Novel Drug Amooranin Induces Apoptosis Through Caspase Activity in Human Breast Carcinoma Cell Lines

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Breast Cancer Research and Treatment 80: 321–330, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.


Novel drug amooranin induces apoptosis through caspase activity in human breast carcinoma cell lines Thangaiyan Rabi1 , Cheppail Ramachandran1 , Hugo B. Fonseca1 , Raveendran P.K. Nair1 , Arturo Alamo1 , Steven J. Melnick2 , and Enrique Escalon3 1 Research

Institute, 2 Division of Pathology, 3 Division of Hematology/Oncology, Miami Children’s Hospital, Miami, FL, USA

Key words: amooranin, apoptosis, breast carcinoma cells, breast epithelial cells

Summary Amooranin (AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India. A. rohituka stem bark is one of the components of a medicinal preparation used in the Indian Ayurvedic system of medicine for the treatment of human malignancies. We investigated the mechanism of cell death associated with AMR cytotoxicity in human mammary carcinoma MCF-7, multidrug resistant breast carcinoma MCF-7/TH and breast epithelial MCF-10A cell lines. AMR IC50 values ranged between 3.8–6.9 µg/ml among MCF-7, MCF-7/TH and MCF-10A cells. AMR induced oligonucleosome-sized DNA ladder formation characteristic of apoptosis when tumor cells were treated with 1–8 µg/ml AMR for 48 h. In situ cell death detection assay indicated that AMR caused 37.3–72.1% apoptotic cells in MCF-7, 32–48.7% in MCF-7/TH and 0–37.1% in MCF-10A cells at 1–8 µg/ml concentrations. The induction of apoptosis in AMR treated cells was accompanied by the elevation of total caspase and caspase-8 activities. Flow cytometric analysis showed that AMR induced caspase-8 activation in 40.8–71% MCF-7, 28.5–43.2% MCF-7/TH and 4–32.8% MCF-10A cells at 1–8 µg/ml concentrations. Our results suggest that AMR is a novel drug having potential for clinical development against human malignancies.

Introduction Amooranin (AMR) is a triterpenoid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India [1]. One of the herbal preparations used in the Indian Ayurvedic system of medicine against human malignancies contain A. rohituka stem bark, Semicarpus anacardium fruits and Glycyrrhiza glabra roots [2]. This herbal preparation is marketed under the trade name ‘CANARIB’ in India. We have reported earlier on the antitumor activity of AMR in tumor cell lines and rat tumor models [1, 3]. AMR IC50 value for HeLa human uterine cervix carcinoma cells is 3.4 µg/ml compared with 6.2 µg/ml for Chang liver cells. Intraperitoneal administration of AMR at 10– 20 mg/kg/day has also prolonged the mean survival time of animals with a significant reduction in tumor size [1].

Information on the molecular mechanism of drug action is quite essential for the development of any potential drug for chemotherapy. In this context, several cancer drugs have been shown to induce apoptosis in tumor cells. Apoptosis has been characterized biochemically by the cleavage of DNA into nucleosomal size fragments of 180–200 base pairs or multiples thereof, which can be detected as DNA ladder by gel electrophoresis [4–7]. Several cancer drugs also cause a G2 /M arrest before tumor cells undergo apoptosis [8]. Pro-apoptotic genes are activated and antiapoptotic genes suppressed during the process of cell death, leading to the activation of caspases and nucleases that serve to degrade protein and genomic DNA, respectively, within the cell [9]. Caspases are aspartate-specific cysteine proteases, existing as latent intracellular zymogens [10, 11]. Once activated by apoptotic signals, they can systematically dismantle the


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cell by cleaving key cellular and nuclear proteins with defined substrate specificities [12, 13]. Based on the sequence of action in apoptosis signaling, more than 14 caspases have been identified and organized into apoptotic initiator and processor caspases [10–13]. In this investigation, we have analyzed the molecular details underlying AMR cytotoxicity of breast cancer cells, mainly its effect on cell cycle, apoptosis and also the role of caspases in AMR-induced cell death.

Student’s t-test was used to determine the significant difference between IC50 values. Cell cycle analysis The number of cells in each stage of the cell cycle was monitored by flow cytometric analysis of cellular DNA content after staining with 1 mg/ml propidium iodide [15]. Data was analyzed to determine the percentage of cells at each phase of the cell cycle (G0 + G1 , S, and G2 + M).

Materials and methods Chemicals and reagents The isolation procedure of AMR has been described earlier [1]. 3-(4,5-Dimethylthiozal-2yl)-2,5 diphenyl tetrazolium bromide (MTT) survival assay kit and in situ cell death detection kit were purchased from Roche Molecular Biochemicals, Indianapolis (IN). Fluorescein Caspase (VAD) Activity Kit and Caspase8 (LETD) Activity Kit were purchased from Intergen Company (NY).

Analysis of DNA fragmentation by agarose gel electrophoresis To assess the pattern of DNA cleavage caused by AMR, agarose gel electrophoresis was performed as described by Cohen and Duke [16]. Briefly, control and drug-treated cells (2 × 106 cells) were lysed with 0.5 ml lysis buffer containing 0.2% Triton X-100 at

Cells and cell culture Human breast adenocarcinoma MCF-7 cells, its multidrug resistant subclone MCF-7/TH and mammary epithelial MCF-10A cells were cultured in Dulbecco’s Modified Essential Medium (Life Technologies, Inc., MD) containing 10% Fetal Bovine Serum and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin sulfate), in a 5% CO2 incubator at 37◦ C.

Figure 1. Chemical structure of amooranin.

Cytotoxicity assay MTT assay was used to determine drug sensitivity [14]. Cells (1 × 104 ) were grown in 96-well plates and after 48 h, the medium was replaced with new medium. The cells were incubated with varying concentrations of AMR for 48 h at 37◦C. AMR was dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO in the medium ranged from 0.1–1%. Cell viability was then assayed by adding MTT dye (500 µg/ml). Six hours after incubation period, MTT containing medium was removed. The formazan crystals were dissolved in 100 µl/well dimethyl sulfoxide, and the plates were read in Bio-Rad Benchmark Microplate Reader at 570 nm wavelength. All experiments were repeated thrice with three replications in each and the mean values were estimated.

Figure 2. Effect of AMR on the growth of MCF-7, MCF-7/TH and MCF-10A cells. Data points are mean ± SD estimates from three independent experiments performed in triplicate. AMR IC50 value of MCF-7 cell line is significantly different from that of MCF-10A and MCF-7/TH cell lines (p < 0.05).

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Figure 3. AMR induces G2 /M phase arrest and apoptosis. MCF-7, MCF-7/TH and MCF-10A cells were treated with the 0–2.5 µg/ml AMR for 48 h at 37◦ C. The cells were stained with propidium iodide and analyzed for cell cycle distribution by DNA flow cytometry.


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Figure 4. (A) TUNEL assay showing dose-dependent induction of apoptosis by AMR. MCF-7, MCF-7/TH and MCF-10A cells were treated with 0, 1, 2, 4 and 8 µg/ml concentrations of AMR for 48 h. After labeling with FITC-conjugated deoxynucleosides in the presence of terminal transferase, cells were analyzed for DNA fragmentation by flow cytometry. (B) Percentage of apoptotic cells induced by AMR is plotted against drug concentrations. Results are mean ± SD values.

room temperature for 30 min. The supernatant fractions were collected by centrifugation at 12, 000 × g for 30 min. The DNA in these fractions was precipi-

tated overnight with 100 µl 5 M sodium chloride and 0.5 ml 2-isopropanol at −20◦C. The DNA was dissolved in 20 µl of 10 mM Tris–HCl (pH 7.4) and

Caspase activity and apoptosis induced by the novel drug amooranin


8 (LETD) Activity Kit according to manufacturer’s instructions [18].

Results Cytotoxicity of AMR

Figure 4. (continued).

1 mM EDTA buffer. RNase (10 units) was added to the samples before incubating at 60◦ C for 1 h. An equal volume of loading buffer was added afterwards and the samples were subjected to electrophoresis on a 2% agarose gel in Tris borate buffer at 50 V for 3 h. The agarose gel was stained with ethidium bromide, and DNA fragmentation pattern was visualized in a UV transilluminator. AMR-induced DNA fragmentation was also analyzed by TUNEL assay using in situ cell detection kit according to manufacturer’s instructions. Briefly, cells were permeabilized in a solution containing 0.1% Triton X-100 and 0.1% (w/v) sodium citrate for 2 min. After washing in PBS, cells were incubated with label solution and terminal transferase. Cells were incubated without terminal deoxynucleotidyl transferase was used as negative control. DNase (1 µg/ml) treated cells incubated with label solution in the presence of terminal deoxynucleotidyl transferase (TUNEL reaction mixture) was used as positive control and the details of TUNEL assay have been described earlier [17]. The percentage of apoptotic cells induced by AMR was determined by overlaying flow histograms of AMR treated cells on to the histograms of untreated cells. If the histograms of treated cells are farther apart from untreated cells, a high estimate was obtained for percentage of apoptotic cells. Detection of apoptosis and caspase activity Breast carcinoma or breast epithelial cells were incubated for 48 h in the presence or absence of AMR (1– 8 µg/ml). AMR-induced apoptosis was evaluated by Fluorescein Caspase (VAD) Activity Kit and caspase-

Figure 1 shows the structure of AMR. The IC50 values were determined by MTT assay after exposing MCF-7, MCF-7/TH and MCF-10A cells to 0–8 µg/ml AMR for 48 h. AMR dose–response curves of cell lines are given in Figure 2. The results have indicated that AMR is cytotoxic to breast cancer cells. MCF-7, MCF-7/TH and MCF-10A cells incubated with AMR demonstrated a dose-dependent reduction of cell viability that reached maximal effects at 8 µg/ml (Figure 2). MCF-7 cell line was more sensitive to AMR (IC50 = 3.8 µg/ml) than MCF-7/TH (IC50 = 6.8 µg/ml) and MCF-10A (IC50 = 6.9 µg/ml) cell lines (p = 0.0004 by Student’s t-test). AMR induces cell cycle arrest MCF-7 and MCF-7/TH cells treated with 1 µg/ml AMR showed accumulation in G2 + M phase population (12.4–32.2%) with a concomitant decrease in G0 + G1 phase cells suggesting a possible cell cycle arrest at G2 + M phase (Figure 3). However, 1 µg/ml AMR does not induce any G2 /M phase arrest in MCF10A cells. AMR also induced a higher percentage of sub G0 − G1 population, indicative of apoptosis, in both breast cancer cell lines (MCF-7 and MCF-7/TH) than human breast epithelial cell line (MCF-10A) at every concentration of drug tested in the present investigation (Figure 3). AMR-induced apoptosis Apoptosis induced by AMR was investigated by different techniques such as DNA fragmentation, general caspase activity and proteolytic activation of caspase-8. The results of TUNEL assay are given in Figure 4(A) and (B). AMR caused DNA fragmentation in 37.3–72.1% MCF-7 cells, 32–48.7% MCF7/TH cells, as compared with 0–37.1% MCF-10A cells at 1–8 µg/ml concentrations. DNase-treated positive control samples showed more than 95% apoptotic cells in all three cell lines. Drug sensitive (MCF-7) and multidrug resistant (MCF-7/TH) cell lines were more sensitive to AMR than breast epithelial cell line (MCF-10A) with respect to induction of apoptosis.


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Figure 5. DNA fragmentation induced by AMR. (A) MCF-7 cells were treated for 48 h with 0, 1, 2, 4 and 8 µg/ml AMR (lanes 2–6). (B) MCF-7/TH cells were treated for 48 h with 0, 1, 2, 4 and 8 µg/ml AMR (lanes 2–6). (C) MCF-10A cells were treated for 48 h with 0, 1, 2, 4 and 8 µg/ml AMR (lanes 2–6). Lane 1 in A, B and C, φX174 DNA marker digested with Hae III.

Furthermore, MCF-7 cell line was more sensitive to AMR than MCF-7/TH. Analysis of internucleosomal DNA fragmentation induced by AMR in MCF-7, MCF-7/TH and MCF10A cells was detected by gel electrophoresis of low molecular weight DNA (Figure 5). AMR has induced charecteristic DNA ladder pattern, a hallmark of apoptosis, when cells were treated for 48 h. AMR induced distinct ladder pattern in both cancer cell lines (MCF-7 and MCF-7/TH), although the ladder was less distinct in human breast epithelial cell lines (MCF-10A). Moreover, DNA fragmentation was almost undetectable in MCF-10A cells treated with 1 µg/ml AMR for 48 h, whereas at the same concentration ladder pattern was highly visible in MCF-7 and MCF-7/TH cell lines. To analyze the mechanism of AMR-induced apoptosis, activation of general caspase was examined. General caspase activity was generally higher in AMR-treated MCF-7 and MCF-7/TH cell lines than MCF-10A cell line (data not shown). To characterize the specific caspase further, activation of initiator caspase-8, a key component of caspase, was analyzed. Both MCF-7 and MCF-7/TH breast cancer cell lines showed higher percentage of caspase-8 positive cells at 1–8 µg/ml AMR than MCF-10A human breast epithelial cell line (Figure 6(A) and (B)). AMR caused caspase activation in 40.8–71% MCF-7 cells, 28.5–43.2% MCF-7/TH cells as compared with only 4–32.8% MCF-10A cells at 1–8 µg/ml concentrations.

Discussion A. rohituka is a wild tropical medicinal tree indigenous to India, Malaysia and Sri Lanka and is known

locally as ‘rohera’ in India. The stem bark and the seeds of the plant are used indigenously as medicines to treat diseases of spleen, liver and abdomen, including cancer [19]. In our early studies, ethyl acetate soluble extract of the A. rohituka stem bark was found to be significantly cytotoxic to Dalton’s lymphoma ascites and MCF-7 tumor cells [1]. AMR was isolated as a major active constituent of A. rohituka stem bark and characterized as the novel compound 25-hydroxy-3-oxoolean-12-en-28-oic acid [1]. In the present investigation, we found that AMR has a more potent effect on inhibiting the growth of MCF-7 breast cancer cells as compared with that on multidrugresistant MCF-7/TH breast cancer cells and MCF-10A breast epithelial cells. MCF-7/TH is a MDR cell line developed from MCF-7 by repetitive exposure to adriamycin and it has an active P-glycoprotein pump. In a separate study we showed that AMR is a substrate for P-glycoprotein and AMR can increase adriamycin accumulation in tumor cells [20]. Therefore a higher AMR IC50 for MCF-7/TH than MCF-7 cell line can be expected. However the reason for higher AMR IC50 value of MCF-10A cells is not clear other than its almost normal cell phenotype. Plant derived cancer drugs such as taxol, podophyllotoxin, vinblastine and vincristine, are important anti-mitotic drugs, currently used for the treatment of human malignancies. These lead compounds have been used as models for the development of novel anticancer agents [21, 22]. AMR induces accumulation of cells at G2 + M phase during cell cycle traverse at 1 µg/ml. Alteration in the cell cycle phase distribution by AMR, described herein is encouraging for the sensitivity of tumor cells to DNA targeting agents. Terpenoids have been shown to inhibit proliferation

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Figure 6. (A) Flow cytometric analysis of caspase-8 activity induced by AMR. MCF-7, MCF-7/TH and MCF-10A cells were treated with the 0, 1, 2, 4 and 8 µg/ml concentrations of AMR for 48 h. After staining with FAM-LETD-FMK and propidium iodide, cells were analyzed for caspase-8 activity. (B) Percentage of caspase-8 positive cells induced by AMR treatment is plotted against drug concentrations. Results are mean ± SD values.


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Figure 6. (continued).

and induce apoptosis in tumor cells [23–26]. AMR is a triterpene acid having a similar basic chemical structure as other plant terpenoids like oleanolic acid [27]. Terpenoids, biosynthesized in plants by the cyclization of squalene, are reported to have anticarcinogenic and antiinflammatory properties [28, 29]. Recently Dr Michael Sporn’s group [23, 26, 27] has reported on the antiproliferative, antiinflammatory and differentiating effects of 2-cyano-3,12-dioxoolean-1,9-dien28-oic acid (CDDO) and its methyl ester CDDO-Me. Both these synthetic triterpenoids induced apoptosis by activation of caspase-8, caspase-3 and induction of mitochondrial cytochrome c release in leukemic cell lines and osteosarcoma cells [30, 31]. The primary mechanism of AMR induced cell death appears to be by induction of apoptosis as shown by propidium iodide staining, DNA fragmentation and TUNEL labeling. Breast cancer cells exhibited discrete DNA fragmentation patterns as being associated with apoptosis. AMR is more specific to tumor cells, since apoptotic cell death induced by AMR in normal mammary epithelial MCF-10A cells is significantly lower than in breast cancer cells. Several reports have demonstrated that the caspase family plays an important role in apoptosis [32–37]. In this investigation, our results also show that AMR causes caspase-8 activation during the apoptotic process in a dose-dependent manner. The breast cancer cells have shown higher caspase-8 activity after 48 h of AMR treatment than MCF-10A breast epithelial cells. Of the 14 caspases cloned to date, caspase-8 has been grouped under apical caspases along with caspase 2, 9 and 10. These apical caspases are reported to activate effector caspases 3, 6 and 7 [38, 39]. MCF-7 cells have a deletion mutation on exon 3

of the caspase-3 gene [40]. Recombinant caspase-8 is able to process/activate all known caspases, including caspase-1 to -7 and caspase-9 and -10 [41] and it lies at the apex of the apoptotic cascade [42]. The importance of the FADD-like prodomains of caspase-8 in directly linking CD95 and TNFR-1 mediated apoptosis has already been emphasized [42]. AMR induction of apoptosis by activation of caspase-8 in breast carcinoma cells can be expected and corroborates the effects already reported with synthetic triterpenoids like CDDO and CDDO-Me. Bedner et al. reported a correlation between the apoptotic index estimated by the presence of DNA strand breaks (TUNEL assay) and the activation of caspases in cultures treated with anticancer agents [43]. We have observed a strong correlation between levels of caspases and DNA strand breaks (TUNEL assay) in cells treated with AMR for 48 h (r = 0.95, p < 0.05) by correlation analysis. This suggests that the estimates of frequency of cells undergoing apoptosis thus, at least in the cell systems described in this study, are almost similar whether it is based on caspases activation, or DNA fragmentation (TUNEL assay). In conclusion, we demonstrate that AMR induces cell cycle arrest, subsequently inducing apoptosis in MCF-7, MCF-7/TH and MCF-10A cells indicating the anticancer effect and clinical potential of this natural triterpene compound.

Acknowledgement This work was supported by funds for Alternative Medicine from Miami Children’s Hospital Foundation.

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Address for offprints and correspondence: Cheppail Ramachandran, PhD, Research Institute, Miami Children’s Hospital, 3100 SW 62 Avenue, Miami, FL 33155, USA; Tel.: +1-305-663-8510; Fax: +1-305-669-6452; E-mail: [email protected]

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