Geraniol, an Inhibitor of Mevalonate Biosynthesis ...

Biochemical and Molecular Roles of nutrients

Geraniol, an Inhibitor of Mevalonate Biosynthesis, Suppresses the Growth of Hepatomas and Melanomas Transplanted to Rats and Mice1Â»2 SUZAHNE Department

G. YÃœ,LESLIE

A. HILDEBRAHDT3

of Nutritional Sciences,

(Jniuersity of Wisconsin,

Madison, Madison,

Wl 53706

â€¢meualonate biosynthesis â€¢tumor growth â€¢raÃ¯s â€¢mice â€¢chemopreuention

dietary geraniol (6.5 mmol/kg). The sensitivity of B16 melanoma cells to geraniol was fivefold that of the P388 cells (Shoff et al. 1991). These findings led us to investigate the impact of dietary geraniol on the

End products of plant mevalonate metabolism in cluding assorted monoterpenes, sesquiterpenes, diterpenes, triterpenes, carotenoids and tocotrienols sup press cholesterol synthesis and tumor cell proliferation (Elson 1995, Elson and Yu 1994). These seemingly di verse actions converge in a metabolic sequence yield ing geranyl pyrophosphate, the precursor shared by all isoprenoid end products of plant mevalonate metab olism, cholesterol and by two intermediate products of the isoprenoid pathway, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. The pathway provides targets through which physiological and synthetic

' Supported by NIH grant CA 49416'and

the College of Agri

cultural and Life Sciences, University of Wisconsin. 1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact. 3 Present address: Department

of Food and Nutrition,

Arizona University, Flagstaff, AZ 86011. 4 To whom correspondence should be addressed.

1 March 1995. Revision accepted 10 July 1995

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KEY WORDS:

0022-3166/95 $3.00 Â©1995 American Institute of Nutrition. Manuscript received 30 January 1995. Initial review completed

E. ELSON4

agents modulate the growth of insects, plants, yeasts, fungi and bacteria and the synthesis of cholesterol. The pathway potentially offers targets for cancer che motherapy because the two intermediate products are required for the posttranslational modification of the G proteins including p21 ras, prelamin A and lamin B, each of which plays an essential role in cell prolif eration (Cuthbert and Lipsky 1995, Goldstein and Brown 1990, Maltese 1990). Inhibition of farnesyl pyrophosphate synthesis by lovastatin, a competitive inhibitor of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity and concomitantly of cholesterol synthesis, inhibits the isoprenylation (and function) of ras and the nuclear lamins. However, the inhibitory dose of lovastatin is toxic to normal cells (Shack et al. 1994). Dietary geraniol suppresses hepatic HMG-CoA re ductase activity and concomitantly lowers serum cho lesterol levels of experimental animals (Fitch et al. 1989) and humans (Elson et al. 1989). This generally regarded as safe (GRAS) substance is approved for use as a food additive (FEMA 2624). The monoterpene had a concentration-dependent, mevalonate-reversed im pact on the proliferation of murine P388 cells. The survival time of mice after the intraperitoneal trans plant of the ~ras P388 cells was extended by 50% with

ABSTRACT Farnesyl-pyrophosphate is required for the posttranslational modification of G proteins includ ing p21 ras, prelamin A and lamin B, each of which plays an essential role in cell proliferation. As a con sequence, competitive inhibitors of mevalonate syn thesis, the rate-limiting substrate for the synthesis of the prenyl-pyrophosphates, arrest cultured cells at the Gl/S interface of the cell cycle and initiate apoptotic cell death. Geraniol, an acyclic monoterpenoid alcohol, suppresses 3-hydroxy-3-metnylglutaryl coenzyme A (HMG-CoA) reductase activity and concomitantly ar rests the growth of cultured tumor cells. We evaluated the impact of dietary geranio! on the growth of two tumors. In the first study, geranio! (23 mmol/kg diet, 350 Â¿tmol/d)was fed to male buffalo rats for 14 d before and for 42 d after the transplant of Morris 7777 hepatomas. Tumor growth was suppressed (P < 0.001). In the second study, the dose-dependent im pact of geranio! on the growth of B16 melanomas was assessed. Dietary geranio! (0.65, 6.5 and 65 mmol/kg diet) was fed to female C57BL mice for 14 d before and for 21 d after tumor transplant. Tumor growth was suppressed (P < 0.02) by 6.5 and 65 mmol geranio!/ kg diet. J. Nutr. 125: 2763-2767, 1995. INDEXING

AND CHARLES

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YU ET AL.

growth pattern of two aggressive, +ras, solid tumors, the Morris hepatoma 7777 and B16 melanoma. MATERIALS AND METHODS

RESULTS Morris hepatoma 7777. Palpable tumors (0.29 Â±0.09 cm3) were detected in all control rats and in four (0.07 Â±0.4 cm3) of the six rats in the geraniol group on d 10 after tumor transfer. The linear regres sion coefficients (Fig. 1) suggest that the growth of tu mors in rats receiving geraniol was 20% that of the controls (P < 0.001). Exponential growth curves for the tumors are plotted on Figure 1. One control rat, moribund on d 24, was euthanized at the recommen dation of the animal caretaker. The estimated volumes of the hepatomas in the control and geraniol groups on d 27 after transplant were 22.1 Â±2.7 cm3 (n = 5) and 3.7 Â±0.9 cm3 (n = 6). Rats with small tumors (< 20 cm3; two controls and all expÃ©rimentais)were continued on the study. On d 36 after transplant, the estimated volumes of the tumors grown in the two control and six experimental rats were 24.7 Â±7.1and 14.9 Â±3.22 cm3. Three experimental rats were con tinued on the study; the tumor volume on d 42 was 16.4 Â±2.2 cm3. At the time of tumor transfer the body weight of the rats in the geraniol group was 97% that of the weight of the control group (P < 0.10). This difference in body weight was maintained through d 20 after transplant. In the absence of quantitative measures, the animal caretaker noted that the experimental rats were more active and sleeker in appearance than the controls. Control rats developed rough hair coats and tended to remain at the rear of their cages when feed cups were replaced. B16 melanoma. Groups of mice were fed diets containing 0, 0.65, 6.5 or 65 mmol geraniol/kg for 14 d before and for 21 d after tumor transplant. The body weight of the high geraniol group was 10% lower (P < 0.01) than those of the other groups at the time of tumor transplant and at the termination of the trial (Table 1). All mice survived the 35-d feeding period


Morris hepatoma 7777. Viable tissue, dissected from 10 g Morris hepatoma 1111, was minced and passed through a 500-^m screen by gentle rubbing with a rubber policeman and an Eagle's modified essential medium (GIBCOBRL, Grand Island, NY) rinse. The slurry was gently centrifuged (3 min at 10 X g). The soft pellet was resuspended in Eagle's modified essen tial medium (0.16 mL pellet/mL). Aliquots of the tis sue suspensions (0.2 mL; 2 X IO10cells/L) were injected subcutaneously using a 22-gauge needle into the left hind limbs of 150-g male buffalo rats (HarÃ-anSpragueDawley, Madison, WI). The individually caged rats were fed a control (AIN-76A, Teklad Test Diets, Mad ison, WI) or experimental (AIN-76A with 23 mmol geraniol/kg diet) diet for 14 d before and after tumor transplant. Geraniol (98%) was purchased from Aidrich Chemical, Milwaukee, WI. Tumor growth was assessed daily with caliper measures of cross-sectional axes, and tumor volume was calculated with the for mula for a cylinder. On d 27 after tumor transplan tation, rats with tumors estimated to be 20 cm3 or larger were killed by CO2 overdose and tumors re moved and weighed. Animals with tumors less than 20 cm3 were continued on the study. The study was terminated at d 42 after tumor transplant. The pro tocol was reviewed and approved by College of Agri cultural and Life Sciences Animal Care Committee. In accord with the protocol, a rat showing signs of distress was killed by CO2 overdose. Murine BlÃ©melanoma. Weanling C57BL female mice (Harlan-Sprague Dawley, Madison, WI) were housed in groups of four on wood shavings in plastic cages. The mice were fed AIN-76A diet for 14 d before and for 21 d after the transplantation of B16(F10) mel anoma cells. The experimental groups received 0.65, 6.5 or 65 mmol geraniol/kg diet. The cells, cultured and harvested as previously described (Shoff et al. 1991), were washed twice with RPMI 1640 (GIB COBRL) containing 10% fetal bovine serum (GIB COBRL). Cell counts were made (98% viable) after a 1:20 dilution in 10% trypan blue. The cells were fur ther diluted in RPMI 1640 (1 X IO8 cells/L), and 0.1 mL of the suspension (1 X IO4cells) was injected sub cutaneously into the flank of each mouse. The cross-sectional axes of the tumors were mea sured daily (d 15-20) with calipers. The mice were killed by CO2 overdose and the tumors were excised and weighed. The protocol was reviewed and approved by College of Agricultural and Life Sciences Animal Care Committee. Statistical methods. StatView software (Abacus Concepts, Berkeley, CA) was used for the analysis of

treatment-mediated effects. Treatment-mediated dif ferences in body and tumor weights were identified with ANOVA and Fisher's Protected Least Significant Difference test (Abacus Concepts 1992). Estimates of tumor surface area, spherical volume and cylindrical volume were calculated. The area and volume esti mates calculated for each tumor on the day the host was killed were plotted against the weight of the ex cised tumor. The estimates of cylindrical volume and surface area were most highly correlated, respectively, with hepatoma (r = 0.80, P < 0.01) and melanoma (r = 0.75, P < 0.01) weight. Estimates of hepatoma vol ume and melanoma area for individual tumors were plotted over time and treatment-mediated differences in linear regression coefficients were evaluated by ANOVA.

GERANIOL

SUPPRESSES TUMOR

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DISCUSSION

1

is

5 -

Dayspost transfer

A NO VA of the linear a geraniol-mediated

Geraniol 0 23

n

regression suppression

coefficients of tumor

revealed growth1

Intercept 0.869 Â±0.173a 0.754 Â±0.044 -10.44 Â±2.11 -2.74 Â±0.780 0.177Â±0.070b 0.638 Â±0.051

1 Values are means Â±SEM.Mean regression values not sharing a superscript are significantly different |P < 0.001). ^ One control rat was euthanized after the tumor measurement on d 24.

free of other signs of geraniol toxicity. Geraniol slowed the growth of the transplanted tumors (Table 1). On determining that the threshold for the dietary geraniol action falls between 0.65 and 6.5 mmol/kg diet, we combined the control and low geraniol groups and re peated the analysis of variance. Group differences were significant (P < 0.02). Tumor surface area at 21 d after transplant was sig nificantly correlated with tumor weight (r = 0.75, P < 0.01). Using tumor surface area as a predictor of tumor mass we examined the dose-dependent impact of dietary geraniol on the rate of tumor growth (Fig. 2). It should be noted that the plot of tumor growth in controls might reflect swelling or investigator bias. The linear growth plot is consistent with the finding that 0.01% dietary geraniol had no impact on final tumor weight (Table 1). A comparison of the plots confirms that tumor growth in mice receiving 6.5 mmol geraniol/kg diet was significantly slower than that of tumors in mice receiving 0.65 mmol geraniol/ kg diet.

aniol. Inhibition of the isoprenylation of the nuclear lamins triggers apoptosis (Perez-Sala and Mollinedo 1994). A key feature of the DNA histogram of apoptotic cells is the presence of a sub-Gl peak (Hotz et al. 1994). The presence of a prominent sub-Gl peak on the DNA histogram of B16 melanoma cells treated with geraniol (Fig. 7, Shoff et al. 1991) suggests that the tumor-suppressive action of this isoprenoid, like that of lovastatin (Perez-Sala and Mollinedo 1994), involves the initiation of apoptotic tumor cell death. Other than modestly reducing blood cholesterol levels, these isoprenoids have no impact on tumor-free animals and humans (Elson 1995).

TABLE 1

Impact of dietary geraniol on the growth of Blf melanomas in female CS7BLmice. The diets were fed for 14 d before and for 21 d after tumor transplant1 Mice Geraniolmmol/kg

dg17.08

dg21.

diet00.65

6.50 65.0n138

0.40a17.08 + 0.55a20.83 62 Â± 0.39a2.55 Â± Â±0.42a Â±0.39a + 0.50a 8121417.20 + 0.28a 21.36 Â±0.47a 1.63Â±0.40b 15.38 + 0.34b35 18.73 Â±0.50bTumorsg2.35 1.33Â±0.17b

Values are means Â±SEM.Means in a column not sharing a su perscript are significantly different (P < 0.01 for body weight; P < 0.02 for tumor weight)


FIGURE 1 The suppression of the growth of Morris 7777 hepatomas transplanted to the flanks of male buffalo rats by dietary geraniol (23 mmol/kg diet, 350 mmol/d for 14 d before and after tumor transplant). The exponential curves are generated using the mean estimates hepatoma volume (Â±SEM).

Plant foods are uniquely rich in anutrient constit uents that inhibit carcinogenesis. Wattenberg (1990) divides these inhibitors into two categories: blocking agents and suppressing agents. Although blocking agents tend to be carcinogen-specific, some provide either a pleiotrophic or a separate suppressing action as a fail-safe means of protection. This dual action interferes with the delineation of the metabolic activ ities of the suppressing agents. Our use of transplant models permitted us to focus on the suppressing action of dietary geraniol. We chose to test the tumor-suppressive impact of geraniol on the growth of two ag gressive and lethal tumors. The melanomas seeded with 1 X IO4cells and hepatomas seeded with 4 X IO6 cells accounted for ~ 10 and 4 % of the body weights of the control hosts at 21 and 27 d, respectively, after transplant. Our finding that dietary geraniol signifi cantly suppresses the growth of transplanted solid +ras tumors (Figs. 1 and 2) adds to the initial finding that the monoterpene increases the survival time of mice bearing ~ras ascites tumors (Shoff et al. 1991). This study does not support the hypothesis that +ras tumors will be more sensitive than ^ras tumors to dietary ger

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YU ET AL. O control â€¢ 0.65 mmol geranio! A A

e 2 ÃŽ I

6.50 mmo! geranio! 65.0 mmol geranio)

scriptionally hepatic HMG-CoA reducÃ-ase activity (Clegg et al. 1982, Moreno et al. 1994, Parker et al. 1993). The dual actions, the suppression of tumorigenesis and HMG-CoA reducÃ-aseaclivily, have been reported for d-limonene, menthol, the tocolrienols, ÃŸionone and geraniol (Elson 1995, Elson and Yu 1994).

LITERATURE CITED


Abacus Concepts, StatView (1992) Abacus Concepts, Inc., Berkeley, CA. Azrolan, N. I. & Coleman, P. S. (1989) A discoordinant increase in the cellular amount of 3-hydroxy-3-methylglutaryl-CoA reductase results in the loss of rate-limiting control over cholesterogenesis in a tumor cell-free system. Biochem. J. 258: 42114 15 16 17 18 425. 19 20 Bennis, F., Favre, G., Le Gaillard, F. & SoÃ»la,G. (1993) Importance Days post transplant of mevalonate-derived products in the control of HMG-coA reductase activity and growth of human lung adenocarcinoma cell FIGURE 2 The suppression of the growth of B16 mel line A549. Int. ]. Cancer 55: 640-645. anomas transplanted to the flanks of female C57BL mice by Brown, J. T., Hegarty, P. K. &. Charlwood, B. V. (1987) The toxdietary geraniol. The values are the mean estimates of mel icity of monoterpenes to plant cell cultures. Plant Sci. 48: 195anoma surface areas (Â±SEM)over the final 6 d of the feeding 201. trial. Bruscalalupi, G., Leoni, S., Mangiantini, M. T., Minieri, M., Spagnuolo, S. & Trentlanc, A. (1985) True uncoupling between ANOVA of the linear regression coefficients cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme revealed treatment-mediated differences in tumor growth rate1 A reducÃ-asein an early stage of liver generation. Cell. Mol. Biol. 31:365-368. Clegg, R.J.,Middleton,B., Bell, G.D.&. White, D. A. (1982) The Geranioln066565500n138812Intercept-0.375-0.068-0.234-0.271Â±0.127Â± mechanism of cyclic monoterpene inhibition of 3-hydroxy-3methylglutaryl coenzyme A reducÃ-ase in vivo in the rat. J. Biol. 0.052aÂ±0.011abÂ± Chem. 257: 2294-2299. 0.087Â± Cuthbert, J. A. &. Lipsky, P. E. (1995) Suppression of the prolif 0.040bÂ± 0.078Â± eration of ras-transformed cells by fluoromevalonate, an inhibitor 0.031b0000910890843846r2Â±Â¿Â¿+0000023032033023 0.0700.5460.3870.2730.261bÂ± of mevalonate metabolism. Cancer Res. 55: 1732-1740. Elson, C. E. (1995) Suppression of mevalonate pathway activities 1 Values are means + SEM.Mean regression values not sharing a by dietary isoprenoids: protective roles in cancer and cardiovas common superscript are significantly different (P < 0.01). cular disease. J. Nutr. 125: 1666S-1672S. Elson, C. E., Underbakke, G. L., Hanson, P., Shrago, E., Wainberg, R. & Qureshi, A. (1989) Impact of lemongrass oil, an essential oil, on serum cholesterol. Lipids 24: 677-679. The inhibition of mevalonate pathway activities Elson, C. E. & Yu, S. G. (1994) The chemoprevention of cancer targeted to tumor cells may offer an approach to che by mevalonate-derived constituents of fruits and vegetables. J. motherapy (Cuthbert and Lipsky 1995, Elson 1995, Nutr. 124: 607-614. Elson and Yu 1994, Goldstein and Brown 1990). Engstrom, W. & Schofield, P. N. (1987) Expression of the 3-hyHMG-CoA reducÃ-aseactivity in sterologenic cells is droxy-3-methyl-glutaryl coenzyme A-reductase and LDL-receptor genes in human embryonic tumors and in normal fetal tissues. modulated by an interplay involving the primary reg Anticancer Res. 7: 337-342. ulatory action, the sterol-mediated repression of tran Fitch, M. E., Mangels, A. R., Altmann, W. A., El Hawry, M., Qureshi, scription and secondary posttranscriptional actions A. A. &. Elson, C. E. (1989) Microbiological screening of mesignaled by a nonsterol mevalonate-derived substance valonate-suppressive minor plant constituents. J. Agrie. Food (Goldstein and Brown 1990). ReducÃ-aseactivities in Chem. 37:687-691. Giron, M. D., Havel, C. M. & Watson, J. A. (1993) Isopentenoid neoplastic cells (Azrolan and Coleman 1989, Bennis synthesis in eukaryotic cells. An initiating role for post-transet al. 1993, Bruscalalupi et al. 1985, Engstrom and lational control of 3-hydroxy-3-methylglutaryl coenzyme A reSchofield 1987, Gregg et al. 1986, Siperstein and Fagan ductase. Arch. Biochem. Biophys. 302: 265-271. 1964, Yachnin et al. 1984) and in nonsterologenic cells, Goldstein, J. L. &. Brown, M. S. (1990) Regulation of the meval e.g., insects, yeast, fungi, bacteria, are resistant to steonate pathway. Nature (Lond.) 343: 425-430. rol-feedback control. Nonsterol, mevalonate-derived Gregg, R. G., Davidson, M. & Wilce, P. A. (1986) CholestÃ©rol synthesis and HMG CoA reducÃ-ase activity during hepatocarsubstances suppress the growth of insects (Giron et cinogenesis in rats. Int. J. Biochem. 18: 389-393. al. 1993), plant cells (Brown et al. 1987), yeast (Janssen Hotz, M. A., Gong, J., TrÃ¡ganos, F. & Darzynkiewicz, Z. (1994) et al. 1987), fungi (Janssen et al.1987) and bacteria Flow cytometric detection of apoptosis: comparison of the assays (Fitch et al. 1989, Janssen et al. 1987). Nonsterol prod of in situ DNA degradation and chromatin changes. Cytometry ucts of plant mevalonate pathways suppress posttran15: 237-244.

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SUPPRESSES TUMOR

Janssen, A. M., Scheffer, J. J. C. & Baerheim-Svendsen, A. |1987) Antimicrobial activities of essential oils. A 1976-1986 literature review on possible applications. Pharm. Weekbl. [Sci.]. 7: 193197. Maltese, W. A. (1990) Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J. 4: 3319-3328. Moreno, F., Rossiello, M. R., Manjeshwar, S., Nath, R., Rao, P. M., Rajalakshmi, S. &. Sama, D. S. R. (1994) Effect of b-carotene on the expression of 3-hydroxy-3-methylglutaryl coenzyme A reducÃ-asein rat liver. Proc. Am. Assoc. Cancer Res. 35: 142 (abs.). Parker, R. A., Pearce, B. C., Clark, R. W., Gordan, D. A. & Wright, J. J. K. (1993) Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reducÃ-ase. J. Biol. Chem. 268: 11230-11238. Perez-Sala, D. &. Mollinedo, F. (1994) Inhibition of isoprenoid biosynlhesis induces apoplosis in human promelocylic HL-60 cells. Biochem. Biophys. Res. Commun. 199: 1209-1215. Shack, S., Prasanna, P., Hudgins, W. R., Liu, L., Myers, C. E. & Samid, D. (1994) Experimental therapies for malignant gli-

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omas: largeling the mevalonale palhway of choleslerol synihesis. Proc. Am. Assoc. Cancer Res. 35: 409 (abs.). Shoff, S. M., Grummer, M., Yalvin, M. B. & Elson, C. E. (1991) Conceniralion-dependenl increase in murine P388 and B16 popul.it u ni doubling lime by ihe acyclic monoierpene geranio!. Can cer Res. 51: 37-42. Sinenski, M. & Logel, J. (1985) Defective macromolecule biosyn thesis and cell cycle progression in a mammalian cell starved for mevalonate. Proc. Nati. Acad. Sci. U.S.A. 82: 3257-3261. Siperstein, M. D. & Fagan, V. M. (1964) Deletion of ihe cholesterol-negalive feedback syslem in liver tumors. Cancer Res. 24: 1108-1115. Wattenberg, L.W. (1990) Inhibilion of carcinogenesis by minor anutrieni consiiiuents of ihe diel. Proc. Nuir. Soc. 49: 173-183. Yachnin, S., Toub, D. B. & Mannickarottu, V. (1984) Divergence in cholesterol biosynthelic rales and 3-hydroxy-3-melhylgluiarylCoA reducÃ-ase aclivily as a consequence of granulocyle versus monocyie-macrophage differenliation in HL-60 cells. Proc. Nati. Acad. Sci. U.S.A. 81: 894-897.
