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Effects of gamma interferon on mice bearing P388 or friend leukemias as sensitive or doxorubicin-resistant variants
195
stimulating in vitro clonal growth of human SCLC in a dose-dependent manner (Davis et al., 1989). To &e&e if growth factor processing is a requirement for SCLC clonal growth we tested the effect of specific protease inhibitors on SCLC growth using the clonogenic assay developed by Hamburger and Salmon (1977). Protease inhibitors at varying concentrations were mixed with human SCLC cells, media and agarose. This mixture was then applied to pre-prepared underlayers in plastic petri dishes. The cells were allowed to grow at 5% CO* for 9-14 days at which time colonies2 42 p were counted by an automated colony counter. Shown below in Table 1 is the effect of the aminopeptidase inhibitor-bestatin, chymotrypsin/trypsin inhibitor-Bowman-Birk inhibitor (BBI), serine proteasc inhibitor-aprotinin and neutral endopeptidase-EC. 3.4.24.11 (NEP) inhibitors-phosphoramidon and thiorphan on SCLC (NCI-N417) growth. Specific enzyme assays were also developed for trypsin-like activity, NEP carboxypeptidase E (E.C. 3.4.17.10) and metalloendopeptidase E.C. 3.4.24.15 in SCLC cell lines. No evidence of NEP was found in N417 which agrees with the lack of growth inhibition by thiorphan and phosphoramidon, however, significant levels of trypsin-like activity were noted which may have contributed to the positive BBI effect after the single application of 100 tg/ml (Table 1). In conclusion, we have shown that specific protease inhibitors inhibit the in vitro clonal growth of human SCLC in a dosedependent manner. These data provide evidence that protease inhibitors may be interfering with the processing of SCLC growth factors and the expression of the autocrine response. Supported by USPHS CA44869 and DA06284. Table 1 Effect of protease inhibitors on SCLC (NCI-N417) colony growth. Data presented is mean colony count. Vehicle
Bestatin
BBI
0.05 pM 448k64.8
220f20.3
50 CM + 144f
Aprotinin
0.10 as/ml 7.9 * 258f19.6
100 /@III 172f
0.01 tg/mJ
7.3 * 421zt59.1
Thiorphan 10 pg/ml
0.01 PM
166f42.5
* 330f83.3
Phosphoramidon 10 pM
0.001 CM 1.0 &tM
332k36.9
350f16.8
234kll.O
(x + S.B.M.; n = 3-8 plates), * p c 0.05 by Newman-Keuls
Refferences Davis, T.P.. H.S. Burgess, S. Crowell. T.W. Moody, A. Culling-Berglund, and R.H. Liu. 1989, &Endorphin and neurotensin stimulate in vitro clonal growth of human SCLC cells, Eur. J. Pharmacol. 161.283. Hamburger, A.W. and S.E. Salmon. 1977, Primary bioassay of human tumor stem cells, Science 197,461. Kennedy. A.R. and J.B. Lit&, 1981, Effects of protease inhibitors on radiation transformation in vitro. Cancer Res. 41, 2103.
Effects of gamma interferon on mice or doxorubicin-resis Crescimanno
*, M., Armata
*, M.G., Rausa
*, L., Tapiero
* *, H. and D’Alessandro
* * *, N.
* Istituto di Farmacologia, Policlinico P. Giaccone, 90127 Palermo, Italy * * Institut de Canchologie et d’lmmunog~nhpe,
Virreilcif.
France and * * * Istituto di Farmacologia, Piazza XX Settembre 4, 98100 Messina, Italy
Considering the need for a more rational integrated approach to cancer treatment, we think that an important and open question regards the sensitivity to biological response modifiers of tumour populations resistant to chemotherapy. Here we report on the effects of murine recombinant gamma interferon (GI, obtained from Holland biotechnology bv) in female DBA/t mice bearing P388 or Friend (Friend erythroleukemia, FLC) as sensitive or Doxorubicin (DXR)-resistant variants (P388/DXR 70 fold resistant to DXR, FLC/DXR 35 fold resistant to DXR, both with multidrug resistance characteristics).
th 1 x lo4 cells of P388 or P388/DXR leukemias. Other mice received ip 1 x mias. P388 and P388/DXR mice received vehicle (controls) or GI ip and up to This dose was the most effective on the x 7 days and starting from day p < 0.01 in P388, increased by 4 not significant in P388/DXR). FLC and ved vehicle or GI ip at the dose of 1 X lo4 unit daily for 10 days and starting from day the survival of FLC/DXR mice ( + 33%, p < 0.05 with respect to controls) and has produced a that of FLC mice (we are still checking the survival of some animals). s of the antioxidant defenses in P388 and P388/DXR cells suggested that the levels of glutathione P388) and of some enzymes related to it (glutathione peroxidase and respectively, in P388/DXR) could confer resistance to toxic oxygen also by macrophages activated by GI. However, a treatment with buthionine7 days), which efficiently reduced the glutathione of P388 cells (3% of the base f the base line) did not significantly modify the activity of GI against P388 and , buthionine-sulfoximine improved the effects of DXR (10 mg/Kg ip at day 4) on P388 bearing animals (+ 109% with buthionine-sulfoximine + DXR, + 75% with DXR alone, p < 0.05). not obsetved in the P388/DXR ones. Although limit& to only two experimental models, our data seem to indicate that some cross-resistance exists chemotherapeutic agents and GI. Further study is in progress in order to: widen the spectrum of our ons to other tumour models; better define the mechanistic aspects of our results. Supported by Associaxione Italiana Per La Ricerca Sul Cancro.
C
ice
mary carcino
Sava, G., Ceschia, V., Pacer, S. and Bregant, F. Insriture of phdog~,
School of Phatmaq,
Universiry of Trieste, via A. Valerio 32, 34127 Trieste, Ita&
Egg-white lysozyme was shown capable to exert antineoplastic effects in mice bearing solid metastasizing tumors (Sava et al., 19882; Sava et al., 19888; Sava et al., 1989). The antitumor action, mainly directed to tumor metastases, was achievc;d by oral administration of lysozyme with the food. With the present study, some aspects of the action of lysoxyme in the MCa mammary carcinoma tumor of the CBA mouse are analyzed (see table 1). Lysoxyme, given at the daily dose of 100 mg/kg, was administered in amounts of 3.5 g of powdered food per day. Treatment of female CBA mice of 20-22 g for 7 consecutive days prior to inoculation of 3.4 X lo4 MCa mammary carcinoma cells causes a significant reduction of tumor growth by 52% (p c 0.05, t-test for grouped data) and increases Table 1 Evidences of indirect antitumor activity of orally administered lysozyme. Treatment Oral lyso~e prior to tumor inocuiation Plasma from lysozyme-treated tumored hosts b fn vitro with phna Sampki (withoutcaching) Envitro with plasma samples (with washing)
’
Primary tumor
Lung metastases
48.0 f 20.7 +
122.8& 8.5 4* 16.5* 6.5 * 20.5 f 15.6 * 174.8 f 82.8
69.6i 4.8 * 145.9 f 30.7
Each value is the mean percent ratio (treated over controls) f S.E. obtained with groups of at least 8 mice. a: average survival time; b: ahquots of 50 ~1 of whole plasma given i.v. 24 hr after removal of primary tumor (day 12 from implantation); c: aliquots of 100 pl of a mixture containing 100 ~1 of whole plasma and 10 tumor cells/ml injected S.C. to intact hosts; d: cells washed twice by centrifuging at 5OOXg per 10 min in cold and resuspending the pellet in phosphate buffered saline; *: means statistically different from the relevant controls.
stimulating in vitro clonal growth of human SCLC in a dose-dependent manner (Davis et al., 1989). To &e&e if growth factor processing is a requirement for SCLC clonal growth we tested the effect of specific protease inhibitors on SCLC growth using the clonogenic assay developed by Hamburger and Salmon (1977). Protease inhibitors at varying concentrations were mixed with human SCLC cells, media and agarose. This mixture was then applied to pre-prepared underlayers in plastic petri dishes. The cells were allowed to grow at 5% CO* for 9-14 days at which time colonies2 42 p were counted by an automated colony counter. Shown below in Table 1 is the effect of the aminopeptidase inhibitor-bestatin, chymotrypsin/trypsin inhibitor-Bowman-Birk inhibitor (BBI), serine proteasc inhibitor-aprotinin and neutral endopeptidase-EC. 3.4.24.11 (NEP) inhibitors-phosphoramidon and thiorphan on SCLC (NCI-N417) growth. Specific enzyme assays were also developed for trypsin-like activity, NEP carboxypeptidase E (E.C. 3.4.17.10) and metalloendopeptidase E.C. 3.4.24.15 in SCLC cell lines. No evidence of NEP was found in N417 which agrees with the lack of growth inhibition by thiorphan and phosphoramidon, however, significant levels of trypsin-like activity were noted which may have contributed to the positive BBI effect after the single application of 100 tg/ml (Table 1). In conclusion, we have shown that specific protease inhibitors inhibit the in vitro clonal growth of human SCLC in a dosedependent manner. These data provide evidence that protease inhibitors may be interfering with the processing of SCLC growth factors and the expression of the autocrine response. Supported by USPHS CA44869 and DA06284. Table 1 Effect of protease inhibitors on SCLC (NCI-N417) colony growth. Data presented is mean colony count. Vehicle
Bestatin
BBI
0.05 pM 448k64.8
220f20.3
50 CM + 144f
Aprotinin
0.10 as/ml 7.9 * 258f19.6
100 /@III 172f
0.01 tg/mJ
7.3 * 421zt59.1
Thiorphan 10 pg/ml
0.01 PM
166f42.5
* 330f83.3
Phosphoramidon 10 pM
0.001 CM 1.0 &tM
332k36.9
350f16.8
234kll.O
(x + S.B.M.; n = 3-8 plates), * p c 0.05 by Newman-Keuls
Refferences Davis, T.P.. H.S. Burgess, S. Crowell. T.W. Moody, A. Culling-Berglund, and R.H. Liu. 1989, &Endorphin and neurotensin stimulate in vitro clonal growth of human SCLC cells, Eur. J. Pharmacol. 161.283. Hamburger, A.W. and S.E. Salmon. 1977, Primary bioassay of human tumor stem cells, Science 197,461. Kennedy. A.R. and J.B. Lit&, 1981, Effects of protease inhibitors on radiation transformation in vitro. Cancer Res. 41, 2103.
Effects of gamma interferon on mice or doxorubicin-resis Crescimanno
*, M., Armata
*, M.G., Rausa
*, L., Tapiero
* *, H. and D’Alessandro
* * *, N.
* Istituto di Farmacologia, Policlinico P. Giaccone, 90127 Palermo, Italy * * Institut de Canchologie et d’lmmunog~nhpe,
Virreilcif.
France and * * * Istituto di Farmacologia, Piazza XX Settembre 4, 98100 Messina, Italy
Considering the need for a more rational integrated approach to cancer treatment, we think that an important and open question regards the sensitivity to biological response modifiers of tumour populations resistant to chemotherapy. Here we report on the effects of murine recombinant gamma interferon (GI, obtained from Holland biotechnology bv) in female DBA/t mice bearing P388 or Friend (Friend erythroleukemia, FLC) as sensitive or Doxorubicin (DXR)-resistant variants (P388/DXR 70 fold resistant to DXR, FLC/DXR 35 fold resistant to DXR, both with multidrug resistance characteristics).
th 1 x lo4 cells of P388 or P388/DXR leukemias. Other mice received ip 1 x mias. P388 and P388/DXR mice received vehicle (controls) or GI ip and up to This dose was the most effective on the x 7 days and starting from day p < 0.01 in P388, increased by 4 not significant in P388/DXR). FLC and ved vehicle or GI ip at the dose of 1 X lo4 unit daily for 10 days and starting from day the survival of FLC/DXR mice ( + 33%, p < 0.05 with respect to controls) and has produced a that of FLC mice (we are still checking the survival of some animals). s of the antioxidant defenses in P388 and P388/DXR cells suggested that the levels of glutathione P388) and of some enzymes related to it (glutathione peroxidase and respectively, in P388/DXR) could confer resistance to toxic oxygen also by macrophages activated by GI. However, a treatment with buthionine7 days), which efficiently reduced the glutathione of P388 cells (3% of the base f the base line) did not significantly modify the activity of GI against P388 and , buthionine-sulfoximine improved the effects of DXR (10 mg/Kg ip at day 4) on P388 bearing animals (+ 109% with buthionine-sulfoximine + DXR, + 75% with DXR alone, p < 0.05). not obsetved in the P388/DXR ones. Although limit& to only two experimental models, our data seem to indicate that some cross-resistance exists chemotherapeutic agents and GI. Further study is in progress in order to: widen the spectrum of our ons to other tumour models; better define the mechanistic aspects of our results. Supported by Associaxione Italiana Per La Ricerca Sul Cancro.
C
ice
mary carcino
Sava, G., Ceschia, V., Pacer, S. and Bregant, F. Insriture of phdog~,
School of Phatmaq,
Universiry of Trieste, via A. Valerio 32, 34127 Trieste, Ita&
Egg-white lysozyme was shown capable to exert antineoplastic effects in mice bearing solid metastasizing tumors (Sava et al., 19882; Sava et al., 19888; Sava et al., 1989). The antitumor action, mainly directed to tumor metastases, was achievc;d by oral administration of lysozyme with the food. With the present study, some aspects of the action of lysoxyme in the MCa mammary carcinoma tumor of the CBA mouse are analyzed (see table 1). Lysoxyme, given at the daily dose of 100 mg/kg, was administered in amounts of 3.5 g of powdered food per day. Treatment of female CBA mice of 20-22 g for 7 consecutive days prior to inoculation of 3.4 X lo4 MCa mammary carcinoma cells causes a significant reduction of tumor growth by 52% (p c 0.05, t-test for grouped data) and increases Table 1 Evidences of indirect antitumor activity of orally administered lysozyme. Treatment Oral lyso~e prior to tumor inocuiation Plasma from lysozyme-treated tumored hosts b fn vitro with phna Sampki (withoutcaching) Envitro with plasma samples (with washing)
’
Primary tumor
Lung metastases
48.0 f 20.7 +
122.8& 8.5 4* 16.5* 6.5 * 20.5 f 15.6 * 174.8 f 82.8
69.6i 4.8 * 145.9 f 30.7
Each value is the mean percent ratio (treated over controls) f S.E. obtained with groups of at least 8 mice. a: average survival time; b: ahquots of 50 ~1 of whole plasma given i.v. 24 hr after removal of primary tumor (day 12 from implantation); c: aliquots of 100 pl of a mixture containing 100 ~1 of whole plasma and 10 tumor cells/ml injected S.C. to intact hosts; d: cells washed twice by centrifuging at 5OOXg per 10 min in cold and resuspending the pellet in phosphate buffered saline; *: means statistically different from the relevant controls.