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Antitumor activity of bestatin and tiorphan — inhibitors of enzymatic degradation of enkephalins in mice
Pharmacological Research, Vol . 25, Supplement 2, 1992
310 ANTITUMOR
ACTIVITY
OF
BESTATIN
AND
TIORPHAN -
INHIBITORS
OF
ENZYMATIC DEGRADATION OF ENKEPHALINS IN MICE Jan Kowalski, Zbigniew S . Herman Department of Clinical Pharmacology, Silesian School of Medicine Medyk6w 18, 40-752 Katowice, Poland Key words : bestatin, tiorphan, tumor, NK, macrophages There are evidence that methionine-enkephalin inhibited the local growth of B16-B16 melanoma 111, prolonged survival time of mice bearing leucemia L1210 121 and neuroblastoma S20Y, but the role of endogenous enkephalins in cancer growth is unclear . In order to study their influence on cancer process we chose the specific inhibitors of degradation of enkephalins - bestatin and tiorphan 131 . Material and methods BDF mice wer6 given s .c . 1x10 5 Bl6 melanoma cells or 1xlO 5 LLC cells or 5x10 B16 melanoma cells . The substances were given i .p for 5 consecutive days, from the 8-th day after the inoculation of cancer cells in the following doses : bestatin 1, 10, 50 pg ; tiorphan 0 .5, 5, 20, 50 pg ; naloxone 20 pg per mouse . The subcutaneous tumors were measured using the Vernier caliper . The measurements on any given day were calculated as x width 2 2 NK and macrophages activity in spleen C57B16 mice in vivo were determined using the typical 51 Cr - released methods using YAC-1 as target cells . v =
lenqht
Results The treatment with bestatin in a dose of 1 pg suppressed growth LLC (30-52% of control) ; 10 pg - (21-52%) . Moreover the injection of bestatin with tiorphan inhibited LLC growth in doses : bestatin 1 pg + tiorphan 0 .5 pg (34-67%) ; bestatin 10 pg + tiorphan 5 pg (57-65%) . The administration of bestatin inhibited growth of B16 melanoma (lx105 cells) in a dose of 1 pg (61-78%) ; 10 pg (6-18%) ; bestatin 1 pg + tiorphan 0 .5 pg (60-69%) ; bestatin 10 pg + tiorphan 5 pg (62-70%) . Bestatin with tiorphan pro- beared B16 melanoma (lxl0 5 longed survival period in mice cells) in a dose dependent manner : bestatin 1 pg - 3 .5% ; bestatin 10 pg - 3 .5% ; bestatin 1 pg + tiorphan 0 .5 pg - 9 .3% ; bestatin 10 pg + tiorphan 5 pg - 35 .8% . Tiorphan administration caused a cure of mice - beared B16 melanoma (5xl05 cells) in a dose-dependent manner : control - 25%, tiorphan 0 .5 pg - 50%, tiorphan 5 pg - 62 .5%, tiorphan 20 pg - 87 .5% . The treatment with tiorphan blocked the tumor growth in mice beared B16 melanoma (5xl05 cells) in doses : tiorphan 0 .5 pg (39-69%), tiorphan 5 pg (12-57%), tiorphan 50 pg + bestatin 50 pg (60-79%) . The administration of 10 pg bestatin + 5 pg tiorphan diminished B16 melanoma (5x105 cells) growth (45-53%) but pretreatment with 20 pg of naloxone blocked the action of bestatin 10 pg + tiorphan 5 pg (0-5%) . Naloxone in a dose of 20 pg shortened survival period in mice - beared B16 melanoma (5xl05 cells) by 20% . As
1043-6618/92/25110310-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol. 25, Supplement 2, 1992
far as NK cells activity is concerned we obtained the following results : 4 days after a one dose of tiorphan (5 pg) or bestatin (10 pg) or bestatin 10 pg with tiorphan 5 pg there was 1 .5%, 9 or 12 - fold increase respectively . Single administration of tiorphan or bestatin did not change the activity of macrophages from mice spleens . Discussion Bestatin is know microbial-origin substance with anticancer action in mice 141 and man . Our study partially supported this data . Bestatin suppressed tumor growth but did not prolong survival period or 'effect a cure animals beared cancer . Tiorphan evidently augmented anticancer action of bestatin and it made longer the survival time . The most interesting result of our study is that tiorphan was found to be able to cure animals bearing B16 melanoma . Naloxone blocked the action of bestatin + tiorphan . It may suggest that the endogenous enkephalins take part in anticancer mechanisms . The possibility of a cure animals bearing tumor suggest direct action of tiorphan on proliferation of B16 melanoma cells . References i . Murgo AJ : Inhibition of B16-B16 melanoma growth in mice by methionine-enkephalin . INCI 1985, 75 : 342-4 . 2 . Plotnikoff NP, Miller GC : Enkephalins as immunomodulators . Int J Immunopharmacol 1983, 5 : 437-41 . 3 . Zhang AZ, Yang HYT and Costa E : Nociception, enkephalin content and dipeptidyl carboxypeptidase activity in brain of mice treated with exopeptidase inhibitors . Neuropharmacology 1982, 21 : 625-30 . 4 . Ishizuka M, Masuola T, Kobayashi N, Fukusawa 5, Takeuchi T, Aoyagi T and Umezawa H : Effect of bestatin on mouse immune system and experimental murine tumors . J Antibiotics 1980, 33 : 642-52 .
311
310 ANTITUMOR
ACTIVITY
OF
BESTATIN
AND
TIORPHAN -
INHIBITORS
OF
ENZYMATIC DEGRADATION OF ENKEPHALINS IN MICE Jan Kowalski, Zbigniew S . Herman Department of Clinical Pharmacology, Silesian School of Medicine Medyk6w 18, 40-752 Katowice, Poland Key words : bestatin, tiorphan, tumor, NK, macrophages There are evidence that methionine-enkephalin inhibited the local growth of B16-B16 melanoma 111, prolonged survival time of mice bearing leucemia L1210 121 and neuroblastoma S20Y, but the role of endogenous enkephalins in cancer growth is unclear . In order to study their influence on cancer process we chose the specific inhibitors of degradation of enkephalins - bestatin and tiorphan 131 . Material and methods BDF mice wer6 given s .c . 1x10 5 Bl6 melanoma cells or 1xlO 5 LLC cells or 5x10 B16 melanoma cells . The substances were given i .p for 5 consecutive days, from the 8-th day after the inoculation of cancer cells in the following doses : bestatin 1, 10, 50 pg ; tiorphan 0 .5, 5, 20, 50 pg ; naloxone 20 pg per mouse . The subcutaneous tumors were measured using the Vernier caliper . The measurements on any given day were calculated as x width 2 2 NK and macrophages activity in spleen C57B16 mice in vivo were determined using the typical 51 Cr - released methods using YAC-1 as target cells . v =
lenqht
Results The treatment with bestatin in a dose of 1 pg suppressed growth LLC (30-52% of control) ; 10 pg - (21-52%) . Moreover the injection of bestatin with tiorphan inhibited LLC growth in doses : bestatin 1 pg + tiorphan 0 .5 pg (34-67%) ; bestatin 10 pg + tiorphan 5 pg (57-65%) . The administration of bestatin inhibited growth of B16 melanoma (lx105 cells) in a dose of 1 pg (61-78%) ; 10 pg (6-18%) ; bestatin 1 pg + tiorphan 0 .5 pg (60-69%) ; bestatin 10 pg + tiorphan 5 pg (62-70%) . Bestatin with tiorphan pro- beared B16 melanoma (lxl0 5 longed survival period in mice cells) in a dose dependent manner : bestatin 1 pg - 3 .5% ; bestatin 10 pg - 3 .5% ; bestatin 1 pg + tiorphan 0 .5 pg - 9 .3% ; bestatin 10 pg + tiorphan 5 pg - 35 .8% . Tiorphan administration caused a cure of mice - beared B16 melanoma (5xl05 cells) in a dose-dependent manner : control - 25%, tiorphan 0 .5 pg - 50%, tiorphan 5 pg - 62 .5%, tiorphan 20 pg - 87 .5% . The treatment with tiorphan blocked the tumor growth in mice beared B16 melanoma (5xl05 cells) in doses : tiorphan 0 .5 pg (39-69%), tiorphan 5 pg (12-57%), tiorphan 50 pg + bestatin 50 pg (60-79%) . The administration of 10 pg bestatin + 5 pg tiorphan diminished B16 melanoma (5x105 cells) growth (45-53%) but pretreatment with 20 pg of naloxone blocked the action of bestatin 10 pg + tiorphan 5 pg (0-5%) . Naloxone in a dose of 20 pg shortened survival period in mice - beared B16 melanoma (5xl05 cells) by 20% . As
1043-6618/92/25110310-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol. 25, Supplement 2, 1992
far as NK cells activity is concerned we obtained the following results : 4 days after a one dose of tiorphan (5 pg) or bestatin (10 pg) or bestatin 10 pg with tiorphan 5 pg there was 1 .5%, 9 or 12 - fold increase respectively . Single administration of tiorphan or bestatin did not change the activity of macrophages from mice spleens . Discussion Bestatin is know microbial-origin substance with anticancer action in mice 141 and man . Our study partially supported this data . Bestatin suppressed tumor growth but did not prolong survival period or 'effect a cure animals beared cancer . Tiorphan evidently augmented anticancer action of bestatin and it made longer the survival time . The most interesting result of our study is that tiorphan was found to be able to cure animals bearing B16 melanoma . Naloxone blocked the action of bestatin + tiorphan . It may suggest that the endogenous enkephalins take part in anticancer mechanisms . The possibility of a cure animals bearing tumor suggest direct action of tiorphan on proliferation of B16 melanoma cells . References i . Murgo AJ : Inhibition of B16-B16 melanoma growth in mice by methionine-enkephalin . INCI 1985, 75 : 342-4 . 2 . Plotnikoff NP, Miller GC : Enkephalins as immunomodulators . Int J Immunopharmacol 1983, 5 : 437-41 . 3 . Zhang AZ, Yang HYT and Costa E : Nociception, enkephalin content and dipeptidyl carboxypeptidase activity in brain of mice treated with exopeptidase inhibitors . Neuropharmacology 1982, 21 : 625-30 . 4 . Ishizuka M, Masuola T, Kobayashi N, Fukusawa 5, Takeuchi T, Aoyagi T and Umezawa H : Effect of bestatin on mouse immune system and experimental murine tumors . J Antibiotics 1980, 33 : 642-52 .
311