Rapid

Communication

J . Biochem. 11-13 (1995) 117,

Geranylgeraniol
Hidekazu and *School Products Kazuyasu of Ohizumi,*

Is a Potent
Yutaka Nakaya*1 Pharmaceutical Formulation Sciences Research , Showa Laboratory Masuda,*

Inducer
Shigeo

of Apoptosis
Nakajo,* Itaru Sakai,t

in Tumor
Shigemitsu

Cells
Ohsawa,t

University, , Eisai Co.,

1-5-8 Ltd.,

Hatanodai, 2-3-14 Minami,

Shinagawa-ku, Honjo, Saitama

Tokyo 367

142;

and

Received

for

publication,

October

24,

1994

We screened various isoprenoids to find inducers of apoptosis for human leukemia HL-60 cells, and found that GGO (geranylgeraniol) had the most potent apoptosis-inducing activity, as judged from DNA fragmentation in HL-60 cells. The apoptosis-inducing activity of GGO is concentration and time-dependent. DNA synthesis by HL-60 cells was selectively inhibited on treatment with GGO. Besides HL-60 cells, apoptosis was induced by GGO in various tumor cell lines, including human myeloid multipotential leukemia K562, lymphoblastic leukemia Molt3, and colon adenocarcinoma COLO320 DM. Key words: apoptosis, geranylgeraniol, isoprenoid, tumor cell.

We previously found various differentiation-inducers for a broad range of myeloid leukemia cell lines, including camptothecin (1), VP16 (2), bufalin (3), geranylgeranyl acetone (4), and daidzein (5). Recently, some differentia tion-inducers for cancer cells, such as camptothecin (6-8), VP16 (6, 8, 9), and bufalin (10), were reported to have the ability to induce apoptosis in cancer as well as normal cells. In addition, diverse anticancer drugs used clinically, such as cisplatin (6, 9, 11), camptothecin 11 (12), and retinoic acid (13), have been proved to induce apoptosis in cancer cells. Since cells that have undergone apoptosis are rapidly recognized and engulfed by macrophages before cell lysis,

they can be removed without inducing inflammation. Therefore, apoptosis-inducing agents are expected to be ideal anticancer drugs. In the present study, we examined the apoptosis inducing abilities of various isoprenoids and found that GGO exhibits potent apoptosis-inducing activity toward human tumor cell lines, including myeloid leukemia cells, lymphoblastic leukemia cells, and colon adenocarcinoma cells. Polyprenylalcohols were synthesized and supplied by Eisai Chemical, Tokyo. HL-60, K562, Molt3, and COLO320 DM cells were provided by the Japanese Cell Research Resources Bank, and cultured in RPMI 1640 medium (Gibco, Glasgow, UK) supplemented with 10% fetal calf serum. Cellular DNA was extracted as reported previously (10). Electrophoresis was performed in 1% agarose gel in 40mM Tris-acetate buffer (pH 7.4) at 50 V. After electrophoresis, DNA was visualized by ethidium bromide staining. The extent of DNA fragmentation was

Fig. HL-60 cohols. cells

1.

Agarose cells treated

gel for DNA with cells; lane 5,

electrophoresis 3 h with was extracted the lane following 2, geraniol; 50M

of

DNA

extracts polyprenylal in the text.

from

of various

Cellular were treated HL-60

as described

HL-60 Lane 1, 4,

polyprenylalcohols. lane lane 6, 3, farnesol;

untreated geranylgeraniol; 1 To whom

lane

geranylfarnesol; should be

farnesylfarnesol.

correspondence DAPI, VP16,

addressed. GGO, geranyl

Abbreviations: geraniol; - glucoside.

4L,6-diamidine-2-phenylindole; 4L-demethylepipodophyllotoxin

ethylidene--D

Fig. 2. Quantification of fragmented DNA in HL-60 cells treat ed with various polyprenylalcohols. HL-60 cells were treated with 50 k M of various polyprenylalcohols for 3 h and then the percentage of DNA fragmented was determined using DAPI. The polyprenylalcohols used are: 1, geraniol; 2, farnesol; 3, geranylger aniol; 4, geranylfarnesol; 5, farnesylfarnesol. 11

Vol. 117, No. 1, 1995

12

H. Ohizumi

et al.

determined slight lysis

by modifications.

the

method Briefly, Tris-HCl, 20min on

described the pH ice. cells 7.4, Then

by

Wyllie were

(14), suspended EDTA,

with in 0.5% was

various above. exhibited among with or 1 a

polyprenylalcohols The results the the isoprenyl most shown potent

were in Fig.

calculated 2 indicate

as

described that GGO activity

buffer

(5mM for at

1mM the and

apoptosis-inducing tested. than the Polyprenylalcohols geranylgeranyl group and were cells dependence (data vitamin also not of less were K2,

Triton~100) centrifuged DNA remaining min at 45 was

suspension the fragmented The sonicated determined fragmentation DNA were to the

polyprenylalcohols units than the shorter

27,000~g from centrifugation amount using ratio and HL-60

for

20min the

group less which in

recovered in W. the The method as the

supernatant. tube was was The DNA

pellet for by

longer

geranylfarnesol

effective. have inducing a

Geranylgeranylacetone geranylgeranyl apoptosis 3 shows in HL-60 group, in HL-60 the time cells of on

of

DNA

effective

fluorometric was DNA. after defined DNA, treatment

DAPI.

shown). the induction 50M induced 85% of 3 h GGO. very the after with within treated (Fig. was in of

of the protein cells and 1Ci/ml,

fragmented syntheses with GGO

total

Figure apoptosis DNA rapidly DNA treatment time. 24h. of The in

RNA, of [3H]

determined adding [3H] to a for

treatment cells Approximately

with was

by

fragmentation by GGO-treatment. cells 50M

HL-60

thymidine, final 30min. tic acid

uridine, of

[3H]leucine, followed with determined

respectively, by 25% incubation trichloroace as described

concentration The and cells then

HL-60 with DNA

was GGO

fragmented and was nuclei condensed that typical increased almost of

within

were

sedimented was

gradually complete cells

radioactivity

fragmentation the were

previously Figure HL-60 side

(10). 1 shows cells treated of for

Morphologically, 100M These by of results GGO in the GGO

HL-60 and

the

results for 3 h lengths. cells

of DNA with

gel

electrophoresis

with 4). duced To protein

fragmented apoptosis

polyprenylalcohols DNA with fragmentation farnesol The treated

with was (C,50H), percent with

indicate HL-60 effects HL-60

chains

various HL-60 and

cells. of cells GGO were on treated DNA, with RNA, and

observed GGO ages

treated

examine syntheses,

(C200H), of

geranylfarnesol DNA in HL-60

(C250H). cells

different

fragmented

Fig. Fig. tion GGO was assays. 3. in for Time HL-60 various dependency cells times by by GGO. and staining then of the HL-60 the with induction cells percentage DA-PI. were of DNA treated of DNA Means}SE fragmenta with 50 ,UM HL-60 GGO

5.

Effect cells.

of The DNA

GGO cells (),

on were

DNA, treated (),

RNA, with and

and

protein

syntheses concentrations

in of were [3H]

various protein of ()

for

3 h. by and

RNA the

syntheses

fragmentation for triplicate

determined - uridine, for

measuring

incorporation Each

[3H]thymidine, value is the

determined

[3H]leucine, determinations.

respectively.

mean}SD

triplicate

Fig. pearance with treated min

4.

Morphological of HL-60 GGO. HL-60 with and 100M then (B).

ap cells treated cells were GGO stained A, for 30

with untreated

Wright-Giemsa HL-60 cells.

J. Biochem.

Geranylgeraniol

Induces

Apoptosis

13

TABLE I. Apoptosis-inducing effect of GGO on tumor cell lines. Cells were treated for 3 h with GGO . At the concentrations indicated, 50% of DNA was fragmented. The percentage of DNA fragmentation was determined by staining with DAPI.

concentrations of GGO for 3 h, and then the incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine was measured (Fig. 5). GGO showed a marked inhibitory effect on DNA synthesis, while RNA and protein syntheses were inhibited only slightly by the GGO-treatment. Cyclohex imide, an inhibitor of protein synthesis, had no effect on the induction of apoptosis in HL-60 cells by GGO, suggesting that the apoptotic process induced by GGO does not require the synthesis of new proteins. Table I shows the effects of GGO on other human tumor cell lines. As shown in this table, GGO also induced apoptosis in myeloid multipotential leukemia K562, lymphoblastic leukemia Molt3, and colon adenocarcinoma COL0320 DM, as judged from the DNA fragmentation induced. The apoptosis-inducing activity of isoprenoids critically depends on the length of the side chains of polyprenylalco hols. Polyprenylalcohols with a geranylgeranyl group (GGO) and a geranylisopropyl group exhibited potent apoptosis-inducing activity. Interestingly, DNA synthesis was inhibited almost selectively on treatment with GGO, suggesting that the inhibition of DNA synthesis may trigger the induction of apoptosis. The mechanism underlying of the induction of apoptosis by GGO in tumor cells is worth further study. REFERENCES 1. Chou,S., Kaneko,M., Nakaya,K., and Nakamura, (1990) Y. Induction differentiation humanand mouse of of leukemiacells by camptothecin. Biochem. Biophys. Commun. 160-167 Res. 166, 2. Nakaya,K., Chou,S., Kaneko,M., and Nakamura, (1991) Y. Topoisomerase inhibitors have potent differentiation-inducing

activity for human and mouse myeloid leukemia cells. Jpn. J. Cancer Res. 82, 184-191 3. Zhang, L., Nakaya, K., Yoshida, T., and Kuroiwa, Y. (1992) Induction by bufalin of differentiation of human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte cells and its potent synergistic effect on the differentiation of human leukemia cells in combination with other inducers. Cancer Res. 52,4634-4641 4. Sakai, I., Tanaka, T., Osawa, S., Hashimoto, S., and Nakaya, K. (1993) Geranylgeranylacetone used as an antiulcer agent is a potent inducer of differentiation of various human myeloid leukemia cell lines. Biochem. Biophys. Res. Commun. 191, 873 -879 5. Jing, Y., Nakaya, K., and Han, R. (1993) Differentiation of promyelocytic leukemia cells HL-60 induced by daidzein in vitro and in vivo. Anticancer Res. 13, 1049-1054 6. Kaufman, S.H. (1989) Induction of endonucleolytic DNA cleav age in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: A cautionary note. Cancer Res. 49, 5870-5878 7. Onishi, Y., Azuma, Y., Sato, Y., Mizuno, Y., Tadakuma, T., and Kizaki, Y. (1993) Topoisomerase inhibitors induce apoptosis in thymocytes. Biochim. Biophys. Acta 1175, 147-154 8. Bertrand, R., Solary, E., Jenkins, J., and Pommier, Y. (1993) Apoptosis and its modulation in human promyelocytic HL-60 cells treated with DNA topoisomerase I and II inhibitors. Exp. Cell Res. 207, 388-397 9. Barry, M.A., Behnke, C.A., and Eastman, A. (1990) Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxin and hyperthermia. Biochem. Pharmacol. 40, 2353 -2362 10. Jing, Y., Ohizumi, H., Kawazoe, N., Hashimoto, S., Masuda, Y., Nakajo, S., Yoshida, T., Kuroiwa, Y., and Nakaya, K. (1994) Selective inhibitory effect of bufalin on growth of human tumor cells in vitro: Association with the induction of apoptosis in leukemia HL-60 cells. Jpn. J. Cancer Res. 85, 645-651 11. Evans, M.A. and Dive, C. (1993) Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and non proliferating immature thymocytes. Cancer Res. 53, 2133-2139 12. Yoshida, A., Ueda, T., Wano, Y., and Nakamura, T. (1993) DNA damage and cell killing by camptothecin and its derivative in human leukemia HL-60 cells. Jpn. J. Cancer Res. 84, 566-573 13. Martin, S.J., Bradley, J.G., and Cotter, T.G. (1990) HL-60 cells induced to differentiation towards neutrophils subsequently die via apoptosis. Clin. Exp. Immunol. 79, 448-453 14. Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284,555-556

Vol. 117, No. 1, 1995