Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells

Damnacanthal, an anthraquinone compound, is isolated from the roots of Morinda citrifolia L. (noni), which has been used for traditional therapy in several chronic diseases, including cancer. Although noni has long been consumed in Asian and Polynesian countries, the molecular mechanisms by which it exerts several benefits are starting to emerge. In the present study, the effect of damnacanthal on MCF-7 cell growth regulation was investigated. Treatment of MCF-7 cells with damnacanthal for 72 h indicated an antiproliferative activity. The MTT method confirmed that damnacanthal inhibited the growth of MCF-7 cells at the concentration of 8.2 μg/ml for 72 h. In addition, the drug was found to induce cell cycle arrest at the G1 checkpoint in MCF-7 cells by cell cycle analysis. Damnacanthal induced apoptosis, determined by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) dual-labeling, acridine-orange/PI dyeing and caspase-7 expression. Furthermore, damnacanthal-mediated apoptosis involves the sustained activation of p21, leading to the transcription of p53 and the Bax gene. Overall, the present study provided significant evidence demonstrating that p53-mediated damnacanthal induced apoptosis through the activation of p21 and caspase-7.


Introduction
Breast cancer is the most common type of malignant tumor and the second highest mortality among females with cancer (1). Survival rates for breast cancer have greatly increased with significant improvement in surgical technology and therapy regimens over the last three decades, particularly for early-stage breast cancer. However, no effective treatments currently exist for metastatic breast cancer (2,3). As cancer development largely results from the uncontrolled growth of malignant cells, in which cell proliferation surpasses cell death, deregulation of apoptosis, which occurs frequently in a vast majority of cancer types, has become a non-negligible target for anticancer strategies (4,5). Proapoptotic compounds, derived from synthetic chemistry or natural sources, are also under active investigation for their therapeutic effects and for their mode of actions against various types of cancer. Over the past decades, considerable effort has been directed to using natural products as a source of novel anticancer drugs in the fight against the challenge that a number of types of cancer remain incurable by currently available therapeutic approaches.
Morinda citrifolia L. (Rubiaceae), commonly known as noni, is a small evergreen tree or shrub that is widely distributed throughout the pacific islands, Southeast Asia and other tropical and semitropical areas. It has been widely used in therapeutic preparations for centuries, owing to its anti-inflammatory, antibacterial, antiviral, antifungal and antitumor properties (6)(7)(8)(9). Damnacanthal, an anthraquinone compound, was isolated from the roots of Morinda citrifolia L. and identified as a potent inhibitor of different types of human tumor cells, including human T-lymphoblastic and acute promyelocytic leukemia (10) and breast carcinoma (11). These anticancer effects of damnacanthal were identified through interfering with the cell cycle, inducing apoptosis and inhibiting the invasive potential of cancer (12)(13)(14). However, the underlying molecular mechanisms of these anticancer effects remain under investigation. In the present study, the Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells human breast cancer MCF-7 cell line was used to investigate the effects of damnacanthal on the breast cancer cell proliferation, apoptosis, cell cycle arrest and apoptotic gene expression.

Materials and methods
Compound. Damnacanthal was obtained from the Faculty of Applied Sciences, Mara University of Technology (Shah Alam, Malaysia) and used as received. The structure of damnacanthal is shown in Fig. 1. Quantification of apoptosis. Damnacanthal-induced cell death in MCF-7 cells was quantified using propidium iodide (PI) (Sigma-Aldrich) and acridine-orange (AO, Sigma-Aldrich) double staining according to standard procedures and examined under fluorescence microscope (Eclipse Ti, Nikon, Melville, NY, USA). Briefly, treatment was performed in a 25-ml culture flask. MCF-7 cells were plated at a concentration of 1x10 6 cells/ml and treated with damnacanthal at IC 50 concentration. Flasks were incubated in an atmosphere of 5% CO 2 at 37˚C for 72 h. The cells were then spun down at 1,000 x g for 10 min. Supernatant was discarded and the cells were washed twice using phosphate-buffered saline (PBS) following centrifugation at 1,000 x g for 10 min to remove the remaining media. In total, 10 µl fluorescent dyes, AO (10 µg/ml) and PI (10 µg/ml), were added into the cellular pellet at equal volumes. Total RNA preparation and reverse transcription-polymera se ch ain rea ct ion (RT-PCR) a n alysis. T he apoptosis-related genes were analyzed following the treatment of the MCF-7 cells with or without damnacanthal at IC 50 concentration for 72 h. The cells were trypsinized and washed twice with PBS. Total RNA was prepared using a Qiagen RNA extraction kit (Qiagen, Hilden, Germany). The RNA concentration was determined by reading the absorbance at 260 and 280 nm with a UV spectrophotometer (DU730; Beckman Coulter, Petaling Jaya, Selangor, Malaysia). The total RNA was transcribed to cDNA using the GenomeLab™ GeXP start kit (Beckman Coulter, Miami, FL, USA) for RT-PCR, according to the manufacturer's instructions. The following primers were designed from the known sequences: 5'-CCCTTTTGCTTCAGGGTTTC-3' a nd 5'-ACA A AGTAGA A A AG G G CGACA A-3' for Ba x; 5'-T GT G GAC C T GT CAC T GT C T T G -3' a n d 5'-TAG G G CT TCCTCT TG GAGA A-3' for p21Cip1; 5'-CAGACCGGTCCTCGTTTGTA-3' and 5'-ACCTCG GCATCTTTGTCTGTT-3' for caspase-7; and 5'-AAGGTG AAGGTCGGAGTCAA-3' and 5'-AGATCTCGCTCCTGG AAGATG-3' for GAPDH. The amplification profile was as follows: Denaturing at 94˚C for 30 sec; annealing at 55˚C for 30 sec; and extension at 68˚C for 1 min. The cDNA was ampli-fied using MJ Research PTC-225 analyzer (MJ Research Inc., St. Bruno, QC, Canada) for 35 cycles, followed by a step of 10 min at 72˚C to extend the partially amplified products. These cycling conditions were established empirically to provide a linear increase in product intensity proportional to the amount of template. The PCR products with fluorescently labeled fragments were separated by capillary gel electrophoresis (One Capillary array, BD Biosciences, Franklin Lake, NJ, USA), at 6.0 kV and 50˚C for 35 min, according to their product size, and the results were analyzed using GenomeLab GeXP system software (Beckman Coulter). GADPH was selected as the reference gene for normalizing all results of the targeted genes. Statistical analysis. Data are presented as the mean ± standard deviation. P-values were determined by analysis of variance followed by Student-Newman-Keuls test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Effect of damnacanthal on the viability of MCF-7 cells.
To examine the effects of damnacanthal on MCF-7 cell viability, MTT assay was performed. MCF-7 cells were plated onto 96-well plates and treated with various concentrations of damnacanthal (0-30 µg/ml) for 72 h. As shown in Fig. 2, damnacanthal dose-dependently (P<0.05) inhibited cell viability. These inhibitory effects were observed following incubation with 8.2 µg/ml damnacanthal, reducing cell growth by 50% (IC 50 ).

Damnacanthal induced apoptotic cell death in MCF-7.
AO and PI dyes were used to differentiate viable, apoptotic and necrotic cells under fluorescence microscope. Fig. 3A shows the intact viable cells and apoptotic and necrotic cells following the treatment of MCF-7 cells with damnacanthal at IC 50 concentration for 72 h. The apoptotic event of damnacanthal-treated cells was increased significantly: ≤4-fold higher than the vehicle-treated cells (Fig. 3B). A fraction of necrotic cells were also detected in the treatment group.

Induction of apoptosis by damnacanthal in MCF-7 cells.
To determine whether the loss of cell viability induced by damnacanthal was associated with apoptosis, Annexin V-FITC/PI binding assay was performed. The assay evaluates phosphatidylserine turnover from the inner to the outer lipid layer of the plasma membrane, an event typically associated with apoptosis. Flow cytometric analysis revealed that the percentage of apoptotic cells with Annexin V-positive but PI-negative cells increased gradually with concentration in damnacanthal-treated cells. As shown in Fig. 4, following 72 h of treatment with damnacanthal at IC 50 concentrations, the numbers of apoptotic MCF-7 cells, as revealed by Annexin V binding, increased in a dose-dependent manner, indicating a proapoptotic activity of damnacanthal. The proportion of MCF-7 cells in early apoptosis was 80.6% after 72 h (P<0.05, vs. vehicle-treated cells at the same time). For cells that were in the late apoptosis, the proportion was 8.1% at 72 h, while the proportion of the cells in necrosis was 2% at 72 h (P<0.05, vs. vehicle-treated cells at the same time). For the vehicle-treated MCF-7 cells, the percentage of cells that underwent necrosis was <4% at 72 h incubation time (Fig. 4).
Damnacanthal induces G1 cell cycle arrest. Following the treatment of MCF-7 cells with 8.2 µg/ml damnacanthal for 72 h, the cell population in G1 phase increased to 80% which was accompanied with a decrease in the S (5%) and G2 (8%) phases (Fig. 5). These results clearly indicated that damnacanthal induces post G1-arrest and apoptosis among the treated cells.   levels of BAX, p21 and caspase-7 genes (Fig. 6). For the p21 gene, all treatments exhibited a significant increase (P<0.05) compared with the control.
Involvement of apoptotic proteins in damnacanthal-induced apoptosis. Expression levels of proteins that are involved in pro-and anti-apoptosis, such as Bcl-2, p53, ER-α and XIAP, were evaluated using a multicolor flow cytometer. Fig. 7 shows the percentage changes on the expression level of protein following the treatment of MCF-7 cells with damnacanthal at 8.2 µg/ml for 72 h. The percentage expres-

Discussion
Cancer has become an increasing public health issue for its high rates of morbidity and mortality. In the current study, the anticancer activity of damnacanthal, an anthraquinone which is extracted from the noni plant, was investigated. Considering that little is known concerning the anticancer activities and related mechanisms of damnacanthal, the current study performed further investigations to elucidate the antitumor activities of damnacanthal in the human breast cancer MCF-7 cell line and the possible mechanisms involved.
The experimental results showed that damnacanthal exhibits potent cytotoxicity to MCF-7 cells, with an IC 50 of 8.2 µg/ml (Fig. 2). Apoptosis assay showed that apoptotic cells induced by damnacanthal exhibited cellular alterations, determined by counting chromatin condensation and nuclear fragmentation by AO/PI ( Fig. 3A and C). Annexin V/PI double-staining assay further confirmed the results of the AO/PI staining by showing the important membrane alterations associated with apoptosis in MCF-7 cells and the percentage of apoptosis increase in damnacanthal treatment (Fig. 4). The cell cycle results demonstrated that damnacanthal induced G1 arrest and apoptosis among the damnacanthal-treated cells (Fig. 5). All these results showed that damnacanthal increases antitumorigenic activity by increasing the expression of p53, followed by p21. It has been previously reported that damnacanthal induces apoptosis in a number of cells, including HL-60, MOLT-4, CEM-SS, HT-29, Hela, 3T3 and peripheral blood mononucleated cells (10,15,16). Proposed mechanisms for the proapoptotic effect include activation of caspases, induction of cytochrome c release, regulation of protein kinase C isoform expression, inhibition of NF-κB and suppression of activator protein 1 (17)(18)(19). The results of the current study were consistent with our previous study, as damnacanthal induced apoptosis in HL-60 and Wehi-3B cells (10). Further investigations were performed to highlight the apoptotic pathways involved in the apoptosis induced by damnacanthal in MCF-7 cells.
Previous studies have revealed that caspases are critical in executing apoptosis (20). In order to gain further insight into the mechanism of the signaling cascade, the present study examined the molecular sequence of events in damnacanthal-induced apoptosis. Apoptosis may occur via two fundamental pathways: i) death receptor or extrinsic pathway; and ii) mitochondrial or intrinsic pathway. The present study demonstrated the considerable role of the mitochondrial apoptotic pathways in apoptosis induced by damnacanthal in MCF-7 cells. Damnacanthal-mediated activation of Bax, p21 and caspase-7 was identified in MCF-7 cells. The activation of p21 and caspase genes stimulates p53 phosphorylation (21). Although multiple pathways contribute to the modulation of p53 (22), the current study investigated the expression of p21 as one of the upstream molecules of p53. The results demonstrated that p21-p53 signaling is one of the key pathways in mediating damnacanthal-induced apoptosis. In addition, the role of p21 in the transcription of the p53-regulated Bax gene is likely to involve p53 phosphorylation (23). The increased damnacanthal-dependent p53 protein levels are consistent with the damnacanthal-dependent transcriptional induction of Bax. Extensive analyses of damnacanthal-dependent modifications of p53 are in progress to link p21 activity with p53 function in damnacanthal-mediated apoptosis. Although modulation of p21 and p53 signaling is common, the current study established connections between well-known proapoptotic molecules in the damnacanthal-induced apoptosis.
In conclusion, damnacanthal, a bioactive compound from noni roots, enhanced the expression of p21 and caspase-7. Overexpression of p21 directly activates transcription and expression of p53 and, subsequently, increases apoptosis in human breast cancer MCF-7 cells. These results are likely to highlight the potential benefits of damnacanthal for further preclinical or clinical practice and damnacanthal may be a useful cancer prevention/therapeutic agent in human breast carcinoma.