ANTICANCER RESEARCH 25: 313-318 (2005)
A Sesquiterpenelactone from Inula britannica Induces Anti-tumor Effects Dependent on Bcl-2 Phosphorylation MOHAMED M. RAFI1, NAI-SHENG BAI1, CHI-TANG HO1, ROBERT T. ROSEN1, EILEEN WHITE2,3, DENISE PEREZ2 and ROBERT S. DIPAOLA3 1Department
of Food Science, New Jersey Agricultural Experimentation Station, Cook College, Rutgers University, 65 Dudley Road, New Brunswick, 08901-8520, New Jersey; 2Howard Hughes Medical Institute, Center for Advanced Biotechnology, Rutgers University, 679 Hoes Lane West, Piscataway, 08854, New Jersey; 3The Cancer Institute of New Jersey, Robert Wood Johnson Medical School/UMDNJ, 195 Albany Street, New Brunswick, 08901-8520, New Jersey, U.S.A.
Abstract. Background: The over-expression of the anti-apoptotic protein Bcl-2 in cancer is associated with resistance to chemotherapeutic drugs. The phosphorylation of Bcl-2 is one mechanism by which anti-microtubule agents, such as paclitaxel or docetaxel, may inactivate Bcl-2. Although initially active in clinical studies, current anti-microtubule agents are only temporarily effective and the discovery of new agents is warranted. Materials and Methods: We isolated and identified two known sesquiterpenelactones, O, O-diacetylbritannilactone (OODABL) and O-acetylbritaanilactone (OABL) from the flowers of the medicinal plant Inula britannica and studied their mechanism of anti-tumor effects. To determine the biological significance of Bcl2 phosphorylation, we used a baby rat kidney (BRK-p53) cell line that was transformed with E1A and a temperature-sensitive mutant p53. The BRK-p53 cell line was transfected with either a vector with wild type Bcl-2 or a vector in which Bcl-2 had mutations in the paclitaxel phosphorylation sites (pcDNA3.1 V5/His Bcl-2 S70, 87 A). Results: OODABL and OABL induced phosphorylation of Bcl-2 in breast, ovary and prostate cancer cell lines and induced G2/M cell cycle arrest. Using the BRK cells with mutant Bcl-2 (BRK-Bcl-2-mt) and control (BRK-Bcl-2-wt), we found that OODABL induced phosphorylation of Bcl-2 at sites similar to paclitaxel. Phosphorylation of Bcl-2 was important for OODABL-induced cytotoxicity, since the abrogation of phosphorylation in BRK-Bcl-2-mt cells decreased OODABLinduced cytotoxicity. Conclusion: We concluded that OODABL
Correspondence to: Dr. Mohamed M. Rafi, Asst. Professor, Department of Food Science, Cook College, Rutgers University, New Brunswick, NJ, 08901-8520, U.S.A., Tel: 732-932-9611 x 258, Fax: 732-932-6776, e-mail:
[email protected] Key Words: Bcl-2, Bcl-2 phosphorylation, sesquiterpenelactones, taxol, apoptotic proteins, anti-tumor.
0250-7005/2005 $2.00+.40
is cytotoxic in multiple tumor cell lines, and the cytotoxicity is dependent, at least in part, on the phosphorylation of Bcl-2. Despite initial local therapy with surgery or radiation, many patients with cancer develop recurrence as evidenced by metastasis (1). Although chemotherapy is initially effective in many patients with metastatic disease, this response is usually temporary, and is secondary to the development of tumor resistance mechanisms (2). One mechanism of tumor resistance is the over-expression of the anti-apoptotic protein Bcl-2, that is associated with resistance to chemotherapy and hormonal therapy in many tumors (3-6). For example, transfection of Bcl-2 induces resistance to androgen ablation in hormone-sensitive prostate cancer cells and chemotherapy in multiple tumor cell lines (5-6). Agents thought to abrogate the function of Bcl-2 are in use clinically against many types of cancer (3). Paclitaxel, docetaxel, vincristine and vinblastine alter microtubule structure and induce apoptosis in association with the phosphorylation of Bcl2 (7,8). For example, Haldar et al. demonstrated that paclitaxel induced apoptosis in Bcl-2-expressing PC-3 prostate cancer cells, and that apoptosis was associated with the phosphorylation of Bcl-2 (7). Since Bcl-2 can protect cells from apoptosis by dimerizing with Bax, and phosphorylation of Bcl2 interferes with dimerization, Bcl-2 phosphorylation may be a mechanism of inducing apoptosis (8). Consistent with this mechanism, deletion of a putative negative regulatory loop that contains serine/threonine paclitaxel-induced phosphorylation sites enables Bcl-2 to promote cell survival despite treatment with paclitaxel (9). In contrast, other investigators demonstrated that the phosphorylation of Bcl-2 might be associated with cytotoxicity induced by paclitaxel-induced mitotic cell cycle arrest (10). Since Bcl-2 phosphorylation is a mechanism of cytotoxicity, or a marker for drug-induced cell cycle arrest by clinically effective anti-mitotic agents, the study of this molecule
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ANTICANCER RESEARCH 25: 313-318 (2005) capable of Bcl-2 phosphorylation may lead to additional options for cancer therapy. Sesquiterpenes are a promising class of derivatives that have been recently isolated and demonstrate cytotoxic activity in vitro and anti-tumor effects in xenograft models (11-13). Despite potential anti-tumor effects, the mechanism of cytotoxicity is poorly understood, but may involve effects on microtubules, and therefore Bcl-2 phosphorylation. Prior studies demonstrated that sesquiterpenes might inhibit mitosis through inhibition of microtubules (13,14). For example, Muller et al. demonstrated that novel sesquiterpenoid hydroquinone isolated from the sponge Dysidea avara was cytotoxic and inhibits assembly of microtubule protein (15). White et al. also demonstrated that elatone, a halogenated sesquiterpene, inhibited cell division and microtubule assembly (16). Pesando et al. demonstrated that caulerpenyne, a sesquiterpene isolated from green alga, inhibits mitosis in sea urchin eggs (14). Clinical studies have demonstrated that some of these agents are safe and clinically active (11). For example, irofulven (MGI 114), a novel sesquiterpene, has antitumor activity in a variety of solid tumors in vivo (11). MG114 was recently studied in forty-six patients in a phase I trial and was found to be safe and active (12). We recently isolated two sesquiterpenelactones 1-Oacetylbritannilactone (OABL) and 1,6-O, O-diacetylbritannilactone (OODABL) from Inula britannica (Figure 1), a plant that grows in Inner Mongolia and provinces of China and is used as a dietary supplement for the treatment of inflammatory disorders with potent cytotoxicity in vitro (1719). Given the anti-tumor activity of sesquiterpenes with known effects on mitosis and microtubules, and the effect of known anti-microtubule agents to phosphorylate Bcl-2, we characterized the cytotoxic effect of the novel sesquiterpenes OODABL and OABL in tumor cells, and tested the hypothesis that at least one mechanism of cytotoxic activity requires Bcl-2 phosphorylation.
Materials and Methods Reagents and cell culture. All cell culture media and supplements were obtained from Life Technologies, (Grand Island, NY, USA). All chemicals were obtained from Sigma (St. Louis, MO, USA). MCF-7, T47D, PA-1, OVCAR and Du-145 cells were obtained from the ATCC. DUPro-1 cells were a gift from Dr. S. Ward (20). Primary Fisher rat kidney (BRK) epithelial cells prepared from 6day-old baby rats were cultured in Dulbecco’s modified eagle medium (DMEM, Life Technologies, Grand Island, NY, USA) with 5% fetal calf serum (Gemini BioProducts, Woodland, CA, USA). Transformed BRK cells were derived from transfection of primary BRK cells with plasmids encoding E1A (pCMVE1A) and the temperature-sensitive p53 mutant p53 (val135) (pLTRcGval135), which is in the mutant conformation at the non-permissive temperature of 38.5ÆC (21-23). Stable transfectants were made with these transformed BRK cells, which were electroporated (220V, 950uF) with 1 Ìg of pcDNA 3.1Bcl-2, or Bcl-2 with mutations at the
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Figure 1. Structure of compounds: A, 1 O-acetylbritannilactone (OABL), and B, 1,6-O,O-diacetylbritannilactone (OODABL).
known Bcl-2 phosphorylation sites, serine 70 and 87 (pcDNA3.1Bcl2S70, 87A), as described below. Cells were selected at 48 hours post-transfection in DMEM containing 0.5 mg/ml G418 (Life Technologies) for 14 days. Individual colonies were cloned using cloning cylinders and expanded into cell lines. Mutagenesis of Bcl-2. The Bcl-2 phosphorylation mutant pcDNA3.1Bcl-2S70, 87A was constructed using a 5’ primer along with a mutagenic primer to PCR to amplify parts of Bcl-2 with the specific mutation, as previously described (24). The PCR product was run on a 1% agarose gel and purified using the Qiagen DNA purification kit (QIAEX II, Qiagen, Valenca, CA, USA) according to the manufacturer’s recommendations. This PCR product containing the desired mutation was used as a 5’ primer along with a 3’ primer to amplify the entire Bcl-2 cDNA with the respective mutations. This 878 bp mutated Bcl-2 PCR product was gel purified and ligated into the TA cloning vector pcDNA3.1-V5/His (Invitrogen, Carlsbad, CA). For the first mutation, wild type Bcl-2 in pcDNA3.1 was used as the DNA template in PCR. For the double mutant Bcl-2S70, 87A, the DNA template was pcDNA3.1V5/HisBcl-2S70A. The Bcl-2 5’ sense primer used with a HindIII site was 5’-GAGAAGCTTGTGCCGTTGGCCCCC-3’ and the 3’ primer with an Xba I site was 5’-CCTGCGCTCGCCCCGG TGCCACC-3’. The following primers were used for mutagenesis: for S70A, 5’-GGTCGCCAGGACCGCGCCACTACAGACCC-3’, and for S87A, 5’-CTGCGCTCGCCCCGGTGCCACC-3’. Extraction and isolation. 1-O-acetylbritannilactone (OABL), and 1,6O,O-diacetylbritannilactone (OODABL) were extracted and identified from Inula britannica var. chinensis, which was collected from Yan-An Country, Shan-Xi Province, People’s Republic of China, as previously described (17). The flowers of the plant were percolated with 95% EtOH at room temperature. The CHCl3soluble part of the EtOH extract was subjected to silica gel column chromatography, eluted with CHCl3-Me2CO mixture to yield OABL and OODABL, which was confirmed by NMR. The fractions were screened for purity using silica gel TLC plates and analytical HPLC. Bcl-2 expression and phosphorylation. Analysis of Bcl-2 protein by Western blots was determined as previously described (25). Protein identification was made using a monoclonal Bcl-2 primary antibody and secondary goat anti-mouse horseradish peroxidase conjugated antibody (Bio-Rad Laboratories, Richmond, CA, USA). The
Rafi et al: Sesquiterpenelactone Induces Bcl-2 Phosphorylation in Tumor Cell Lines
Figure 2. HPLC chromatogram of OODABL. The OODABL peak at 25 minutes represents > 99% purity.
phosphorylation of Bcl-2 was monitored by a shift in mobility of Bcl-2 in Western blot (7,26).
Table I. Inhibitory effect of OODABL and OABL on colony formation of various tumor cell lines.
Cell cycle analysis. T47D cells were treated for 12-24 hours, incubated with 10 ÌM BrdU for 45 minutes at 37ÆC. The cells were then washed with ice-cold PBS, resuspended in 200 ÌL PBS and fixed with cold 70% ethanol. The cells were resuspended, incubated for 30 min in 2 N hydrochloric acid /0.5% Triton X-100 in PBS, and neutralized by rinsing once in 0.1 M sodium tetra borate (pH 8.5). Fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (Becton-Dickinson) was added (10 Ìg per sample) in 50 ÌL of 0.5% Tween 20/1% BSA in PBS and incubated for 30 minutes. The cells were washed and resuspended in 1 mL of PBS containing 5 Ìg/ml propidium iodide. Flourescence intensity was determined by quantitative flow cytometry and profiles were generated on Becton Dickinson FACScan. A minimum of 10,000 cells were analyzed using Modifit LT (Verily Software House, Inc.).
Cell line
Clonogenic assay. Monolayer cultures of MCF-7, T47D, PA-1, OVCAR, Du-145, DuPro-1, BRK-Bcl-2-mt and BRK-Bcl-2-wt cell lines were harvested by treatment with Trypsin-EDTA and washed in medium. Five hundred cells were seeded in 60 x 15 mm tissue culture petri dishes with various concentrations of OODABL and OABL and incubated for 14 days at 37ÆC. Thereafter, cells were incubated for 3 minutes in methylene blue (2.5 g methylene blue trihydrate in 50% ethanol) Colonies were counted by hand and confirmed using Bio-Rad Quantity one software (Bio-Rad Laboratories Inc, Hercules, CA, USA). The IC50 values were obtained by extrapolation by linear regression analysis.
Results Effect of OODABL on cell growth. OODABL was purified and assessed for cytotoxicity by clonogenic assay. OODABL was confirmed to be >99% pure by HPLC (Figure 2).
MCF-7 T47D PA-I OVCAR DUPro-I DU-145
IC50 OODABL (ÌM) 5.00 7.50 8.00 10.00 5.00 10.00
IC50 OABL (ÌM) 10.00 13.00 12.00 17.00 10.00 20.00
Cells were treated with various concentrations of OODABL and OABL and incubated for 14 days at 37ÆC. The IC50 for each cell line was determined by plotting the number of individual colonies vs concentration of OODABL and OABL. This experiment was repeated three times with similar observations.
Although similar in structure, as shown in Figure 1, OODABL was more cytotoxic than OABL (Table I) and therefore OODABL was used for further mechanistic studies. Treatment with OODABL and OABL for 14 days inhibited clonogenic growth of MCF-7, T47D, OVCAR, PA-1, Du-145 and DuPro-1cells with an IC50 of approximately 5 to 20 ÌM (Table I). Effect of OODABL on cell cycle and Bcl-2. Since prior studies demonstrated that anti-mitotic agents such as paclitaxel and vinca alkaloids induce G2/M cell cycle arrest and Bcl-2 phosphorylation, cancer cells were treated with OODABL and assessed for effects on cell cycle and Bcl-2 by flow cytometry and immunoblot, respectively. Treatment of cells with 1 ÌM of paclitaxel for 12 hours, which is known to induce a mitotic
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ANTICANCER RESEARCH 25: 313-318 (2005)
Figure 3. Effect of OODABL on cell cycle. Cells were treated with vehicle (3A), 1 mM paclitaxel (3B), 15 mM OODABL (3C), and 30 mM OODABL (3D) for 12 hours. Cell cycle was assessed after treatment with BrdU and fluorescein isothiocyanate (FITC)conjugated anti-BrdU antibody. Fluorescence intensity was determined by quantitative flow cytometry and profiles were generated on Becton Dickinson FACScan. A minimum of 10,000 cells were analyzed using Modifit LT (Verily Software House, Inc.).
arrest, demonstrated an increase in G2/M peak by FACS analysis as compared to vehicle control (Figure 3A and 3B). Treatment of cells for 12 hours with OODABL (15 and 30 ÌM) induced a similar G2/M cell cycle arrest (Figure 3C, 3D). To assess the effect on Bcl-2, cells were treated with different concentrations (2.5-20 ÌM) of OABL and OODABL for 12 hours and cell lysates probed for Bcl-2. In Figure 4A, Taxol, which is known to induce Bcl-2 phosphorylation, caused a slower migrating band characteristic of phosphorylated Bcl-2 (8). Also shown in Figure 4, phosphorylation of Bcl-2 occurred in MCF-7 (Figure 4A), OVCAR (Figure 4B), DUPro-1 (Figure 4C), and PA-1 (Figure 4D) cells, as evidenced by a slower migrating band, similar to the Taxol used as a positive control (Figure 4A). Effect of site-directed mutation of Bcl-2. To determine if OODABL induced Bcl-2 phosphorylation at sites induced by known anti-mitotic agents, we assessed the effect of OODABL in a cell line with Bcl-2 mutated at serine residues in position 70 and 87, which are known paclitaxel-induced phosphorylation sites (24). As shown in Figure 5, paclitaxel (1 mM) induced phosphorylation in BRK cells transfected with a wildtype bcl-2 expression vector (BRK Bcl-2-wt) and not in BRK cells transfected with a vector producing bcl-2
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mutated at serine 70 and 87 (BRK Bcl-2-mt). Similarly, OODABL (40 mM) induced phosphoryation of Bcl-2 in BRK–Bcl-wt cells and not in BRK-Bcl-2–mt cells. Effect of Bcl-2 phosphorylation on OODABL induced cytotoxicity. To determine if the phosphorylation of Bcl-2 was important for the cytotoxicity of OODABL, we treated both BRK-Bcl-2-wt and BRK-Bcl-2-mt cells with OODABL and assessed for cytotoxicity by clonogenic assay. OODABL decreased cell growth in a dose dependent manner in both BRK-Bcl-2-wt and BRK-Bcl-2-mt cells (Figure 6). Mutation at the serine 70 and 87 site of Bcl-2 increased BRK cell resistance to OODABL (Figure 6). The IC50 of OODABL was increased approximately 10 fold in Bcl-2 mutant cell line. Specifically, the IC50 was 1 ÌM in BRK-Bcl-2-wt cells compared to 10 ÌM in BRK-Bcl-2-mt cells. Thus, the phosphorylation of Bcl-2 caused, directly or indirectly, by OODABL is important for cytotoxicity.
Discussion The present work demonstrated that the sesquiterpenelactone, OODABL, isolated from Inula Britannica was cytotoxic, induced the phosphorylation of Bcl-2, and induced G2/M cell
Rafi et al: Sesquiterpenelactone Induces Bcl-2 Phosphorylation in Tumor Cell Lines
Figure 4. Effect of OODABL and OABL on Bcl-2 protein phosphorylation in MCF-7 (4A), OVCAR (4B), DUPro-1 (4C) and PA1 (3D) cancer cells. Phosphorylation of Bcl-2 was detected by immunoblot using anti-Bcl-2 monoclonal antibody. The slower migrating band represents Bcl-2 phosphorylation. Taxol was used as a positive control for Bcl-2 phosphorylation, represented by a slower migrating band (4A). Vehicle treated cells were used as control.
Figure 5. Effect of OODABL on Bcl-2 protein in wild-type (Bcl-2-wt) and mutant (Bcl-2-mt) Bcl-2 transfected BRK cells. Mutant and wild-type cells were treated with vehicle, 1mM paclitaxel or 40 mM OODABL for 12 hours. Cells were lysed and Bcl-2 phosphorylation was assessed by antiBcl-2 monoclonal antibody.
cycle arrest in cancer cells. The cytotoxicity of OODABL was dependent, at least in part, on Bcl-2 phosphoryation at serine 70 and 87, sites known to be phosphorylated following treatment with the clinically active agent paclitaxel. Prior studies have suggested that sesquiterpene compounds may be a promising class of anticancer agents, although the mechanism of cytotoxicity of these agents is poorly understood. Whan Han et al. reported that a sesquiterpene from Inula inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 through inactivation of NFkappaB in macrophages, but did not examine cancer cell cytotoxicity (19). Eckhardt et al. demonstrated that irofulven, a mushroom-derived sesquiterpene was safe and active in patients with advanced malignancies (12). Hammond et al. studied irofulven in combination with topotecan or paclitaxel in nude mice bearing MV522 xenografts (21). Irofulven was synergistic with topotecan and enhanced paclitaxel activity. Recent studies have also found that parthenolide, a sesquiterpenelactone derivative of feverfew, is cytotoxic and enhances the sensitivity of breast cancer cells to paclitaxel.
Figure 6. Effect of OODABL on clonogenicity of wild-type (Bcl-2-wt) and mutant (Bcl-2-mt) Bcl-2 transfected BRK cells. Cells were treated with various doses of OODABL over 14 days. The IC50 for each cell line was determined by plotting the total number of individual colonies vs concentration of OODABL. This experiment was repeated three times with similar observations. Each data point represents mean ± SEM of three replicates.
Parthenolide is now being studied in clinical trials for antitumor activity (22,23). The potential mechanisms of cytotoxicity of parthenolide include effects on NF-kappa B and inhibition of adhesion molecule-1 expression (23,27). Thapsigargin, also a sesquiterpenelactone that has cytotoxicity against tumors, has also been studied without a clear understanding of the mechanism of cytotoxicity (28,29). In an effort to define the mechanism of cytotoxicity of OODABL, we studied the importance of Bcl-2 phosphorylation. Prior studies of anti-microtubule agents demonstrated that Bcl-2 phosphorylation is associated with cytotoxicity (7, 8). Haldar et al. demonstrated that paclitaxel induces Bcl-2 phosphorylation, which abrogates Bcl-2-Bax heterodimers and decreases the apoptotic threshold (8). Deletion of the loop region of Bcl-2 decreased paclitaxel induced cytotoxicity, suggesting that drug-induced phosphorylation of Bcl-2 is important for paclitaxel-induced cell death (9). In contrast, other investigators have demonstrated that Bcl-2 phosphorylation may be a marker of mitotic cell cycle arrest, and possibly not a mechanism of cytotoxicity by anti-microtubule agents (10). Our current study demonstrated the importance of Bcl-2 phosphorylation by OODABL-induced cytotoxicity in a Bcl2 mutant cell line compared to control (Figures 5 and 6). These data support the importance of Bcl-2 phosphorylation by OODABL. In conclusion, these data demonstrate, for the first time, that OODABL has biological activity capable of inducing Bcl-2 phosphorylation and G2/M cell cycle arrest. These data also demonstrated that OODABL induces phosphorylation of Bcl-2 similarly to paclitaxel, and phosphorylation of Bcl-2 increases the sensitivity of the cell to OODABL. Further studies of this compound and analogs are warranted to determine their clinical usefulness.
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ANTICANCER RESEARCH 25: 313-318 (2005) Acknowledgements This study was supported by the New Jersey Agricultural Experimentation Station Project and the New Jersey Commission on Cancer Research 5030CA072720-0751-CAM and 5P30C40727200651-CAM
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Received October 5, 2004 Accepted November 22, 2004
