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Antiproliferative effects of curcuphenol, a sesquiterpene phenol
Fitoterapia 81 (2010) 762–766
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Antiproliferative effects of curcuphenol, a sesquiterpene phenol Gloria Rodrigo a,c,d, Giovanna R. Almanza b, Yajun Cheng c, Jiangnan Peng e, Mark Hamann e, Rui-Dong Duan c, Björn Åkesson d,⁎ a b c d e
Molecular Biology and Biotechnology Institute, University Major of San Andres, La Paz, Bolivia Chemistry Research Institute, University Major of San Andres, La Paz, Bolivia Gastroenterology Laboratory, Department of Clinical Sciences, Lund University, Lund, Sweden Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, Lund, Sweden Department of Pharmacognosy, The University of Mississippi, Oxford, USA
a r t i c l e
i n f o
Article history: Received 14 October 2009 Accepted in revised form 30 March 2010 Available online 10 April 2010 Keywords: Curcuphenol Cell proliferation Apoptosis Caspase
a b s t r a c t Curcuphenol is a sesquiterpene isolated from sponges and plants having several significant biological activities. The present study explored its effect on cell proliferation and apoptosis in Caco-2 human colon cancer cells. It was demonstrated that curcuphenol in concentrations in the range of 29–116 µg/ml inhibited cell proliferation and DNA replication and induced cell death in a dose-dependent manner. The induction of apoptosis was associated with a stimulation of the activity of caspase-3. The findings presented here suggest that curcuphenol has antiproliferative and pro-apoptotic properties. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Herbal preparations have long been used as remedies for infectious and other diseases and they are used in primary health care in several countries [1]. Chemical and pharmacological investigations have indicated that sesquiterpene phenols are important bioactive components in several plants [2]. Curcuphenol (5-methyl-2-[(2S)-6-methylhept-5-en-2yl]phenol) was isolated and identified from several sources such as the sponges Didiscus flavus [3,4], Arenochalina [5]), Myrmekioderma [6], Pseudopterogorgia species [7] and Epipolasis species [8], and plants including Baccharis penningtonii Heering [9], Valeriana wallichii DC. [10], Ruta graveolens [11], Protium heptaphyllum (Aubl.) Marchand [12], Curcuma longa L. [13] and Achillea sudetica (Asteraceae) [14]. Curcuphenol was reported to have antimicrobial activity against Staphylococcus aureus and Cryptococcus neoformans [3], antifungal activity against Candida albicans and antima-
⁎ Corresponding author. Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, POBox 124, SE-221 00 Lund, Sweden. Tel.: +46 46 222 45 23; fax: +46 46 222 46 11. E-mail address: [email protected] (B. Åkesson). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.04.001
larial activity against Plasmodium falciparium [8] and antileishmania activity [15]. (S)-(+)-Curcuphenol has also been shown to inhibit proton–potassium ATPase [3] and to activate GPR40, a cell surface receptor for free fatty acids [16]. Curcuphenol works synergistically with cyanthiwigin B as an antibiotic and both are found in good yields in Myrmekioderma [17]. A preliminary indication of antitumor activity of curcuphenol has been reported for murine leukaemia cells (P-388) and several human cancer cell lines, lung (A-549), colon (HCT-8) and mammary (MDAMB) cells [4] and it showed cytotoxic activity in a manner independent of a p53 mechanism [18]. To get further information about its antitumour properties, we investigated the effects of curcuphenol on cell proliferation and apoptosis in this report. 2. Experimental 2.1. General Dulbecco's Modified Eagle's Medium (DMEM) and fetal calf serum (FCS) were purchased from Gibco, and benzamidine, phenylmethylsulphonyl-fluoride (PMSF), taurocholate, dithiothreitol (DTT) were purchased from Sigma-Aldrich.
G. Rodrigo et al. / Fitoterapia 81 (2010) 762–766
2.2. Sources of curcuphenol Curcuphenol was isolated from a Bolivian plant Baccharis genistelloides [16] and from the marine sponge Myrmekioderma styx [6]. The structure was assigned by 1D and 2D NMR (Bruker DRX 400) and comparison with literature values. Samples isolated from the two sources were shown to be the same by identical proton and carbon NMR data (Table 1). Curcuphenol was dissolved in DMSO to a final concentration of 116 mg/ml. Serial dilutions to final concentrations ranging from 7 to 116 µg/ml (33–531 µmol/l) were prepared in cell culture medium. 2.3. Cell culture Caco-2 cells were cultured in DMEM with L-glutamine, containing 100 IU/mL penicillin, 10 µg/ml streptomycin and 10% (v/v) heat-inactivated FCS. They were maintained in 25 cm2 culture flasks at 37 °C in a humidified incubator containing 95% air and 5% CO2. For assays of cell proliferation, DNA replication and apoptosis, the cells were detached with 0.05% trypsin/0.02% EDTA, resuspended to a concentration of 1 × 105/ml and seeded into 96-well plates (Falcon). For the caspase-3 assay, the cells were seeded at 0.6 × 106 cells/ml in 25 cm2 culture flasks for 72 h. They were then treated by the
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different concentrations of curcuphenol for 6 h, and then detached with a rubber policeman and harvested by centrifugation.
2.4. Cell proliferation assay The cell proliferation rate was assayed by use of the reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3benzene disulfonate (WST-1) (Roche). It is metabolized by mitochondrial dehydrogenases to a formazan, a dye that stains dark red. The formation of the formazan is proportional to the total mitochondrial dehydrogenase activity in the sample, which in turn correlates with the total number of viable cells. Briefly, the Caco-2 cells, 2 × 104 in 200 µl medium were incubated with curcuphenol at concentrations from 7 to 116 µg/ml in a 96-well microplate for 24 h, and 20 μl WST-1 reagent was then added in each well. After incubation for 1 h at 37 °C, the plate was shaken thoroughly for 1 min and the optical density (OD) was read at 405 nm using 655 nm as background. The cell proliferation rate was expressed as a percentage of the control. All determinations were carried out in sixtuplicate wells in three different experiments.
2.5. DNA replication Table 1 13 C NMR data of curcuphenol measured in DMSO at 100 MHz. Data are shown for curcuphenol isolated from A, Myrmekioderma styx, B, Baccharis genistelloides and the (+)-curcuphenol structure is shown below. No.
A
B
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15
17.9 130.9 125.1 37.0 26.2 31.4 130.3 154.9 116.0 135.7 120.1 126.9 21.1 21.4 26.0
17.9 130.9 125.1 37.0 26.2 31.4 130.3 154.9 116.0 135.7 120.1 126.9 21.1 21.4 26.0
DNA synthesis was measured by the amount of [3H] thymidine incorporation. Caco-2 cells were seeded in 96-well plates and preincubated for 20 h with different concentrations of curcuphenol. Then [3H]thymidine (0.5 µCi/well; American Radiolabeled Chemicals, St. Louis, MO, USA) was added to the cells followed by incubation for 4 h. Cells were washed with ice cold PBS, then treated with 5% trichloroacetic acid for 20 min at 4 °C, followed by one wash with ice cold PBS. Finally, the cells were lysed in 0.5 M NaOH and 0.1% SDS for 15 min at room temperature and 100 µL was collected into scintillation vials and counted. DNA synthesis was expressed as percentage of control. Triplicate analyses in two different experiments were performed.
2.6. Assay of apoptosis Apoptosis was assayed by a Cell Death Detection ELISAPLUS kit (Roche). Briefly, 1 × 104 cells were seeded in 96-well plates in 200 µl DMEM with L-glutamine, 100 IU/mL penicillin, 10 µg/mL streptomycin and 10% (v/v) heat-inactivated FCS. After treatment by curcuphenol for 6 h, the cells were lysed and the nuclei were separated from the cytoplasmatic fraction by centrifugation (10 min, 200 g). A sample from the supernatant was transferred to a streptavidin-coated microtiter plate provided by the kit. The relative enrichment of cytoplasmic histone-associated DNA fragments to the streptavidin-coated plate was measured. After removal of unbound antibodies by washing, the amount of nucleosomes was determined photometrically at 415 nm with 490 nm as background. The enrichment factor was calculated as absorbance of the sample (dying and dead cells)/absorbance of the corresponding control (cells without treatment). Triplicate analyses in three different experiments were performed.
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Fig. 1. Antiproliferative effect of curcuphenol on colon cancer cells. Cells were treated with different concentrations of curcuphenol for 24 h, and then the cell viability was determined by WST-1 assay. The growth inhibitory activity was calculated as % of inhibition compared with the control.
2.7. Caspase-3 activity
Fig. 3. Effect on apoptosis of different concentrations of curcuphenol on Caco-2 cells after exposure for 6 h. The values represent means of three separate experiments with standard errors depicted by vertical bars, each was done using triplicate samples.
3. Results Caspase-3 activity was measured by proteolytic cleavage of the fluorogenic substrate Ac-DEVD-pNA (VWR International AB, Stockholm, Sweden) using a microplate reader [19]. Caco-2 cells (1 × 105 cells/well) were treated with curcuphenol at concentrations of 29, 58 and 116 µg/ml. After incubation for 6 h, the cells were harvested and then washed with cold PBS. The pellets were lysed using 100 µl of lysis buffer (50 mM Tris, 2 mM EDTA, pH 7.4, taurocholate 6 mM, PMSF 1 mM, DTT 100 mM, leupeptin 10 mg/ml, aprotinin 10 mg/ml) on ice for 15 min and then 100 µl of assay buffer (50 mM Hepes, 0.1 M NaCl, 0.1% Chaps, 10 mM DTT, 0.1 mM EDTA, 10% glycerol, pH 7.4) and 10 µl of substrate solution (2 mM substrate in assay buffer) were added. After incubation at 37 °C for 30 and 60 min, the absorbance was measured at 405 nm. Triplicate analyses in three different experiments were performed.
3.1. Effect of curcuphenol on the growth of colon cancer cells When Caco-2 cells were treated with different concentrations (7–116 µg/ml) of curcuphenol, an inhibition of cell proliferation was observed (Fig. 1). The inhibition of cell growth was 32% at 116 µg/ml, 33% at 58 µg/ml, 29% at 29 µg/ml and 9% at 14 µg/ml. The antiproliferative effect of curcuphenol was further confirmed by measuring DNA replication. Curcuphenol was added at concentrations of 29, 58 and 116 µg/ml and it reduced [3H]thymidine incorporation into Caco-2 cells in a dose-dependent manner, with an IC50 of 43 µg/ml (Fig. 2). 3.2. Effects of curcuphenol on apoptosis
The results are presented as mean (SE). The statistical significance of differences between groups was determined by the Student t-test and P b 0.05 was considered to be statistically significant.
Proliferation and apoptosis are two important mechanisms related to the fate of the cells and the effect of curcuphenol on apoptosis was therefore further examined. After treatment of Caco-2 cells with curcuphenol (14–116 µg/ml) for 6 h the rates of apoptosis were significantly increased at all concentrations of curcuphenol (Fig. 3). It increased significantly from 14 to
Fig. 2. Effect on DNA synthesis after exposure to 29–116 µg/ml of curcuphenol on Caco-2 cells. The values are expressed as % of control and represent means (S.E.) of two separate experiments performed with sextuplicate samples. The absolute thymidine incorporation into DNA of untreated Caco-2 cells was 0.4 fmol/h/106 cells.
Fig. 4. Increase of caspase-3 activity induced by curcuphenol in Caco-2 cells. The values represent means (S.E.) of three independent experiments at 6 h of exposure to curcuphenol.
2.8. Statistical analysis
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58 µg/ml and then reached a plateau. The stimulation of apoptosis was 2.6-, 3.3- and 3.0-fold at 29, 58 and 116 µg/ml, respectively, as compared to untreated cells. After treatment of Caco-2 cells with curcuphenol (14–58 µg/ml) for 6 h, it also increased the caspase-3 activity dose-dependently (Fig. 4) at 30 min of incubation. A 6.6- and 8-fold increase was obtained as compared to untreated cells at 29 and 58 µg/ml, respectively. No increase in the activity was demonstrated by curcuphenol at 14 µg/ml. Thus, the results suggest that the curcuphenol killed Caco-2 cells through an apoptosis mechanism via the activation of caspase-3.
4. Discussion 4.1. Curcuphenol and cell proliferation Curcuphenol isolated from sponges has been reported to have antifungal activity against Candida albicans and antimalarial activity against Plasmodium falciparium [8]. There is also preliminary evidence of antitumor activity against human cancer cell lines [4] and moderate activity against HCT-116 cells [18]. Since curcuphenol has moderate antiradical activity [20] and strong antioxidant activity [21], it may also have actions related to antioxidants which may suppress carcinogenesis during the initiation phase, and affect the activity of xenobiotic metabolizing enzymes [22]. In this paper it was observed that curcuphenol isolated from two sources inhibited cell proliferation and DNA synthesis in Caco-2 cells, suggesting that curcuphenol may inhibit the progress of the cell cycle, resulting in inhibition of cell growth. The inhibition seemed to be larger on DNA synthesis than on cell proliferation and we have no obvious explanation for this discrepancy. Regarding the effects of other natural sources of related compounds, the oil of Photinia serrulata was shown to have strong cytotoxic activity against cancer cell lines, and it contains high amounts of sesquiterpenes, including α-bisabolol, γ-curcumene, ar-curcumene, β-bisabolene, β-curcumene and (E)-γ-bisabolene [23,24]. Turmeric extracts also contain compounds similar in structure to curcuphenol (ar-curcumene, β-bisabolene, α- and γ-atlantone, ar-turmerol) and showed strong anticancer activity in cell lines, e.g. HT-29 colon cancer cells [25], and at least in part these compounds were responsible for the cytotoxic activity. Piochon et al. [26] found that α-bisabolol, a similar compound to curcuphenol, showed cytotoxic activity against U-87 glioma cells with an IC50 of 130 µM (29 µg/ml) which is similar to the results found in our work. Moreover, this compound showed weak activity against human lung carcinoma and ovary teratocarcinoma cells [26], and affected the cell viability in transformed cells [27]. This drop in cell viability was mainly due to cell death due to apoptosis [27]. Finally, curcuphenol showed moderate cytotoxic activity against HCT-116 colon cancer cells [18] between 27 and 35 µg/ml, which is similar to the results of our work. Although the effects are consistent it must be noted that the lowest effective concentration (29 µg/ml = 133 µmol/l) is rather high. Further work is necessary to possibly find more active analogues of curcuphenol and to study the mechanisms of cell uptake of these compounds. To some extent this research is hampered by the limited availability of the compounds in question in pure form.
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4.2. Effects of curcuphenol on apoptosis The killing of tumor cells may be mediated through induction of apoptosis in target cells [27,28]. Our data strongly suggest that curcuphenol exerts some of its action through activation of apoptosis, similar to previous findings with αbisabolol [27,29,30]. The conclusion of our study is supported by data from both oligonucleosome determination and caspase-3 determination. Further detailing of the mechanisms involved will require additional studies. Also other compounds having a sesquiterpene structure may cause both cell cycle arrest and apoptosis. Like curcuphenol, ursolic acid can induce G1 arrest by the inhibition of DNA replication [31], and in addition ursolic acid induces apoptosis via inhibition of the NF-ΚB induced bcl-2 mediated anti-apoptotic pathway and subsequent activation of the p53mediated and TNF-α-induced caspase-3 mediated pro-apoptotic pathways [32]. α-bisabolol, a closely similar compound to curcuphenol exhibited strong apoptotic activity probably by the intrinsic and extrinsic pathways [27,29]. 4.3. Effects of curcuphenol on caspase activity Curcuphenol may promote collapse of the mitochondrial membrane potential, release of cytochrome C, and activation of caspase-3 (Fig. 4). Some germacranolide sesquiterpene lactones also induced DNA fragmentation dose-dependently [33]. Biochemical characterization of the apoptotic DNA damage revealed that the sesquiterpene lactones promoted the internucleosomal degradation of DNA, resulting in the formation and eventual release of oligonucleosomal DNA fragments [33]. Also other sesquiterpenes than curcuphenol have been shown to affect caspase activity. Like curcuphenol, diacetyl taridin A acted after a short time [33]. The trigger for curcuphenol-induced apoptosis remains to be determined. 4.4. Concluding remarks Curcuphenol exerts antiproliferative and pro-apoptotic effects in the Caco-2 colon cells. It also induces biochemical changes that are characteristic of apoptosis and we can conclude that curcuphenol induced caspase-dependent cell death. A survey of the literature revealed that no studies on the cytotoxic activity of curcuphenol had previously been undertaken, although several related sesquiterpenes have been found to have antiproliferative and other activities. Apoptosis plays a vital role in controlling cell number in many developmental and physiological stages, tissue homeostasis, and regulation of immune system, while inadequate apoptosis is an integral part of cancer development [34]. Agents, which suppress or inhibit the development of malignant cells by inducing apoptosis, may represent a useful mechanistic approach to both chemoprevention and chemotherapy of cancer. Acknowledgements This research was supported by the Swedish International Development Agency (Sida) for a project on Plant biodiversity as a collaboration between University Major of San Andres, La Paz, Bolivia and Lund University, Sweden. Lund University is a
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member of the EU NoE ECNIS, which partly supported the study. This work was also supported by NIH (1R01A136596) and CDC (U01/CI00211-02). The Natural Resource Conservation Authority, Jamaica and Discovery Bay Marine Laboratory are gratefully acknowledged for assistance with sample collections. References [1] Sokmen A, Jones BM, Erturk M. The in vitro antibacterial activity of Turkish medicinal plants. J Ethnopharmacol 1999;67:79–86. [2] Ciavatta ML, Lopez Gresa MP, Gavagnin M, Romero V, Melck D, Manzo E, et al. Studies on puupehenone-metabolites of a Dysidea sp.: structure and biological activity. Tetrahedron 2007;63:1380–4. [3] El Sayed KA, Yousaf M, Hamann MT, Avery MA, Kelly M, Wipf P. Microbial and chemical transformation studies of the bioactive marine sesquiterpenes (S)-(+)-curcuphenol and curcudiol isolated from a deep reef collection of the Jamaican sponge Didiscus oxeata. J Nat Prod 2002;65:1547–53. [4] Wright AE, Pomponi SA, McConnell OJ, Kohmoto S, McCarthy PJ. (+)Curcuphenol and (+)-curcudiol, sesquiterpene phenols from shallow and deep water collections of the marine sponge Didiscus flavus. J Nat Prod 1987;50:976–8. [5] Butler MS, Capon RJ, Nadeson R, Beveridge AA. Aromatic bisabolenes from an Australian marine sponge, Arenochalina sp. J Nat Prod 1991;54: 619–23. [6] Peng J, Franzblau SG, Zhang F, Hamann MT. Novel sesquiterpenes and a lactone from the Jamaican sponge Myrmekioderma styx. Tetrahedron Lett 2002;43:9699–702. [7] Harvell CD, Fenical W, Greene C. Chemical and structural defenses of Caribbean gorgonians (Pseudopterogorgia spp.). I. Development of an in situ feeding assay. Mar Ecol Prog Ser 1988;49:287–94. [8] Fusetani N, Sugano M, Matsunaga S, Hashimoto K. (+)-Curcuphenol and dehydrocurcuphenol, novel sesquiterpenes which inhibit H, KATPase, from a marine sponge Epipolasis sp. Experientia 1987;43: 1234–5. [9] Retta D, Gattuso M, Gattuso S, Di Leo NP, van Baren C, Bandoni A. Volatile constituents of five Baccharis species from Northeastern Argentina. J Braz Chem Soc 2009;20:1379–84. [10] Sadyrbekov DT, Suleimenov EM, Tikhonova EV, Atazhanova GA, Tkachev AV, Adekenov SM. Component composition of essential oils from four species of the genus Achillea growing in Kazakhstan. Chem Nat Comp 2006;42:294–7. [11] Naguib YN, Hussein MS, El-Sherbeny SE, Khalil MY, Lazari D. Response of Ruta graveolens L. to sowing dates and foliar micronutrients. J Appl Sci Res 2007;3:1534–43. [12] Pontes WT, de Oliveira JCG, da Camara CAG, Lopes ACHR, Gondim Jr MGC, de Oliveira JV, et al. Chemical composition and acaricidal activity of the leaf and fruit essential oils of Protium heptaphyllum (Aubl.) Marchand (Burseraceae). Acta Amazonica 2007;37:103–10. [13] Ma X, Gang DR. Metabolic profiling of turmeric (Curcuma longa L.) plants derived from in vitro micropropagation and conventional greenhouse cultivation. J Agric Food Chem 2006;54:9573–83. [14] Bos R, Woerdenbag HJ, Hendriks H, Smit HF, Wikström HV, Scheffer JJC. Composition of the essential oil from roots and rhizomes of Valeriana wallichii DC. Flavour Fragance J 1997;12:123–31. [15] Gul W, Hammond NL, Yousaf M, Peng J, Holley A, Hamann MT. Chemical transformation and biological studies of marine sesquiterpene (S)-(+)-curcuphenol and its analogs. Biochim Biophys Acta 2007;1770: 1513–9.
[16] Valdivia C, Boketoft Å, Olde B, Owman C, Vilaseca A, Fuentes L, et al. Activation of the free fatty acid receptor GPR40 by (+)-curcuphenol and related synthetic compounds. Rev Boliv Quim 2009;26:77–82. [17] Peng J, Kasanah N, Stanley CE, Chadwick J, Fronczek FR, Hamann MT. Microbial metabolism studies of cyanthiwigin B and synergetic antibiotic effects. J Nat Prod 2006;69:727–30. [18] Tasdemir D, Bugni T, Mangalindan G, Concepcion G, Harper M, Ireland C. Bisabolane type sesquiterpenes from a marine Didiscus sponge. Turk J Chem 2003;27:273–9. [19] Andersson D, Liu JJ, Nilsson Å, Duan R-D. Ursolic acid inhibits proliferation and stimulates apoptosis in HT29 cells following activation of alkaline sphingomyelinase. Anticancer Res 2003;23:3317–22. [20] Utkina NK, Makarchenko AE, Shchelokova OV, Virovaya MV. Antioxidant activity of phenolic metabolites from marine sponges. Chem Nat Compd 2004;40:373–7. [21] Takamatsu S, Hodges TW, Rajbhandari I, Gerwick WH, Hamann MT, Nagle DG. Marine natural products as novel antioxidant prototypes. J Nat Prod 2003;66:605–8. [22] Gomes CA, Giraõ da Cruz T, Andrade JL, Milhazes N, Borges F. Marques MPM. Anticancer activity of phenolic acids of natural or synthetic origin: a structure–activity study. J Med Chem 2003;46:5395–401. [23] Hou J, Sun T, Hu J, Chen SY, Cai XQ, Zou GL. Chemical composition, cytotoxic and antioxidant activity of the leaf essential oil of Photinia serrulata. Food Chem 2007;103:355–8. [24] Sylvestre M, Legault J, Dufour D, Pichette A. Chemical composition and anticancer activity of leaf essential oil of Myrica gale L. Phytomedicine 2005;12:299–304. [25] Leal PF, Braga MEM, Sato DN, Carvalho JE, Marquez MOM, Meireles MAA. Functional properties of spice extracts obtained via supercritical fluid extraction. J Agric Food Chem 2003;51:2520–5. [26] Piochon M, Legault J, Gauthier C, Pichette A. Synthesis and cytotoxicity evaluation of natural α-bisabolol β-D-fucopyranoside and analogues. Phytochemistry 2009;70:228–36. [27] Darra E, Lenaz G, Cavalieri E, Fato R, Mariotto S, Bergamini C, Carcereri de Prati A, Perbellini L, Leoni S, Suzuki H. α-bisabolol: unexpected plant-derived weapon in the struggle against tumour survival? Ital J Biochem 2007;56:323–8. [28] Woynarowska BA, Woynarowski JM. Preferential targeting of apoptosis in tumor versus normal cells. Biochim Biophys Acta 2002;1587:309–17. [29] Cavalieri E, Mariotto S, Fabrizi C, Carcereri de Prati A, Gottardo R, Leone S, et al. α-Bisabolol, a nontoxic natural compound, strongly induces apoptosis in glioma cells. Biochem Biophys Res Commun 2004;315: 589–94. [30] Cavalieri E, Bergamini C, Mariotto S, Leoni S, Perbellini L, Darra E, et al. Involvement of mitochondrial permeability transition pore opening in α-bisabolol induced apoptosis. FEBS J 2009;276:3990–4000. [31] Kim DK, Baek JH, Kang CM, Yoo MA, Sung JW, Kim DK, et al. Apoptotic activity of ursolic acid may correlate with the inhibition of initiation of DNA replication. Int J Cancer 2000;87:629–36. [32] Bonaccorsi I, Altieri F, Sciamanna I, Oricchio E, Grillo C, Contartese G, et al. Endogenous reverse transcriptase as a mediator of ursolic acid's anti-proliferative and differentiating effects in human cancer cell lines. Cancer Lett 2008;263:130–9. [33] Rivero A, Quintana J, Eiroa JL, López M, Triana J, Bermejo J, et al. Potent induction of apoptosis by germacranolide sesquiterpene lactones on human myeloid leukemia cells. Eur J Pharmacol 2003;482:77–84. [34] Arepalli SK, Sridhar V, Venkateswara Rao J, Kavin Kennady P, Venkateswarlu Y. Furano-sesquiterpene from soft coral, Sinularia kavarittiensis: induces apoptosis via the mitochondrial-mediated caspase dependent pathway in THP-1, leukemia cell line. Apoptosis 2009;14:729–40.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Antiproliferative effects of curcuphenol, a sesquiterpene phenol Gloria Rodrigo a,c,d, Giovanna R. Almanza b, Yajun Cheng c, Jiangnan Peng e, Mark Hamann e, Rui-Dong Duan c, Björn Åkesson d,⁎ a b c d e
Molecular Biology and Biotechnology Institute, University Major of San Andres, La Paz, Bolivia Chemistry Research Institute, University Major of San Andres, La Paz, Bolivia Gastroenterology Laboratory, Department of Clinical Sciences, Lund University, Lund, Sweden Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, Lund, Sweden Department of Pharmacognosy, The University of Mississippi, Oxford, USA
a r t i c l e
i n f o
Article history: Received 14 October 2009 Accepted in revised form 30 March 2010 Available online 10 April 2010 Keywords: Curcuphenol Cell proliferation Apoptosis Caspase
a b s t r a c t Curcuphenol is a sesquiterpene isolated from sponges and plants having several significant biological activities. The present study explored its effect on cell proliferation and apoptosis in Caco-2 human colon cancer cells. It was demonstrated that curcuphenol in concentrations in the range of 29–116 µg/ml inhibited cell proliferation and DNA replication and induced cell death in a dose-dependent manner. The induction of apoptosis was associated with a stimulation of the activity of caspase-3. The findings presented here suggest that curcuphenol has antiproliferative and pro-apoptotic properties. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Herbal preparations have long been used as remedies for infectious and other diseases and they are used in primary health care in several countries [1]. Chemical and pharmacological investigations have indicated that sesquiterpene phenols are important bioactive components in several plants [2]. Curcuphenol (5-methyl-2-[(2S)-6-methylhept-5-en-2yl]phenol) was isolated and identified from several sources such as the sponges Didiscus flavus [3,4], Arenochalina [5]), Myrmekioderma [6], Pseudopterogorgia species [7] and Epipolasis species [8], and plants including Baccharis penningtonii Heering [9], Valeriana wallichii DC. [10], Ruta graveolens [11], Protium heptaphyllum (Aubl.) Marchand [12], Curcuma longa L. [13] and Achillea sudetica (Asteraceae) [14]. Curcuphenol was reported to have antimicrobial activity against Staphylococcus aureus and Cryptococcus neoformans [3], antifungal activity against Candida albicans and antima-
⁎ Corresponding author. Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, POBox 124, SE-221 00 Lund, Sweden. Tel.: +46 46 222 45 23; fax: +46 46 222 46 11. E-mail address: [email protected] (B. Åkesson). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.04.001
larial activity against Plasmodium falciparium [8] and antileishmania activity [15]. (S)-(+)-Curcuphenol has also been shown to inhibit proton–potassium ATPase [3] and to activate GPR40, a cell surface receptor for free fatty acids [16]. Curcuphenol works synergistically with cyanthiwigin B as an antibiotic and both are found in good yields in Myrmekioderma [17]. A preliminary indication of antitumor activity of curcuphenol has been reported for murine leukaemia cells (P-388) and several human cancer cell lines, lung (A-549), colon (HCT-8) and mammary (MDAMB) cells [4] and it showed cytotoxic activity in a manner independent of a p53 mechanism [18]. To get further information about its antitumour properties, we investigated the effects of curcuphenol on cell proliferation and apoptosis in this report. 2. Experimental 2.1. General Dulbecco's Modified Eagle's Medium (DMEM) and fetal calf serum (FCS) were purchased from Gibco, and benzamidine, phenylmethylsulphonyl-fluoride (PMSF), taurocholate, dithiothreitol (DTT) were purchased from Sigma-Aldrich.
G. Rodrigo et al. / Fitoterapia 81 (2010) 762–766
2.2. Sources of curcuphenol Curcuphenol was isolated from a Bolivian plant Baccharis genistelloides [16] and from the marine sponge Myrmekioderma styx [6]. The structure was assigned by 1D and 2D NMR (Bruker DRX 400) and comparison with literature values. Samples isolated from the two sources were shown to be the same by identical proton and carbon NMR data (Table 1). Curcuphenol was dissolved in DMSO to a final concentration of 116 mg/ml. Serial dilutions to final concentrations ranging from 7 to 116 µg/ml (33–531 µmol/l) were prepared in cell culture medium. 2.3. Cell culture Caco-2 cells were cultured in DMEM with L-glutamine, containing 100 IU/mL penicillin, 10 µg/ml streptomycin and 10% (v/v) heat-inactivated FCS. They were maintained in 25 cm2 culture flasks at 37 °C in a humidified incubator containing 95% air and 5% CO2. For assays of cell proliferation, DNA replication and apoptosis, the cells were detached with 0.05% trypsin/0.02% EDTA, resuspended to a concentration of 1 × 105/ml and seeded into 96-well plates (Falcon). For the caspase-3 assay, the cells were seeded at 0.6 × 106 cells/ml in 25 cm2 culture flasks for 72 h. They were then treated by the
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different concentrations of curcuphenol for 6 h, and then detached with a rubber policeman and harvested by centrifugation.
2.4. Cell proliferation assay The cell proliferation rate was assayed by use of the reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3benzene disulfonate (WST-1) (Roche). It is metabolized by mitochondrial dehydrogenases to a formazan, a dye that stains dark red. The formation of the formazan is proportional to the total mitochondrial dehydrogenase activity in the sample, which in turn correlates with the total number of viable cells. Briefly, the Caco-2 cells, 2 × 104 in 200 µl medium were incubated with curcuphenol at concentrations from 7 to 116 µg/ml in a 96-well microplate for 24 h, and 20 μl WST-1 reagent was then added in each well. After incubation for 1 h at 37 °C, the plate was shaken thoroughly for 1 min and the optical density (OD) was read at 405 nm using 655 nm as background. The cell proliferation rate was expressed as a percentage of the control. All determinations were carried out in sixtuplicate wells in three different experiments.
2.5. DNA replication Table 1 13 C NMR data of curcuphenol measured in DMSO at 100 MHz. Data are shown for curcuphenol isolated from A, Myrmekioderma styx, B, Baccharis genistelloides and the (+)-curcuphenol structure is shown below. No.
A
B
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15
17.9 130.9 125.1 37.0 26.2 31.4 130.3 154.9 116.0 135.7 120.1 126.9 21.1 21.4 26.0
17.9 130.9 125.1 37.0 26.2 31.4 130.3 154.9 116.0 135.7 120.1 126.9 21.1 21.4 26.0
DNA synthesis was measured by the amount of [3H] thymidine incorporation. Caco-2 cells were seeded in 96-well plates and preincubated for 20 h with different concentrations of curcuphenol. Then [3H]thymidine (0.5 µCi/well; American Radiolabeled Chemicals, St. Louis, MO, USA) was added to the cells followed by incubation for 4 h. Cells were washed with ice cold PBS, then treated with 5% trichloroacetic acid for 20 min at 4 °C, followed by one wash with ice cold PBS. Finally, the cells were lysed in 0.5 M NaOH and 0.1% SDS for 15 min at room temperature and 100 µL was collected into scintillation vials and counted. DNA synthesis was expressed as percentage of control. Triplicate analyses in two different experiments were performed.
2.6. Assay of apoptosis Apoptosis was assayed by a Cell Death Detection ELISAPLUS kit (Roche). Briefly, 1 × 104 cells were seeded in 96-well plates in 200 µl DMEM with L-glutamine, 100 IU/mL penicillin, 10 µg/mL streptomycin and 10% (v/v) heat-inactivated FCS. After treatment by curcuphenol for 6 h, the cells were lysed and the nuclei were separated from the cytoplasmatic fraction by centrifugation (10 min, 200 g). A sample from the supernatant was transferred to a streptavidin-coated microtiter plate provided by the kit. The relative enrichment of cytoplasmic histone-associated DNA fragments to the streptavidin-coated plate was measured. After removal of unbound antibodies by washing, the amount of nucleosomes was determined photometrically at 415 nm with 490 nm as background. The enrichment factor was calculated as absorbance of the sample (dying and dead cells)/absorbance of the corresponding control (cells without treatment). Triplicate analyses in three different experiments were performed.
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Fig. 1. Antiproliferative effect of curcuphenol on colon cancer cells. Cells were treated with different concentrations of curcuphenol for 24 h, and then the cell viability was determined by WST-1 assay. The growth inhibitory activity was calculated as % of inhibition compared with the control.
2.7. Caspase-3 activity
Fig. 3. Effect on apoptosis of different concentrations of curcuphenol on Caco-2 cells after exposure for 6 h. The values represent means of three separate experiments with standard errors depicted by vertical bars, each was done using triplicate samples.
3. Results Caspase-3 activity was measured by proteolytic cleavage of the fluorogenic substrate Ac-DEVD-pNA (VWR International AB, Stockholm, Sweden) using a microplate reader [19]. Caco-2 cells (1 × 105 cells/well) were treated with curcuphenol at concentrations of 29, 58 and 116 µg/ml. After incubation for 6 h, the cells were harvested and then washed with cold PBS. The pellets were lysed using 100 µl of lysis buffer (50 mM Tris, 2 mM EDTA, pH 7.4, taurocholate 6 mM, PMSF 1 mM, DTT 100 mM, leupeptin 10 mg/ml, aprotinin 10 mg/ml) on ice for 15 min and then 100 µl of assay buffer (50 mM Hepes, 0.1 M NaCl, 0.1% Chaps, 10 mM DTT, 0.1 mM EDTA, 10% glycerol, pH 7.4) and 10 µl of substrate solution (2 mM substrate in assay buffer) were added. After incubation at 37 °C for 30 and 60 min, the absorbance was measured at 405 nm. Triplicate analyses in three different experiments were performed.
3.1. Effect of curcuphenol on the growth of colon cancer cells When Caco-2 cells were treated with different concentrations (7–116 µg/ml) of curcuphenol, an inhibition of cell proliferation was observed (Fig. 1). The inhibition of cell growth was 32% at 116 µg/ml, 33% at 58 µg/ml, 29% at 29 µg/ml and 9% at 14 µg/ml. The antiproliferative effect of curcuphenol was further confirmed by measuring DNA replication. Curcuphenol was added at concentrations of 29, 58 and 116 µg/ml and it reduced [3H]thymidine incorporation into Caco-2 cells in a dose-dependent manner, with an IC50 of 43 µg/ml (Fig. 2). 3.2. Effects of curcuphenol on apoptosis
The results are presented as mean (SE). The statistical significance of differences between groups was determined by the Student t-test and P b 0.05 was considered to be statistically significant.
Proliferation and apoptosis are two important mechanisms related to the fate of the cells and the effect of curcuphenol on apoptosis was therefore further examined. After treatment of Caco-2 cells with curcuphenol (14–116 µg/ml) for 6 h the rates of apoptosis were significantly increased at all concentrations of curcuphenol (Fig. 3). It increased significantly from 14 to
Fig. 2. Effect on DNA synthesis after exposure to 29–116 µg/ml of curcuphenol on Caco-2 cells. The values are expressed as % of control and represent means (S.E.) of two separate experiments performed with sextuplicate samples. The absolute thymidine incorporation into DNA of untreated Caco-2 cells was 0.4 fmol/h/106 cells.
Fig. 4. Increase of caspase-3 activity induced by curcuphenol in Caco-2 cells. The values represent means (S.E.) of three independent experiments at 6 h of exposure to curcuphenol.
2.8. Statistical analysis
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58 µg/ml and then reached a plateau. The stimulation of apoptosis was 2.6-, 3.3- and 3.0-fold at 29, 58 and 116 µg/ml, respectively, as compared to untreated cells. After treatment of Caco-2 cells with curcuphenol (14–58 µg/ml) for 6 h, it also increased the caspase-3 activity dose-dependently (Fig. 4) at 30 min of incubation. A 6.6- and 8-fold increase was obtained as compared to untreated cells at 29 and 58 µg/ml, respectively. No increase in the activity was demonstrated by curcuphenol at 14 µg/ml. Thus, the results suggest that the curcuphenol killed Caco-2 cells through an apoptosis mechanism via the activation of caspase-3.
4. Discussion 4.1. Curcuphenol and cell proliferation Curcuphenol isolated from sponges has been reported to have antifungal activity against Candida albicans and antimalarial activity against Plasmodium falciparium [8]. There is also preliminary evidence of antitumor activity against human cancer cell lines [4] and moderate activity against HCT-116 cells [18]. Since curcuphenol has moderate antiradical activity [20] and strong antioxidant activity [21], it may also have actions related to antioxidants which may suppress carcinogenesis during the initiation phase, and affect the activity of xenobiotic metabolizing enzymes [22]. In this paper it was observed that curcuphenol isolated from two sources inhibited cell proliferation and DNA synthesis in Caco-2 cells, suggesting that curcuphenol may inhibit the progress of the cell cycle, resulting in inhibition of cell growth. The inhibition seemed to be larger on DNA synthesis than on cell proliferation and we have no obvious explanation for this discrepancy. Regarding the effects of other natural sources of related compounds, the oil of Photinia serrulata was shown to have strong cytotoxic activity against cancer cell lines, and it contains high amounts of sesquiterpenes, including α-bisabolol, γ-curcumene, ar-curcumene, β-bisabolene, β-curcumene and (E)-γ-bisabolene [23,24]. Turmeric extracts also contain compounds similar in structure to curcuphenol (ar-curcumene, β-bisabolene, α- and γ-atlantone, ar-turmerol) and showed strong anticancer activity in cell lines, e.g. HT-29 colon cancer cells [25], and at least in part these compounds were responsible for the cytotoxic activity. Piochon et al. [26] found that α-bisabolol, a similar compound to curcuphenol, showed cytotoxic activity against U-87 glioma cells with an IC50 of 130 µM (29 µg/ml) which is similar to the results found in our work. Moreover, this compound showed weak activity against human lung carcinoma and ovary teratocarcinoma cells [26], and affected the cell viability in transformed cells [27]. This drop in cell viability was mainly due to cell death due to apoptosis [27]. Finally, curcuphenol showed moderate cytotoxic activity against HCT-116 colon cancer cells [18] between 27 and 35 µg/ml, which is similar to the results of our work. Although the effects are consistent it must be noted that the lowest effective concentration (29 µg/ml = 133 µmol/l) is rather high. Further work is necessary to possibly find more active analogues of curcuphenol and to study the mechanisms of cell uptake of these compounds. To some extent this research is hampered by the limited availability of the compounds in question in pure form.
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4.2. Effects of curcuphenol on apoptosis The killing of tumor cells may be mediated through induction of apoptosis in target cells [27,28]. Our data strongly suggest that curcuphenol exerts some of its action through activation of apoptosis, similar to previous findings with αbisabolol [27,29,30]. The conclusion of our study is supported by data from both oligonucleosome determination and caspase-3 determination. Further detailing of the mechanisms involved will require additional studies. Also other compounds having a sesquiterpene structure may cause both cell cycle arrest and apoptosis. Like curcuphenol, ursolic acid can induce G1 arrest by the inhibition of DNA replication [31], and in addition ursolic acid induces apoptosis via inhibition of the NF-ΚB induced bcl-2 mediated anti-apoptotic pathway and subsequent activation of the p53mediated and TNF-α-induced caspase-3 mediated pro-apoptotic pathways [32]. α-bisabolol, a closely similar compound to curcuphenol exhibited strong apoptotic activity probably by the intrinsic and extrinsic pathways [27,29]. 4.3. Effects of curcuphenol on caspase activity Curcuphenol may promote collapse of the mitochondrial membrane potential, release of cytochrome C, and activation of caspase-3 (Fig. 4). Some germacranolide sesquiterpene lactones also induced DNA fragmentation dose-dependently [33]. Biochemical characterization of the apoptotic DNA damage revealed that the sesquiterpene lactones promoted the internucleosomal degradation of DNA, resulting in the formation and eventual release of oligonucleosomal DNA fragments [33]. Also other sesquiterpenes than curcuphenol have been shown to affect caspase activity. Like curcuphenol, diacetyl taridin A acted after a short time [33]. The trigger for curcuphenol-induced apoptosis remains to be determined. 4.4. Concluding remarks Curcuphenol exerts antiproliferative and pro-apoptotic effects in the Caco-2 colon cells. It also induces biochemical changes that are characteristic of apoptosis and we can conclude that curcuphenol induced caspase-dependent cell death. A survey of the literature revealed that no studies on the cytotoxic activity of curcuphenol had previously been undertaken, although several related sesquiterpenes have been found to have antiproliferative and other activities. Apoptosis plays a vital role in controlling cell number in many developmental and physiological stages, tissue homeostasis, and regulation of immune system, while inadequate apoptosis is an integral part of cancer development [34]. Agents, which suppress or inhibit the development of malignant cells by inducing apoptosis, may represent a useful mechanistic approach to both chemoprevention and chemotherapy of cancer. Acknowledgements This research was supported by the Swedish International Development Agency (Sida) for a project on Plant biodiversity as a collaboration between University Major of San Andres, La Paz, Bolivia and Lund University, Sweden. Lund University is a
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member of the EU NoE ECNIS, which partly supported the study. This work was also supported by NIH (1R01A136596) and CDC (U01/CI00211-02). The Natural Resource Conservation Authority, Jamaica and Discovery Bay Marine Laboratory are gratefully acknowledged for assistance with sample collections. References [1] Sokmen A, Jones BM, Erturk M. The in vitro antibacterial activity of Turkish medicinal plants. J Ethnopharmacol 1999;67:79–86. [2] Ciavatta ML, Lopez Gresa MP, Gavagnin M, Romero V, Melck D, Manzo E, et al. Studies on puupehenone-metabolites of a Dysidea sp.: structure and biological activity. Tetrahedron 2007;63:1380–4. [3] El Sayed KA, Yousaf M, Hamann MT, Avery MA, Kelly M, Wipf P. Microbial and chemical transformation studies of the bioactive marine sesquiterpenes (S)-(+)-curcuphenol and curcudiol isolated from a deep reef collection of the Jamaican sponge Didiscus oxeata. J Nat Prod 2002;65:1547–53. [4] Wright AE, Pomponi SA, McConnell OJ, Kohmoto S, McCarthy PJ. (+)Curcuphenol and (+)-curcudiol, sesquiterpene phenols from shallow and deep water collections of the marine sponge Didiscus flavus. J Nat Prod 1987;50:976–8. [5] Butler MS, Capon RJ, Nadeson R, Beveridge AA. Aromatic bisabolenes from an Australian marine sponge, Arenochalina sp. J Nat Prod 1991;54: 619–23. [6] Peng J, Franzblau SG, Zhang F, Hamann MT. Novel sesquiterpenes and a lactone from the Jamaican sponge Myrmekioderma styx. Tetrahedron Lett 2002;43:9699–702. [7] Harvell CD, Fenical W, Greene C. Chemical and structural defenses of Caribbean gorgonians (Pseudopterogorgia spp.). I. Development of an in situ feeding assay. Mar Ecol Prog Ser 1988;49:287–94. [8] Fusetani N, Sugano M, Matsunaga S, Hashimoto K. (+)-Curcuphenol and dehydrocurcuphenol, novel sesquiterpenes which inhibit H, KATPase, from a marine sponge Epipolasis sp. Experientia 1987;43: 1234–5. [9] Retta D, Gattuso M, Gattuso S, Di Leo NP, van Baren C, Bandoni A. Volatile constituents of five Baccharis species from Northeastern Argentina. J Braz Chem Soc 2009;20:1379–84. [10] Sadyrbekov DT, Suleimenov EM, Tikhonova EV, Atazhanova GA, Tkachev AV, Adekenov SM. Component composition of essential oils from four species of the genus Achillea growing in Kazakhstan. Chem Nat Comp 2006;42:294–7. [11] Naguib YN, Hussein MS, El-Sherbeny SE, Khalil MY, Lazari D. Response of Ruta graveolens L. to sowing dates and foliar micronutrients. J Appl Sci Res 2007;3:1534–43. [12] Pontes WT, de Oliveira JCG, da Camara CAG, Lopes ACHR, Gondim Jr MGC, de Oliveira JV, et al. Chemical composition and acaricidal activity of the leaf and fruit essential oils of Protium heptaphyllum (Aubl.) Marchand (Burseraceae). Acta Amazonica 2007;37:103–10. [13] Ma X, Gang DR. Metabolic profiling of turmeric (Curcuma longa L.) plants derived from in vitro micropropagation and conventional greenhouse cultivation. J Agric Food Chem 2006;54:9573–83. [14] Bos R, Woerdenbag HJ, Hendriks H, Smit HF, Wikström HV, Scheffer JJC. Composition of the essential oil from roots and rhizomes of Valeriana wallichii DC. Flavour Fragance J 1997;12:123–31. [15] Gul W, Hammond NL, Yousaf M, Peng J, Holley A, Hamann MT. Chemical transformation and biological studies of marine sesquiterpene (S)-(+)-curcuphenol and its analogs. Biochim Biophys Acta 2007;1770: 1513–9.
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