Article pubs.acs.org/JAFC
Isophilippinolide A Arrests Cell Cycle Progression and Induces Apoptosis for Anticancer Inhibitory Agents in Human Melanoma Cells Hui-Min David Wang,*,† Chung-Yi Chen,‡ and Pei-Fang Wu† †
Department of Fragrance and Cosmetic Science, Graduate Institute of Natural Products, Kaohsiung Medical University, 100 Shih-Chuan First Road, Kaohsiung City 807, Taiwan, Republic of China ‡ School of Medical and Health Sciences, Fooyin University, 151 Ching-Hsueh Road, Kaohsiung City 83102, Taiwan, Republic of China ABSTRACT: Three new butanolides, isophilippinolide A, philippinolide A, and philippinolide B, and an amide, cinnaretamine, were isolated from the roots of Cinnamomum philippinense to be identified by spectroscopic analysis. Four isolated compounds were screened to examine their radical-scavenging ability, metal-chelating power, and ferric-reducing antioxidant power assay (FRAP). Cinnaretamine showed powerful antioxidative properties in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and a reducing activity; all compounds presented minor inhibition of metal-chelating capacities. The effects of anti-tyrosinase of C. philippinense constituents were determined by the level of the suppression of hydroxylation that turned from L-tyrosine to L-dopa through an in vitro mushroom tyrosinase assay, and all testing samples illustrated slight mushroom tyrosinase inhibitory properties. Isophilippinolide A exhibited inhibitory effectivenesses against the A375.S2 melanoma cell line in a cell viability assay at concentrations ranging from 0 to 200 μM for 24 h. Propidium iodide staining and flow cytometry analyses were applied to assess cell cycle accumulative distribution. It was discovered that isophilippinolide A caused sub-G1 phase accumulation in positive correlation for apoptosis to inhibit cell growth. Further investigation revealed that isophilippinolide A induced A375.S2 cells with an increase of caspase-dependent apoptotic proteins to trigger correlated pathway mechanisms according to Western blotting results. Finally, isophilippinolide A displayed only low cytotoxicities to human normal epidermal cells (melanocytes) and dermal cells (fibroblasts). Altogether, the results implied C. philippinense compounds could be considered functional ingredients in cosmetics, foods, and pharmaceutical products, particularly for their anticancer ability on human skin melanoma cells. KEYWORDS: Cinnamomum philippinense, melanoma, antiproliferation, cell cycle arrest, caspase-dependent apoptosis
■
INTRODUCTION Cinnamomum philippinense (Merr.) Chang (Lauraceae) is a medium-sized tree distributed mainly in the Philippines and in southern Taiwan. The secondary metabolites of this species have been studied by Professor Wu et al. and were isolated.1 To further understand the chemotaxonomy of the genus Cinnamomum and to continue searching for new bioactive agents from Formosan Lauraceous plants, C. philippinense was chosen for the present phytochemical investigation. In this paper, we report the isolation and characterization of four compounds, including three new butanolides, isophilippinolide A, philippinolide A, and philippinolide B, along with one known amide, cinnaretamine. The generation of free radicals has been linked to many chronic diseases. It is well-known that antioxidants feature great capability in the prevention and treatment of many human diseases, including cancer, neuropathy, inflammation, and agerelated conditions.2 Various natural phytochemicals are attributed medical properties to prevent various diseases and promote human health. Antioxidant properties are directly related to redox abilities that are essential to the absorption and neutralization of free radicals, ligands for singlet and triplet oxygen, and decomposition of peroxides. These reactions depend on the supply of hydrogen, which combines with radicals to produce stable complex molecules, thus ending © 2013 American Chemical Society
radical chain reactions. For this reason, healthy foods and natural cosmetics are recommended for reducing oxidative stress and preventing disease.3 Herbal medicines, fruits, vegetables, legumes, oats, grains, and cereals with antioxidative properties are able to scavenge free radicals or terminate excess oxidative biochemical reactions in the human body.4 As a result, natural antioxidants used as preservatives for cosmetics or food contain exceptional possibilities for enhancing product quality and consumer satisfaction. Skin problems, including acne scars, lentigos, melasmas, and senile freckles, can cause great concern to both women and men all over the world.5 Hyperpigmentation treatments usually employ therapeutic medicines or medicinal cosmetics with agents that can reduce skin pigmentation or whiten the skin. Tyrosinase belongs to oxidases and is recognized as the first of two rate-limiting enzymes in melanin synthesis. It catalyzes two distinctive reactions in the production of melanin pigments: the hydroxylation of L-tyrosine to 3,4-dihydroxy-L-phenylalanine (LDOPA) and the oxidation of L-DOPA to dopaquinone, which is a chemical precursor to melanin. In molecular biology, it is Received: Revised: Accepted: Published: 1057
August 23, 2013 December 16, 2013 December 20, 2013 December 20, 2013 dx.doi.org/10.1021/jf403730z | J. Agric. Food Chem. 2014, 62, 1057−1065
Journal of Agricultural and Food Chemistry
Article
and visualized with 50% H2SO4. Flow cytometry analysis was done by a Coulter Epics XL-MCL (Beckman Coulter, CA, USA) flow cytometer. Labeling dyes, such as PI, were used to investigate the events involved in apoptosis. Extraction and Isolation. The roots (3.7 kg) of C. philippinense were air-dried and extracted repeatedly with MeOH (5 L × 6) at room temperature. The combined MeOH extracts (54.3 g) were obtained by concentration under reduced pressure and partitioned to yield CHCl3 (34.4 g) and H2O (8.8 g) extracts. The concentrated CHCl3 extract (34.4 g) was chromatographed over silica gel using n-hexane−EtOAc− MeOH as eluent to produce six fractions. Fraction 1 (6.33 g) was separated by silica gel CC with increasingly polar mixtures of nhexane−EtOAc to give 10 fractions (1-1 to 1-10). Fraction 1-1 (2.15 g) was resubjected to silica gel CC, eluting with n-hexane−EtOAc (60:1) and enriched gradually with EtOAc to obtain five fractions (11-1 to 1-1-5). Fraction 1-1-2 (0.85 g) was further purified by another silica gel CC using n-hexane−EtOAc to obtain philippinolide B (16 mg). Fraction 1−-2 (1.85 g) was further separated by silica gel CC (nhexane−EtOAc (60:1)) and preparative TLC (n-hexane−EtOAc (100:1)), giving philippinolide A (24 mg) and isophilippinolide A (37 mg). Fraction 2 (4.93 g) was separated by silica gel CC with increasingly polar mixtures of n-hexane−EtOAc to give five fractions (2-1 to 2-5). Fraction 2-1 (2.52 g) was resubjected to silica gel CC, eluted with CHCl3−MeOH (100:1), and gradually enriched with MeOH to obtain 10 fractions (2-1-1 to 2-1-10). Fraction 2-1-1 (0.11 g) was resubjected to silica gel CC and purified by preparative TLC to yield cinnaretamine (5 mg). Three new compound structures (see Figure 1) were identified as the following.
necessary for tyrosinase to contain two copper ions in the active site, catalyzing colorful pigments or melanin by the above oxidations. One of the major protocols for treating these pigmentation-related dermatological disorders is to inhibit tyrosinase activity. Human skin is under daily oxidative stress from both external and internal sources, such as free radicals, reactive oxygen species, or ultraviolet (UV) radiation.6 It has been well reported that skin exposure to oxidative stress or UV radiation is the cause for skin aging or tumorigenesis. At present, the main lethal reason of ineffective treatment is tumor proliferation, which may lead to death. Within the past several decades, these are etiological factors responsible for producing melanoma, which is a form of malignant tumor of uncontrollable melanocytes and is among the most fatal of skin cancers.7 Melanoma is one of the most aggressive cancers, resistant to medical treatments such as surgery, radiotherapy, or chemotherapy.8 Treatments these days for melanoma are not effective enough to reduce mortalities and enhance survival rate of patients with melanoma because of a lack of therapeutic remedy. For this reason, to improve the efficacy of cancer treatment, it is essential to develop novel natural medicinal compounds for anticancer therapies. The purpose of biological aspects of this study was therefore to elucidate the antioxidative, tyrosinase-inhibitory, and anticancer properties of C. philippinense compounds. We further analyzed the cytotoxicity of isophilippinolide A in a human skin melanoma cancer cell line, A375.S2, to discuss the apoptotic genetic and molecular mechanisms.
■
MATERIALS AND METHODS
Plant Material. The roots of C. philippinense were collected from Taipei County, in May 2008. A voucher specimen was identified by Dr. Yen-Ray Hsui of the Chungpu Research Center, Taiwan Forestry Research Institute, and deposited in the Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan. Reagents. 2,2′-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), butylated hydroxyanisole (BHA), 2,2-diphenyl-1-(2,4,6trinitrophenyl)hydrazyl (DPPH), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), ferrozine, FeCl3, FeCl2·4H2O, kojic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), mushroom tyrosinase, vitamin C, peroxidase type I from horseradish (HRPase), potassium ferricyanide [K3Fe(CN)6], propidium iodide (PI), trichloroacetic acid, and L-tyrosine were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), minimum essential medium (MEM), and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from GIBCO BRL (Gaithersburg, MD, USA). An Annexin V/Dead Cell Apoptosis Kit was purchased from Invitrogen (Carlsbad, CA, USA). All buffers and other reagents were of the highest purity commercially available. Instruments. Melting points were determined using a Yanagimoto micromelting point apparatus and are uncorrected. Optical rotations were measured with a JASCO DIP-370 digital polarimeter. UV spectra were obtained on a JASCO V-530 spectrophotometer in MeCN. IR spectra were measured on a Hitachi 260-30 spectrophotometer (Hitachi, Tokyo, Japan). 1H (400 MHz, using CDCl3 as solvent for measurement), 13C (100 MHz), DEPT, HETCOR, COSY, NOESY, and HMBC spectra were obtained on a Varian (Unity Plus) NMR spectrometer. Low-resolution FABMS and ESIMS spectra were obtained on an API 3000 (Applied Biosystems) and high-resolution FABMS and ESIMS spectra on a Bruker Daltonics APEX II 30e spectrometer (Bruker, Bremen, Germany). Silica gel 60 (Merck, 70− 230 mesh, 230−400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254), 0.20 and 0.50 mm, were used for analytical TLC and preparative TLC, respectively,
Figure 1. Chemical structures of four compounds from C. philippinense. Isophilippinolide A: pale yellowish liquid; [α]25D −24.6 (c 0.05, CHCl3); UV λmax (MeCN) (log ε) 225 (4.12) nm; IR (neat) νmax 3450 (br, OH), 1770, 1670 (α,β-unsaturated γ-lactone), 1465, 1360, 1090 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.88 (3H, t, J = 6.8 Hz, H18′), 1.25 (28H, br s, H-4′−17′), 1.52 (2H, m, H-3′), 2.45 (2H, m, H2′), 4.72 (1H, dd, J = 2.8, 1.2 Hz, H-6a), 4.94, (1H, dd, J = 2.8, 1.2 Hz, H-6b), 5.24 (1H, br s, H-4), 7.07 (1H, td, J = 7.6, 2.4 Hz, H-1′); 13C NMR (100 MHz, CDCl3) δ 14.0 (C-18′), 22.8 (C-17′), 28.2−29.7 (C-2′−15′), 32.0 (C-16′), 66.6 (C-4), 91.5 (C-6), 127.2 (C-3), 150.2 (C-1′), 157.7 (C-5), 165.5 (C-2); FABMS m/z 365 [M + H]+, 211 (15), 167, 149 (16), 139 (14), 125 (33), 111 (100), 97 (70), 83 (81), 69 (90); HRFABMS m/z 365.3055 [M + H]+ (calcd for C23H41O3, 365.3056). Philippinolide A: pale yellowish liquid; [α]25D −22.3 (c 0.05, CHCl3); UV λmax (MeCN) (log ε) 225 (4.11) nm; IR (neat) νmax 3450 (br, OH), 1770, 1670 (α,β-unsaturated γ-lactone), 1465, 1365, 1090 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.88 (3H, t, J = 7.2 Hz, H18′), 1.25 (28H, br s, H-4′−17′), 1.48 (2H, m, H-3′), 2.77 (2H, m, H2′), 4.67 (1H, dd, J = 2.8, 1.6 Hz, H-6a), 4.89, (1H, dd, J = 2.8, 1.6 Hz, H-6b), 5.11 (1H, br s, H-4), 6.68 (1H, td, J = 7.6, 2.0 Hz, H-1′); 13C NMR (100 MHz, CDCl3) δ 14.0 (C-18′), 22.6 (C-17′), 28.3−29.8 1058
dx.doi.org/10.1021/jf403730z | J. Agric. Food Chem. 2014, 62, 1057−1065
Journal of Agricultural and Food Chemistry
Article
(C-2′−15′), 31.9 (C-16′), 68.8 (C-4), 90.4 (C-6), 126.7 (C-3), 151.3 (C-1′), 157.5 (C-5), 165.5 (C-2); FABMS m/z 365 [M + H]+ (5), 211 (21), 149 (11), 139 (13), 125 (46), 111 (100), 97 (75), 83 (80), 69 (92); HRFABMS m/z 365.3053 [M + H]+ (calcd for C23H41O3, 365.3056). Philippinolide B: colorless oil; [α]25D −21.8 (c 0.05, CHCl3); UV λmax (MeCN) (log ε) 265 (4.05) nm; IR (neat) νmax 3500 (br, OH), 1780, 1680 (α,β-unsaturated γ-lactone), 1290 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.87 (3H, t, J = 6.8 Hz, H-17′), 1.25 (26H, br s, H4′−16′), 1.60−1.77 (4H, m, H-2′, 3′), 3.34 (3H, s, OMe-1′), 4.11 (1H, dd, J = 7.4, 4.8 Hz, H-1′), 4.87, 5.19 (each 1H, d, J = 2.4 Hz, H6a, b), 7.21 (1H, br s, H-4); 13C NMR (100 MHz, CDCl3) δ 14.2 (C17′), 22.6 (C-16′), 25.5 (C-3′), 29.1−30.3 (C-4′−14′), 31.6 (C-15′), 35.1 (C-2′), 57.5 (OMe-1′), 77.4 (C-1′), 98.0 (C-6), 137.7 (C-4), 137.7 (C-3), 155.0 (C-5), 169.8 (C-2); HRESIMS m/z 387.2877 [M + Na]+ (calcd for C23H40O3Na, 387.2875). DPPH• Radical Scavenging Capacity Assay. The antioxidant activity of the tested compounds was determined as their ability to donate hydrogen or remove radicals by utilizing the stable DPPH• method.9 Briefly, various concentrations of C. philippinense compounds were added to 0.1 mL of aqueous stable DPPH• (60 μM) solution. When DPPH• reacts with an antioxidant compound that can donate hydrogen, it is reduced, resulting in a decrease in the absorbance at 517 nm. The analyzed time interval was 10 min per point, totaling up to 30 min, using a UV−vis spectrophotometer (BioTek Co., Winooski, VT, USA). Vitamin C acted as a positive control. According to the preliminary investigations, the antioxidative results of using four compounds failed to reach high inhibitions toward the values of IC50. We presented the inhibitory data at suitable concentrations in all of the antioxidative assays. The DPPH• radical scavenging activity (%) was determined as scavenging activity (%) =
(Acontrol − A sample) Acontrol
× 100%
tyrosinase inhibition activity (%) =
[(A − B) − (C − D)] × 100% (A − B)
(2)
In this equation, A is the optical density (OD475) without the tested substance, B is the OD475 without the tested substance, but with tyrosinase, C is the OD475 with the tested substance, and D is the OD475 with the tested substance, but without tyrosinase. Cell Cultures. The A375.S2 (Bioresource Collection and Research Center, BCRC no. 60039, ROC) human melanoma cell line was maintained in monolayer culture within MEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 μg/mL amphotericin B. Neonatal foreskin primary human epidermal melanocytes were bought from Cascade Biologics and cultured in Medium 254 (Cascade Biologics, Life Technologies, Portland, OR, USA) supplemented with human melanocyte growth supplement. Human skin fibroblasts were grown from foreskin primary culture that was obtained from Chung-Ho Memorial Hospital, Kaohsiung Medical University. All of the cell types were incubated at 37 °C under a humidified atmosphere of 5% CO2 in air and routine passage by trypsinization.11,12 Cell Cytotoxicity Assays. The effects of compounds on cell growth were evaluated using a MTT assay.13 In short, cells were seeded in 96-well plates at a density of 7 × 103 cells/well. The medium was changed, and cells were maintained either in medium by themselves (control cells) or in the presence of the indicated drug concentrations in a final volume of 100 μL in 10% FBS culture medium. The testing samples were dissolved in sterile DMSO to create a working concentration of 1, 10, 25, 50, 100, or 200 μM. After 24 h of incubation, the medium was substituted with 100 μL of fresh medium including 0.5 mg/mL MTT. The plate was then cultured in a 37 °C incubator filled with 5% CO2 for 2 h. Each precipitate in a specific well was treated with 100 μL of DMSO to dissolve the purple formazan crystals. After the dishes were gently shaken for 10 min in the dark to ensure maximal dissolution of the formazan crystals, the absorbance (A) values of the supernatant were measured at 595 nm. Cell viability was calculated as
(1)
Metal-Chelating Activity. The ferrous ion chelating potentials of all compounds were probed according to the method described previously.10 In short, various tested concentrations of samples dissolved in DMSO were added to a solution of 2 mM FeCl2·4H2O (0.01 mL). Ferrozine (5 mM; 0.02 mL) was added to the reaction, and the mixture was strongly shaken and left standing at room temperature for 10 min. The absorbance of the mixture was read at 562 nm against a blank. EDTA was used as a positive control, and the formula for calculation of the chelating activity was similar to eq 1. Reducing Power Assay. The reducing power of the four compounds was measured on the basis of the previous method.10 Briefly, different concentrations of each test sample were mixed with 0.085 mL of 67 mM phosphate buffer (pH 6.8) and 2.5 μL of 20% potassium ferricyanide (K3Fe(CN)6). The mixture was incubated at 50 °C for 20 min, and 0.16 mL of trichloroacetic acid (10%) was then added to the mixture, which was then centrifuged for 10 min at 3000g. The upper layer of the solution (75 μL) was mixed with 2% FeCl3 (25 μL), and the absorbance was measured at 700 nm. BHA was used as a positive control. In this assay, a higher absorbance demonstrated a higher reductive capability. Inhibition of Mushroom Tyrosinase Activity. Tyrosinase activity inhibition was determined spectrophotometrically according to the previously described method, with minor modifications.10 Assays were carried out with an optics plate reader used to determine the absorbance at 475 nm. Kojic acid was used as a positive control for the tyrosinase inhibitory assay. The substances tested were dissolved in aqueous DMSO and incubated with L-tyrosine (2.5 mg/mL) in a 50 mM phosphate buffer (pH 6.8). All samples were dissolved in DMSO, because DMSO did not affect tyrosinase activity when
