Anti‑Breast Cancer Potential of Frullanolide from Grangea Maderaspatana Plant by Inducing Apoptosis
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ONCOLOGY LETTERS 17: 5283-5291, 2019 Anti‑breast cancer potential of frullanolide from Grangea maderaspatana plant by inducing apoptosis SIRIPHORN CHIMPLEE1, POTCHANAPOND GRAIDIST1,2, THEERA SRISAWAT3, SUCHADA SUKRONG4, RASSANEE BISSANUM1 and KANYANATT KANOKWIROON1,2 1Department of Biomedical Sciences, Faculty of Medicine; 2The Excellent Research Laboratory of Cancer 3 Molecular Biology, Prince of Songkla University, Hat Yai, Songkhla 90110; Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, Surat Thani 84000; 4Research Unit of DNA Barcoding of Thai Medicinal Plants, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Received June 21, 2018; Accepted February 6, 2019 DOI: 10.3892/ol.2019.10209 Abstract. Breast cancer is the leading cause of female demonstrated that frullanolide may exert anticancer activity mortality worldwide. Although there are several modern on breast cancer cell lines by inducing apoptosis. Frullanolide treatments for breast cancer, there is a high rate of recur- offers a possible novel approach to breast cancer therapy. rence for the majority of treatments; therefore, the search for effective anticancer agents continues. The present study Introduction aimed to investigate the anti-breast cancer potential of frul- lanolide, a compound which is isolated and purified from the Cancer is one of the leading causes of human mortality world- Grangea maderaspatana plant, for selected human breast wide, with an estimated 14 million new cancer cases projected cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). for 2030 (1). In women, breast cancer is quickly becoming the The MTT assay was used to assess cytotoxic activity in breast leading cause of mortality worldwide, and novel therapeutic cancer cell lines of treatment with frullanolide at 1.25, 2.5, avenues are constantly being explored (2). One such line of 5.0, 10.0 and 20.0 µg/ml. Additionally, the apoptotic induc- investigation involves evaluating natural products extracted tion ability of frullanolide at various concentrations [0.5x, from plants and endophytic fungi, such as vincristine and 1x and 2x half maximal inhibitory concentration (IC50)] was vinblastine, which have been demonstrated to exhibit anti- investigated by flow cytometry and western blot analysis. cancer activities, including the inhibition of breast cancer cell Frullanolide exhibited strong anti-breast cancer activity growth (3,4). Studies are continuously being conducted in the against MDA-MB-468 (IC50, 8.04±2.69 µg/ml) and weak search for novel effective and nontoxic anticancer compounds cytotoxicity against the MCF-7 (IC50, 10.74±0.86 µg/ml) and from various medicinal plants. MDA-MB-231 (IC50, 12.36±0.31 µg/ml) cell lines. The IC50 The genus Grangea belongs to the Compositae of frullanolide was high in the human normal epithelial breast (Asteraceae) family and comprises only six species, which are cell line (MCF‑12A) and mouse fibroblast cell line (L‑929). mostly native and distributed throughout Africa, South Asia Density plot diagrams revealed that frullanolide induced and Southeast Asia (5,6). G. maderaspatana (L.) Poir. (Phayaa apoptosis in MCF-7, MDA-MB-468 and MDA-MB-231 cells. Mutti) is one of the most common medicinal plants used in Notably, a plausible anticancer mechanism was elucidated via traditional Thai medicine in various therapeutic approaches, cellular apoptosis by p53-independence in the treated MCF-7 including ingestion of the whole plant to stimulate digestion, cell line and p53-dependence in the treated MDA-MB-468 reduce pain and inflammation, and regulate menses, while and MDA-MB-231 cell lines. In conclusion, the present study the leaf is used for reducing spasms (7). Although anesthetic, antioxidant, antibacterial and topoisomerase I and II (Top I and II) inhibitory activities have been reported previously for compounds derived from G. maderaspatana (8-12), to the best Correspondence to: Dr Kanyanatt Kanokwiroon, Department of our knowledge, no previous studies have assessed whether of Biomedical Sciences, Faculty of Medicine, Prince of Songkla these compounds possess anticancer activity. University, 15 Karnjanavanich Road, Hat Yai, Songkhla 90110, A previous study in our laboratory revealed the presence Thailand of sesquiterpene lactones (SLs) in G. maderaspatana (12). SLs E-mail: [email protected] are compounds which have several significant cancer‑asso- ciated implications and are used in targeted therapy against Key words: breast cancer cell lines, apoptosis, natural compounds, cancer cells, their stem cells and specific signaling path- frullanolide, sesquiterpene lactone ways (13). Various SLs, including thapsigargin, artemisinin and parthenolide, have been demonstrated to exhibit potent action against certain types of cancer (13). SL extracts, known 5284 CHIMPLEE et al: ANTI-BREAST CANCER POTENTIAL OF FRULLANOLIDE as eudesmanolides, derived from frullanolide have been spectral data were compared with previous reports (9,12). The demonstrated to possess anticancer activity in oral, non-small compound was identified as the sesquiterpene lactone, frullano- cell lung and breast cancer cell lines (12). However, no scien- lide (Fig. 1), with the chemical formula C15H20O2 (parent peak tific studies have been conducted concerning frullanolide and at m/z 232, colorless solid). The compound was stored at ‑20˚C its anticancer activities or its cytotoxic mechanisms against prior to testing with cancer cells. breast cancer cells. Therefore, in the present study, the cyto- toxic effects of frullanolide and its mode of action on breast MTT assay. All cell lines were seeded in 96-well plates at a cancer cell inhibition were explored. density of 2x104 cells/well in 100 µl culture medium/well. The cells were treated with different concentrations of the dimethyl Materials and methods sulfoxide (DMSO)-dissolved frullanolide compound (1.25, 2.5, 5.0, 10.0 and 20.0 µg/ml). After 72 h of incubation at 37˚C, Cell cultures. The breast cancer cell lines MCF-7 (HTB-22™), 100 µl MTT reagent (0.5 mg/ml) was added to each well and MDA-MB-468 (HTB-132™) and MDA-MB-231 (HTB-26™), the cultures were incubated for an additional 30 min. The MTT and a normal epithelial breast cell line (MCF-12A; reagent was removed and replaced by DMSO to ensure that CRL-10782™) were purchased from the American Type solubilization was complete. Absorbance at 570 and 650 nm Culture Collection (ATCC, Manassas, VA, USA), and main- (reference wavelengths) was measured on a microplate reader. tained at the Department of Biomedical Sciences, Faculty of Half-maximal inhibitory concentration (IC50) values were Medicine, Prince of Songkla University (Hat Yai, Thailand). calculated from fitted response curves of the concentration The mouse fibroblast L-929 (CCL-1™; ATCC) cell line and viability (%). Determination of cytotoxic activity followed was provided by Professor Teerapol Srichana, Department the criterion; <5 µg/ml represented highly active; 5-10 µg/ml of Pharmaceutical Technology, Faculty of Pharmaceutical represented strongly active; and >10 represented weak cyto- Sciences, Prince of Songkla University. The culture conditions toxicity (15,16). Normal breast cells (MCF-12A) and L-929 of the cell lines followed methods described previously (14). fibroblasts were treated using the aforementioned procedure. Briefly, MCF-7 cells were grown in RPMI-1640 (Gibco; Selectivity index (SI) values, indicating selectivity for tested Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the cell lines, were calculated from the ratio of IC50 values of the MDA-MB-468, MDA-MB-231 and L-929 cells were cultured compounds obtained for normal vs. cancer cells. An SI score in Dulbecco's modified Eagle's medium (DMEM) (Gibco; >3 represented good selectivity (17). This experiment was Thermo Fisher Scientific, Inc). The cultures were supple- performed in triplicate. mented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), with 100 U/ml of penicillin and streptomycin. Flow cytometry for cell cycle analysis and apoptosis detection. The MCF-12A cell line was cultured in DMEM and Ham's The MCF-7 and MDA-MB-468 cells were seeded at densities F12 medium (GE Healthcare, Chicago, IL, USA), supple- of 4x105 cells/well in 12-well plates. The MDA-MB-231 cells mented with 100 ng/ml cholera toxin (Sigma-Aldrich; Merck were seeded at 3x105 cells/well. The cells were treated for KGaA, Darmstadt, Germany), 500 ng/ml hydrocortisone 24 h with three concentrations of frullanolide (0.5x, 1x and (Sigma-Aldrich; Merck KGaA), 0.01 mg/ml bovine insulin 2x IC50). Following removal of the compound, the treated cells (Sigma-Aldrich; Merck KGaA), 20 ng/ml human epidermal were fixed with 70% ethanol at 4˚C for 4 h and washed three growth factor (Invitrogen; Thermo Fisher Scientific, Inc.) and times with cold PBS. Harvesting and fluorochrome binding 5% horse serum (Invitrogen; Thermo Fisher Scientific, Inc.). of the cells were carried out as described previously (2). For All cultures were incubated at 37˚C in a humidified atmo- cell cycle analysis, the fixed cells were resuspended in 400 µl 6 sphere containing 5% CO2. propidium iodide (PI; 50 µg/ml) solution/1x10 cells and incu- bated at RT for 5-10 min in the dark. A total of 5,000 cells Plant material and isolation. Dried G. maderaspatana (L.) were analyzed for each condition with a fluorescence‑activated Poir plants were purchased from the Chaokromper Drug cell sorting (FACS) Calibur flow cytometer (BD Biosciences, Store (Bangkok, Thailand). Their identity was confirmed by San Jose, CA, USA), and histograms of cell population ratios Dr Nijsiri Ruangrungsri, Department of Pharmacognosy and in each phase of the cell cycle were acquired. For apoptosis Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, detection, the apoptotic cells in the treated conditions were Chulalongkorn University (Bangkok, Thailand). A voucher stained with Annexin V‑fluorescein isothiocyanate (FITC)/ specimen (no. 5182) was deposited at the Museum of Natural PI following the manufacturer's protocol (FITC Annexin V Medicine, Chulalongkorn University.