Cytotoxicity of phenolic acid phenethyl esters on oral cancer cells

Cancer Letters 223 (2005) 19–25 www.elsevier.com/locate/canlet

Cytotoxicity of phenolic acid phenethyl esters on oral cancer cells Ya-Ting Leea, Ming-Jaw Donb, Pei-Shih Hungc, Yuh-Chiang Shenb, Yin-Shen Loc, Kuo-Wei Changc, Chieh-Fu Chenb, Li-Kang Hoa,* a

Department of Pharmacology, School of Medicine, National Yang-Ming University, 155, Sec. 2, Li-Nung St. Peitou, Taipei 112, Taiwan, ROC b National Research Institute of Chinese Medicine, Taipei 112, Taiwan, ROC c School of Dentistry, National Yang-Ming University, Taipei 112, Taiwan, ROC

Received 20 August 2004; received in revised form 24 September 2004; accepted 26 September 2004

Abstract Many phenolic acid phenethyl esters possess diverse biological effects including anti-cancer activity. A series of 14 derivatives were synthesized for the evaluation of their cytotoxic effect on oral cancer cells. These derivatives were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and trypan blue dye exclusion assay on the growth of oral squamous cell carcinoma (SAS), oral epidermoid carcinoma-Meng 1 (OEC-M1), and normal human oral fibroblast (NHOF) cells, respectively. Caffeic acid phenethyl esters, 3a (CAPE), and 3b, 3c, and 3d showed cytotoxic effects on the SAS and OEC-M1 cell lines, but not the NHOF cell line at a 5–100 mM dose range. Flow cytometric analysis showed that 3c caused OEC-M1 cell arrest at G2/M phase. Such differential effects on representative cancer and normal cells suggested these compounds might be useful in oral cancer chemotherapy. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Caffeic acid phenethyl ester; Phenolic acid; Oral cancer; Squamous cell carcinoma; SAS; OEC-M1

1. Introduction Phenolic acids such as caffeic acid, ferulic acid, coumaric acid and their analogues are widely distributed in nature and constitute a class of compounds with a broad spectrum of pharmacological properties such as antioxidation, anti-thrombosis, anti-inflammatory, antiviral and inhibition of human * Corresponding author. Tel.: C886 2 28267097; fax: C886 2 28264372. E-mail address: [email protected] (L.-K. Ho).

immunodeficiency virus (HIV) [1–4]. Among them, caffeic acid phenethyl ester (3a, CAPE) an antioxidant from the propolis of honeybee hives has anti-oral and colon cancer activities as well as cyclooxygenase II (COX-2) inhibition effect [5–7]. It is also known that CAPE is cytotoxic to tumor and virally transformed cells but not normal cells [8,9]. Betel quid chewing causes oral cancer and is a problem of epidemic proportions in Taiwan [10–12]. Management-wise currently there is no effective means in combating this socio-medical problem. In our search for anticancer components from Chinese

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.09.048

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herb, we had some interesting observations of phenolic esters and amides [13,14]. Scant literature exists regarding the evaluations of phenolic acid phenethyl esters against oral cancer cells, prompting us to synthesize more phenolic acid phenethyl esters such as ferulic acid, caffeic acid, and isoferulic acid as well as CAPE to evaluate their cytotoxicity against oral cancer cells. Representative cell lines including squamous cell carcinoma (SAS), oral epidermoid carcinoma-Meng 1 (OEC-M1) and normal human oral fibroblast (NHOF) cells were used. Cytotoxicity tests included MTT-colorimetric test [15] and trypan blue dye exclusion assay [16]. The SAS cell line is a human squamous cell carcinoma obtained from the primary lesion of a tongue carcinoma in Japan, which is p53 wild-type with high invasive potential and has higher migration ability [17]. Another oral cancer cell line, OEC-M1 is a human oral epidermoid carcinoma obtained from the primary lesion of an oral carcinoma in Taiwan, which is p53 mutant and resistant to retinoic acid and expresses smaller sized hypophosphorylated Rb proteins compared with normal cells [18]. The objective of this investigation was to study CAPE derivatives on oral cancer cells using cultured cancer cell lines and a normal human buccal mucosa fibroblast cell line and examining their effects on cell growth pattern and their toxicity. Changes in the cell cycle after drug treatment were analyzed by flow cytometry [19].

2. Materials and methods 2.1. Chemicals Caffeic acid, ferulic acid, isoferulic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, benzyl chloride, dimethyl aminopyridine (DMAP), potassium carboxide and methyl iodide were purchased from the Aldrich Chemical Company. 1,3-Dicyclohexylcarbodiimid (DCC), phenethyl alcohol, sodiumsulphate hydrate and 4-hydroxyphenyl alcohol were purchased from the Acros Chemical Company. DMEM, RPMI, 3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT), trypan blue solution, and propidium iodide (PI) were purchased from the Sigma Chemical Company.

2.2. Analytical and spectral equipment Synthesized products were purified on a silical gel column and identified by TLC, NMR, IR and GCMass analysis. Melting points were determined with a Yanaco micromelting point apparatus. Infrared spectra (IR) were obtained on a Nicolet Avatar-320 FTIR spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian INOVA500 spectrometer. CDCl3, CD3OD, and acetone-d6 were used as solvents; chemical shifts are reported in parts per million (d) units relative to internal tetramethylsilane. Mass spectra (MS) were recorded on an EI-MS JEOL JMS-HX 100 mass spectrometer. Thin layer chromatography (TLC) was performed on precoated silical gel F254 plates (Merck) using a 254 nm UV lamp to monitor these reactions. 2.3. Synthesis and identification of the phenolic acid phenethyl esters (3a–3n) The methodology for preparation of phenolic acid phenethyl esters is outlined in Scheme 1 [20]. Compounds 1a–1e were prepared by benzylation with benzyl chloride from hydroxyl and/or methoxy cinnamic acids. Compounds 1f and 1g were prepared from 3-hydroxy- and 4-hydroxy cinnamic acid by methylation with MeI, respectively. Upon treatment of 1a–1g with appropriate phenethyl alcohol (2a, 2b) in the presence of DCC and DMAP gave the corresponding products which were then debenzylated by BCl3 resulted the desired phenolic acid phenethyl esters (3a–3n); the over all yield was about 40–60%. Analytical data for selected compound 3a, white powder; mp 121–123 8C; 1H NMR (CD3OD): d2.93 (2H, t, JZ7.2 Hz), 4.31 (2H, t, JZ7.2 Hz), 4.95 (1H, s), 6.20 (1H, d, JZ15.8 Hz), 6.76 (1H, d, JZ8.4 Hz), 6.89 (1H, dd, JZ8.4, 2.0 Hz), 7.02 (1H, d, JZ2.0 Hz), 7.19 (m), 7.49 (1H, d, JZ15.8 Hz); 13C NMR (CD3OD): d169.2, 149.5, 146.9, 146.7, 139.3, 129.9, 129.4, 127.6, 127.5, 122.9, 116.5, 115.1, 115.0, 66.1, 36.1. Phenethyl (E)-3-[3-hydroxy-4-methoxyphenyl]-2propenoate (3c), product was yellow oil; 1H NMR (CDCl3): d3.0 (2H, t, JZ7.0 Hz), 3.9 (3H, s), 4.4 (2H, t, JZ7.0 Hz), 6.27 (1H, d, JZ16.0 Hz), 6.83 (1H, d, JZ 8.2 Hz), 7.0 (1H, d, JZ2.0 Hz), 7.04 (1H, d, JZ2.0 Hz), 7.12 (1H, d, JZ2.0 Hz), 7.26 (m), 7.57 (1H, d, JZ 16.0 Hz); 13C NMR (CDCl3): d167.1, 148.5, 145.8,

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Scheme 1. Synthesis procedure of phenolic acid phenethyl ester analogues 3a–3n.

144.6, 137.9, 129.0, 128.7, 128.3, 126.5, 123.1, 121.8, 116.1, 113.0, 110.5, 64.3, 60.4, 56.0, 35.1. Phenethyl (E)-3-[3-hydroxyphenyl]-2-propenoate (3d), product was white solid; mp 78–79 8C; 1H NMR (CDCl3): d3.04 (2H, t, JZ7.0 Hz), 4.16 (2H, q, JZ7.0 Hz), 6.41 (2H, t, JZ16.0 Hz), 6.45 (2H, t, JZ 7.0 Hz), 6.91(1H, d, JZ2.5 Hz), 6.93 (1H, d, JZ 2.0 Hz), 7.01 (1H, d, JZ2.0 Hz), 7.08 (1H, d, JZ7.5 Hz), 7.25–7.35 (m), 7.64 (1H, d, JZ16.0 Hz); 13 C NMR (CDCl3): d167.3, 156.5, 145.0, 137.7, 135.7, 130.0, 128.9, 128.5, 126.6, 120.5, 118.0, 117.6, 114.6, 65.2, 35.1. Phenethyl (E)-3-[4-methoxyphenyl]-2-propenoate (3g), product was yellow oil; 1H NMR (CDCl3): d2.98 (2H, t, JZ7.0 Hz), 3.81 (3H, s), 4.09 (2H, q, JZ 7.0 Hz), 4.38 (2H, t, JZ7.0 Hz), 6.27 (1H, d, JZ 16.0 Hz), 6.87 (1H, d, JZ8.0 Hz), 7.21–7.33 (m), 7.46 (1H, d, JZ2.0 Hz), 7.60 (1H, d, JZ16.0 Hz); 13C NMR (CDCl3): d167.2, 161.4, 144.5, 137.9, 129.7, 128.9, 128.5, 128.4, 127.1, 126.5, 115.5, 114.3, 64.8, 55.3, 35.2. 2.4. Cell cultures The SAS cell line was established from a human squamous cell carcinoma of the tongue [21]. Cells were cultured in DMEM medium containing 10% fetal bovine serum and Pen-Strep-Ampho

antibiotics (Penicillin G: 100 units/ml, Streptomycin: 0.1 mg/ml, Amphotericin: 250 ng/ml) in T25 flasks. Cells were incubated at 37 8C in 5% CO2 humidified atmosphere and passaged every 3 days to maintain normal growth (monolayer on the flask). The OEC-M1 is an epidermoid carcinoma cell line derived from human gingiva cells [22]. The cells were grown in RPMI-1640 medium containing 10% fetal bovine serum and Pen-Strep-Ampho antibiotics in T25 flasks. Cells were incubated at 37 8C in 5% CO2 humidified atmosphere and passaged every 3 days to maintain normal growth (monolayer on the flask). Sampling of oral tissues to obtain NHOF cells for cell culture was approved by an institutional review board [23]. After obtaining informed consent, a 0.5 cm3 tissue sample from the gum organization were obtained from a healthy 22 years old male. The tissue was treated with transport medium (Pen-StrepAmpho antibiotics; Penicillin G: 100 units/ml, Streptomycin: 0.1 mg/ml, Amphotericin: 250 ng/ml and Dispase: 2.4 mg/ml in Hank’s Balanced Salt Solution) at 4 8C for 18 h. The epidermis and the connective tissues were then separated. The connective tissues were cultured in DMEM medium containing 10% fetal bovine serum and Pen-Strep-Ampho antibiotics at 37 8C in 5% CO2. After 1 week incubation, the connective tissues were removed and the NHOF cells were attached on the 90 mm dish for further growth.

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2.5. Cell growth-MTT assay

2.8. Statistical analysis

The inhibitory effect of phenolic acid phenethyl esters on the growth of cells were evaluated by a MTT assay. Cells in triplicates (3 wells) were plated onto a series of 96-well culture plates (2!104 cells/100 ml/ well). Following a 24 h incubation at 37 8C, 5% CO2, 100 ml culture medium containing various concentrations of these compounds were added and the cells were further incubated for 24 h. Then the media were removed and, following the addition of culture medium aliquots containing 1 mg/ml MTT substrate, the cells were further incubated at 37 8C for 4 h. The supernatants were then removed and, following the addition of formazan crystals dissolved in 50 ml DMSO, were shaken gently for 10 min. The optical density (OD) at 540 nm (peaks at 503 and 565 nm were determined) with an UV scanning spectrophotometer (Shimadzu UV-260). Dose–response curves were computer plotted after converting the mean data values to percentages of the control response. The 50% inhibitory concentration (IC50) was derived from the dose–response curves.

Concentration dependence was analyzed by simple linear regression analysis of response levels against concentrations of compounds and testing the slope of the regression line against 0 mM by Student’s t test. Values of P!0.05 were considered statistically significant.

2.6. Cytotoxicity-trypan blue dye exclusion It is generally accepted that a viable cell will exclude an acid dye such as trypan blue, and that its uptake is indicative of irreversible membrane damage preceding cell death. The cells were harvested with 0.25% trypsin. Viable cells were determined by trypan blue exclusion and counted on a hemacytometer. 2.7. Flow cytometric analysis of cell cycle Cells were treated with 25 or 50 mM of test compound for 24 h, trypsinized and fixed in 80% ethanol at K20 8C for 30 min. The fixed cells were collected by centrifugation and resuspended in 1.0 ml of 0.1% Triton X-100 at room temperature for 5 min. The permeabilized cells were stained with 40 mg/ml of propidium iodide for 10 min and analyzed immediately by a flow cytometer (FACSCalibur, Becton-Dickinson). Cell cycle analysis was calculated by the ModFit-LTw software (Becton-Dickinson).

3. Results and discussion Taking into considerations of the structure of CAPE, the phenolic acid moiety possesses an a,bunsaturated carbonyl which can be considered as a Michael acceptor, an active moiety often employed in the design of anti-cancer drugs as well as the interesting bioactivity of the CAPE. We prepared 14 phenolic acid phenethyl esters (Scheme 1) in order to evaluate their cytotoxicity against oral cancer cells. Using MTT assay, the cytotoxicity of these compounds on the growth of SAS, OEC-M1, and NHOF cells was monitored, the IC50 values calculated. The results are shown in Table 1. MTT assay is used Table 1 Chemical structures and cytotoxicity of phenolic acid phenethyl ester analogues on the oral cancer cell lines by MTT assay

Compounds

R1

R2

R3

SAS

OEC-M1

3a 3b 3c 3d 3e 3f 3g 3h 3I 3j 3k 3l 3m 3n

OH OMe OH OH H OMe H OH OMe OH OH H OMe H

OH OH OMe H OH H OMe OH OH OMe H OH H OMe

H H H H H H H OH OH OH OH OH OH OH

129.7G4.2 106.3G5.1 189.8G7.6 125.0G6.6 347.3G12.3 O400 O400 282.4G12.1 198.0G5.8 138.3G6.7 296.7G10.2 129.7G8.3 128.1G5.4 O400

159.2G7.2a 87.6G6.0 42.6G4.3 142.5G7.4 O400 O400 205.6G9.6 162.6G8.5 O400 132.6G8.2 O400 160.2G5.6 166.0G7.8 186.4G5.1

a

IC50, data are in mM.

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cytotoxicity than CAPE on both SAS and OEC-M1 cells. Compound 3c exhibited selective cytotoxicity toward OEC-M1 cells about three times higher than that of CAPE, but was less cytotoxic against SAS. These compounds revealed no cytotoxic effect against NHOF cells up to 100 mM (Fig. 3). These results corresponded well to findings of previous studies [24–27] that CAPE and its analogues were not cytotoxic towards normal cells. The cytotoxic activities of compounds (3a–3d) on the SAS and OEC-M1 cell lines were further

Fig. 1. MTT assay of phenolic acid phenethyl ester analogues against oral SAS cells at various concentrations (0, 50, 100, 200 mM) for 24 h, indicating concentration dependency. Each datum represents the meanGSD values from three independent experiments. *P!0.05 by Student’s t test indicating statistically significant differences from their respective controls.

Fig. 3. MTT assay of phenolic acid phenethyl ester analogues against NHOF cells at various concentrations (0, 5, 10, 50, 100, 200 mM) for 24 h, indicating non-toxicity for all compounds up to 100 mM and toxicity for compounds 3a, 3b, 3d, and 3m at high concentrations, 3c remained non-toxic throughout. Each datum represents the meanGSD values from three independent experiments. The value was significantly different (*P!0.05) from the corresponding control values (0 mM), as analyzed by the Student’s t test. Fig. 2. MTT assay of phenolic acid phenethyl ester analogues against OEC-M1 cells at various concentrations (0, 50, 100, 200 mM) for 24 h, indicating concentration dependency. Each datum represents the meanGSD values from three independent experiments. *P!0.05 by Student’s t test indicating statistically significant differences from their respective controls.

extensively to evaluate the dose related cytotoxic activities. CAPE and tested compounds (50–200 mM) showed significant cytotoxicity on SAS and OEC-M1 cells (Figs. 1 and 2). Among them 3b, 3c, and 3j (IC50: 87.6, 42.6, 132.6 mM) exhibited higher cytotoxicity than CAPE (IC50: 159.2 mM) on OEC-M1 cells in a dose-dependent manner. Compound 3b had higher

Fig. 4. Cytotoxicity of selected compounds (3a–3d) on SAS cells at various concentrations (0, 50, 100, 200 mM) for 24 h by trypan blue exclusion. Each datum represents the meanGSD values from three independent experiments. *P!0.05 vs. corresponding controls by Student’s t test.

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Fig. 5. Cytotoxicity of selected compounds (3a–3d) on OEC-M1 cells at various concentrations (0, 50, 100, 200 mM) for 24 h by trypan blue exclusion. Each datum represents the meanGSD values from three independent experiments. *P!0.05 vs. corresponding control by Student’s t test.

no cytotoxicity towards NHOF cells up to 200 mM (Fig. 3). To further study the underlying mechanism, cell cycle analysis was performed on OEC-M1 cells. Treatment with 25 mM of 3c for 24 h arrested cells at G2/M phase (Fig. 6). At a higher concentration (50 mM), 3c induced a sub-G0/G1 peak, an exhibition of genomic DNA degradation into multiple oligonucleosomal fragments, indicated that significant cell apoptosis was induced. In conclusion, a rapid and facile synthetic route for CAPE and its analogues has been achieved. In previous studies, Rao et al. [28,29] have shown that the substitution of dimethoxy on the phenolic acid ring does not impart higher inhibitory activities than 3b on colon carcinogenesis. 3b has a strong growth inhibitory effect against the human colon adenocarcinoma cells than CAPE. Our results also indicated that the substitutions of a methoxy group of the phenolic acid ring affected the cytotoxic effect significantly such as 3b and 3c in OEC-M1 cells, but not in SAS cells. The hydroxy group on phenyl alcohol moiety did not impart any further improvements. The easy accessibility of these compounds should simplify further investigations into its mode of action, and may lead to a better understanding of the underlying mechanisms. Experiments in vivo will be needed to evaluate the efficacy of these compounds in cancer chemotherapy.

Acknowledgements

Fig. 6. Flow cytometric assays of the DNA contents in OEC-M1 cells treated with 25 or 50 mM of 3c for 24 h. Control represents cells without drug treatment. Representative data from three independent experiments are shown.

confirmed by the trypan blue exclusion assay. Treatment with 50–200 mM of these compounds also showed cytotoxicity on SAS and OEC-M1 cells in a dose dependent manner (Figs. 4 and 5). Both MTT and trypan blue dye exclusion assays, despite having different cellular targets, provided similar results. Among the drugs examined, 3c exhibited the most potent cytotoxic effect against OEC-M1 cells with IC50 less than 50 mM (Table 1). In addition, 3c showed

This research was supported by the National Science Council of Republic of China and National Research Institute of Chinese Medicine.

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