Alantolactone inhibits growth of K562/adriamycin cells by downregulating Bcr/Abl and P-glycoprotein expression

Authors

  • Chunhui Yang,

    1. Department of Laboratory Hematology, College of Laboratory Medicine, Dalian Medical University, Dalian, People's Republic of China
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  • Jingbo Yang,

    1. Key Laboratory for Molecular and Chemical Genetics of Critical Human Diseases of Jilin Province, Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun, People's Republic of China
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  • Meiyan Sun,

    1. Key Laboratory for Molecular and Chemical Genetics of Critical Human Diseases of Jilin Province, Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun, People's Republic of China
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  • Jiangzhou Yan,

    1. Department of Laboratory Hematology, College of Laboratory Medicine, Dalian Medical University, Dalian, People's Republic of China
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  • Xiuxiang Meng,

    Corresponding author
    1. Department of Laboratory Hematology, College of Laboratory Medicine, Dalian Medical University, Dalian, People's Republic of China
    • Department of Laboratory Hematology, College of Laboratory Medicine, Dalian Medical University, Dalian 116044, People's Republic of China
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    • Tel.:+ +086-0411-86110389. Fax:+86-0411-86110392

  • Tonghui Ma

    1. Key Laboratory for Molecular and Chemical Genetics of Critical Human Diseases of Jilin Province, Central Research Laboratory, Jilin University Bethune Second Hospital, Changchun, People's Republic of China
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Abstract

Alantolactone, a sesquiterpene lactone containing an α-methylene-γ-lactone group, is the active component of Inula helenium (Compositae), a traditional Chinese medicinal herb. It has been reported that alantolactone has the capacity to inhibit tumor cell growth through induction of apoptosis. The purpose of this study was to assess the effects of alantolactone in the adriamycin (ADR)-resistant human erythroleukemia cell line K562/ADR, and provide evidence that it might function as a potent therapeutic agent in chronic myelogenous leukemia (CML) patients with Bcr/Abl and the multidrug-resistance phenotype. Our results showed that alantolactone significantly inhibited K562/ADR cell growth by downregulating Bcr/Abl and P-glycoprotein expression. Alantolactone also induced apoptosis via modulation of protein levels of Bcl-2 family members, caspase activation, poly ADP ribose polymerase cleavage, and cytochrome C release. We also observed that alantolactone induced cell-cycle arrest in the G2/M phase, downregulated cyclin B1 and cyclin-dependent protein kinase 1, and upregulated the cyclin-dependent kinase inhibitor p21. Together, these results demonstrate that alantolactone may be a potent therapeutic agent against CML, and a potential Bcr/Abl inhibitor. © 2013 IUBMB Life., 65(5):435–444, 2013.

Introduction

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell disorder characterized by the expression and abnormal tyrosine kinase activity of the Bcr/Abl protein. Tyrosine kinase activity is necessary for cellular transformation by Bcr/Abl, and a hallmark of Bcr/Abl transformation is extreme resistance to chemotherapeutic agent-induced apoptosis, a phenotype that makes CML difficult to manage clinically. CML results from translocation of the Abl tyrosine kinase gene from the long arm of chromosome 9 to the breakpoint cluster region of chromosome 22, the Philadelphia chromosome (1). This translocation results in the production of a constitutively active tyrosine kinase fusion gene product, Bcr/Abl (2). Bcr/abl has been shown to be the sole transforming oncogene in animal models of CML (3, 4), and is generally considered to be the causative agent in CML carcinogenesis. The most common form of Bcr/Abl in CML is the 210-kDa p210bcr/abl gene, and the initiation, maintenance, and progression of CML are driven by the Bcr/Abl oncoprotein (5, 6). Several studies have shown that Bcr/Abl can significantly inhibit apoptosis through effects on DNA damage and repair, and can also mediate the resistance of tumor cells to multiple anticancer agents, resulting in drug resistance (7). Imatinib is an anticancer agent that was found to inhibit Bcr/Abl tyrosine kinase activity with high selectivity (8), and hence its discovery was revolutionary; however, amplification of the bcr/abl gene and P-glycoprotein (P-gp)-mediated drug efflux has been shown to play major roles in imatinib resistance (9). Therefore, the discovery of novel therapeutic agents and an understanding of their molecular mechanisms are necessary for improving the outcome of CML.

Sesquiterpene lactones are plant-derived compounds used in traditional medicine to treat inflammatory diseases (10). In recent years, many studies have shown that sesquiterpene lactones possess anticancer properties against various human cancer cell lines (11, 12). Alantolactone, a sesquiterpene lactone containing an α-methylene-γ-lactone group, is the active component of Inula helenium (Compositae), a traditional Chinese medicinal herb that has been used to treat tuberculotic enterorrhea, bronchitis, and chronic enterogastritis. It has been reported that alantolactone inhibits the growth of various solid tumor cells (13, 14), such as leukemic cells, by induction of apoptosis and/or cell-cycle arrest (15–17). However, the detailed molecular mechanism underlying its ability to inhibit leukemic cell proliferation has not been elucidated.

In this study, we observed the effects of alantolactone in the human chronic myelocytic leukemia drug-resistant cell line, K562/adriamycin (ADR), which overexpress multidrug resistance (MDR)1 and the Bcr/Abl oncogene. Here, we report for the first time that alantolactone induces apoptosis and cell growth inhibition of K562/ADR cells, and that these effects are mediated via inhibition of Bcr/Abl, Bcl-2, P-gp, and activation of Bax, caspase-9, caspase-3, and poly ADP ribose polymerase (PARP). These data indicate that alantolactone is an interesting natural compound that may be useful in treating CML patients exhibiting the functional MDR phenotype.

Abbreviations

CDK, cyclin-dependent protein kinase; P-gp, P-glycoprotein; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PARP, poly ADP ribose polymerase; caspases, cysteine-dependent aspartate-directed proteases; PBS, phosphate buffered saline; CML, chronic myelogenous leukemia; PI, propidium iodide; DMSO, dimethyl sulfoxide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MDR, multidrug resistance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4′,6-diamidino-2-phenyindole, dilactate.

Materials and Methods

Reagents

Alantolactone was purchased from The National Institute for the Control of Pharmaceutical and Biological Products (Shanghai, China) and purity (>99%) was detected by high-performance liquid chromatography. Doxorubicin hydrochloride, RNase A, propidium iodide (PI), 4′,6-diamidino-2-phenyindole, dilactate (DAPI), dimethyl sulfoxide (DMSO), RPMI-1640 medium, fetal bovine serum (FBS), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma (USA). Apoptosis assay kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Primary antibodies for Western were as follows: anti-Bax, anti-cytochrome C, anti-Bcr/Abl, anti-cyclin B1, anti-cleaved-caspase-3, anti-cleaved-caspase-9, anti-cleaved-PARP were purchased from Cell Signaling Technology (USA); anti-Bcl-2 mouse monoclonal was purchased from Abcam (USA); anti-cyclin-dependent protein kinase 1 (CDK1) was purchased from Boster Bio-Engineering (Wuhan, China). Antibodies specific to P-gp, β-actin, and all secondary antibodies (goat-anti-rabbit, goat-anti-mouse) were obtained from Santa Cruz Biotechnology (Beijing, China).

Cell Culture

The human CML cell line K562 and drug-resistant cell line K562/ADR were obtained from the Institute of Hematology & Blood Diseases Hospital Chinese Academy of Medical Sciences & Peking Union Medical College (Tianjin, China). The cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37 °C with 5% CO2 in humidified atmosphere. In addition, 2 μg/mL doxorubicin hydrochloride was added to the medium of K562/ADR until 2 weeks before tests.

Determination of Cell Viability

The cellular viability was assessed by MTT assay. K562/ADR cells were seeded in 96-well plates at a density of 5 × 104/mL. Then, the cells were treated with DMSO or various concentrations of alantolactone (0, 1, 2, 4, 6, 8, and 10 μM) for different time points (12, 24, 36, and 48 h). after treatment, 20 μL MTT reagent was added (5 mg/mL) and the cells were further incubated at 37 °C for 4 h, after that, 150 μL DMSO was added to each well and mixed thoroughly and absorbance was measured at 570 nm using a microplate reader (Thermo Scientific, USA). All our experiments were performed in triplicate and repeated three times. The percentage of cellular viability was calculated as follows:

equation image

Cell-Cycle Analysis

K562/ADR cells were treated with different concentrations of alantolactone for 24 h. After treatment, the cells were collected in tubes, supernatant was discarded, then cells were washed with ice-cold PBS for three times, and then they were fixed with 70% ethanol at 4 °C for overnight. The cells were spined down, ethanol was discarded, then cells were washed three times with ice-cold PBS and were stained with a solution containing 100 μg/mL RNase A, 50 μg/mL PI, and then they were put in the dark place for 30 min at room temperature. The DNA contents for cell-cycle phase distribution were analyzed by flow cytometry (Beckman Coulter, Epics XL, USA) using Cell Quest software.

Apoptosis Assay

Apoptosis was determined by Annexin V-FITC and PI staining. K562/ADR cells were treated with alantolactone in a dose-dependent manner. After treatment, the cells were harvested, washed twice with ice-cold PBS, then suspended in 200 μL of binding buffer containing 5 μL Annexin V-FITC, and then put it in the dark place for 10 min at room temperature according to the kit instructions (Beyotime, Shanghai, China). After incubation, the cells were centrifuged at 1,000g for 5 min, supernatant was discarded, and resuspended the pellet in the remaining volume with 200 μL of binding buffer containing 10 μL PI, and samples were immediately analyzed by flow cytometry (Beckman Coulter, Epics XL).

Determination of Doxorubicin Accumulation

The effect of alantolactone on the intracellular accumulation of doxorubicin was detected as described previously (18). To determine drug accumulation, the cells were treated with different concentrations of alantolactone at 37 °C for 3 h. Then, 10 μM doxorubicin was added to the cells and incubated for additional 3 h, and then the cells were collected, washed three times with ice-cold PBS, and analyzed with flow cytometric analysis (Beckman Coulter, Epics XL).

Immunofluorescence Microscopy

K562/ADR cells were treated with various concentrations of alantolactone for 24 h. Then, the cells were collected and washed with ice-cold PBS for three times. The cells were resuspended and fixed with 4% paraformaldehyde for 15 min, washed three times with ice-cold PBS, and then permeabilized with 0.1% Triton X-100 in PBS for 15 min, washed three times with PBS, the cells were then incubated for 2 h at room temperature with 3% bovine serum albumin in PBS, then incubated with antibody (Cell Signaling Technology, USA) at 4 °C for overnight. The cells were then washed three times with PBS and fluorescent secondary antibodies were added for 1 h at room temperature in the dark. The nuclei were stained with DAPI. Fluorescent images were acquired at room temperature under an Olympus IX71-SL microscope equipped with a 400 objective and an Olympus DP 72 digital camera connected to a computer.

Immunoblotting

Total proteins were extracted from control and alantolactone-treated cells by lysing cells in RIPA buffer (50 mM Tris-HCl, pH 7.4, 5 mM ethylenediaminetetraacetic acid, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, sodium orthovanadate, sodium fluoride, and leupeptin). Protein concentrations in the lysates were detected using BCA protein assay kit (KeyGEN Biotech, China). Forty micrograms of proteins was electrophoresed on 8–12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane. After blocking with 5% (w/v) nonfat dried milk in Tris-buffered saline containing 0.5% Tween-20 (TBST), membranes were incubated at 4 °C for overnight with Bcl-2 (1:500), Bax (1:1,000), cytochrome C (1:1,000), cleaved caspase-3 (1:1,000), cleaved caspase-9, cleaved-PARP (1:2,000), P-gp (1:100), Bcr/Abl (1:1,000), p21 (1:500), cyclin B1 (1:2,000), CDK1 (1:400), and β-actin (1:500) antibodies, respectively. After washing three times, the blots were probed with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (1:5,000) for 2 h at room temperature. After washing with TBST, signal detection was performed using Amersham ECL Prime Western Blotting Detection Reagent (GE, USA) with Fluor Chem HD2 (Alphal Innotech, USA) and bands were quantified by densitometry using Gel-Pro analyzer.

Statistical Analysis

All assays were repeated at least three times. The results were expressed as mean ± SEM. The significance was analyzed using the Student's t-test and one-way ANOVA compared with control group. Statistical significance was set at P < 0.05.

Results

Assessment of Resistance Characteristics of K562/ADR Cells

To investigate the main drug-resistance characteristics of K562/ADR cells, parental K562 cells and K562/ADR cells were treated with doxorubicin at various concentrations, and IC50 values were determined by the MTT viability assay (Fig. 1A). The IC50 of K562/ADR cells was 60-fold greater than that of K562 cells. We also investigated the differences in P-gp and Bcr/Abl protein levels in K562 cells and K562/ADR cells, and found remarkably increased expression of P-gp in K562/ADR cells, but almost no expression was observed in K562 cells. Furthermore, Bcr/Abl expression in K562/ADR cells was approximately threefold greater than in K562 cells (Fig. 1B). These results are in accordance with the data from the previous studies (19).

Figure 1.

Assessment of resistance characteristics of K562/ADR cells. (A) Cytotoxicity of doxorubicin in K562 and K562/ADR cells was detected by MTT. The drug-resistance index of K562/ADR cells was about 60-fold calculated with the IC50 of K562 and K562/ADR cells. Data are expressed as mean ± SEM (n=3). (B) The differential expressions of Bcr/Abl and P-gp in K562 and K562/ADR cells were confirmed by Western blotting. K562/ADR cells expressed a threefold increase in Bcr/Abl protein, and with an overexpression of P-gp. Relative signal intensities of protein levels were shown against β-actin, quantified by Gel-Pro analyzer software. The values are expressed as mean ± SEM. Three individual experiments were performed. *P < 0.05.

Alantolactone Inhibits K562/ADR Cells Proliferation

To determine the effect of alantolactone on K562/ADR cell growth, an MTT assay was performed. Alantolactone treatment inhibited the growth of K562 and K562/ADR cells in a dose-dependent manner (Fig. 2). The IC50 values of alantolactone in K562 and K562/ADR cells were 2.7 and 4.7 μM, respectively, and the drug-resistance index of alantolactone was 1.7-fold. We also determined the drug-resistance index of imatinib in K562/ADR cells, and found it to be about 13-fold (data not shown). The IC50 values of imatinib in K562 and K562/ADR cells were 1.2 and 16 μM, respectively. These data indicate that K562/ADR is more sensitive to alantolactone than imatinib.

Figure 2.

Alantolactone inhibits K562/ADR cell proliferation. (A) The cells (5 × 104/mL) were treated with various concentrations of alantolactone for 12, 24, 36, and 48 h; growth inhibition was detected by MTT. Data are expressed as mean ± SEM (n=3). (B) Chemical structure of alantolactone.

Effect of Alantolactone on the Cell Cycle and Cell Cycle-Related Proteins in K562/ADR Cells

The cell cycle is a crucial regulator of cell proliferation; thus, we were interested in determining if alantolactone could cause cell-cycle arrest. K562/ADR cells were treated with different concentrations of alantolactone for 24 h, and cell-cycle phase distribution was investigated using flow cytometry. As shown in Fig. 3A, there was a trend toward cell distribution in the G2/M phase of the cell cycle in K562/ADR cells; the percentage of accumulated cells in the G2/M phase increased from 15.8% in the control group to 21, 26.4, and 30.5% in cells treated with 2.5, 5, and 7.5 μM of alantolactone for 24 h, respectively. There was also a corresponding decrease in the percentage of cells in the G0/G1 phase from 29.9 to 27% and from 20.2 to 14% in cells treated with 2.5, 5, and 7.5 μM of alantolactone for 24 h, respectively. This provides evidence that G2/M phase cell-cycle arrest is one of the mechanisms by which alantolactone induces cytotoxicity in K562/ADR cells. To further understand the molecular mechanisms underlying the effect of alantolactone on cell-cycle arrest, we investigated the expression of cell cycle-related proteins at various alantolactone concentrations for 24 h, and found that expression of the CDK inhibitor p21 was significantly enhanced in a dose-dependent manner. In addition, the expression of cyclin B1 and CDK1 gradually decreased with increasing concentrations of alantolactone (Fig. 3B).

Figure 3.

Effect of alantolactone on the cell cycle and cell cycle-related proteins in K562/ADR cells. (A) Effects of alantolactone on cell cycle in K562/ADR cells were detected by flow cytometry analysis. Alantolactone induced G2/M phase arrest in K562/ADR cells in a dose-dependent manner. (B) CDK inhibitor p21, levels of cyclin B1 and CDK1 were also performed by Western blotting and β-actin was used as a loading control. (C, D) Relative signal intensities of protein levels were shown against β-actin, quantified by Gel-Pro analyzer software. The values are expressed as mean ± SEM. Three individual experiments were performed. *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Alantolactone Induces Apoptosis in K562/ADR Cells

Cell-cycle arrest and apoptosis are two major mechanisms involved in the induction of cell death. Several studies have shown that cell-cycle arrest might be caused by the induction of apoptosis. To further study the mechanism underlying alantolactone-induced cell death, K562/ADR cells were treated with 2.5, 5, and 7.5 μM alantolactone for 24 h, and cells undergoing apoptosis or necrosis were detected by flow cytometry after staining with annexin V-FITC and PI. As shown in Fig. 4A, alantolactone induced apoptosis in a dose-dependent manner. Furthermore, high concentrations of alantolactone increased necrotic cell death. Alantolactone-mediated apoptotic cell death was further substantiated with DAPI staining. K562/ADR cells were treated with different concentrations of alantolactone for 24 h, and nuclear morphological changes were determined by fluorescence microscopy. As shown in Fig. 4B, compared to the control group, nuclear morphological changes in alantolactone-treated cells showed condensed and fragmented nuclei in a dose-dependent manner. Taken together, these data demonstrate that alantolactone can induce apoptosis in K562/ADR cells, which is accompanied by necrotic cell death when cells are exposed to higher concentrations of alantolactone.

Figure 4.

Alantolactone induces apoptosis in K562/ADR cells. (A) Apoptosis of K562/ADR cells treated with various concentrations of alantolactone were detected by Annexin V-FITC and PI staining using flow cytometry analysis. Apoptosis rate increased with the increase of drug concentration. (B) The nuclear morphological changes of K562/ADR cells were observed by DAPI staining under fluorescence microscope. The morphology of cell nucleus has significantly changed with increased alantolactone. (C, D) The values are expressed as mean ± SEM. Three individual experiments were performed. *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Alantolactone Induces the Intrinsic Apoptotic Pathway Involved in Reduction of Bcr/Abl Levels

To gain better insight into the mechanism underlying alantolactone-mediated apoptosis, we examined the effects of alantolactone on the expression of major proapoptotic proteins after exposing cells to alantolactone at various concentrations for 24 h. Our results showed that alantolactone significantly decreased Bcl-2 protein expression and increased Bax expression and the associated release of cytochrome C from the mitochondria to cytosol. This was followed by caspase-9 and caspase-3 activation, and cleavage of PARP in a dose-dependent manner, suggesting that caspase activation and PARP cleavage are mechanisms by which alantolactone induces the mitochondrial apoptosis pathway in K562/ADR cells (Fig. 5B). As CML is characterized by expression of the bcr/abl gene, which has been shown to play a significant role in the resistance of CML cells to apoptosis (20, 21), we examined the expression of p210 protein, which is the product of the bcr/abl hybrid gene. We found that the protein levels of p210Bcr/Abl in K562/ADR cells were significantly downregulated at low concentrations of alantolactone (Figs. 5A and 5C).

Figure 5.

Alantolactone induces the intrinsic apoptotic pathway involved in reduction of Bcr/Abl levels. (A, C) Effects of alantolactone on Bcr/Abl level were detected by Western blotting and Immunofluorescence, alantolactone could significantly downregulate the expression of Bcr/Abl. (B) The expressions of major related apoptotic proteins Bcl-2, Bax, cytochrome C, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP were analyzed after exposing cells to alantolactone at various concentrations for 24 h and β-actin was used as a loading control. (D) Relative signal intensities of protein levels were shown against β-actin, quantified by Gel-Pro analyzer software. The values are expressed as mean ± SEM. Three individual experiments were performed. *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Alantolactone Increases Doxorubicin Accumulation and Decreases P-gp Expression

P-gp is an apical transmembrane protein, an efflux pump transporting a variety of therapeutic agents to outside of cell (22). The reduction of intracellular drug concentrations as a result of anticancer drug efflux from tumor cells is believed to be caused primarily by multidrug resistance. To investigate whether alantolactone inhibits MDR1 function, the intracellular accumulation of doxorubicin in the presence or absence of alantolactone was examined in K562/ADR and K562 cells. As shown in Fig. 6A, the intracellular accumulation of doxorubicin in drug-resistant K562/ADR cells was decreased compared to parental cells. Moreover, alantolactone enhanced the intracellular accumulation of doxorubicin in K562/ADR cells, but not parental K562 cells, in a dose-dependent manner. In addition, P-gp expression in K562/ADR cells treated with various concentrations of alantolactone was also determined by immunoblotting, and we observed that P-gp levels in K562/ADR cells gradually decreased with increasing concentrations of alantolactone (Fig. 6B).

Figure 6.

Alantolactone increases doxorubicin accumulation and decreases P-gp expression. (A) Effects of alantolactone on doxorubicin accumulation were measured by flow cytometry analysis. Alantolactone could enhance the intracellular accumulation of doxorubicin in K562/ADR cells in a dose-dependent manner, but not in its parental sensitive cells. (B) Alantolactone could decrease P-gp expression and β-actin was used as a loading control. Relative signal intensities of protein levels were shown against β-actin, quantified by Gel-Pro analyzer software. (C, D) The values are expressed as mean ± SEM. Three individual experiments were performed. *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Discussion

Cell-cycle control is one of the major regulatory mechanisms of cell growth (23, 24). Many anticancer agents have been reported to arrest the cell cycle at a specific checkpoint, and thereby induce apoptotic cell death (25, 26). We found that alantolactone acts as an inhibitor of K562/ADR cell growth. This growth inhibition might result from cell-cycle arrest, apoptosis, or necrosis, as those processes represent effective mechanisms involved in the induction of cell death (27). To determine if alantolactone inhibited cell growth via cell-cycle arrest or apoptotic induction, flow cytometric analyses of the cell cycle and apoptosis was performed, and revealed that alantolactone arrested the cell cycle at the G2/M phase, accompanied by a reduction in the G0/G1 phase in a dose-dependent manner. These data indicate that the cytotoxic effects of alantolactone result from its ability to induce cell-cycle arrest. Cyclins play an important role in cell-cycle events, and is upregulated in a variety of cancers. A number of studies have shown that cyclin B1 generally emerges during the G2/M phase transition of the cell cycle, during which cyclin B1 binds to CDK1 (cdc2) to form mitosis-promoting factor, which promotes the transition from G2 to the M phase of the cell cycle (28). Therefore, downregulated levels of cyclin B1 can attenuate CDK1 activation, finally resulting in cell-cycle arrest in the G2/M phase. Consistent with this phenomenon, our results showed a significantly increased percentage of alantolactone-treated K562/ADR cells in the G2/M phase, suggesting that alantolactone may trigger G2/M cell-cycle arrest by downregulating cyclin B1 and CDK1. CDK inhibitors also play a crucial part in controlling cell-cycle progression by negatively regulating the CDKs activities in cell cycle. p21 is the major member of CDK inhibitors. It has been reported that overexpression of p21 results in inhibited proliferation of mammalian cells and inactivation of all cyclin–CDK complexes including cdc2 and activation of p21 has been shown to participate in G2/M phase arrest of cell cycle (29, 30). Our present data showed increasing expression of p21 in K562/ADR cells treated with different doses of alantolactone for 24 h, which supports the finding of cell-cycle arrest effect in G2/M phase in this cell line. In addition to cell-cycle arrest, alantolactone also exerts its cytotoxic effect by the induction of apoptosis in K562/ADR, via modulation of Bcl-2 and Bax protein levels. Moreover, Bcr/Abl and P-gp also play important roles in alantolactone-induced apoptosis in K562/ADR cells.

Apoptosis, a type of programmed cell death, is a major mechanism for cell death following many types of chemotherapy. There are two principal apoptosis pathways; namely, the extrinsic and intrinsic pathways. The mitochondrial (or intrinsic) apoptotic pathway is mainly regulated by the interplay between members of the Bcl-2 protein family (31), including a wide variety of antiapoptotic proteins such as Bcl-2 and proapoptotic proteins such as Bax. It was reported that changes in the mitochondrial membrane potential and the release of cytochrome C from the mitochondria are associated with the ratio of Bax/Bcl-2 proteins (32). Bcl-2 suppresses apoptosis by stabilizing the mitochondrial membrane, whereas Bax induces apoptosis by increasing the mitochondrial membrane permeability, which leads to the release of cytochrome C from the mitochondria. The balance between these two classes of proteins is critical for determining whether a cell undergoes apoptosis (33). Release of cytochrome C from the mitochondria to cytosol is a hallmark of intrinsic apoptosis.

Cytochrome C binds to Apaf-1, thereby forming an oligomeric Apaf-1–cytochrome C complex (apoptosome). This apoptosome then binds and activates caspase-9, which in turn, activates caspase-3. Finally, as a main executioner of the apoptotic response, activated caspase-3 cleaves its effector protein, PARP, which eventually leads to cell death (34). Here, we observed that alantolactone significantly downregulated Bcl-2 protein and upregulated the protein levels of Bax, cytochrome C, cleaved-caspase-9, and cleaved-caspase-3, suggesting the involvement of an intrinsic apoptotic pathway by which alantolactone induces apoptosis in K562/ADR cells. It has been shown that Bcr/Abl induces resistance to apoptosis induced by various chemotherapeutic agents through positively modulating its downstream target gene Bcl-2 (35, 36). Silencing of Bcr/Abl by siRNA downregulates Bcl-2 expression (37). Therefore, the effective inhibition of Bcr/Abl could be critical in providing a targeted pathway for leukemia with the fusion gene prevention and treatment. We found that treatment of K562/ADR with alantolactone led to a dose-dependent decrease in Bcr/Abl protein levels, suggesting that this decrease may lead to apoptosis of K562/ADR cells.

MDR has been regarded as one of the major problems in cancer chemotherapy, and is often the result of overexpression of the drug efflux protein, P-gp, a 170-kDa glycoprotein, which is an energy-dependent drug efflux pump that maintains intracellular drug concentrations below cytotoxic levels, thereby decreasing the cytotoxic effects of many chemotherapeutic agents (38). In addition, P-gp also plays a special role in inhibiting the caspase-dependent apoptosis pathway in MDR cancer cells (39, 40). Therefore, effective inhibition of P-gp could be critical in providing a targeted site for cancer treatment for patients with MDR. We observed that treatment of K564/ADR with alantolactone led to a dose-dependent decrease in P-gp protein levels, accompanied by elevated intracellular concentrations of doxorubicin, which may result in apoptosis and cytotoxic effects in K562/ADR cells.

Conclusions

In conclusion, the ability of alantolactone to inhibit cell growth and induce apoptosis by a multicascade of events, including decreased Bcr/Abl, P-gp, and Bcl-2 expression levels, and increased Bax, cytochrome C, cleaved-caspase-9, and cleaved-caspase-3 levels, suggests that it might be a potent therapeutic agent for CML patients with Bcr/Abl and the MDR phenotype.

Acknowledgements

This research was supported by a grant from Liaoning Provincial Funding of Natural Science (grant no. 20072169), Dalian Municipal Science and Technology Fund (No. 2008E11SF162).

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