Cancer Letters, 30 (1986) 143-151 Elsevier Scientific Publishers Ireland Ltd.

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SEARCH FOR POSSIBLE ANTITUMOR PROMOTERS BY INHIBITION OF 12.O-TETRADECANOY LPHORBOL-1SACETATEINDUCED EPSTEIN-BARR VIRUS ACTIVATION; URSOLIC ACID AND OLEANOLIC ACID FROM AN ANTI-INFLAMMATORY CHINESE MEDICINAL PLANT, GLECHOMA HEDERACEAE L.

HAJIME OHIGASHIa, HITOSHI TAKAMURAa, KOICHI KOSHIMIZ@* , HARUKUNI TOKUDAb and YOHEI ITOb

=Department of Food Science and Technology, Faculty of Agriculture and bDepartment of Microbiology, Faculty of Medicine, Kyoto University, Kyoto 606 (Japan)

(Received 31 October 1985) (Accepted 27 November 1985)

SUMMARY

From an anti-inflammatory Chinese medicinal plant, Glechoma hederuceae L., two triterpene carboxylic acids, ursolic acid (UA) and oleanolic acid (OA) have been isolated as inhibitors of 12-0-tetradecanoylphorbol-13- acetate (TPA) induced Epstein-Barr virus (EBV) activation in Raji cells. Both acids significantly inhibited the activation at a lOOO-fold molar ratio to TPA, and also teleocidin B-4. The dose responses of the acids were very similar to those of the antitumor promoters, retinoic acid (RA) and glycyr- rhetinic acid (GA). However, a characteristic property that UA and OA possess, far higher cell viability to the Raji cells than RA to the Raji cells, has been pointed out. Furthermore, enhancement of the inhibitory activity was found in 3-keto derivatives of UA and OA, while either loss of oxygen functionality at C-3 position of UA or oxidation at C-3 of GA led to reduc- tion of the activity. Binding assay suggested that the inhibitory activity should be exhibited by some event caused after binding of TPA to the receptor in the cells.

INTRODUCTION

Recent estimation of short-term detection methods of tumor promoters has led to finding tumor promoters in the human environment [1,6,8,9,11, 13,17,19,23,27]. Concurrently, compounds which act as inhibitors in each method have been found and some of them have been identified as anti-

*To whom a11 correspondence should be addressed.

0304-3835/86/$03.50 0 1986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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tumor promoters. Antitumor promoters include the following compounds: (1) retinoic acid (RA) and related compounds [25,26] ; (2) anti-inflam- matory steroids (fluocinolone acetonide etc.) [ 261; (3) some protease inhi- bitors (leupeptins etc.) [lo] ; (4) polyamine synthesis inhibitors (difluoro- methylornithine (DFMO) etc.) [21]; (5) prostaglandin synthesis inhibitors (indomethacin, naproxen etc.) [24]; (6) analogues of promoters such as phorbol esters with short chain fatty acid residues [20]; (7) others [2,3, 15,161. Thus, antitumor promoters extend over several types of compounds, and hence the mode of action must vary from compound to compound. Therefore, the preparation of novel antitumor promoters has been antici- pated not only in a clinical sense but also in the elucidation of the mech- anism of the complex promotion stages.

To search for possible antitumor promoters, we have seen that using a’ test system in which inhibition of TPA-induced Epstein-Barr virus (EBV) activation in Raji cells [ 131 is measured, is very effective, as reported by Okamoto et al. [ 181. Our first trial of the screening for the inhibitors was directed toward Chinese medicinal plants with anti-inflammatory action, because most antitumor promoters cause inflammation in animal skin [ 20,221. Systematic studies with this test system were successful to purify possible antitumor promoters from one of the Chinese medicinal plants, Glechoma hederaceae L. (Labiatae). This paper reports on the inhibitors of the plant and their effect on EBV activation.

MATERIALS AND METHODS

Chemicals and chromatographic materials TPA and phorbol-12,13-dibutyrate (PDBu), RA, GA and n-butyric acid,

and [ 3H]phorbol-l 2,13-dibutyrate ( 3H-PDBu) were purchased from Chemi- cal Carcinogenesis, Inc., (MN, U.S.A.), Sigma Chemical Co., Ltd., (MO, U.S.A.), Nakarai Chemical Ltd., (Kyoto, Japan) and New England Nuclear (MS, U.S.A.), respectively. Ursolic acid (la) and oleanolic acid (2a) sub- mitted to derivatization were those isolated from Arctostupylos uva-ursi L. Sprenel, in which the content of la was found to be high 1141, and was kindly given by Dr. Hiroyuki Inoue at the Department of Pharmaceutical Science, Kyoto University, respectively. Teleocidin B-4 was isolated from Streptoverticillium blastmyceticum NA34-17 as described previously [ 121. The following chromatographic materials were used: Wako gel C-100 (Wako Pure Chemical Industries, Ltd., (Kyoto, Japan), ODS gel (Yamamura Chro- matograph Co. Ltd., Kyoto, Japan) and silica gel H (Type 60, Merck, Darmstadt, F.R.G.).

Bioassay The EBV genome-carrying human lymphoblastoid cells, Raji, were culti-

vated in RPM1 1640 medium (Nissui) as described previously [ 131. The inhi- bition of EBV activation was assayed basically using the same method as

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for short-term detection of promoters. The cells were incubated for 48 h in 1 ml of a medium containing n-butyric acid (88 Mg), TPA (40 ng) and a known amount of test compound. The experiments were repeated twice. The average EA induction was compared to that of control experiments only with n-butyric acid (88 fig) and TPA (40 ng), in which EA induction was ordinarily around 30%.

Isolation of the inhibitors Isolation of inhibitors was guided by the bioassay. Dried whole plant of

G. hederacea L. (150 g) was extracted with methanol for 2 weeks at room temperature. After evaporation of the solvent in vacua, the concentrate was partitioned with ethyl acetate and water. The ethyl acetate soluble fraction (4.0 g) was chromatographed on Wako gel C-100 with benzene/ ethyl acetate and then on ODS gel with 90% methanol to give a solid which was treated with diazomethane followed by acetic anhydride/pyridine. The mixture thus obtained was separated by column chromatography on silica gel H eluted with cyclohexane/benzene to give two crystalline com- pounds, Id and 2d. Hydrolysis of both compounds with t-BuOK in di- methylsulfoxide (DMSO) [ 51 (lb and 2b) followed by methanolic KOH gave pure free hydroxy-acids, ursolic acid (UA, la) (25 mg) and oleanolic acid (OA, 2a) (54 mg). UA was also isolated from the extract of A. uua-ursi L. Sprengel [ 141 by recrystallization from the fraction which was obtained by purification on Wako gel and ODS gel, and used for the derivatization.

Binding assay Binding assay was performed using a modified method on Blumberg’s

[4]. Duplicate experiments for each sample were run in a final volume of 1 ml of the binding buffer (pH 7.5), consisting of 100 mM Hepes, 120 mM NaCl, 5 mM KCl, 1.2 mM MgS04, 15 mM CH&OONa, 10 mM glucose, 1 mM EDTA and 1 mg BSA, in disposable tubes (17 X 100 mm). The cells (5 X 106), washed once with the buffer, were incubated with various con- centrations of unlabelled compound for 30 min at 4°C. In the case of TPA plus inhibitor, cells were first incubated with various concentrations of inhibitor for 30 min at 4% and then 5 pg of TPA was added to the medium and incubated for 30 more min at 4°C. Five nanograms of 3H-PDBu was then added to the tube and further incubated for 3 h at 4°C. Immediately after incubation, 0.3 ml of the binding cell suspension was transferred to an Eppendorf tube, containing 0.5 ml of phosphate buffered saline. The cells were pelleted at 3000 rev./min for 10 min in a table centrifuge. The super- natant was carefully removed by decantation,.and the tip of the tube was cut off into a scintillation vial. Three milliliters of Biofluor scintillation liquid (New England Nuclear, MS, U.S.A.) was added and the vial was vigorously shaken. Radioactivity was determined in an Aloka LSC 903. All values were corrected for quenching and non-specific binding of 3H- PDBu was determined in the presence of 30 pg of unlabelled PDBu.

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Deriva tization of ursolic acid, oleanolic acid and glycyrrhetinic acid [5] Acetylation of UA and OA with acetic anhydride-pyridine, and methyl-

ation of UA and OA with diazomethane afforded lb and 2b, and lc and 2c, respectively. Oxidation of UA, OA and GA with Cornforth reagent yielded 3-keto-UA (lh), 3-keto-OA (2h) and 3-keto-GA (3h), respectively. Treat- ment of lh with aluminium tri-isopropoxide in isopropanol gave 3-epi-UA (lg). Reduction of lc with lithium aluminium hydride gave If. Wolf-Kishner reduction of methyl 3-keto-UA followed by treatment with t-BuOK-DMSO gave 3-deoxy-UA (li), and pyrolysis of 3-0-tosyl-UA in pyridine yielded dehydro-2,3-ene-UA (lj). Hydrolysis of methyl 3-0-methyl-UA which was prepared by methylation of UA with methyl iodide/sodium hydride in dimethyl formamide, gave le. All of the derivatives were chromato- graphically purified and identified by MS, IR and ‘H-NMR.

RESULTS

The inhibitors isolated from G. hederaceae L., a Chinese medicinal plant with anti-inflammatory action, were identified as ursolic acid (UA, la) and oleanolic acid (OA, 2a), respectively, by the direct comparison of the spectral data with those of authentic specimens [ 71.

\ R2

RI J:-‘ll I\ \

lo, Rl=/+OH. H. R2=COOH If, RI-a-ONH. R2=CH2OH

lb, R.p-OAc,H, R2.COOH 19, RI~~-OH*H* R~=COOH

:;; ;::;;;;;;, ;:zz;: ::: ;:::.H,

R2=COOH

R2=COOH

la, W=jFOMe.H. R2=COOH

20, RI =/J-OH. H , RI-COOH

2b. RI -p-0Ac.H. R2=COOH

2~. RI =j-0H.H. Rr-COOMe

2d, RI =)I -OAc, H , R2-COOMe

2h. RI =O, W-COOH R2.

31, RI=/MH.H. R2=COOH

3h, RI-O. R2=COOH

147

10-l I IO IO2 IO3

Mot ratio (test cornpound/rPA)

IO-’ I IO Id 103

Mel rotiobst compoundfleleocidin B-4)

Fig. 1. Inhibitory activity and cell viability of the inhibitors. (a) Against EA-induction by TPA at 40 ng/ml (65 nM). (b) Against EA-induction by teleocidin B-4 at 40 ng/ml (89nM). UA(o);OA (o);GA(a);RA(r).

The inhibitory effects of UA and OA on EBV EA-induction caused by TPA at 40 ng/ml (65 nM) are shown in Fig. la. Application of’40 r.lg (88 PM) of either UA or OA completely inhibited EA-induction and the inhi- bitory activities reduced in a dose-dependent manner. Both acids also inhi- bited the EA-induction caused by another potent tumor promoter, teleo- cidin B-4 [ 121 at 40 ng/ml(89 nM), in a similar manner as shown in Fig. lb. The inhibitory activities of UA and OA are comparable with those of known antitumor promoters, RA [25] and GA (3a) [16,18]. UA, OA and GA preserved high viability of the cells even at high dose (40 pg), while the viability of RA was very low at that dose (see the broken lines in Fig. la,b).

The inhibitory activities of the derivatives of UA, OA and GA were also tested. Covering the hydroxy or/and carboxyl group(s) (lb-le, 2b-2d), reduction of the carboxyl group to a primary alcohol (If) and epimeriz- ation at C-3 (lg) showed no remarkable change in the activity from the original acids (data not shown). However, oxidation of the hydroxy group at C-3 of UA and OA enhanced the activity 100 times, as shown in Fig. 2a, though the cell viabilities fell. Interestingly, an oxidative product (3h) of GA and deoxygenated compounds (li and lj) at C-3 of UA resulted in the reduction of the activity (Fig. 2b).

To approach the inhibition-mechanism of the acids, UA, OA and their oxidative products (lh and 2h) were tested against binding effect [4] to TPA receptor in Raji cells. As shown in Fig. 3a, none of the acids inhibited the specific binding of 3H-PDBu at the concentrations tested (2.2 mM- 2.2 nM), while TPA significantly inhibited it (I& = 8.1 PM). Furthermore, the inhibition of 3H-PDBu binding by TPA at a concentration of 5 @g/ml

148

. IO' I IO IO2 lo3

Mol rotio hat compound/Tl%) Mel rotb (test canpound/TPA)

Fig. 2. Inhibitory activity and cell viability of the derivatives. (a) Against EA-induction by TPA at 40 ng/ml(65 nM). 3-Keto-UA (0); 3-keto-OA (0). (b) Against EA-induction by TPA at 40 ng/ml (65 nM). Dehydro-2,3-ene-UA (0); 3-deoxy-UA (0); 3-keto-GA (a).

2 ioo-

5 q -m~a+e,*_&

(a) cl

’ SO- 55 \ cl

@ 5 5 60- I \

8 cl

a. I 40-

I *I \

:g

1 20

\ cl ‘O-0

I I I I I I I I

10-3 10-Z to-1 I IO I02 103

Concsntrotion (PM)

I- FIOC

&

= 80 35

c” 5 ~60

i4o

,I 0 E Y 20 I%

(b)

~-0-p-0-9

I L I I ,

lo-2 lo-1 I IO I02

Mol ratio (test compound/TPA)

Fig. 3. Binding effect of the inhibitors to Raji cells. (a) Specific 3H-PDBu-binding in the presence of either TPA or inhibitor. TPA (0); UA (0); OA (0); 3-keto-UA (a); 3-keto- OA (A). (b) Specific ‘H-PDBu-binding in the presence of both TPA (5 pg) and various amounts of inhibitor. UA (0); OA (0).

149

(I(&,) was measured in the presence of several amounts of UA and OA. The results, shown in Fig. 3b, indicated that UA and OA did not affect the inhibition of 3H-PDBu binding by TPA at all. The data described above were in agreement with those of RA and GA (data not shown).

DISCUSSION

The triterpene carboxylic acids, UA and OA, which have been isolated from G. hederuceae, significantly inhibited the EBV EA-induction by tumor promoters, TPA and teleocidin B-4. The levels of the activity were almost equal to those of RA and GA, estimated as antitumor promoters. Present experiments, however, reveal that the inhibitors classified as triterpene carboxylic acid possess high viability at least to Raji cells, which is far superior than RA to Raji cells.

On the basis of the inhibitory activities of UA-, OA- and GA-derivatives, the following structural requirements for the activity on EA-induction have been proposed; the oxygen functionality at C-3 should be important and elevation of oxidation-stage at C-3 (lh and 2h) enhances the activity, while further oxidation on other ring system(s) (3h) should be unfavorable.

The facts that UA and OA never inhibited the specific binding of 3H- PDBu to the cells and did not affect the inhibition of 3H-PDBu-binding by TPA at all, indicated that UA and OA should act on some event(s) after binding of tumor promoters to the receptor(s).

The present investigation by an in vitro short-term assay strongly suggest- ed that UA and OA may be valuable antitumor promoters. Since the distri- bution of triterpene carboxylic acids, such as UA and OA are ubiquitous in the plant kingdom, antitumor promoters may also be rich in the human environment.

Initiation-promotion tests in vivo are now under study and the results will be reported elsewhere.

ACKNOWLEDGEMENT

We thank Dr. Hiroyuki Inoue, Faculty of Pharmaceutical Sciences, Kyoto University for his kind supply of authentic UA and OA. This study was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan (No. 60015034 for K.K.).

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