2015 Volume 21 Issue 6 Pages 879-882
We previously reported on meyerin (trans-4-hydroxycinnamic acid derivative) and 7-methoxy-5-prenyloxycoumarin, which are abundantly found in Meyer lemon (Citrus meyeri) relative to other citrus fruits. In the present study, the antitumor-promoting activities of these compounds were examined using the in vitro Epstein-Barr virus early antigen (EBV-EA) activation test and the in vivo two-stage mouse skin carcinogenesis test. 7-Methoxy-5-prenyloxycoumarin was suggested to have inhibitory activity on tumor promotion in the EBV-EA test, whereas meyerin exhibited weak activity. Furthermore, 7-methoxy-5-prenyloxycoumarin significantly inhibited mouse skin tumor promotion relative to control in the two-stage carcinogenesis test. These results indicate that 7-methoxy-5-prenyloxycoumarin has antitumor-promoting activity in vitro and in vivo.
Chemotherapy to inhibit tumor promotion has been regarded as one means of cancer control. It has been the focus of attention because tumor promotion is long-term process and is the only reversible process during the multiple stages of carcinogenesis (Hennings et al., 1993; Wattenberg et al., 1993). The Epstein-Barr virus early antigen (EBV-EA) activation test is a convenient in vitro assay for detecting naturally occurring antitumor-promoting activity. Furthermore, effective compounds have also been evaluated in vivo for their antitumor-promoting activities using mouse skin tumor promotion in the two-stage carcinogenesis test (Tokuda et al., 1986).
Citrus fruits have been reported to contain numerous bioactive compounds (González-Molina et al., 2010). The compounds in Meyer lemon (Citrus meyeri) have also attracted attention, and we have reported on the isolation of meyerin (trans-4-hydroxycinnamic acid derivative) as a novel antioxidant (Miyake et al., 2012) and 7-methoxy-5-prenyloxycoumarin (Mityake et al., 2013), both of which are abundantly contained in Meyer lemon compared to other citrus fruits. The present study employed the EBV-EA activation test and the in vivo two-stage mouse skin carcinogenesis test to evaluate the ability of meyerin and 7-methoxy-5-prenyloxycoumarin to inhibit tumor promotion.
Test compounds Meyerin and 7-methoxy-5-prenyloxycoumarin were isolated from Meyer lemon following the procedures described in previous reports (Miyake et al., 2012; 2013). Their chemical structures are shown in Fig. 1.
Chemical Structures of Meyerin and 7-Methoxy-5-prenyloxycoumarin.
EBV-EA activation test The inhibition of EBV-EA activation was assayed using the method described previously (Itoigawa et al., 2004). Raji cells were grown to a density of 106 cells/mL, harvested by centrifugation and resuspended in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% FCS containing 4 mM n-butyric acid as an inducer, 32 pmol of 12-O-tetradecanoylphorbol-13-acetate (TPA), and 32, 16, 3.2 or 0.32 nmol of the test compound (a DMSO solution). Each DMSO sample solution was put into an assay tube and incubated at a final concentration of 0.5% DMSO medium throughout this experiment. The cells were incubated at 37°C for 48 h, and cell number and viability were determined after 48 h with a hemocytometer using the Trypan Blue staining method. A minimum 60% survival rate for the Raji cells after treatment with the compounds was required for an accurate result. EBV activation was estimated by detection of EA using the indirect immunofluorescence method. The EBV-EA inhibitory activity of each test compound was estimated on the basis of the percentage of positive cells compared to that observed in a control without the test compound. The IC50 (50% inhibitory concentration) value in each assay was estimated by the probit transformation technique. Each assay was repeated three times for each test compound.
Two-stage mouse skin carcinogenesis test The two-stage mouse skin carcinogenesis test was assayed using the method described previously (Itoigawa et al., 2002). Female ICR mice were obtained at 5 – 6 weeks of age from SLC Co. (Shizuoka, Japan). Groups of animals (15 animals per group) were housed in subgroups of five in polycarbonate cages. The mice were permitted free access to an MF solid diet (Oriental Yeast Co., Chiba, Japan) and drinking water at all times during the study. The back of each mouse was shaved with surgical clippers before the first day of initiation. A tumor was initiated on the back of each mouse with dimethylbenz[a]anthracene (DMBA; 390 nmol) in acetone (0.1 mL). One week after initiation, the tumor was promoted twice a week by applying TPA (1.7 nmol) in acetone (0.1 mL). The mice in the treatment groups were treated with the compounds (85 nmol) in acetone (0.1 mL) for 1 h before each TPA treatment. The incidence of papillomas was observed weekly for 20 weeks. The animal experiment protocol was approved by the Institute for Experimental Animals of Kanazawa University Advanced Science Research Center. Statistical significance was evaluated using a t-test and differences at p < 0.05 were considered to be significant.
EBV-EA activation test The inhibitory effects of meyerin and 7-methoxy-5-prenyloxycoumarin based on the in vitro EBV-EA activation test are shown in Table 1. A minimum 60% survival rate for the Raji cells is shown for samples with a concentration of less than 1,000 (mol ratio/32 pmol TPA). The IC50 value for 7-methoxy-5-prenyloxycoumarin (IC50; 361) was lower than that for meyerin (IC50; 534). While the IC50 value for 7-methoxy-5-prenyloxycoumarin was approximately that for curcumin (IC50; 341), a typical antitumor-promoting agent (Nakamura et al., 1998), the IC50 value for 7-methoxy-5-prenyloxycoumarin was slightly higher. 7-Methoxy-5-prenyloxycoumarin was suggested to exhibit antitumor-promoting activity in the in vitro EBV-EA test. We previously reported that the prenyl group on benzaldehyde (Miyake et al., 2010) and terpenoid coumarins (Miyake et al., 1999; Ito et al., 2005; Iranshahi et al., 2008) was related to the antitumor-promoting activity in the EBV-EA activation test. We consider that the O-prenyl group at the C-5 position on 7-methoxy-5-prenyloxycoumarin is related to the expression of activity in a similar manner to the reported compounds.
| EBV-EA-positive cells (% viability) | |||||
|---|---|---|---|---|---|
| Compound concentration (mol ratio/32 pmol TPA) | IC50b | ||||
| Compound | 1000 | 500 | 100 | 10 | (mol ratio/32 pmol TPA) |
| Meyerin | 11.6 ± 0.7c (60d) | 60.0 ± 1.7 (>80) | 83.8 ± 2.5 (>80) | 100 ± 0.2 (>80) | 534 |
| 7-Methoxy-5-prenyloxycoumarin | 0 ± 0.3 (60) | 42.5 ± 1.3 (>80) | 67.4 ± 2.3 (>80) | 100 ± 0.3 (>80) | 361 |
| Curcuminc | 0 ± 0.2 (60) | 22.8 ± 0.6 (>80) | 81.7 ± 1.8 (>80) | 100 ± 0.3 (>80) | 341 |
Two-stage mouse skin carcinogenesis test The inhibitory effect of meyerin and 7-methoxy-5-prenyloxycoumarin on tumor promotion in the in vivo two-stage mouse skin carcinogenesis test was examined (Fig. 2). The 7-methoxy-5-prenyloxycoumarin–treated group showed a significant suppressive effect on the average number of papillomas per mouse after 20 weeks relative to the control group (p < 0.05). 7-Methoxy-5-prenyloxycoumarin showed antitumor-promoting activity in the in vivo two-stage mouse skin carcinogenesis test. While the meyerin–treated group did not exhibit a significant suppressive effect on papilloma expression relative to the control, the tendency for a suppressive effect was observed. Reactive oxygen species (ROS) are known to play an important role in mutagenesis and carcinogenesis, particularly in tumor promotion, and antioxidants are shown to have antitumor-promoting activity in mouse skin (Perchellet et al., 1989). In the present study, meyerin, an antioxidant, exhibited weak inhibitory activity on tumor promotion. It is proposed that meyerin may have weak scavenging activity against ROS.
Inhibitory Effect of Meyerin and 7-Methoxy-5-prenyloxycoumarin on DMBA-TPA Mouse Skin Carcinogenesis.
Tumors were initiated in all mice with DMBA (390 nmol) and promoted with TPA (1.7 nmol) twice weekly starting 1 week after initiation. After 20 weeks of tumor promotion, the number of papillomas per mouse differed between the 7-methoxy-5-prenyloxycoumarin-treated group and the control group (p < 0.05).
*Significantly different (p < 0.05) compared to the control group by a Student's t-test.
Meyerin was reported to be present in the flavedo (1.47 mg/g) and alvedo (3.31 mg/g) of Meyer lemon peel; while 7-methoxy-5-prenyloxycoumarin is contained in the flavedo (0.087 mg/g) (Miyake et al., 2012; 2013). The potential intake of this compound from Meyer lemon is high because meyerin is abundantly found compared to 7-methoxy-5-prenyloxycoumarin, although meyerin exhibited weak inhibitory activity on tumor promotion in the present study. 7-Methoxy-5-prenyloxycoumarin is reported to be a bioactive compound, with inhibitory activity on acetylcholinesterase (Kang et al., 2001). In the present study, 7-methoxy-5-prenyloxycoumarin was shown to have an antitumor-promoting effect for the first time using the in vitro EBV-EA activation test and the in vivo two-stage mouse skin carcinogenesis test.
Epstein-Barr virus early antigen
TPA12-O-tetradecanoylphorbol-13-acetate
