancer compounds such as trifolin acetate , fucoidan  and 3,6-dihydroxyflavone  activated ERK, JNK and/or p38, in human acute myeloid leukemia HL60 cells. The activation of MAPKs was related to apoptosis of HL60 cells [7–10]. Hence, our and other data suggest that MAPKs regulates the matter of life and death in leukemia cells.
Jun family proteins have dual roles in neoplasia and tumor suppression and their roles need to be considered in a context-dependent manner [21–23]. For example, JunB was shown to repress cell proliferation when c-Jun:JunB heterodimers were formed [24, 25]. JunB was also reported to have tumor suppressor function in chronic myeloid leukemia [26, 27] and B cells . More recently, JunB was shown to inhibit autophagy and induce apoptosis [29, 30]. Consistently, AP-1 was shown to be implicated in HL-60 cell apoptosis mediated by JNK [7–10]. In this study, our results showed that c-Jun and JunB are involved in the curcumin-induced apoptosis in THP1 cells (Figures 3B, 4D and 5), suggesting the tumor suppressor role of c-Jun and JunB in THP-1 cells.
Collectively, this study showed that curcumin induces THP-1 apoptosis via the activation of ERK/JNK pathways and its downstream mediators, c-Jun and JunB. The data are in good agreement with the publications indicating that MAPK/AP1 pathways regulated cell death in acute myeloid leukemia HL60 cells. Moreover, our and other data support the notion that the MAPKs and the downstream molecule, AP-1, are the major mediators that regulate cell death of AML tumors.
Leukemic cells are aberrant immature blood cells. Differentiation of leukemic cells is thought as an anti-leukemia approach. PMA, a PKC activator, is known to promote the differentiation of immature THP-1 monocytic cells to mature THP-1 macrophages. Interestingly, the apoptotic effect of curcumin was abolished in PMA-treated THP-1 cells. Surprisingly, phosphorylation of ERK, JNK and Jun by curcumin decreased in PMA-treated THP-1 cells (Figure 5A). Phosphorylation of AKT seemed to increase (Figure 5A). The data suggest that apoptotic effect of curcumin is more effective against immature leukemic cells than mature cells. Put together, curcumin induces human monocytic leukemic THP-1 cell apoptosis via the activation of MAPK/AP1 pathways.
This work shows the pro-apoptotic effect and mechanism of curcumin in THP-1 cells. Its apoptotic action involves the activation of JNK/ERK/AP1 pathways. Besides, our data imply the novel use of curcumin as anti-leukemia agent.
This work was supported in part by the National Science Council grants (NSC 96-2320-B-031-001-MY2 and NSC 98-2321-B-031-001) to C.W. Yang.
- Rubnitz JE, Gibson B, Smith FO: Acute myeloid leukemia. Hematol Oncol Clin North Am. 2010, 24 (1): 35-63. 10.1016/j.hoc.2009.11.008.View ArticlePubMedGoogle Scholar
- Deschler B, Lubbert M: Acute myeloid leukemia: epidemiology and etiology. Cancer. 2006, 107 (9): 2099-2107. 10.1002/cncr.22233.View ArticlePubMedGoogle Scholar
- Dhillon AS, Hagan S, Rath O, Kolch W: MAP kinase signalling pathways in cancer. Oncogene. 2007, 26 (22): 3279-3290. 10.1038/sj.onc.1210421.View ArticlePubMedGoogle Scholar
- Torres F, Quintana J, Diaz JG, Carmona AJ, Estevez F: Trifolin acetate-induced cell death in human leukemia cells is dependent on caspase-6 and activates the MAPK pathway. Apoptosis. 2008, 13 (5): 716-728. 10.1007/s10495-008-0202-0.View ArticlePubMedGoogle Scholar
- Jin JO, Song MG, Kim YN, Park JI, Kwak JY: The mechanism of fucoidan-induced apoptosis in leukemic cells: involvement of ERK1/2, JNK, glutathione, and nitric oxide. Mol Carcinog. 2010, 49 (8): 771-782.PubMedGoogle Scholar
- Chang H, Lin H, Yi L, Zhu J, Zhou Y, Mi M, Zhang Q: 3,6-Dihydroxyflavone induces apoptosis in leukemia HL-60 cell via reactive oxygen species-mediated p38 MAPK/JNK pathway. Eur J Pharmacol. 2010, 648 (1-3): 31-38. 10.1016/j.ejphar.2010.08.020.View ArticlePubMedGoogle Scholar
- Mollinedo F, Gajate C, Modolell M: The ether lipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine induces expression of fos and jun proto-oncogenes and activates AP-1 transcription factor in human leukaemic cells. Biochem J. 1994, 302 (Pt 2): 325-329.View ArticlePubMedPubMed CentralGoogle Scholar
- Kondo T, Matsuda T, Kitano T, Takahashi A, Tashima M, Ishikura H, Umehara H, Domae N, Uchiyama T, Okazaki T: Role of c-jun expression increased by heat shock- and ceramide-activated caspase-3 in HL-60 cell apoptosis. Possible involvement of ceramide in heat shock-induced apoptosis. J Biol Chem. 2000, 275 (11): 7668-7676. 10.1074/jbc.275.11.7668.View ArticlePubMedGoogle Scholar
- Sawai H, Okazaki T, Yamamoto H, Okano H, Takeda Y, Tashima M, Sawada H, Okuma M, Ishikura H, Umehara H: Requirement of AP-1 for ceramide-induced apoptosis in human leukemia HL-60 cells. J Biol Chem. 1995, 270 (45): 27326-27331. 10.1074/jbc.270.45.27326.View ArticlePubMedGoogle Scholar
- Chen SH, Lin JK, Liang YC, Pan MH, Liu SH, Lin-Shiau SY: Involvement of activating transcription factors JNK, NF-kappaB, and AP-1 in apoptosis induced by pyrrolidine dithiocarbamate/Cu complex. Eur J Pharmacol. 2008, 594 (1-3): 9-17. 10.1016/j.ejphar.2008.07.024.View ArticlePubMedGoogle Scholar
- Fresco P, Borges F, Marques MP, Diniz C: The anticancer properties of dietary polyphenols and its relation with apoptosis. Curr Pharm Des. 2010, 16 (1): 114-134. 10.2174/138161210789941856.View ArticlePubMedGoogle Scholar
- Huang WY, Cai YZ, Zhang Y: Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. Nutr Cancer. 2010, 62 (1): 1-20.View ArticlePubMedGoogle Scholar
- Das T, Sa G, Saha B, Das K: Multifocal signal modulation therapy of cancer: ancient weapon, modern targets. Mol Cell Biochem. 2010, 336 (1-2): 85-95. 10.1007/s11010-009-0269-0.View ArticlePubMedGoogle Scholar
- Ravindran J, Prasad S, Aggarwal BB: Curcumin and cancer cells: how many ways can curry kill tumor cells selectively?. AAPS J. 2009, 11 (3): 495-510. 10.1208/s12248-009-9128-x.View ArticlePubMedPubMed CentralGoogle Scholar
- Anuchapreeda S, Limtrakul P, Thanarattanakorn P, Sittipreechacharn S, Chanarat P: Inhibitory effect of curcumin on WT1 gene expression in patient leukemic cells. Arch Pharm Res. 2006, 29 (1): 80-87. 10.1007/BF02977473.View ArticlePubMedGoogle Scholar
- Anuchapreeda S, Thanarattanakorn P, Sittipreechacharn S, Tima S, Chanarat P, Limtrakul P: Inhibitory effect of curcumin on MDR1 gene expression in patient leukemic cells. Arch Pharm Res. 2006, 29 (10): 866-873. 10.1007/BF02973907.View ArticlePubMedGoogle Scholar
- Liao YF, Hung HC, Hour TC, Hsu PC, Kao MC, Tsay GJ, Liu GY: Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells. Life Sci. 2008, 82 (7-8): 367-375. 10.1016/j.lfs.2007.11.022.View ArticlePubMedGoogle Scholar
- Pae HO, Jeong SO, Jeong GS, Kim KM, Kim HS, Kim SA, Kim YC, Kang SD, Kim BN, Chung HT: Curcumin induces pro-apoptotic endoplasmic reticulum stress in human leukemia HL-60 cells. Biochem Biophys Res Commun. 2007, 353 (4): 1040-1045. 10.1016/j.bbrc.2006.12.133.View ArticlePubMedGoogle Scholar
- Mukherjee Nee Chakraborty S, Ghosh U, Bhattacharyya NP, Bhattacharya RK, Dey S, Roy M: Curcumin-induced apoptosis in human leukemia cell HL-60 is associated with inhibition of telomerase activity. Mol Cell Biochem. 2007, 297 (1-2): 31-39. 10.1007/s11010-006-9319-z.View ArticlePubMedGoogle Scholar
- Burgering BM, Medema RH: Decisions on life and death: FOXO Forkhead transcription factors are in command when PKB/AKT is off duty. J Leukoc Biol. 2003, 73 (6): 689-701. 10.1189/jlb.1202629.View ArticlePubMedGoogle Scholar
- Bhoumik A, Ronai Z: ATF2: a transcription factor that elicits oncogenic or tumor suppressor activities. Cell Cycle. 2008, 7 (15): 2341-2345.View ArticlePubMedGoogle Scholar
- Lopez-Bergami P, Lau E, Ronai Z: Emerging roles of ATF2 and the dynamic AP1 network in cancer. Nat Rev Cancer. 2010, 10 (1): 65-76. 10.1038/nrc2681.View ArticlePubMedPubMed CentralGoogle Scholar
- Shaulian E: AP-1-The Jun proteins: Oncogenes or tumor suppressors in disguise?. Cell Signal. 2010, 22 (6): 894-899. 10.1016/j.cellsig.2009.12.008.View ArticlePubMedGoogle Scholar
- Deng T, Karin M: JunB differs from c-Jun in its DNA-binding and dimerization domains, and represses c-Jun by formation of inactive heterodimers. Genes Dev. 1993, 7 (3): 479-490. 10.1101/gad.7.3.479.View ArticlePubMedGoogle Scholar
- Passegue E, Wagner EF: JunB suppresses cell proliferation by transcriptional activation of p16(INK4a) expression. EMBO J. 2000, 19 (12): 2969-2979. 10.1093/emboj/19.12.2969.View ArticlePubMedPubMed CentralGoogle Scholar
- Passegue E, Jochum W, Schorpp-Kistner M, Mohle-Steinlein U, Wagner EF: Chronic myeloid leukemia with increased granulocyte progenitors in mice lacking junB expression in the myeloid lineage. Cell. 2001, 104 (1): 21-32. 10.1016/S0092-8674(01)00188-X.View ArticlePubMedGoogle Scholar
- Passegue E, Wagner EF, Weissman IL: JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell. 2004, 119 (3): 431-443. 10.1016/j.cell.2004.10.010.View ArticlePubMedGoogle Scholar
- Szremska AP, Kenner L, Weisz E, Ott RG, Passegue E, Artwohl M, Freissmuth M, Stoxreiter R, Theussl HC, Parzer SB: JunB inhibits proliferation and transformation in B-lymphoid cells. Blood. 2003, 102 (12): 4159-4165. 10.1182/blood-2003-03-0915.View ArticlePubMedGoogle Scholar
- Yogev O, Goldberg R, Anzi S, Shaulian E: Jun proteins are starvation-regulated inhibitors of autophagy. Cancer Res. 2010, 70 (6): 2318-2327. 10.1158/0008-5472.CAN-09-3408.View ArticlePubMedGoogle Scholar
- Yogev O, Shaulian E: Jun proteins inhibit autophagy and induce cell death. Autophagy. 2010, 6 (4): 566-567. 10.4161/auto.6.4.11950.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/22/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.