J. Agric. Food Chem. 2011, 59, 755–762
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DOI:10.1021/jf103904b
Antiangiogenic Potential of Three Triterpenic Acids in Human Liver Cancer Cells )
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CHUN-CHE LIN,†,‡,§ CHUN-YIN HUANG,§, MEI-CHIN MONG,^ CHIEN-YI CHAN, MEI-CHIN YIN*, ,^ †
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Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung City, Taiwan, ‡ Institute of Medicine, Chung Shan Medical University, Taichung City, Taiwan, Department of Nutrition, China Medical University, Taichung City, Taiwan, and ^Department of Health and Nutrition Biotechnology, Asia University, Taichung County, Taiwan. § These two authors contributed equally to this study
Three triterpenic acids, oleanolic acid, ursolic acid and maslinic acid, at 2 or 4 μmol/L were used to study their antiangiogenic potential in human liver cancer Hep3B, Huh7 and HA22T cell lines. The effects of these compounds upon the level and/or expression of hypoxia-inducible factor (HIF)-1R, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin (IL)-8, urokinase plasminogen activator (uPA), reactive oxygen species (ROS), nitric oxide (NO) and cell invasion and migration were examined. Results showed that these triterpenic acids at 4 μmol/L significantly suppressed HIF-1R expression in three cell lines (P < 0.05); and these compounds at test doses failed to affect bFGF expression (P > 0.05). Three triterpenic acids dose-dependently decreased production and expression of VEGF and IL-8, retained glutathione level, lowered ROS and NO levels, and declined cell invasion and migration in test cell lines (P < 0.05). These compounds also dose-dependently reduced uPA production and expression in Hep3B and Huh7 cell lines (P < 0.05); but these agents only at 4 μmol/L significantly suppressed uPA production and expression in HA22T cells (P < 0.05). These findings suggest that these triterpenic acids are potent antiangiogenic agents to retard invasion and migration in liver cancer cells. KEYWORDS: Angiogenesis; triterpenic acid; liver cancer cell; hypoxia-inducible factor; vascular endothelial growth factor
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(NO) play important roles in cancer angiogenesis because these free radicals enhance oxidative stress and stimulate VGEF production, which in turn favors cancer progression (8, 9). Obviously, these factors provide vascular support for the growth of already established malignant tumor. Thus, any agents with suppressive effects on the production or expression of these angiogenic factors may be beneficial in retarding tumor progression. Oleanolic acid, ursolic acid and maslinic acid are pentacyclic triterpenic acids naturally occurring in many herbs and plant foods such as glossy privet fruit (Ligustrum lucidum Ait.), hawthorn fruit (Crataegi Pinnatifidae Fructus) and olive (10, 11). The inhibitory effects of oleanolic acid and ursolic acid against the growth of human bladder, prostate and colorectal cancer cells have been observed (12, 13). Our previous study (14) also found that these two triterpenic acids at 8 μmol/L caused markedly apoptotic effects in four human liver cancer HepG2, Hep3B, Huh7 and HA22T cell lines via increasing DNA fragmentation, decreasing mitochondrial membrane potential and lowering Naþ-Kþ-ATPase activity. Juan et al. (15) reported that maslinic acid, another triterpenic acid, could inhibit cell proliferation in human colon cancer cell, HT-29. These studies suggest that these triterpenic acids are potent anticancer agents to cause apoptosis or retard metastasis for liver cancer. Therefore, a further study was conducted to examine the antiangiogenic potential of these
© 2010 American Chemical Society
Published on Web 12/22/2010
INTRODUCTION
Angiogenesis, also called as neovascularization, is an important process for tumor growth and metastasis. Liver cancer, also called hepatocellular carcinoma (HCC), is a hypervascular tumor characterized by massive angiogenesis (1). Increased circulating levels and/or expression of several angiogenic factors such as vascular endothelial growth factor (VEGF) and interleukin (IL)-8 in patients with HCC have been reported (2, 3). Hypoxiainducible factor (HIF)-1R and basic fibroblast growth factor (bFGF) are two crucial activators responsible for cancer angiogenesis, in which HIF-1R regulates the essential adaptive responses of cancer cells to hypoxia, the major pathophysiological condition promoting angiogenesis; and bFGF mediates extracellular matrix degradation (4, 5). In addition, VEGF through increasing vascular permeability could enhance endothelial cell migration and proliferation, which are required for vascular remodeling and sprouting development of new blood vessels (6). Overexpression of urokinase plasminogen activator (uPA), a gene related to invasion, also contributed to HCC angiogenesis (7). On the other hand, reactive oxygen species (ROS) and nitric oxide
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triterpenic acids. The major purpose of our present study was to investigate the effects of triterpenic acids on the production and/ or expression of HIF-1R, bFGF, VEGF, IL-8 and uPA in liver Table 1. Content (mg/100 g dry weight) of Oleanolic Acid (OA), Ursolic Acid (UA) and Maslinic Acid (MA) in Eleven Vegetables, Carrot, Celery, Okra, Eggplant, Spinach, Leaf Mustard, Gynura, Chinese Chive, Fennel, Basil and Daylilya
carrot celery okra eggplant spinach leaf mustard gynura Chinese chive fennel basil daylily a
OA
UA
MA
25 ( 4 17 ( 2 61 ( 6 53 ( 4 167 ( 13 135 ( 11 54 ( 8 96 ( 7 118 ( 10
31 ( 6 23 ( 3 32 ( 3 133 ( 8 92 ( 6 45 ( 6 17 ( 5 83 ( 12 76 ( 5
-b 40 ( 4 84 ( 7 126 ( 11 174 ( 12 37 ( 5 38 ( 4 35 ( 4 105 ( 9
Data are mean ( SD, n = 6. b Too low to be detected.
cancer cell lines. The influence of these compounds upon the production of ROS and NO in these cell lines was also evaluated. On the other hand, ursolic acid and maslinic acid present in apple peel have been considered as major compounds responsible for the anticancer activity of apple (16). So far, less information is available regarding the presence of these triterpenic acids in fresh vegetables. In order to enrich the food component database, eleven locally available fresh vegetables in Taiwan were used to analyze the content of oleanolic acid, ursolic acid and maslinic acid. MATERIALS AND METHODS Chemicals. Oleanolic acid (OA, 99%), ursolic acid (UA, 98%) and maslinic acid (MA, 98.5%) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Medium, plates, antibiotics and chemicals used for cell culture were purchased from Difco Laboratory (Detroit, MI, USA). All chemicals used in these measurements were of the highest purity commercially available. Content of Triterpenic Acids. Eleven locally available fresh vegetables, carrot (Daucus carota L.), celery (Apium graveolens L.), okra (Hibiscus esculentus), eggplant (Solanum melongena L.), spinach (Spinacia oleracea L.), leaf mustard (Brassica juncea), gynura (Gynura bicolor DC.), Chinese
Figure 1. HIF-1R mRNA expression in human Hep3B, Huh7 and HA22T cell lines under normoxia and hypoxia condition. Data are mean ( SD (n = 9). Means among bars without a common letter (a, b) differ, P < 0.05.
Figure 2. Effect of oleanolic acid (OA), ursolic acid (UA) and maslinic acid (MA) at 0 (control), 2 or 4 μmol/L upon HIF-1R mRNA expression in human Hep3B, Huh7 and HA22T cell lines. Data are mean ( SD (n = 9). Means among bars without a common letter (a-c) differ, P < 0.05.
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Figure 3. Effect of oleanolic acid (OA), ursolic acid (UA) and maslinic acid (MA) at 0 (control), 2 or 4 μmol/L upon bFGF mRNA expression in human Hep3B, Huh7 and HA22T cell lines. Data are mean ( SD (n = 9). Means among bars without a common letter (a) differ, P < 0.05.
Figure 4. Effect of oleanolic acid (OA), ursolic acid (UA) and maslinic acid (MA) at 0 (control), 2 or 4 μmol/L upon VEGF level (upper part) and mRNA expression (lower part) in human Hep3B, Huh7 and HA22T cell lines. Data are mean ( SD (n = 9). Means among bars without a common letter (a-d) differ, P < 0.05. chive (Allium tuberosum Rottler), fennel (Foeniculum vulgare), basil (Ocimum basilicum L.) and daylily (Hemerocallis fulva L.), were used to analyze the content of OA, UA and MA. These vegetables, harvested in
summer of 2010, were purchased from farms in the central area of Taiwan. The content of three triterpenic acids in these vegetables was analyzed by HPLC methods described in Liu et al. (17).
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Figure 5. Effect of oleanolic acid (OA), ursolic acid (UA) and maslinic acid (MA) at 0 (control), 2 or 4 μmol/L upon IL-8 level (upper part) and mRNA expression (lower part) in human Hep3B, Huh7 and HA22T cell lines. Data are mean ( SD (n = 9). Means among bars without a common letter (a-d) differ, P < 0.05. Cell Culture. Human normal liver cell line (L-02 cell) and liver cancer Hep3B, Huh7 and HA22T cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). All cells were cultured in RPMI 1640 medium, containing 10% fetal bovine serum (BSA), 100 units/mL of penicillin and 100 units/mL of streptomycin (pH 7.4). Hypoxia (1% O2) was created using an anaerobic jar (AnaeroPack Series, Mitsubishi Gas Chemical, Tokyo, Japan) with an AnaeroPack disposable O2-absorbing and CO2-generating agent. The culture medium was changed every three days, and cells were subcultured once a week. A phosphate buffer saline (PBS, pH 7.2) was added to adjust the cell number to 105/mL for various experiments and analyses. Experimental Design. A stock solution of OA, UA or MA was prepared in dimethyl sulfoxide (DMSO) and diluted with medium. An equal volume of DMSO (final concentration 0.05). Three triterpenic acids dosedependently decreased production and mRNA expression of VEGF and IL-8 in test cell lines (Figures 4 and 5, P
