Cytotoxic activity of dihydrochalcones isolated from corema album leaves against HT-29 colon cancer cells

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published in Natural Product Communications on September 2013, available at: https://doi.org/10.1177/1934578x1300800918 .” Cytotoxic Activity of Dihydrochalcones Isolated from Corema album Leaves against HT-29 Colon Cancer Cells Antonio J. León-Gonzáleza, Miguel López-Lázaroa, José L. Esparterob and Carmen Martín-Corderoa,* aDepartment of Pharmacology, School of Pharmacy, University of Seville, Spain bDepartment of Organic Chemistry, School of Pharmacy, University of Seville, Spain [email protected] Received: March 14th, 2013; Accepted: June 3rd, 2013 During our search for cytotoxic compounds from Andalusian vascular plants, the ethyl acetate extract from the leaves of Corema album (L.) D. Don (Ericaceae) was selected for its cytotoxic activity against the HT-29 human colon adenocarcinoma cell line. Two dihydrochalcones, 2’,4’- dihydroxydihydrochalcone (1) and 2’-methoxy-4’-hydroxydihydrochalcone (2), have been isolated from the leaves of C. album. Their structural identification was based on 1H NMR and 13C NMR data, including 2D NMR, and mass spectrometry. These compounds were subjected to the sulfhorhodamine B (SRB) cytotoxic assay against human colon adenocarcinoma cells (HT-29). Compounds 1 and 2 showed higher cytotoxicity than the positive control 5-fluorouracil (5-FU); the IC50 values (M ± SEM) were 1.8 ± 0.4 for compound 1, 8.5 ± 2.1 M for compound 2, and 8.7 ± 4.0 for 5-FU. The cytotoxic activity of 1 and 2 was reduced in the presence of the antioxidants N-acetylcysteine (NAC) and Mn(III) Tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), therefore suggesting that reactive oxygen species generation participates in the cytotoxic activity of these dihydrochalcones. Keywords: Corema album, Ericaceae, Dihydrochalcones, Cytotoxic activity, Reactive oxygen species, Colon adenocarcinoma, HT-29. Corema album (L.) D. Don (Ericaceae), an evergreen dioecious shrub, is endemic to the Atlantic coast of the Iberian Peninsula, growing on sandy soil over coastal dunes and cliffs 1. We have recently reported on the chemical and biological studies of C. album berries, which revealed the presence of phenolic acids, flavonoids and anthocyanins 2 and a chemoprotective effect 3. In a preliminary screening program for cytotoxic compounds in Andalusian vascular plants, the ethyl acetate extract of leaves of C. album was shown to be one of the active extracts. The aim of this study was the isolation and identification of active compounds from the ethyl acetate extract of C. album leaves and to determinate if their cytotoxic activity is mediated by ROS production. The initial cytotoxic testing of the ethyl acetate extract using HT-29 human colon adenocarcinoma cell line against the anticancer agent 5-Fluoruracil clearly demonstrated strong activity (IC50 values 15.8  2.6 and 16.2  2.6 g/mL, respectively). The phytochemical study of the ethyl acetate extract led to the isolation of two dihydrochalcones. Their structures were established by comparison of their 1H NMR, 13C NMR and MS data with literature values 4, 5 and were identified as 2’,4’-dihydroxydihydrochalcone (1) and 2’-methoxy-4’-hydroxydihydrochalcone (2) (Figure 1). These dihydrochalcones were isolated for the first time from Empetrum nigrum, a species of the Empetreae tribe. Compounds 1 and 2 were tested for cytotoxic activity against HT-29 human colon adenocarcinoma cells using the SRB assay. All compounds reduced cell viability at concentrations in the low micromolar range, the IC50 (means ± SEM) values being: 1.8 ± 0.4 M for compound 1, 8.5 ± 2.1 M for compound 2 and 8.7 ± 4.0 M for the positive control, 5-FU. This cytotoxic effect was dose-dependent and was more pronounced for compounds 1 and 2 than for the anticancer agent 5-FU (Figure 2). Chalcones and their derivatives are a group of compounds reported to exhibit promising anticancer activity. Previous studies have Figure 1: Structure of isolated compounds: (1) R= OH, 2’,4’-dihydroxy- dihydrochalcone; (2) R= OCH3, 2’-methoxy-4’-hydroxydihydrochalcone. %viabilidadcelular Concentración(µM) 0 20 40 60 80 100 120 0,1 1 10 100 Concentration (µM) % Cell viability 2',4'-dihidroxydihydrochalcone 2'-hydroxy-4'-methoxydihydrochalcone 5-Fluorouracil Figure 2: Cell viability, expressed as percentage of cells in relation to controls. indicated that natural and synthetic chalcones have strong antiproliferative effects in human cancer cell lines 6. Previous literature suggests that chalcones are capable of inducing apoptosis and also to induce oxidative stress because of their ability to uncouple mitochondrial respiration and thus collapse mitochondrial membrane potential [7]. In order to assess the possible involvement of ROS in the cytotoxic activity of compounds 1 and 2, HT-29 cells were treated with both dihydrochalcones and 5-FU in either the presence or absence of the antioxidants N-acetylcysteine (NAC) and Mn(III) Tetrakis(1- methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP). Figure 2 shows that both antioxidants reduced the cytotoxicity of compound 1 and MnTMPyP reduced the activity of compound 2, suggesting NPC Natural Product Communications 2013 Vol. 8 No. 9 1255 - 1256 1256 Natural Product Communications Vol. 8 (9) 2013 León-González et al. 5-Fluorouracil 0 10 20 30 40 50 60 70 80 90 100 % Cell viability 5-FU 100 100 100 10 10 10 1 1 1 – – NAC – + – – + – – + – + – MnTMPyP ––+ – –+– – +–+ 2',4'-dihydroxydihydrochalcone 0 10 20 30 40 50 60 70 80 90 100 % Cell viability Chalcone 1 1 1 – – NAC – + – + – MnTMPyP – – + – + 2'-hydroxy-4'-methoxydihydrochalcone 0 10 20 30 40 50 60 70 80 90 100 % Cell viability * Chalcone 10 10 10 – – NAC – + – + – MnTMPyP––+–+ % Cell viability Figure 2: Cell viability of HT-29 colon adenocarcinoma cells after 48 hours treatment with 2’,4’-dihydroxydihydrochalcone, 2’-methoxy-4’-hydroxydihydrochalcone and of the positive control 5-fluorouracil (5-FU) in the presence or absence of the antioxidants N-acetylcysteine (NAC) or Mn(III) Tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP). that ROS generation participates in the mechanism of action of these two dihydrochalcones. Neither antioxidant reduced the activity of 5-FU, therefore suggesting that oxidative stress is not involved in the cytotoxic activity of this anticancer agent (Figure 2). Experimental data have revealed that the induction of H2O2 by prooxidant agents may lead to the selective killing of cancer cells [8]. Our results show that the cytotoxic activity of compounds 1 and 2 may be mediated in part by their pro-oxidant activity. Further experiments are required to establish whether or not these dihydrochalcones can kill cancer cells selectively. Experimental General: 1D and 2D NMR, Bruker AVANCE-500; EI and FAB MS, Micromass AUTOSPECQ; UV, Perkin Elmer Lambda 3, TLC, Merck silica gel 60 F254. Plant material: Wild Corema album leaves were harvested in December 2009 at Huelva (Spain) 37º 04´10.15" N - 6º 41´15.45" W, and identified by Dr Mari Cruz Díaz-Barradas, from the Department of Plant Biology and Ecology, University of Seville. A voucher specimen (no. 277798) was deposited in the herbarium of the University of Seville for further reference. Extraction and isolation: The dried and powdered leaves (250 g) of male Corema album plants were extracted at room temperature in an ultrasound bath for 45 min with ethyl acetate. After filtration, the solvent was removed using a rotary evaporator (45ºC) to obtain the crude ethyl acetate extract (4.8% yield). This (3 g) was subjected to silica gel CC (3 x 30 cm) and eluted with gradient mixtures of n-hexane/ethyl acetate from 9:1 to 7:3, v/v (to give 419 fractions of 10 mL). Compound 1 (15 mg) was isolated by crystallization as yellow needles from fractions 57-66 and compound 2 as white needles from fractions 135-154 (13 mg). The spectroscopic data of the isolated compounds were compared with those reported previously in the literature. Compound 1: 2’,4’- dihydroxydihydrochalcone 4, 5 and compound 2: 2’-methoxy-4’- hydroxydihydrochalcone 5. Cell assays: For in vitro cytotoxic assays, cell viability of the HT-29 human colon adenocarcinoma cell line was evaluated after 48 h of treatment with 1 and 2, using the sulforhodamine B (SRB) assay [9]. Cell viability was expressed as a percentage in relation to controls and the half inhibitory concentration (IC50) of each compound was determined. 5-Fluorouracil (5-FU) was included as positive control. Finally, to evaluate the possible involvement of ROS in the cytotoxic activity of compounds, HT-29 cells were treated with 1, 2 and 5-FU in the presence or absence of the antioxidants N-acetylcysteine (NAC) and Mn (III) Tetrakis(1- methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP). After a 48 h drug exposure, cell viability was estimated using the SRB assay. All experiments were carried out in triplicate. Data are expressed as means ± standard deviation. Differences between results and the corresponding control were determined by the Student’s paired t-test at significance levels of p<0.05 (*), p<0.01 (**), and p<0.001 (***). Supporting information: General experimental procedures are available as Supporting Information. Acknowledgments - The authors are grateful to Dr Mari Cruz Díaz- Barradas, from the Department of Plant Biology and Ecology, for identification of plant species; and to Nuclear Magnetic Resonance and Mass Spectrometry services at “Centro de Investigación, Tecnología e Innovación” (CITIUS) of the University of Seville. References [1] Álvarez-Cansino L, Zunzunegui M, Díaz Barradas MC, Esquivias MP. (2010) Gender-specific costs of reproduction on vegetative growth and physiological performance in the dioecious shrub Corema album. Annals of Botany, 106, 989-998. [2] León-González AJ, Truchado P, Tomás-Barberán FA, López-Lázaro M, Díaz Barradas MC, Martín-Cordero C. (2013) Phenolic acids, flavonols and anthocyanins in Corema album (L.) D. Don berries. Journal of Food Composition and Analysis, 29, 58-63. [3] León-González AJ, Mateos R, Ramos S, Martín MA, Sarriá B, Martín-Cordero C, López-Lázaro M, Bravo L, Goya L. (2012) Chemo-protective activity and characterization of phenolic extracts from Corema album. Food Research International, 49, 728-738. [4] Krasnov EA, Ermilova EV, Kadyrova TV, Raldugin VA, Bagryanskaya IY, Gatilov, YV, Druganov AG, Semenov AA, Tolstikov GA. (2000) Phenolic components of Empetrum nigrum extract and the crystal structure of one of them. Chemistry of Natural Compounds, 36, 493-496. [5] Wollenweber E, Dorr M, Stelzer R, Arriaga Giner FJ. (1992) Lipophilic phenolics from the leaves of Empetrum nigrum - chemical structures and exudate localization. Botanica acta, 105, 300-305. 6 Syam S, Abdelwahab SI, Al-Mamary MA, Mohan S. (2012) Synthesis of Chalcones with Anticancer Activities. Molecules, 17, 6179-6195. 7 Lin E, Lin WH, Wang SY, Chen CS, Liao JW, Chang HW, Chen SC, Lin KY, Wang L, Yang HL, Hseu YC. (2012) Flavokawain B inhibits growth of human squamous carcinoma cells: Involvement of apoptosis and cell cycle dysregulation in vitro and in vivo. The Journal of Nutritional Biochemistry, 23, 368-378. [8] Martín-Cordero C, León-González AJ, Calderón-Montaño JM, Burgos-Morón E, López-Lázaro M. (2012) Pro-oxidant natural products as anticancer agents. Current Drug Targets, 13, 1006-1028. [9] Alexandre J, Hu Y, Lu W, Pelicano H, Huang P. (2007) Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species. Cancer Research, 67, 3512-3517.