Induction of Apoptosis in Human Breast Adenocarcinoma Cells MCF-7 by Monapurpyridine A, a New Azaphilone Derivative from Monascus purpureus NTU 568

A new azaphilonidal derivative, monapurpyridine A (MPA), has recently been isolated from the fermented products of Monascus purpureus NTU 568. The structure of MPA was elucidated by nuclear magnetic resonance (1H-NMR, 13C-NMR, COSY, HMQC, and HMBC) and other spectroscopic analyses. Biological evaluation revealed that MPA could induce cell death in human breast adenocarcinoma cells MCF-7, and it has no significant toxicity to normal mammary epithelial cells M10. The MTT assay and flow cytometric analysis were employed to investigate cell viability and cell cycle influenced by MPA. Moreover, we used Western blot and caspase activity assay to demonstrate the activation of caspase-3, -8 and -9 resulted from MPA. All evidence supported that MPA was suitable for developing into a chemotherapeutic or chemopreventive agent against breast cancer.


Introduction
Monascus species have traditionally been used as food additives in Asian countries for thousands of years. Recently, Monascus-fermented rice, also called red mold rice (RMR), has been reported to possess various biological functions, such as: hypolipidemic effects [1], antifatigue activities [2], neuroprotective properties against Alzheimer's disease [3], preventive ability for obesity [4], and prevention of carcinogenesis [5] or tumor progression [6], etc.
Recently, a variety of new azaphilones were isolated and characterized from Monascus-fermented products. For example, monapurones A-C were isolated from the extract of RMR and showed selective cytotoxicity against human lung cancer cell line A549, while exhibiting no significant toxicity to human normal lung cells MRC-5 and WI-38 [14]. Four new pyridine derivatives, monasnicotinates A-D were isolated from Monascus pilosus BCRC 38093 and evaluated for their inhibitory effects against lipopolysaccharide (LPS)-induced nitric oxide production [15].
In our laboratory, five new azaphilone pigments, including two blue fluorescent monapurfluores have been isolated from Monascus purpureus NTU 568. These new azaphilones were reported to be cytotoxic to cancer cell lines or anti-inflammatory on LPS-stimulated Raw 264.7 cells [16,17]. We also executed a large-scale preparation for monaphilone A, one of these new azaphilones, to explore the apoptosis-related and anti-inflammatory properties in inducing death of human laryngeal carcinoma cell line HEp-2 and reducing inflammatory responses on RAW 264.7 cells [18]. Here, we report the structural elucidation of the new isolated compound, monapurpyridine A (MPA; Figure 1), and its apoptosis-related mechanisms. For these purposes, we obtained MPA on a large scale and designed some experiments to induce apoptosis in the human breast cancer cell line MCF-7.

Cytotoxicity of MPA on MCF-7 and M10 Cells
We utilized MTT assay for a two-day course to study the inhibition on cell viability of MCF-7 and M10 cells treated with MPA ( Figure 3). Up to the concentration of 100 M, MPA showed dose-dependent and moderated cytotoxic activity against MCF-7 cells, but no significant cytotoxicity to normal M10 cells. The results suggested that MPA was selectively cytotoxic to breast cancer cell line.

Cell Death Induced by MPA on MCF-7 Cells
To study cell deaths of MCF-7 cells induced by MPA, we utilized flow cytometry (propidium iodide staining) to analyze the ratio of Sub-G  area for 12 and 24 h ( Figure 4). MPA (50 M, 24 h) significantly induced about 10-fold more cell deaths than control group. To make clear that the cell death resulted from apoptosis or necrosis, we designed some apoptotic approaches in the next step.

Caspase Activation of MPA on MCF-7 Cells
MCF-7 cells were treated with 50 and 100 M of MPA for 12 and 24 h, and further analyzed for the cleaved caspase-3 by Western blot ( Figure 5) and enzyme activity of caspase-8 and -9 by colorimetric assay kit ( Figure 6). Treatment of MPA (50 and 100 M, 24 h) exhibited increases of cleaved caspase-3, which were estimated as a down-streamed event of apoptosis. As to the up-streamed caspase-9 and caspase-8, treatment of MPA (50 M, 24 h) exhibited significant increases of caspase-9 activity, but showed no significant increase of caspase-8. Thus, MPA was demonstrated to induce apoptosis through caspase-9 activations.

General
Electrospray ionization mass spectrometry (ESI-MS) data were acquired on a LCQ mass spectrometer (Finnigan MAT LCQ, San Jose, CA, USA). NMR spectra were run on a Bruker Unity Plus 400 MHz NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) using CDCl 3 as the solvent. Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and silica gel 60 (70-230 mesh and 230-400 mesh, Merck, Darmstadt, Germany) were used as chromatographic supports. Silica Gel 60 F254 plates (Merck) were used for thin layer chromatography (TLC). The TLC spots were detected under UV-lamps (254 and 365 nm) and also by using an anisaldehyde-sulphuric acid solution, applied as a spray reagent, followed by heating. The high performance liquid chromatography (HPLC) was performed using a Shimadzu LC-6AD apparatus with a SPD-6AV UV detector that was equipped with a preparative Cosmosil AR-II column (250 × 20 mm i.d., Nacalai Tesque, Inc., Kyoto, Japan).

Cell Lines and Culture Conditions
Human breast adenocarcinoma cells MCF-7 and normal mammary epithelial cells M10 were obtained from Bioresources Collection and Research Center (Hsinchu, Taiwan). Both cell lines were maintained in MEM (5% FBS) in a humidified incubator with 5% CO 2 at 37 °C.

Cytotoxicity Assay
Cells (3 × 10 3 per well) were seeded with MEM (180 L) in 96-well plates. After 4 h, test agents dissolved in PBS solution (20 L) were added at final concentrations of 25, 50 and 100 M and incubated in a 37 °C incubator with 5% CO 2 . After culturing for 24, 48 h, MTT solution (2 mg/mL, 20 L) was added to each well and incubated for 4 h to induce the cellular conversion of the tetrazolium salt into a formazan product. The supernatant was then removed and DMSO (200 L) was added to dissolve the formazan, which can be detected by spectrophotometry at 570 nm and provided a relative estimate of cell viability.

Assay of DNA Contents by Flow Cytometry
MCF-7 cells (5 × 10 4 per well) were seeded with MEM (2 mL) in 6-well plates. After 12 h, test agents dissolved in MEM solution (2 mL) were added at final concentrations of 50 and 100 M. After 12 and 24 h of incubation, the cells were harvested and fixed with 80% ethanol for 30 min. Then the cell pellets were washed three times with PBS and co-incubated the cells with propidium iodide (4 g/mL), Triton X-100 (1%), and RNase (0.1 g/mL) in the dark for 30 min. Finally, the cells can be analyzed by flow cytometry (FACSCalibur, Ser. No. E1577, BD) equipped with Cell Quest software to provide a relative estimate of DNA contents.

Western Blot Analysis
Cells (about 5 × 10 5 ) were seeded with media (10 mL) in a 75 cm 2 flask. After 12 h, test agents dissolved in media (10 mL) of were added. After 12 and 24 h of incubation, the cells were harvested and extracted by RIPA lysis buffer (Millipore, Bellerica, MA, USA) with 1% protease inhibitor (Sigma, St. Louis, MO, USA). The cell lysates were analyzed with primary antibodies, including of caspase-3 antibody (Novus Biologicals, Littleton, CO, USA) and β-actin antibody (Epitomics, Burlingame, CA, USA). The anti-mouse secondary horseradish peroxidase antibodies (Jackson ImmunoResearch, West Grove, PA, USA) was further added. Finally, the detection was performed using the Western lightning chemiluminescence reagent (PerkinElmer Life Sciences, Waltham, MA, USA).

Caspase Activity Assay
Cells (about 5 × 10 5 ) were seeded with media (10 mL) of in a 75 cm 2 cell culture flask. After 12 h, test agents dissolved in media (10 mL) were added. After 12 and 24 h of incubation, the cells were harvested and tested for caspase-8 and caspase-9 activities respectively using a colorimetric assay kit (BioVision, Linda Vista Avenue, Mountain View, CA, USA). Caspase activity was determined according to the manufacturer's protocol.

Data Analysis
Data were presented as mean ± standard deviation (n = 3). The statistical comparisons were performed by one-way analysis of variance (ANOVA) with Duncan's test. The significant differences were indicated as p < 0.05 or 0.01.

Conclusions
Previous studies in our group showed that RMR extracts or red mold dioscorea (RMD) extracts fermented from M. purpureus NTU 568 might prevent carcinogenesis or tumor progression in animal models. We also isolated and confirmed that some azaphilone derivatives were cytotoxic to laryngeal, colon and lung cancer cell lines. In this study, a new azaphilone MPA was isolated from M. purureus NTU 568 fermented red mold rice, and showed moderate cytotoxicity against breast cancer cells. In conclusion, azaphilone derivatives isolated in our studies were moderately cytotoxic but tissue-specific to different cancer cell lines. These results strongly implied that fermented products from M. purpureus NTU 568 are potential candidates for tumor prevention due to the available amounts of azaphilone derivatives.