Alantolactone

Alantolactone induces apoptosis in chronic myelogenous leukemia sensitive or resistant to imatinib through NF-jB inhibition

Introduction

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell disease caused by the t(9;22) (q34;q11) reciprocal translocation and the expression of the Bcr/Abl fusion protein, which exhibits constitutively active kinase activity [1, 2].

The Bcr/Abl kinase signals to multiple downstream survival pathways, including the mitogen-activated protein kinase (MEK)/extracellular signal regulating kinase (ERK) cascade, Akt, signal transducers and activators of tran- scription (STATs), and nuclear factor jB (NF-jB) [3–5]. Therefore, Bcr/Abl+ cells display varying degrees of resistance against conventional cytotoxic drugs [6, 7].

Collectively, these discoveries have prompted the wide search for specific drugs targeting Bcr/Abl. Imatinib mes- ylate (Gleevec, STI571), the first generation of oral Bcr/ Abl kinase inhibitors, blocks the growth of Bcr/Abl-trans- formed cells and is highly effective in inducing remission in patients with CML in the chronic phase with more than 90 % complete remission [8–10].

Unfortunately, the pre- existence or development of imatinib mesylate resistance ultimately leads to disease progression. The resistance of CML to imatinib treatment is most often due to diminished drug uptake, Bcr/Abl amplification, and point mutations in the Bcr/Abl fusion gene [11–14].

Therefore, much effort has been focused on alternative molecular-based strategies, mainly involving Bcr/Abl and its signaling [15–18]. Nil- otinib and dasatinib, the second generation of tyrosine kinase inhibitors (TKIs) [19–21], have gained regulatory approval and are being introduced into clinical application for CML patients who are resistant or intolerant to imati- nib, but their efficacy is sometimes limited, such as in patients with the T315I substitution mutation.

Given the continuing problem of TKI resistance, new approaches to the treatment of Bcr/Abl+ leukemia remain a high priority. Alantolactone, an allergenic sesquiterpene lactone, extracted from the roots of Inula helenium L. is a well- known medicinal plant officially listed in some European pharmacopoeias as elecampane [22].

It has been found to have various pharmacologic activities, such as hepatopro- tective, anti-inflammatory, antibacterial, antifungal, and antitumor effects [22–25]. Interestingly, an in vitro study showed that it has some toxicity against some cancer cells [26, 27], including the colon cancer cell lines Colo-205 and HCT-8, the central nervous system cancer cell line SF-295, and HL-60 cells, at micromolar concentrations.

Alanto- lactone induces apoptosis through intrinsic and extrinsic pathways by generating reactive oxygen species (ROS) intermediates. However, the mode of this action remains largely unknown.

In this study, we preliminarily found that alantolactone could inhibit cell proliferation and induce apoptosis at a micromolar concentration in both imatinib-sensitive and-resistant CML cells. We demonstrated that NF-jB activity inhibition and Bcr/Abl protein depletion contributed in parallel to alantolactone-induced apoptosis. These findings suggested that alantolactone may be a lead compound in the treatment of Bcr/Abl+ leukemia.

Materials and methods

Cell lines, antibodies, and drugs

The imatinib-sensitive and -resistant cell lines, K562 and K562r cells, respectively, were kindly provided by Prof. Junia V. Melo. K562 cells were cultured in RPMI-1640 supplemented with 10 % heat-inactivated fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA) in 5 % CO2/ 95 % air-humidified atmosphere at 37 °C. K562r cells were cultured in 1 lM imatinib to maintain their drug- resistant status.

32D, a murine IL-3-dependent myeloid cell line, was maintained in DMEM supplemented with 10 % FBS and 10 % WEHI-3B conditional medium containing IL-3. Alantolactone was obtained from the Tauto Biotech Company (Shanghai, China), with greater than 98 % purity by HPLC analysis; it was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mM and stored at -20 °C.

Imatinib (STI571) was purchased from Selleck Chemicals (Houston, TX, USA) and prepared as a 1 mM stock solution in DMSO at -20 °C. Annexin V-FITC and PI (propidium iodide) were obtained from BD Biosciences, and the mouse monoclonal antibody against actin was obtained from Cell Signaling Technology (CST).

MG-132 was purchased from Calbio-Aldrich (San Diego, CA). Z-VAD-FMK and epoxomicin were purchased from Sigma-Aldrich. Recombinant human TNFa was obtained from Peprotech (Rocky Hill, NJ). Antibodies against p65, IjBa, c-Abl (C-19), and CrkL (32H4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Mouse anti- bodies against PARP, cleaved caspase-3, phospho-Bcr at Tyr177, phospho-CrkL at Tye207, phospho-IjBa at Ser32, and phosphor-p65 at Ser536 were purchased from CST.

Cell viability assay

K562 and K562r cells were incubated with different con- centrations of alantolactone for 24 and 48 h. Cell prolif- eration was assayed using a Cell Counting Kit (Laboratories, Kumamoto, Japan) according to the manu- facturer’s instructions.

Additionally, peripheral blood samples from 5 normal humans and bone marrow samples from 4 CML-CP patients were collected, and mononuclear cells were isolated through Ficoll-Hypaque centrifugation and cultured with alantolactone for 48 h to assess cell proliferation.

Quantification of apoptosis

Apoptosis was measured using the FITC Annexin V Apoptosis Detection Kit (BD, Biosciences, NJ, USA) according to the manufacturer’s instructions. Annexin V-positive and PI-negative cells were considered to be in the early apoptotic phase, and cells positive for both for Annexin V and PI were deemed to be undergoing late apoptosis or necrosis.

Determination of mitochondrial transmembrane potentials (DWm)

After being washed twice with PBS, 106 cells were incu- bated with 10 mg/L rhodamine 123 (Rh123) at 37 °C for 30 min. Rh123 is a cationic lipophilic fluorochrome that is taken up by mitochondria in proportion to the DWm.

Then, 50 mg/L PI, a membrane-impermeable DNA-binding dye, was added to the cells. The fluorescent intensities were determined with flow cytometry (Becton–Dickinson). Ten thousand cells were analyzed in every sample. All data were collected, stored, and analyzed using LYSIS II soft- ware (Becton–Dickinson).

Western blot analysis

The cells were washed with PBS and lysed with lysis buffer (62.5 mM Tris–HCl, pH 6.8, 100 mM DTT, 2 % SDS, and 10 % glycerol). The cell lysates were centrifuged at 20,0009g for 10 min at 4 °C, and the proteins in the supernatants were quantified.

Protein extracts were equally loaded onto an 8–14 % SDS-polyacrylamide gel, electro- phoresed, and transferred to a nitrocellulose membrane (Amersham Bioscience, Buckinghamshire, United King- dom).

The blots were stained with 0.2 % Ponceau S red to ensure equal protein loading. After blocking with 5 % nonfat milk in PBS, the membranes were probed with primary antibodies. The signals were detected using the chemiluminescence phototope-HRP kit (Cell Signaling) according to the manufacturer’s instructions.

As necessary, the blots were stripped and reprobed with the anti-actin antibody (Oncogene, Fremont, CA) as an internal control. All experiments were repeated three times, with similar results.

Luciferase assay

THP-1 cells were transfected with an NF-jB luciferase vector (pGL4.32[luc2P/NF-jB/Hygro]) through electro- poration. After 24 h, the cells were exposed to hygromycin for 2 weeks to acquire resistant cells (THP-1NFjB-luc).

The THP-1NFjB-luc cells were pretreated with alantolactone for 1 h and subsequently treated with TNFa (10 lg/ml) for 5 h, and the luciferase activities were measured with dual- luciferase assay kits (Promega, Madison, WI) as described previously.

Electrophoretic mobility shift assay (EMSA)

Nuclear extracts of K562 cells pretreated with or without 5 or 10 lM alantolactone for 4 h followed by TNFa (10 lg/ml) for 0.5 h were prepared using the Nuclear and Cytoplasmic Extraction Kit (Pierce Biotech, Rockford, IL, USA).

The oligonucleotides for NF-jB (50-AGT TGA GGG GAC TTT CCC AGG C-30; and 50-GCC TGG GAA AGT CCC CTC AAC T-30) were biotin labeled using the Biotin 30-End DNA Labeling Kit (Pierce Biotech) and annealed with their complementary strands.

Nuclear proteins were incubated with the double-stranded biotinylated oligonucleotide with or without a 50-fold molar excess of unlabeled wild-type oligonucleotide. The reaction mixtures were then separated on a 5 % native polyacrylamide gel, and shifted bands that corresponded to protein–DNA complexes were visualized using a horseradish peroxidase-based detection system.

Real-time quantitative transcriptase polymerase chain reaction (PCR) analysis of Bcr/Abl mRNA

Total RNA was isolated using a TRIzol kit (Invitrogen, Carlsbad, CA, USA), and the RNA was treated with DNase (Promega, Madison, WI, USA). Complementary DNA was synthesized according to the manufacturer’s instructions. Real-time quantitative PCRs for Bcr/Abl and b-actin were performed with SYBR Green PCR Master Mixture Reagents (Applied Biosystems, Foster City, CA, USA) with the ABI PRISM 7900 system (Applied Biosystems).

The specific primers used for detecting p210 Bcr/Abl were the following: forward primer (50-CTG GCC CAA CGA TGG CGA-30) and reverse primer (50-CAC TCA GAC CCT GAG GCT CAA-30). The primers were synthesized by Sangon Biotech (Shanghai, China).

DNA constructs

The cDNA containing the entire coding sequence of wild- type human Bcr/Abl was subcloned from P210 pcDNA3 (Addgene). The MSCV-Bcr/Abl-IRES-GFP expression vector was constructed through the insertion of the entire coding sequence of Bcr/Abl/P210 into the retroviral vector MSCV-IRES-GFP (MIGR1). The pMSCV-p65 expression vector was constructed through the insertion of p65 cDNA into the retroviral vector pMSCV.

Retroviral transduction and generation of stable cell lines

The retrovirus was produced as previously described [28]. The murine IL-3-dependent myeloid progenitor 32Dcl3 cell line (32D cells) was infected with retroviral superna- tants harboring Bcr/Abl/GFP or GFP in culture medium supplemented with polybrene (8 lg/ml; Millipore).

GFP- positive cells were isolated using flow cytometry to gen- erate stable 32D cells expressing Bcr–Abl/GFP or GFP. Bcr/Abl transformed murine 32D cells to cytokine-inde- pendent growth, whereas the GFP control cell populations did not grow in the absence of IL-3.

RNA interference and transfection

Pairs of complementary oligonucleotides against p65 (50- CAAGATCAATGGCTACACA-30), Bcr–Abl (50-AGCAGATCGAGACCATCTT-30) and non-target control shRNA (NC) were synthesized by Sangon Biotech (Shanghai), annealed, and ligated into the PSIREN-RetroQ Vector (Clontech Laboratories). These siRNA-carrying retrovi- ruses, which were produced in 293T cells, were used to infect K562 cells.

Bone marrow colony assay

Bone marrow samples were obtained from 5 CML patients. Informed consent was obtained from all patients. The patients consented to all manipulations, and all manipula- tions were approved by the Medical Science Ethic Com- mittee of SJTU-SM.

Mononuclear cells were isolated through Ficoll-Hypaque centrifugation, and CD34+ cells were positively selected with anti-CD34 magnetic beads in a magnetic-activated cell sorter system (Stem Cell Tech- nologies, Vancouver, BC, Canada). A total of 103 CD34+ cells were mixed with 2.5, 5 or 10 lM alantolactone and plated in semisolid methylcellulose progenitor culture (Methocult H4434; Stem Cell Technologies, Vancouver, BC, Canada).

After 14 days of incubation at 37 °C in a fully humidified atmosphere of 5 % CO2, colonies com- posed of [50 cells were counted as one colony using an inverted phase-contrast microscope. All clonogenic assays were performed in triplicate.

Statistical analysis

All experiments were repeated three or four times, with similar results. The SPSS (Statistical Package for the Social Sciences) 11.0 software package and Student’s t test (Microsoft Excel, Microsoft Corp., Seattle, WA) were used for statistical analysis. A p value of less than 0.05 was considered to be statistically significant.

Results

Alantolactone inhibits the proliferation and induces the apoptosis of leukemic cell lines independent of the IM-sensitive and -resistant status

We first examined the effect of imatinib (IM) on IM-sen- sitive (K562) and IM-resistant (K562r) cells. The IC50 of IM on K562 and K562r cells was 1 and 5 lM, respectively (data not shown), indicating that K562r cells were more resistant to IM treatment, alanto- lactone significantly inhibited the proliferation and induced apoptosis in both K562 and K562r cells in a dose-dependent manner.

Incubation with 5 or 10lM alantolactone for 48 h resulted in 21.41 ± 1.04 % and 40.93 ± 0.98 % Annexin V-positive cells, respectively, in K562 cells compared with the control. Unexpectedly, similar results were observed in the K562r cells. These data indicated that alantolactone could inhibit the proliferation of K562 and K562r cells independent of their IM-sensitive or IM-resistant status.

To further demonstrate the induction of apoptosis, cas- pase-3, an important executing caspase of apoptosis, and PARP, a substrate of caspase-3, were examined through western blotting analysis. As shown in Fig. 1e and 1f, 5 or 10 lM alantolactone induced caspase-3 activation and PARP cleavage in a dose- and time-dependent manner and independent of the IM-sensitive or IM-resistant status.

Because the mitochondrial signaling pathway plays a critical role in apoptosis, we next assessed the effect of alantolactone on the mitochondrial membrane potential (DWm). After incubation with 10 lM alantolactone for 24 h, significant DWm collapse was observed in K562 (51.47 ± 1.12 %, p \ 0.01, Fig. 1g) and K562r (48.19 ± 0.72 %, p \ 0.0001) cells compared with the control. These data indi- cated that DWm collapse contributed to alantolactone- induced apoptosis.

Alantolactone administration results in blockade of the NF-jB pathway

NF-jB activation by the Bcr/Abl oncoprotein mediates proliferation, transformation, and resistance to apoptosis in Bcr/Abl-positive cells. Furthermore, alantolactone (Fig. 2a) contains an a,b-unsaturated ketene residue that may be able to inhibit NF-jB [29].

Thus, we examined whether alantolactone affected NF-jB-dependent reporter gene transcription. As shown in Fig. 2b, TNFa-induced NF-jB reporter activity was strongly inhibited by alanto- lactone pretreatment in a dose-dependent manner.

To fur- ther confirm NF-jB activity inhibition, EMSA analysis was performed. As shown in Fig. 2c, TNF-a-induced NF- jB-DNA complex formation was markedly inhibited in the presence of 5 or 10 lM alantolactone. Furthermore, treat- ment with alantolactone reduced p65 and IjBa phosphor- ylation (Fig. 2d).

Similar results were observed in K562r cells (Fig. 2e). These results indicated that alantolactone could inhibit the NF-jB signaling pathway in both IM- sensitive and IM-resistant CML cells.

Alantolactone downregulates the Bcr/Abl protein independent of the IM-sensitive and IM-resistant status

The Bcr/Abl kinase was shown to play a key role in the pathogenesis of Bcr/Abl+ malignancies by blocking apop- tosis through multiple mechanisms, resulting in increased resistance to various conventional agents. Next, we tested the possible effects of 5 or 10 lM alantolactone on the Bcr/ Abl protein in K562 and K562r cells.

Interestingly, 5 lM alantolactone significantly reduced the Bcr/Abl protein in K562 cells in less than 6 h (Fig. 3a). A more pronounced reduction of the Bcr/Abl protein was observed when the concentration of alantolactone was increased to 10 lM.

The inhibitory activity of alantolactone on the Bcr/Abl kinase was further demonstrated by the decrease in phos- phorylated CrkL protein, a well-known downstream target of Bcr/Abl (Fig. 3a). Similar results were also observed in alantolactone, and 1 or 10 lM STI571 was used as a positive control.

The PARP-1 and caspase-3 proteins were detected by western blotting, with b-actin as a loading control. g, h K562 (left) and K562r cells (right) were exposed to 10 lM alantolactone for 12 and 24 h, and the percentage of cells with low mitochondrial membrane potential was measured.

The values represent the mean ± S.D. of triplicates of independent experiments, which were repeated with similar results. **p \ 0.01 and ***p \ 0.0001, respectively, com- pared with the control K562r cells (Fig. 3b).

These results suggested that alanto- lactone could downregulate the expression of the Bcr/Abl protein independent of IM sensitivity.

To determine whether alantolactone could downregulate Bcr/Abl expression at the transcriptional level, we per- formed reverse transcription quantitative real-time PCR in K562 cells. Interestingly, the mRNA levels of Bcr/Abl in K562 and K562r cells were not affected by alantolactone compared with the control (Fig. 3c, d).

These results suggested that alantolactone may downregulate the Bcr/ Abl protein at the post-transcriptional level. To investigate whether the proteasome pathway was involved in the alantolactone-mediated downregulation of Bcr/Abl, we pretreated K562 cells with MG132 and epoxomicin, two well-known proteasome inhibitors, but found no effect (Fig. 4a–d).

Additionally, z-VAD, a caspase-3 inhibitor, did not lead to Bcr/Abl reduction, although caspase-3 activation was significantly inhibited (Fig. 4e, f). These results indicated that alantolactone downregulated the Bcr/ Abl protein at the post-transcriptional level via a protea- some- or caspase-independent pathway.

Discussion

Accumulating studies have demonstrated that neoplastic cells of hematopoietic origin are susceptible to a strategy of interrupting both oncogene production and survival sig- naling pathways.

In this study, we demonstrated that al- antolactone, a sesquiterpene lactone, could significantly induce apoptosis in Bcr/Abl+ cells by inhibiting NF-jB signaling and depleting the Bcr/Abl protein.

Several lines of evidence suggest that the NF-jB path- way plays an important role in Bcr/Abl+ cells and mediates the proliferation, transformation, and resistance to apop- tosis of Bcr/Abl+ cells [5, 30–32]. Importantly, studies in nude mice and primary bone marrow transformation assays have revealed a requirement for NF-jB in tumorigenesis and Bcr/Abl transformation [5, 30].

Therefore, different types of NF-jB inhibitors have been tested against Bcr/ Abl+ cells. Lu et al. [33] reported that pristimerin potently blocked the NF-jB signaling pathway, inhibited growth, and induced apoptosis in both imatinib-sensitive and-resistant cells.

Parthenolide [34] and triptolide [35], known NF-jB inhibitors, have also been shown to inhibit proliferation and induce apoptosis in CML blast cells independent of the absence or presence of the T315I mutation.

We found that alantolactone significantly inhib- ited the NF-jB signaling pathway during apoptosis in CML cells. The upregulation or silencing of NF-jB signaling significantly inhibited or enhanced, respectively, alanto- lactone-induced apoptosis.

Alantolactone inhibited the phosphorylation and degradation of IjBa, which in turn sequestered NF-jB in the cytoplasm and inhibited its nuclear translocation. The mechanism underlying the ability of alantolactone to block IjBa phosphorylation is currently unknown. During NF-jB activation, IKK com- plex activation phosphorylates IjBa, which marks it for ubiquitination and subsequent degradation, resulting in the release of NF-jB.

As a compound with an a,b-unsaturated ketene residue, alantolactone may directly inhibit IKK activation. Several compounds containing a,b-unsaturated ketene residues have been shown to inhibit IKK activation by covalently binding to IKK. Further investigation is needed to test this possibility.

The degradation of the Bcr–Abl oncoprotein represents a direct strategy for the targeted therapy of CML. Rapid disruptions of the Bcr/Abl protein may be more direct and completely inhibit the diverse signaling pathways activated by Bcr/Abl rather than only inhibiting Bcr/Abl kinase activity.

We found that alantolactone sharply decreased Bcr/Abl protein but not mRNA levels in less than 6 h in IM-sensitive K562 cells. Notably, alantolactone also suc- cessfully triggered apoptosis in IM–resistant cells, which occurred in association with the rapid reduction of the Bcr/ Abl protein and its phosphorylation.

More interestingly, 32D cells transfected with the Bcr/Abl oncogene showed increased sensitivity to alantolactone, indicating the spec- ificity of alantolactone for Bcr/Abl+ cells. However, the underlying mechanisms of alantolactone-induced Bcr/Abl degradation are currently unknown.

Several compounds, such as As4S4 [36] and geldanamycin [37], have been shown to enhance Bcr/Abl degradation through targeting HSP90 or enhancing CHIP E3 ligase activity.

In this study, proteasome inhibitors, including MG132 and epoxomicin, did not rescue the alantolactone-induced decrease in Bcr/Abl, indicating that the Bcr/Abl degradation induced by alantolactone may be proteasome independent. Similar to our results, several other compounds, including WP1130 [38] and adaphostin [39], could reduce Bcr/Abl protein levels in a proteasome-independent manner.

Addi- tionally, p65 silencing did not reduce the Bcr/Abl protein or affect the alantolactone-induced decrease in Bcr/Abl in K562 cells, indicating that alantolactone-induced Bcr/Abl depletion is independent of its NF-jB inhibition activity. A similar situation was observed with other NF-jB inhibitors.

For example, pristimerin [33], a quinone methide triterpe- noid, induced the apoptosis of CML cells through parallel NF-jB inactivation and Bcr/Abl inhibition. Together, our results strongly suggested that alantolactone could selec- tively induce apoptosis in Bcr/Abl+ cells by inhibiting the NF-jB signaling pathway and inducing Bcr/Abl degrada- tion. The mechanism underlying the alantolactone-induced depletion of Bcr/Abl warrants further investigation.

The clinical relevance of drug resistance in CML patients whose disease has progressed during imatinib treatment is currently a subject of intense interest. Most of the resistance to imatinib mesylate in Bcr/Abl+ cells in culture has been attributed to Bcr/Abl gene amplification, increased Bcr/Abl protein levels, or mutations in the IM- binding site (e.g., T315I) that make the contact sites inac- cessible [40, 41] as well as the loss of tumor-suppressor functions and arrest of differentiation.

Thus, therapeutic strategies are needed for the treatment and prevention of resistance and disease progression. Such strategies can include, for example, TKI dose escalation, treatment interruption to stop the selection of resistant cells, alloge- neic stem cell transplantation in eligible patients, and the use of novel TKIs [17–21] or other agents [42–44].

We contend that the key to curing CML will involve strategies beyond targeting Bcr/Abl because primitive human CML stem cells are not fully dependent on Bcr/Abl [45]. Ulti- mately, drug combinations or the exploitation of synthetic lethality may transform responses into definitive cures for CML.

Alantolactone has recently been reported as an anti- cancer agent in solid tumors. Here, micromolar concen- trations of alantolactone alone significantly inhibited pro- liferation and induced apoptosis in IM-sensitive K562 cells, primarily via NF-jB signaling pathway inhibition and direct Bcr/Abl protein depletion.

We believe that this is crucial for successful treatment of K562r cells with alan- tolactone. Importantly, these effects were also proven in primary CML cells. In summary, these results indicate that alantolactone, with its combined effects of NF-jB inhibi- tion and Bcr/Abl protein degradation, may represent a novel strategy to overcome CML drug resistance or a lead compound for drug development, and further studies are warranted to uncover the targets of alantolactone.