Planta Med. 56 (1990) 377
Cytotoxic and Antitumor Constituents in Pericarps of
Mallotusjaponicus Munehisa Arisawa" . Akio Fujita , Naokata Morita', and Saburo Koshimura2 Department of Medicinal Resources, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan 2 Department of Serology, Kanazawa Medical University, 1-1 Daigaku Uchinada-machi Kahokugun, Ishikawa 920-02, Japan 'Address for correspondence
Materials and Methods
Abstract A variety of phloroglucinol derivatives iso-
lated from the pericarps ofMallotusjaponicus were as-
sessed for growth inhibiting activity against human
larynx (HEp-2) and lung (PC-13) carcinoma cells as well as mouse B16 melanoma, leukemia P388, and L5178Y cells. Most of these derivatives were proved to be signifi-
cantly cytotoxic in culture. One of them, 3-(3,3-dimethylallyl)-5-(3-acetyl-2 ,4-dihydroxy-5-methyl-6methoxybenzyl)-phloracetophenone (1) showed an ex-
cellent cytotoxicity against all target tumor cells and a
marked prolongation of life-span in mice bearing L51 78Y leukemia.
Key words
Mallotus japonicus, cytotoxicity, antitumor constituent, combined use, multiple effect.
Introduction We reported previously the isolation of various rottlerin-like compounds from the pericarps of Mal-
lotus japonicus Muell. Arg. (Euphorbiaceae) and their biological properties (1—4). Some of these compounds and
their chemically modified derivatives were found to be cytotoxic against the KB cell line in culture and to be moder-
ately effective in inhibiting the growth of the solid type of
Ehrhch carcinoma in mice. As a part of our continuing
studies on antitumor constituents of this plant, we examined the cytotoxicity of 13 phloroglucinol derivatives isolated against 2 human carcinoma cell lines, larynx (HEp2), and lung (PC-13), and 3 mouse tumor cell lines, B16 melanoma, P388, and L5178Y leukemia. Furthermore, the
antitumor effects of combined use of a higher cytotoxic compound 1 and OK-432 (5, 6) as a BRM (Biological Re-
Test compounds The compounds to be tested were isolated from the pericarps of Mallotus japonicus (1—4). OK-432 was kindly supplied from Chugai Pharmaceutical Co., Ltd., Tokyo.
Animals Male B6D2F1 mice (6.5-week-old) and male ddY mice (5-week-old) were obtained from the Shizuoka Lab. Animal Agricul. Cooper, Assoc., Hamamatsu.
Tumors Human lung carcinoma (PC-13) cells and mouse B16 melanoma and L5178Y leukemia cells were kindly supplied from the Department of Experimental Therapeutics, Cancer Research Institute, Kanazawa University. Mouse P388 leukemia cells were provided by the Research Laboratory, Toyama Chemical Co., Ltd., Toyama, and the human larynx carcinoma (Hep-2) cells were also kindly supplied from the Department of Virology, School of Medicine, Toyama Medical and Pharmacetical University.
Assay for cytotoxic activity in vitro Media used for the propagation and growth of these cell lines were minimum essential medium (MEM, Nissui Co. Tokyo) supplemented with 10% fetal bovine serum (FBS) for HEp-2 cells, RPMI 1640 (Nissui Co.) with 10% FBS for PC-13, B16 and L51 78Y cells, RPMI 1640 medium with 10% FBS and 50 iM 2-mer-
captoethanol for P-388 cells. L5178Y and P388 of 5 x io cells, HEp-2, PC-13 and B16 of 3 x 10.1 cells were suspended in 24-well tissue culture plates (Falcon Co., U.S.A.) containing I ml of each medium respectively, and cultured at 37°C in 5% CO,-incubator. The test compounds were dissolved in dimethylformamide (DMF)
and added to each well to give three graded concentrations. The amount of DMF was adjusted to give a final concentration of 0.1 % in all cases, including control culture. After 72 hours the number of
HEp-2, PC-13 and B16 cells were determined according to the usual way (7, 8) and expressed as the percentages, relative to a control culture. The L5 1 78Y and P388 cells were also determined after
48 hours incubation according to the colorimetric MTT (tetrazolium) assay (9). The IC50 values, i.e. the concentration in ig/ml affording 50% inhibition of cell growth in the control, was calcu-
sponse Modifiers) were also examined in vh'o using L5178Y lated from a semi-log plot of the suhstance concentration vs. the leukemia and Ehrlich ascites carcinoma. The present paper percentage of viable cells. deals with the results obtained in these experiments.
Downloaded by: National University of Singapore. Copyrighted material.
Received: June 26, 1989
378 Pkznta Med. 56(1990)
Mtinehisa ArLsawa et al.
Assay for antitumor effect of compound I onL5l78Yleukemia in vivo L5178Y cells (1.4 x 106 cells/head) were implanted intraperitoneally (i.p.) into B6D2F1 mice. Twenty-four
OCH3
O,,R
Ho.LOH H3
hours after the implantation, compound 1 at a given dose was administered Lp. once a day for 7 consecutive days as a suspension in 0.25% carboxymethyl cellulose (CMC). The mice that survived for
OH
OCH3 1 R
CH3
CH3
2 R n-C3H7 3 R I-C3H7
60 days were observed, and median survival time (MST) and number of survivors were compared with those of the control group.
L5 1 78Y leukemia or Ehrlich carcinoma of 1.4 x 106 cells were implanted ip. to each group of mice, B6D2F1 of ddY, respectivery. Alter 24 hours, compound I was administered i.p. as described above, and OK-432 was given i.p. on days 1,3, 5, and 7 as a suspension in physiologic saline. The mice were observed for 60 days and evaluated in the usual way.
OCH3 O,R HOL1,.,OH Ho).yoHOH H
OH
OCR3
4
R
CH3
CR3
5 R n-C3H7 6 R /-C3H7
Results and Discussion
0R
O CH3
0 CH3
0 CH3 CR3
All the compounds (Fig. 1) isolated from Mallotusjaponicus were tested for their inhibiting activities on the growth of five tumor cell lines including human lung PC- 13 and larynx Hep-2 carcinoma cells, and the results are shown in Table 1. As seen in this Table, most of the substituted phioroglucinol derivatives showed inhibition of tumor cell growth, while the chromanol derivatives 11 and 12 as well as the monomeric derivative 14 were almost inactive.
In structure-activity relationship, a comparison of the cytotoxic activities of compounds 1—9 indicated that acyl radical replacement at the C-i position decreased the potencies in the order of methyl, propyl, isopropyl ketone. Compounds 1 and 4 having a side chain at the 3-position were found to be more active than the others. Especially, 1 showed significant cytotoxicity, having IC50 of
OH
OCH3
OCR3
OCR3
10
7RCH3 8 R = n-C3H7
9R
i-C3H7
HO, H 3C'
12
11
0.60ig/ml for Hep-2 cells, 0.541g/ml for PC-13 cells,
0.70 g/ml for B16 cells, and 0.81 .tg/ml for L5178Y cells. Moreover, antitumor activity of this compound was demonstrated in mice bearing L5178Y leukemia. As shown in Table 2, potent antileukemic activity was observed by 1 giving over 200% of increase of life-span (ILS) at a dose level of
10—40 mg/kg/day. Koshimura et al. (10) indicated previ13 ously that treatment with 5-fluorouracil in combination with a streptococcal preparation OK-432, which is widely used at present in the treatment of human cancer in Japan Table 1 Cytotoxic activities of the compounds isolated from and abroad, exerted a striking synergistic effect against Mallet us japonicus against various cultured cancer cell lines. L1210 leukemia in mice. An attempt of the combined treat816 L5178Y PC-13 HEp-2 ment with I and OK-432 against L5178Y leukemia and Compound carcinoma carcinoma melanoma leukemia Ehrlich carcinoma expectedly gave favorable results as 0.70 0.81 1 0.60' 0.54 shown in Table 3. Further investigations on the antitumor 0.60 2 0.41 0.91 1.08 activities of compound 1 and its action mechanism are now 1.75 2.50 3 1.10 3.05 in progress. 4 1.53 1.01 1.61 1.08
Acknowledgements We are grateful to Dr. M. Tanaka, Department of Experimental Therapeutics. Cancer Research Institute, Kanazawa University. for supply of L5178Y, B16 and PC-13 cell lines, and to Dr. T. Hon. Research Laboratory, Toyama Chemical Co., Ltd., for supply of P388 cell line. Thanks are also due to Assistant Prof. K. Hayasi, Department of Virology, School of Medicine of our University, for supply of Hep-2 cell line.
5
0.91
6
0.93 0.72 1.70 1.08
7
8 9
6.30
10 11
>20
12 13
>20 >20
IC55 (sg/mll.
pericarps of P388 leukemia
1.14 2.85 3.00
0.63
2.38
1.27
2.18 1.22
1.80
1.96
2.50
3.85
0.82
1.08
1.71
1.30 1.77
1.29 1.44 4.80
1.26 2.36 2.78
3.75
>20 >20 >20
>20 >20 >20
3.65
>20 >20 >20
3.40 4.03 10.08
>20 >20 >20
Downloaded by: National University of Singapore. Copyrighted material.
Experiments for antitumor effects of combined use of compound I and OK-432
Planta Med. 56(1990) 379
Cytotoxic and Antitumor Constituents in Pericarps ofMallotusjaponicus
References
Table 2 Effect of compound 1 on mouse L5178Y leukemia in vivo. Mean body weight (g) day 0 day 7
None
22.1
10
23.5
20 40
23.5 22.6
No. of
MST
lLS'
survivors/tested
(day)
(%)
on 60th day
23.6 24.6
16 54.5
240
24.1
60 49
275 206
0/7 3/7 7/7
22.7
3/7 (Toxic)
The experiment was terminated at 60th day after the leukemic cell implantation. a Median survival time. Increase of life-span [MST(treated)/MST(control)] x 100—100.
Table 3 Antitumor effects of combined use of compound 1 and OK-432 in
1 Arisawa, M., Fujita, A., Suzuki, R., Hayashi, T., Morita, N., Kawano, N., Koshimura, S. (1985) J. Nat. Prod. 48, 455—459. 2 Arisawa, M., Fujita, A., Saga, M., Hayashi, T., Morita, N., Kawano, N., Koshimura, S. (1986) J. Nat. Prod. 49, 298—302.
Fujita,A., Hayashi,T.,Arisawa,M., Shimizu,M.,Morita,N.,Kikuchi, T., Tezuka, Y. (1988)J. Nat. Prod. 51, 708—712. " Arisawa, M., Fujita, A., Hayashi, T., Morita, N., Kikuchi, T., Tezuka, Y. (1990) Chem. Pharm. Bull. 38 (No. 3), in press. Okamoto, H., Shoin, S., Koshimura, S., Shimizu, R. (1972) 3-Hemolytic Streptococcus as a Cancer controller —A commentary on PC-B-24. Chugai General Print. Co., Tokyo. 6 Koshimura, S., Murayama, T., Natsuume-Sakai, S. (1986) in: Mechanisms of Antitumor Effects of OK-432, (Ishida, N., ed), pp. 221—226. ExcerptaMedica, Tokyo. Smith. C. G., Lummis, W. L., Grady, J. B. (1959) Cancer Has. 19,843—
853. Grady, J. F., Lummis, W. L., Smith, C. G. (1960) Cancer Res. 20,
mice.
1. Mouse L5l78YIeukemia
No.of Agent
None Comp. 1 OK-432 Comp. 1
+
OK-432
Dose (mg/kg)
MST
ILSb
(day)
(%)
survivors/tested on 60th day
0/8 3/6 2/6
10
16 54.5
1OC
46
240 187
60
275
6/6
MST
ILSb
(day)
(%)
No.of survivors/tested on 60th day
0/8 0/6 0/6 2/6
10
+ 10
2. Ehrlich ascites carcinoma
Agent
Dose (mg/kg)
None
17
18 19.5
5.8 14.7
Comp.1
10 7.5 10
+
33.5
97
OK-432
7.5
Comp. 1 OK-432
+
a, b See footnotes in Table 2.
Corresponds to 100 KE; one KE (Klinische Einheit) of OK-432 contains 0.1 mg of dried streptococcal cells.
10
1114—1117. Mosmann, T. (1983) J. Immuno. Methods 65, 55—63. Koshimura, S., Ryoyama, K. (1977) Cancer Treat. Repts. 61, 17—27.
Downloaded by: National University of Singapore. Copyrighted material.
Dose (mg/kg)
Cytotoxic and Antitumor Constituents in Pericarps of
Mallotusjaponicus Munehisa Arisawa" . Akio Fujita , Naokata Morita', and Saburo Koshimura2 Department of Medicinal Resources, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan 2 Department of Serology, Kanazawa Medical University, 1-1 Daigaku Uchinada-machi Kahokugun, Ishikawa 920-02, Japan 'Address for correspondence
Materials and Methods
Abstract A variety of phloroglucinol derivatives iso-
lated from the pericarps ofMallotusjaponicus were as-
sessed for growth inhibiting activity against human
larynx (HEp-2) and lung (PC-13) carcinoma cells as well as mouse B16 melanoma, leukemia P388, and L5178Y cells. Most of these derivatives were proved to be signifi-
cantly cytotoxic in culture. One of them, 3-(3,3-dimethylallyl)-5-(3-acetyl-2 ,4-dihydroxy-5-methyl-6methoxybenzyl)-phloracetophenone (1) showed an ex-
cellent cytotoxicity against all target tumor cells and a
marked prolongation of life-span in mice bearing L51 78Y leukemia.
Key words
Mallotus japonicus, cytotoxicity, antitumor constituent, combined use, multiple effect.
Introduction We reported previously the isolation of various rottlerin-like compounds from the pericarps of Mal-
lotus japonicus Muell. Arg. (Euphorbiaceae) and their biological properties (1—4). Some of these compounds and
their chemically modified derivatives were found to be cytotoxic against the KB cell line in culture and to be moder-
ately effective in inhibiting the growth of the solid type of
Ehrhch carcinoma in mice. As a part of our continuing
studies on antitumor constituents of this plant, we examined the cytotoxicity of 13 phloroglucinol derivatives isolated against 2 human carcinoma cell lines, larynx (HEp2), and lung (PC-13), and 3 mouse tumor cell lines, B16 melanoma, P388, and L5178Y leukemia. Furthermore, the
antitumor effects of combined use of a higher cytotoxic compound 1 and OK-432 (5, 6) as a BRM (Biological Re-
Test compounds The compounds to be tested were isolated from the pericarps of Mallotus japonicus (1—4). OK-432 was kindly supplied from Chugai Pharmaceutical Co., Ltd., Tokyo.
Animals Male B6D2F1 mice (6.5-week-old) and male ddY mice (5-week-old) were obtained from the Shizuoka Lab. Animal Agricul. Cooper, Assoc., Hamamatsu.
Tumors Human lung carcinoma (PC-13) cells and mouse B16 melanoma and L5178Y leukemia cells were kindly supplied from the Department of Experimental Therapeutics, Cancer Research Institute, Kanazawa University. Mouse P388 leukemia cells were provided by the Research Laboratory, Toyama Chemical Co., Ltd., Toyama, and the human larynx carcinoma (Hep-2) cells were also kindly supplied from the Department of Virology, School of Medicine, Toyama Medical and Pharmacetical University.
Assay for cytotoxic activity in vitro Media used for the propagation and growth of these cell lines were minimum essential medium (MEM, Nissui Co. Tokyo) supplemented with 10% fetal bovine serum (FBS) for HEp-2 cells, RPMI 1640 (Nissui Co.) with 10% FBS for PC-13, B16 and L51 78Y cells, RPMI 1640 medium with 10% FBS and 50 iM 2-mer-
captoethanol for P-388 cells. L5178Y and P388 of 5 x io cells, HEp-2, PC-13 and B16 of 3 x 10.1 cells were suspended in 24-well tissue culture plates (Falcon Co., U.S.A.) containing I ml of each medium respectively, and cultured at 37°C in 5% CO,-incubator. The test compounds were dissolved in dimethylformamide (DMF)
and added to each well to give three graded concentrations. The amount of DMF was adjusted to give a final concentration of 0.1 % in all cases, including control culture. After 72 hours the number of
HEp-2, PC-13 and B16 cells were determined according to the usual way (7, 8) and expressed as the percentages, relative to a control culture. The L5 1 78Y and P388 cells were also determined after
48 hours incubation according to the colorimetric MTT (tetrazolium) assay (9). The IC50 values, i.e. the concentration in ig/ml affording 50% inhibition of cell growth in the control, was calcu-
sponse Modifiers) were also examined in vh'o using L5178Y lated from a semi-log plot of the suhstance concentration vs. the leukemia and Ehrlich ascites carcinoma. The present paper percentage of viable cells. deals with the results obtained in these experiments.
Downloaded by: National University of Singapore. Copyrighted material.
Received: June 26, 1989
378 Pkznta Med. 56(1990)
Mtinehisa ArLsawa et al.
Assay for antitumor effect of compound I onL5l78Yleukemia in vivo L5178Y cells (1.4 x 106 cells/head) were implanted intraperitoneally (i.p.) into B6D2F1 mice. Twenty-four
OCH3
O,,R
Ho.LOH H3
hours after the implantation, compound 1 at a given dose was administered Lp. once a day for 7 consecutive days as a suspension in 0.25% carboxymethyl cellulose (CMC). The mice that survived for
OH
OCH3 1 R
CH3
CH3
2 R n-C3H7 3 R I-C3H7
60 days were observed, and median survival time (MST) and number of survivors were compared with those of the control group.
L5 1 78Y leukemia or Ehrlich carcinoma of 1.4 x 106 cells were implanted ip. to each group of mice, B6D2F1 of ddY, respectivery. Alter 24 hours, compound I was administered i.p. as described above, and OK-432 was given i.p. on days 1,3, 5, and 7 as a suspension in physiologic saline. The mice were observed for 60 days and evaluated in the usual way.
OCH3 O,R HOL1,.,OH Ho).yoHOH H
OH
OCR3
4
R
CH3
CR3
5 R n-C3H7 6 R /-C3H7
Results and Discussion
0R
O CH3
0 CH3
0 CH3 CR3
All the compounds (Fig. 1) isolated from Mallotusjaponicus were tested for their inhibiting activities on the growth of five tumor cell lines including human lung PC- 13 and larynx Hep-2 carcinoma cells, and the results are shown in Table 1. As seen in this Table, most of the substituted phioroglucinol derivatives showed inhibition of tumor cell growth, while the chromanol derivatives 11 and 12 as well as the monomeric derivative 14 were almost inactive.
In structure-activity relationship, a comparison of the cytotoxic activities of compounds 1—9 indicated that acyl radical replacement at the C-i position decreased the potencies in the order of methyl, propyl, isopropyl ketone. Compounds 1 and 4 having a side chain at the 3-position were found to be more active than the others. Especially, 1 showed significant cytotoxicity, having IC50 of
OH
OCH3
OCR3
OCR3
10
7RCH3 8 R = n-C3H7
9R
i-C3H7
HO, H 3C'
12
11
0.60ig/ml for Hep-2 cells, 0.541g/ml for PC-13 cells,
0.70 g/ml for B16 cells, and 0.81 .tg/ml for L5178Y cells. Moreover, antitumor activity of this compound was demonstrated in mice bearing L5178Y leukemia. As shown in Table 2, potent antileukemic activity was observed by 1 giving over 200% of increase of life-span (ILS) at a dose level of
10—40 mg/kg/day. Koshimura et al. (10) indicated previ13 ously that treatment with 5-fluorouracil in combination with a streptococcal preparation OK-432, which is widely used at present in the treatment of human cancer in Japan Table 1 Cytotoxic activities of the compounds isolated from and abroad, exerted a striking synergistic effect against Mallet us japonicus against various cultured cancer cell lines. L1210 leukemia in mice. An attempt of the combined treat816 L5178Y PC-13 HEp-2 ment with I and OK-432 against L5178Y leukemia and Compound carcinoma carcinoma melanoma leukemia Ehrlich carcinoma expectedly gave favorable results as 0.70 0.81 1 0.60' 0.54 shown in Table 3. Further investigations on the antitumor 0.60 2 0.41 0.91 1.08 activities of compound 1 and its action mechanism are now 1.75 2.50 3 1.10 3.05 in progress. 4 1.53 1.01 1.61 1.08
Acknowledgements We are grateful to Dr. M. Tanaka, Department of Experimental Therapeutics. Cancer Research Institute, Kanazawa University. for supply of L5178Y, B16 and PC-13 cell lines, and to Dr. T. Hon. Research Laboratory, Toyama Chemical Co., Ltd., for supply of P388 cell line. Thanks are also due to Assistant Prof. K. Hayasi, Department of Virology, School of Medicine of our University, for supply of Hep-2 cell line.
5
0.91
6
0.93 0.72 1.70 1.08
7
8 9
6.30
10 11
>20
12 13
>20 >20
IC55 (sg/mll.
pericarps of P388 leukemia
1.14 2.85 3.00
0.63
2.38
1.27
2.18 1.22
1.80
1.96
2.50
3.85
0.82
1.08
1.71
1.30 1.77
1.29 1.44 4.80
1.26 2.36 2.78
3.75
>20 >20 >20
>20 >20 >20
3.65
>20 >20 >20
3.40 4.03 10.08
>20 >20 >20
Downloaded by: National University of Singapore. Copyrighted material.
Experiments for antitumor effects of combined use of compound I and OK-432
Planta Med. 56(1990) 379
Cytotoxic and Antitumor Constituents in Pericarps ofMallotusjaponicus
References
Table 2 Effect of compound 1 on mouse L5178Y leukemia in vivo. Mean body weight (g) day 0 day 7
None
22.1
10
23.5
20 40
23.5 22.6
No. of
MST
lLS'
survivors/tested
(day)
(%)
on 60th day
23.6 24.6
16 54.5
240
24.1
60 49
275 206
0/7 3/7 7/7
22.7
3/7 (Toxic)
The experiment was terminated at 60th day after the leukemic cell implantation. a Median survival time. Increase of life-span [MST(treated)/MST(control)] x 100—100.
Table 3 Antitumor effects of combined use of compound 1 and OK-432 in
1 Arisawa, M., Fujita, A., Suzuki, R., Hayashi, T., Morita, N., Kawano, N., Koshimura, S. (1985) J. Nat. Prod. 48, 455—459. 2 Arisawa, M., Fujita, A., Saga, M., Hayashi, T., Morita, N., Kawano, N., Koshimura, S. (1986) J. Nat. Prod. 49, 298—302.
Fujita,A., Hayashi,T.,Arisawa,M., Shimizu,M.,Morita,N.,Kikuchi, T., Tezuka, Y. (1988)J. Nat. Prod. 51, 708—712. " Arisawa, M., Fujita, A., Hayashi, T., Morita, N., Kikuchi, T., Tezuka, Y. (1990) Chem. Pharm. Bull. 38 (No. 3), in press. Okamoto, H., Shoin, S., Koshimura, S., Shimizu, R. (1972) 3-Hemolytic Streptococcus as a Cancer controller —A commentary on PC-B-24. Chugai General Print. Co., Tokyo. 6 Koshimura, S., Murayama, T., Natsuume-Sakai, S. (1986) in: Mechanisms of Antitumor Effects of OK-432, (Ishida, N., ed), pp. 221—226. ExcerptaMedica, Tokyo. Smith. C. G., Lummis, W. L., Grady, J. B. (1959) Cancer Has. 19,843—
853. Grady, J. F., Lummis, W. L., Smith, C. G. (1960) Cancer Res. 20,
mice.
1. Mouse L5l78YIeukemia
No.of Agent
None Comp. 1 OK-432 Comp. 1
+
OK-432
Dose (mg/kg)
MST
ILSb
(day)
(%)
survivors/tested on 60th day
0/8 3/6 2/6
10
16 54.5
1OC
46
240 187
60
275
6/6
MST
ILSb
(day)
(%)
No.of survivors/tested on 60th day
0/8 0/6 0/6 2/6
10
+ 10
2. Ehrlich ascites carcinoma
Agent
Dose (mg/kg)
None
17
18 19.5
5.8 14.7
Comp.1
10 7.5 10
+
33.5
97
OK-432
7.5
Comp. 1 OK-432
+
a, b See footnotes in Table 2.
Corresponds to 100 KE; one KE (Klinische Einheit) of OK-432 contains 0.1 mg of dried streptococcal cells.
10
1114—1117. Mosmann, T. (1983) J. Immuno. Methods 65, 55—63. Koshimura, S., Ryoyama, K. (1977) Cancer Treat. Repts. 61, 17—27.
Downloaded by: National University of Singapore. Copyrighted material.
Dose (mg/kg)
