Cancer Letters 289 (2010) 46–52
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Cucurbitacin B inhibits STAT3 and the Raf/MEK/ERK pathway in leukemia cell line K562 Kin Tak Chan, Kwan Li, Shiu Lam Liu, Kee Hung Chu, Melvin Toh, Wei Dong Xie * Department of Technology and Product Development, CK Life Sciences Int’l., (Holdings) Inc., 2 Dai Fu Street, Tai Po Industrial Estate, Hong Kong SAR, China
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Article history: Received 25 May 2009 Received in revised form 15 July 2009 Accepted 20 July 2009
Keywords: Cucurbitacin B STAT3 Raf/MEK/ERK pathway K562 Leukemia
a b s t r a c t Cucurbitacin B is a natural anti-cancer compound found in Cucurbitaceae. Although the anti-cancer activity of cucurbitacin B in human leukemia cells has been reported, the underlining mechanism is still unclear. To clarify its anti-cancer activity and the mechanism of action, five different leukemia cell lines (CCRF-CEM, K562, MOLT-4, RPMI-8226 and SR) of the National Cancer Institute panel were treated with cucurbitacin B. Leukemia cell growth was inhibited by cucurbitacin B with GI50 ranged from 15.6 nM to 35.3 nM and the growth inhibition effect was attributable to G2/M phase arrest and apoptosis. Western blotting analysis of cell signaling molecules indicated that cucurbitacin B inhibits STAT3 activation and the Raf/MEK/ERK pathway in the K562 cells. Crown Copyright Ó 2009 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cucurbitacins are essential herbs for a large number of traditional Chinese medicines and the bitter principles of Cucurbitaceae [1,2]. Previous studies showed that it has a broad range of biological effects such as hemoprevention and hepotoprotection against CCl4 induced toxicity, as well as anti-inflammatory, anti-microbial and anti-cancer activities [2–4]. Chemically, cucurbitacins are highly diverse and arbitrarily divided into twelve categories, incorporating cucurbitacins A-T [2]. Several types of cucurbitacin compounds have been isolated for anti-cancer studies in vitro and in vivo [2,5–7]. Cucurbitacin B is found in many Cucurbitaceae species and is one of the most abundant forms of cucurbitacins [8]. It has significant anti-inflammatory activity and is used traditionally to treat hepatitis [9,10]. Recently, it has been reported that cucurbitacin B inhibits the growth of numerous human cancer cell lines and tumor xenografts [11–13]. Although the anti-cancer activity of cucurbitacin B has been reported, the
* Corresponding author. Tel.: +852 2126 1194; fax: +852 2126 1399. E-mail address:
[email protected] (W.D. Xie).
mechanism of action is still unclear. Signal transducers and activators of transcription (STATs) belong to a family of transcription factors that relay cytokine receptor-generated signals into nucleus to modulate cell growth and differentiation. STAT3 is a well known oncogene that is constitutively activated in many types of human cancer [14,15]. Inhibition of STAT3 may lead to apoptosis and inhibition of tumor growth [16]. Previous studies suggested that cucurbitacin B may exert its anti-cancer effect through suppression of STAT3 phosphorylation [11,17]. In addition to the STAT3 pathway, the Ras/Raf/MEK/ERK pathway is also important for cell proliferation, differentiation and death in response to growth signals from tyrosine kinase receptor. Aberrant activation of the Ras/Raf/ MEK/ERK pathway may lead to carcinogenesis and is frequently found in leukemia, colon cancer, lung cancer, pancreatic cancer, and even thyroid carcinoma [18–21]. It has been reported that the Ras/Raf/MEK/ERK pathway may also regulate STAT3 phosphorylation through cross talk [22]. Although the importance of the Ras/Raf/MEK/ERK pathway in carcinogenesis is well documented, its response to cucurbitacin B is still controversial. In 2008, Liu found that treatment of cucurbitacin B induces ERK
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K.T. Chan et al. / Cancer Letters 289 (2010) 46–52
phosphorylation in laryngeal cancer cell [23], in contrast, it has been reported to inhibit ERK phosphorylation in brain cancer [17]. Leukemia is a malignant cancer of bone marrow and blood that shows higher mortality rate than other cancers among children and young adults under the age of 20. In 2007, more than 200,000 people in the United States were living with leukemia. Recently, it has been reported that cucurbitacin B inhibits leukemia cell growth through S-phase arrest [24]. In the present study, we attempt to investigate the growth inhibition effect and the anti-cancer mechanism of cucurbitacin B in the leukemia cell lines of the National Cancer Institute (NCI) panel which has been used for anti-cancer drug screening since 1990s [25]. Our results suggested that cucurbitacin B induces cell cycle arrest and apoptosis in the leukemia cells. More importantly, it was revealed that cucurbitacin B may exert its anti-cancer activity through the suppression of STAT3 and the Raf/MEK/ERK pathway, which is important for carcinogenesis.
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bance was measured at 515 nm by FLUOstar OPTIMA equipment. 2.4. Flow cytometry for apoptosis analysis K562 cells were treated with cucurbitacin B in complete medium for 48 h. The cells were harvested and rinsed twice with PBS (pH 7.4) at 4 C. A total of 2.5 105 K562 cells were stained with Annexin V and propidium iodide for 15 min in dark. The stained apoptotic cells were counted by a FACSCalibar Flow cytometer system (FACS Calibur BD Flow Cytometer). 2.5. Flow cytometry for cell cycle analysis K562 cells were serum starved for 24 h and then treated with cucurbitacin B for 48 h. The cells were then washed with PBS (pH 7.4) and fixed with 80% ice-cold ethanol at 4 °C overnight. After fixation, the cells were stained with propidium iodine at 1 mg/ml for 15 min at room temperature. The stained cells were analyzed by flow cytometry (FACS Calibur BD Flow Cytometer).
2. Materials and methods 2.6. Western blot analysis 2.1. Cell lines Human leukemia cell lines (CCRF-CEM, K562, MOLT-4, RPMI-8226 and SR) were obtained from the National Cancer Institute (NCI). The leukemia cells were cultured in RPMI 1640 medium supplemented with 5% (v/v) heatinactivated fetal bovine serum, 100 units/ml penicillin and 100 units/ml streptomycin in a humidified 5% CO2 atmosphere at 37 °C. 2.2. Reagents and antibodies Cucurbitacin B was obtained from ChromaDex, Inc. (2952 S. Daimler St. Santa Ana, CA), all cell culture reagents from Gibco, Trichloroacetic acid (TCA) and Sulforhodamine B sodium salt (SRB) from Sigma, Annexin V-FITC Apoptosis detection kit and PI/RNase Staining Buffer from BD Pharmingen, EZ-Detect Ras Activation kit from Pierce, antibodies specific to phospho-ERK1/2, ERK1/2, phospho-MEK1/2, MEK1/2, phosphor-c-Raf, c-Raf, phosphor-STAT3, STAT3 from Cell signaling technology.
K562 leukemia cells were serum starved for 18 h before cucurbitacin B treatment. Cells were grown in 75-mm culture flasks at 1 105 cells/ml with or without cucurbitacin B for various time intervals. The treatment was terminated by centrifugation at 1500 rpm for 5 min and the cells were rinsed twice with PBS and lysed at 4 °C in lysis buffer (pH 7.5) of 50 mM Tris–HCl, 100 mM NaCl, 5 mM EDTA, 40 mM NaP2O7, 1% Triton X-100, 1 mM dithiothreitol, 200 lM Na3VO4, 100uM phenylmethysufonyl fluoride, 2 lg/ml leupeptin, 4 lg/ml aprotinin and 0.7 lg/ml pepstatin. The insoluble protein lysate was removed by centrifugation at 13,000 rpm for 10 min. Fifteen micrograms of protein lysate was resolved using 8–12% SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to Western blot analysis with antibodies specific for phosphorylated and total STAT3, c-Raf, MEK, and ERK.b The signal was visualized with the Enhanced Chemiluminescence Plus (ECL Plus) detection system (Amersham). 2.7. GTPase pull-down assay
2.3. Cell proliferation assay The human leukemia cell lines were subjected to cell proliferation assay using SRB to quantify protein mass following cucurbitacin B treatment. Briefly, appropriate number of the leukemia cells (depends on their doubling time) were seeded into 96-well tissue culture plates and treated with different dosages of cucurbitacin B for 2 days. Afterwards the cells were fixed with 25 ll 80% TCA and the plates were rinsed with tap water and air dried. The fixed cells were stained with 100 ll SRB reagent (0.4%) for 10 min followed by washing with 1% acetic acid for 4 times. The plates were dried again and the stain was dissolved with 100 ll 10 mM Trizma-base buffer. The absor-
The Ras-GTP content of the curcubitacin B treated cells was determined by glutathione S-transferase (GST)-RBD (Ras-binding domain of Raf) pull-down assay. Briefly, the cells were lysed in lysis buffer of 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM DTT and 5% glycerol for 5 min at 4 °C. The soluble cell lysates were obtained by centrifugation at 17,000g for 5 min. The cell lysates were then incubated with 80 lg GST-Raf1RBD in 50 mM Tris–HCl, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, 5 mM MgCl2, 1 mM DTT and 10% glycerol for 1 h at 4 °C. The beads were washed with lysis buffer for 3 times and boiled in 2 sample buffer for 5 min. Supernatants were collected by centrifugation at 7200g for 2 min and
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then resolved in 15% SDS–PAGE followed by Western blot analysis with anti-Ras antibody. 2.8. Statistical analysis All experiments were repeated at least three times and the data were expressed as the means ± SD. Statistical significance was determined by Student’s t-test. P-value <0.05 was considered statistically significant. 3. Results 3.1. Growth inhibition effect of cucurbitacin B on human leukemia cells The growth inhibition effect of cucurbitacin B on leukemia cells was studied using the SRB assay and the GI50 values were ranged from 15.6 nM to 35.3 nM (Fig. 1). Among the five leukemia cell lines, K562 showed the lowest GI50 value and therefore was selected for further analysis. 3.2. Cucurbitacin B induces apoptosis and cell cycle arrest in K562 cells The most possible cause of growth inhibition is cell cycle arrest and apoptosis. The effect of cucuribitacin B on cell cycle profile and apoptosis was analyzed using flow cytometry. Cucuribitacin B significantly induced K562 cell apoptosis in a dose dependent manner (Fig. 2A) and more than 50% of the cells were apoptotic in response to 80 nM cucurbitacin B treatment (Fig. 2B). Treatment with 40 and 80 nM cucurbitacin B for 48 h dramatically arrested the cells in the G2/M phase (Fig. 3). 3.3. Cucurbitacin B inhibits STAT3 activation in K562 cells STAT3 signaling plays an important role in cell growth, proliferation, and survival. Previous results have demonstrated that cucurbitacin B inhibits STAT3 activation in certain cancer cell types [11,17]. To examine the inhibitory effect of cucurbitacin B on STAT3 in leukemia cells, the K562 cells were treated with cucurbitacin B from 1 lM to 50 lM. Western blot analysis of phosphorylated STAT3 clearly showed that treatment with cucurbitacin B at 50 lM for 4 h inhibited STAT3 activation while no inhibition effect was found at 5 lM (Fig. 4A). To further investigate the time dependence, a time course study was preformed. Inhibition of STAT3 was observed in 1- and 4-h treatment but not in the first 30 min (Fig. 4B). 3.4. Inhibition effect of cucurbitacin B on Raf/MEK/ERK pathway in K562 cells Deregulation of the Raf/MEK/ERK pathway is commonly found in leukemia cells. To investigate the effect of cucurbitacin B on the Raf/MEK/ ERK pathway, phosphorylation status of c-Raf, MEK1/2 and ERK1/2 in cucurbitacin B treated K562 cells was studied. Activation of c-Raf,
GI50 (nM)
50 40 30 20 10 0
CCRF-CEM
K562
MOLT-4
RPMI-8226
SR
Fig. 1. Growth inhibition of cucurbitacin B in human leukemia cells. Human leukemia cells were seeded into 96-well plates and treated with different concentrations of cucurbitacin B for 2 days. The growth inhibition effects of curcurbitacin B were determined by SRB assay. Each bar represents the means of three independent experiments and error bars indicate standard deviation.
MEK1/2 and ERK1/2 was inhibited by 5-min cucurbitacin B treatment. These inhibition effects increased with time and phosphorylation of ERK1/2 was completely abrogated in 4-h treatment. However, no significant impact on the Ras-GTP level was observed, suggesting that cucurbitacin B inhibits the Raf/MEK/ERK pathway without affecting Ras in the K562 cells (Fig. 5).
4. Discussion Development of small molecules for cell signaling inhibition is an important aspect of anti-cancer drug research. The anti-cancer activity of cucurbitacin B has been shown in various cancer cell types, including liver, breast and laryngeal cancers [11,12,26]. In 2008, it was shown that cucurbitacin B also inhibits the growth of leukemia cell lines and primary leukemia cells in vitro [24]. The US National Cancer Institute (NCI) 60-cell line panel has been using as a standard model for pre-clinical anti-cancer drug screening since 1990s [25]. To further clarify the anti-cancer activity of cucurbitacin B against human leukemia cells and to explore its molecular mechanism for cell growth inhibition, the NCI cell line panel was used in the present study. Cell growth inhibition is an important indicator of the anti-cancer activity. Treatment with cucurbitacin B for 48 h showed an obvious growth inhibition on the five NCI panel leukemia cell lines with GI50 ranged from 15.6 nM to 33.3 nM that is comparable to Haritunian’ finding [24]. To find out the cause of cell growth inhibition, we have performed several experiments on cell cycle distribution and apoptosis. Flow cytometry analysis showed that treatment with cucurbitacin B arrested the K562 cells at G2/M phase and induced apoptosis that may contribute to the anti-cancer activity of cucurbitacin B, but the underlining molecular mechanism is still not known. STAT3 is an oncogene that is strictly controlled in normal cell to prevent aberrant cell proliferation [14,15]. Constitutively activated STAT3 is commonly found in cancer cells that may lead to tumor development through up-regulation of genes encoding apoptosis inhibitors (such as Bcl-2) and cell cycle regulators (such as Cyclins) [27]. For this reason, the STAT3 signaling pathway is regarded as a suitable target for cancer therapy [28]. Previous study suggested that curcubitacin B might block the STAT3 signaling by suppressing its phosphorylation in various cancer cell lines [11,13,17]. Treatment with curcubitacin B reduces the level of phosphorylated STAT3 and its downstream targets, cyclin B1 and Bcl-2, in the human laryngeal cell line Hep-2 [13]. In the present study, phosphorylation of STAT3 was suppressed by cucurbitacin B in dosage and time dependent manners, suggesting that cucurbitacin B is a potential STAT3 inhibitor in leukemia treatment. Although inhibition of STAT3 is believed to be the underlining anti-cancer mechanism of cucurbitacin B, it was reported that cucrbitacin B could inhibit breast cancer cell growth without suppressing STAT3 phosphylation [29], suggesting that STAT3 is not the only target of cucurbitacin B. Phosphorylation of STAT3 can be mediated by activated ERK and there is an interaction between the Raf/MEk/ERK and the JAK/STAT3 pathways [30,31]. In certain renal carcinoma cell lines, cell proliferation is working through a cross-talking between
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Fig. 2. Apoptotic effect of cucurbitacin B on K562 cells. K562 leukemia cells were seeded into T-25 flasks and treated with different concentrations of curcurbitacin B for 2 days. (A) Apoptotic effects of cucurbitacin B were assessed by Flow Cytometry analysis after staining with Annexin V-FITC. Annexin V staining is represented on the x-axis and PI staining is represented on the y-axis. The most representative result of three independent experiments is shown. (B) Percentage of living cells, necrotic cells and apoptotic cells after cucurbitacin B treatment were calculated from the Flow Cytometry results. Each bar represents the mean of three independent experiments and error bars indicate standard deviation.
the JAk-STAT3 and Raf/MEK/ERK1/2 pathways and the phosphorylation of STAT3 can be completely abrogated by inhibition of the ERK1/2 activity [31]. In addition to its effect on STAT3 activation, the Raf/MEK/ERK pathway can directly promote cell growth and survival. Upon activation, the ERK translocates to the nucleus to induce transcription of the genes, such as Bcl-2, caspase 9 and cyclin, for growth and survival [32,33]. Over the past decade, the Ras/Raf/MEK/ERB pathway has been involved in the etiology of tumogenesis and the induction of chemotherapeutic drug resistance. Recently, it has been shown that inhibition of the Raf/MEK/ERB pathway may suppress cancer cell growth through an epigenetic mechanism to down-regulate the anti-apoptotic molecule Bcl-2 [34]. In the present study, the effect of cucurbitacin B on the Ras/Raf/MEK/ ERB pathway was studied in the human leukemia cell line K562. The results showed that cucurbitacin B suppressed c-Raf, MEK1/2 and ERK1/2 phosphorylation in the first 5 to 10 min of treatment that was earlier than the inhibition of STAT3, suggesting that cucurbitacin B may suppress STAT3 phosphylation through ERK1/2 inactivation. Our
result is consistent to Yin’s finding on brain cell that treatment with cucurbitacin B inhibits ERK phosphorylation, while it is opposite to Liu’s finding in laryngeal cancer cell that treatment with cucurbitacin B resulted in a significant activation (phosphorylation) of ERK. The discrepancy of ERK activation among different cancer cell types suggests that the effect of cucirbitacin B is cell type specific. Aberrant activation of the Raf/MEK/ERK pathway is frequently observed in leukemia patients and associates with a poor prognosis than patients lacking these changes [35]. Several attempts have been made to suppress both the STAT3 and Raf/MEK/ERK pathways for cancer treatment. For example, high-dose of IFN alpha2 was used to down-regulate the level of phosphorylated STAT3 and MEK1/2 in melanoma [36]. In the present study, cucurbitacin B is shown to be a drug candidate to suppress both the STAT3 and Raf/ MEK/ERK pathways in the treatment of leukemia. It would be interesting to investigate the effect of cucurbitacin B on Raf/MEK/ERK inhibition in other cancers. Ras is the most upstream factor of the Ras/Raf/MEK/ERK pathway and constitutive active Ras mutation is commonly found in human
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Fig. 3. Cell cycle profile of K562 cells after 48-h cucurbitacin B treatment. (A) The cells were serum starved for 24 h prior to treatment with different doses of cucurbitacin B for 2 days. The cell cycle profile of the treated cells were analyzed with Flow Cytometry after fixation with 80% ice-cold ethanol at 4 °C and stained with PI for 15 min. Representative results of the actual cell cycle profile are shown. (B) Cell cycle distribution was calculated by ModFit LT Version 3.0. Each bar represents the mean of three independent experiments and error bars indicate standard deviation.
Fig. 4. Suppression of STAT3 in the K562 cells by cucurbitacin B. (A) The cells were serum starved for 18 h followed by treatment with 1 lM, 5 lM and 50 lM cucurbitacin B for 4 h while control cells were treated with 2% DMSO. (B) To assess the time dependency of STAT3 inhibition by cucurbitacin B, the cells were serum starved for 18 h followed by treatment with 50 lM cucurbitacin B for 10 min, 30 min, 1 h and 4 h. After treatment 15 lg cell lysate of each sample was separated in 8% SDS–PAGE followed by immuno-blotting with anti-STAT3 antibodies to assess the phosphorylated status of STAT3 as well as its total expression level.
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Fig. 5. Cucurbitacin B inhibits activation of the Raf/MEK/ERK pathway in K562 cells. K562 cells were treated with 50 lM cucurbitacin B for the indicated time intervals. After treatment 15 lg cell lysates were subjected to Western blot analysis of activated Ras with the GTPase pull-down assay described in Section 2, and the total and phosphorylated forms of c-Raf, MEk1/2, ERK1/2.
cancers [37]. Surprisingly, the level of activated Ras is not changed in the curcubitacin B treated cells, suggesting that Ras is not the upstream target of cucurbitacin B for Raf inactivation. Possible target of cucurbitacin B including the Raf protein itself and the PI3K/PTEN/Akt/mTOP pathway which is intimately linked with the Raf/MEK/EEK pathway in leukemia [38,39]. Cucurbitacin B is a potential chemotherapic agent against various types of human cancer. The present study indicates that cucurbitacin B suppresses not only the STAT3 pathway but also the Raf/MEK/ERK pathway in the human leukemia cells that provides valuable information on the molecular mechanism of its anti-cancer activity and strongly suggests that cucurbitacin B is a promising drug candidate for the treatment of human leukemia. Conflict of interest The results of the present invention of using cucurbitacin B for leukemia therapy has been filed for the patent application entitled ‘‘Cucurbitacin B and uses thereof” (WO 2008/071968 and US2008/0234244 A1). References [1] O.L. Chambliss, C.M. Jone, Cucurbitacins: specific insect attractants in Cucurbitaceae, Science 153 (3742) (1996) 1392–1393. [2] J.C. Chen, M.H. Chiu, R.L. Nie, G.A. Cordell, S.X. Qiu, Cucurbitacins and cucurbitane glycosides: structures and biological activities, Nat. Prod. Rep. 22 (3) (2005) 386–399.
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