fmmunupharmacology.23 (1992) 199-204
0 1992Elsevier Science Publishers B.V. All rights reserved. 0162-3109/92/805.00
199
IMPHAR 00590
Augmentation of in combined use recombinant human tumor necrosis factor K. Matsunaga’,
H. Mash&a’, Y. Seo2, S. Wada3 and K. Hata3
‘Divisionof Immunology.2Depamnem of Gamo-enterologiical Swgmy and ‘Department of Radiatiwti%ewpy. Nationtz!Kyrepkrr Cancer Center. F&&a. Japatt
(Accepted 23 January 1992)
Abstract: The effect of combined use of a derivative ofnitrosourea, ACNU, with recombinant human tumor necrosis factor (rhTNF) on the radiation-induced antiprolifwative effect was examined using Metb A tumor ce!ls. The radiation-induced antiproliferative effect was sliitly augmented by the simultaneous addition ofACNU at IO&cl in 5 Gy aud 15 Gy Irradiated groups. The antiproliferative elfect was augmented in parahel with the radiation dose by the addition of rhTNF. Further mgmentation of the proliferation inhibition was observed when both ofACNU (lOug/ml) and 10 U/m: ofrhTNF were added in combination with irradiation. Similar augmentation was observed when the target cells were treated with ACNU prior to irradiation and the addition of rhTNF. Key worda: Nimustine hydrochloride (ACNU); Tumor necrosis factor: Radiosensitizing effect; Meth A turnor cd
Introduction
Activated macrophages and lymphocytes can be stimulated to release materials which are called tumor necrosis factor (TNF) and lymphotoxin (LT), respectively (Granger et al., 1969; Carswell et al., 1975). It has heen reported that TNF and LT exhibit antiproliferative effect on tumor cells in addition to the role in inflammation and the regu-
Cwespondence to: Keiko Matsunaga, Division of Immunology, National Kyushu Cancer Center, 3-I-l Notame, Minami-ku. Fukuoka 815. Japan Ab&~ialions: ACNU, nimustine hydrochloride; rhTNF, recombinant human tumor necrosis factor; Gy, gray; LT. lymphotoxin; BSS, balanced salt solution; DDC, diethyldithiocarbamate; SOD, superoxidedismutase.
latory role on other cell function (Granger and Williams, 1968; Urban et al., 1986). It has been demonstrated that the antitumor activity of TNF and LT can be potentiated synergistically by interferons (Williams and Bellanti, 1983; Fransen et al., 1986) and in combination with antitumor drugs (Matsunaga and Mashiba, 1983). As molecular basis for the mechanisms of cytotoxininduced direct cytotoxicity, the involvement of oxygen Free radicals have been suggested (Mattews et al., 1987). BCNU has been reported to inactivate glutathione reductase (F&her and Ahmad, 1977). As a resuh of the inhibition of the ghrtathione redox cycle, oxygen free radicals, such as hydrogen peroxide and hydroperoxide, can be increased. By using a derivative of nitrosourea,
ACNU. we have observed that the simultaneous addition of ACNU with cytotoxins or the pretreatment oftarget ceils with ACNU can augment cytotoxin-induced cytotoxicity (Matsunaga and Mashiba, 1990). The augmentation of the antiproliferative effect on tumor cells could be induced through the combined use of LT with ACNW by lowering the intracellular level of glutathione (Mashiba et al., 1991). On the other hand, highly reactive superoxide has been reported to be responsible for the major component of radiation-induced oxygen effects (Oberley et al., 1976). In the present study, the effect of the combined use of ACNU with rhTNF on the radiationinduced antiproliferative effect was examined.
1 x lo5 cells/ml at 37 “C for 1 h. The cells were washed and adjusted to the same concentration as described above. The plates were incubated at 37 “C for I h before irradiation. After the termination of irradiation, the plates were incubated without washing at 37 ‘C for 48 h and the cultures were pulsed with 0.05 ml of serum-free [3H]thymidine 24 h before harvesting. Uptake of [ ‘Hlthymidine by the target cells was determined by scintitlation counting.
Materials and Metbods
Results
Cdl Meth A tumor cell line derived from Balb/c mouse was used as target cells and the cells were maintained in RPM1 1640 supplemented with 10% fetal bovine serum until use.
Eflect of the addition of ACNU on the radiationinduced inhibition of cell prolt$eration It was examined whether the addition of ACNU could augment the radiation-induced antiproliferative effect. Meth A tumor cells were irradiated in the presence of ACNU at 2&nl or 10 pg/ml and cultured in the continuous presence of ACNU for 48 h. As shown in Fig. 1, the proliferation of Meth A tumor cells was inhibited by the irradiation in a dose-dependent fashion, and the rate of inhibition of Meth A tumor cell proliferation at S Gy, 10 Gy and 15 Gy was 3.3%, 32.3 % and 43.6% compared with that in the nonirradiated group, respectively. The addition of ACNU at 2 pg/ml did not have a significant effect with the non-irradiated group. However, a slight degree of the augmented inhibition was observed through the addition of ACNU at 10 pg/ml in the 5 Gy, 10 Gy and 15 Gy irradiated groups, and the rates of inhibition compared with that in each medium control group were 27.3%, 18.8% and 26.1x, respectively. On the other hand, the rate of inhibition in the non-irradiated group was 17.4%.
Agent A derivative of nitrosourea, nimustine hydrochloride (ACNU) (Sankyo Co., Ltd., Japan) was used. As a cytokine, recombinant human tumor necrosis factor (rhTNF) (As&i Chemical Japan) was used. Assay for inhibition of cell proliferation Meth A tumor cells were washed with Hanks’ balanced salt solution (BSS) and adjusted to a concentration of 1 x IO4 cells/ml in RPM1 1640 supplemented with 10% fetal bovine serum. Onetenth of 1 ml of the cell suspension was plated in the well of U-bottomed microculture plates. ACNU and rhTNF at various concentrations were added to each well in a total volume of 0.2ml/weU. As controls, medium or each agent alone was added. In the experiment to examine the effect of pretreatment with ACNU, the target cells were incubated with ACNU (25 &ml) in a 15 x 60 mm plastic dish at a concentration of
Irradiation Irradiation was performed using Telecobalt at a dose rate of 0.513 Gy/min. Source-surface distance was 78.5 cm and field size was 2 1 x 2 1 cm.
201
C
5
10
15
Fig. I. Antiproliferative effect ofACNU in combined use of irradiation. Meth A tumor cells were incubated with medium alone (white bars) or ACNU at a final concentration of 2 @ml (stippled bars) and IO &nl (black bars) at 37 “C for I h in the wells of microplates, and irradiated without washing. The plates were incubated at 37 “C for 48 h and the cultures were pulsed with rH]thymidine 24 h before harvesting. Vertical bars indicate standard deviation. Significant at 0.01 rp < 0.025 (*), 0.005 -z p c 0.01 (**) and p -c 0.001 (I**) compared with the value in each medium control.
Augmentation of the radiation-induced antiprolrerative e#ect by addition of rhTNF Effect of rhTNF on the radiation-induced inhibition of the cell proliferation was examined. As
shown in Fig. 2a, the antiproliferative e&t was augmented by the addition of rhTNF in parallel with the dose of irradiation. The rate of inhibition through the addition of rhTNF in the 5 Gy, 10 Gy
“1
lrredkth
0
5
lb
15
b)
0
5
10
15
WY)
Fig. 2. Radiosensitizing effect ofTNF and ACNU. Meth A tumor cegs were incubated with medium alone (white bars) or TNF (IO U/ml) (stippled bars) in the absence (a) or the presence (b) of ACNU (10 pgiml) at 37 “C for I h in the wells of micmplates, and irradiated without washing at the dose of 5 Gy. 10 Gy or 15 Gy. Cytostatic assay was performed aa described in Fii. 1. Significant at 0.001 < p < 0.005 (*) and p
0 1992Elsevier Science Publishers B.V. All rights reserved. 0162-3109/92/805.00
199
IMPHAR 00590
Augmentation of in combined use recombinant human tumor necrosis factor K. Matsunaga’,
H. Mash&a’, Y. Seo2, S. Wada3 and K. Hata3
‘Divisionof Immunology.2Depamnem of Gamo-enterologiical Swgmy and ‘Department of Radiatiwti%ewpy. Nationtz!Kyrepkrr Cancer Center. F&&a. Japatt
(Accepted 23 January 1992)
Abstract: The effect of combined use of a derivative ofnitrosourea, ACNU, with recombinant human tumor necrosis factor (rhTNF) on the radiation-induced antiprolifwative effect was examined using Metb A tumor ce!ls. The radiation-induced antiproliferative effect was sliitly augmented by the simultaneous addition ofACNU at IO&cl in 5 Gy aud 15 Gy Irradiated groups. The antiproliferative elfect was augmented in parahel with the radiation dose by the addition of rhTNF. Further mgmentation of the proliferation inhibition was observed when both ofACNU (lOug/ml) and 10 U/m: ofrhTNF were added in combination with irradiation. Similar augmentation was observed when the target cells were treated with ACNU prior to irradiation and the addition of rhTNF. Key worda: Nimustine hydrochloride (ACNU); Tumor necrosis factor: Radiosensitizing effect; Meth A turnor cd
Introduction
Activated macrophages and lymphocytes can be stimulated to release materials which are called tumor necrosis factor (TNF) and lymphotoxin (LT), respectively (Granger et al., 1969; Carswell et al., 1975). It has heen reported that TNF and LT exhibit antiproliferative effect on tumor cells in addition to the role in inflammation and the regu-
Cwespondence to: Keiko Matsunaga, Division of Immunology, National Kyushu Cancer Center, 3-I-l Notame, Minami-ku. Fukuoka 815. Japan Ab&~ialions: ACNU, nimustine hydrochloride; rhTNF, recombinant human tumor necrosis factor; Gy, gray; LT. lymphotoxin; BSS, balanced salt solution; DDC, diethyldithiocarbamate; SOD, superoxidedismutase.
latory role on other cell function (Granger and Williams, 1968; Urban et al., 1986). It has been demonstrated that the antitumor activity of TNF and LT can be potentiated synergistically by interferons (Williams and Bellanti, 1983; Fransen et al., 1986) and in combination with antitumor drugs (Matsunaga and Mashiba, 1983). As molecular basis for the mechanisms of cytotoxininduced direct cytotoxicity, the involvement of oxygen Free radicals have been suggested (Mattews et al., 1987). BCNU has been reported to inactivate glutathione reductase (F&her and Ahmad, 1977). As a resuh of the inhibition of the ghrtathione redox cycle, oxygen free radicals, such as hydrogen peroxide and hydroperoxide, can be increased. By using a derivative of nitrosourea,
ACNU. we have observed that the simultaneous addition of ACNU with cytotoxins or the pretreatment oftarget ceils with ACNU can augment cytotoxin-induced cytotoxicity (Matsunaga and Mashiba, 1990). The augmentation of the antiproliferative effect on tumor cells could be induced through the combined use of LT with ACNW by lowering the intracellular level of glutathione (Mashiba et al., 1991). On the other hand, highly reactive superoxide has been reported to be responsible for the major component of radiation-induced oxygen effects (Oberley et al., 1976). In the present study, the effect of the combined use of ACNU with rhTNF on the radiationinduced antiproliferative effect was examined.
1 x lo5 cells/ml at 37 “C for 1 h. The cells were washed and adjusted to the same concentration as described above. The plates were incubated at 37 “C for I h before irradiation. After the termination of irradiation, the plates were incubated without washing at 37 ‘C for 48 h and the cultures were pulsed with 0.05 ml of serum-free [3H]thymidine 24 h before harvesting. Uptake of [ ‘Hlthymidine by the target cells was determined by scintitlation counting.
Materials and Metbods
Results
Cdl Meth A tumor cell line derived from Balb/c mouse was used as target cells and the cells were maintained in RPM1 1640 supplemented with 10% fetal bovine serum until use.
Eflect of the addition of ACNU on the radiationinduced inhibition of cell prolt$eration It was examined whether the addition of ACNU could augment the radiation-induced antiproliferative effect. Meth A tumor cells were irradiated in the presence of ACNU at 2&nl or 10 pg/ml and cultured in the continuous presence of ACNU for 48 h. As shown in Fig. 1, the proliferation of Meth A tumor cells was inhibited by the irradiation in a dose-dependent fashion, and the rate of inhibition of Meth A tumor cell proliferation at S Gy, 10 Gy and 15 Gy was 3.3%, 32.3 % and 43.6% compared with that in the nonirradiated group, respectively. The addition of ACNU at 2 pg/ml did not have a significant effect with the non-irradiated group. However, a slight degree of the augmented inhibition was observed through the addition of ACNU at 10 pg/ml in the 5 Gy, 10 Gy and 15 Gy irradiated groups, and the rates of inhibition compared with that in each medium control group were 27.3%, 18.8% and 26.1x, respectively. On the other hand, the rate of inhibition in the non-irradiated group was 17.4%.
Agent A derivative of nitrosourea, nimustine hydrochloride (ACNU) (Sankyo Co., Ltd., Japan) was used. As a cytokine, recombinant human tumor necrosis factor (rhTNF) (As&i Chemical Japan) was used. Assay for inhibition of cell proliferation Meth A tumor cells were washed with Hanks’ balanced salt solution (BSS) and adjusted to a concentration of 1 x IO4 cells/ml in RPM1 1640 supplemented with 10% fetal bovine serum. Onetenth of 1 ml of the cell suspension was plated in the well of U-bottomed microculture plates. ACNU and rhTNF at various concentrations were added to each well in a total volume of 0.2ml/weU. As controls, medium or each agent alone was added. In the experiment to examine the effect of pretreatment with ACNU, the target cells were incubated with ACNU (25 &ml) in a 15 x 60 mm plastic dish at a concentration of
Irradiation Irradiation was performed using Telecobalt at a dose rate of 0.513 Gy/min. Source-surface distance was 78.5 cm and field size was 2 1 x 2 1 cm.
201
C
5
10
15
Fig. I. Antiproliferative effect ofACNU in combined use of irradiation. Meth A tumor cells were incubated with medium alone (white bars) or ACNU at a final concentration of 2 @ml (stippled bars) and IO &nl (black bars) at 37 “C for I h in the wells of microplates, and irradiated without washing. The plates were incubated at 37 “C for 48 h and the cultures were pulsed with rH]thymidine 24 h before harvesting. Vertical bars indicate standard deviation. Significant at 0.01 rp < 0.025 (*), 0.005 -z p c 0.01 (**) and p -c 0.001 (I**) compared with the value in each medium control.
Augmentation of the radiation-induced antiprolrerative e#ect by addition of rhTNF Effect of rhTNF on the radiation-induced inhibition of the cell proliferation was examined. As
shown in Fig. 2a, the antiproliferative e&t was augmented by the addition of rhTNF in parallel with the dose of irradiation. The rate of inhibition through the addition of rhTNF in the 5 Gy, 10 Gy
“1
lrredkth
0
5
lb
15
b)
0
5
10
15
WY)
Fig. 2. Radiosensitizing effect ofTNF and ACNU. Meth A tumor cegs were incubated with medium alone (white bars) or TNF (IO U/ml) (stippled bars) in the absence (a) or the presence (b) of ACNU (10 pgiml) at 37 “C for I h in the wells of micmplates, and irradiated without washing at the dose of 5 Gy. 10 Gy or 15 Gy. Cytostatic assay was performed aa described in Fii. 1. Significant at 0.001 < p < 0.005 (*) and p
