胍丁胺抗肿瘤作用的药效学评价与机制探讨
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摘要
胍丁胺(agmatine)是左旋精氨酸脱羧基的产物。目前，大多数学者认为它
是一种新的神经递质和／或神经调质，是咪唑啉受体的内源性配体。国内外研究
已证实胍丁胺具有多种生物学功能，主要包括神经元保护、影响激素或递质的释
放、降低血糖浓度、促进肾脏水钠排泄、减少心肌细胞坏死、抑制血管内皮细胞
过度增生、刺激胃酸分泌、抗炎、抗抑郁、调节阿片功能等等。作为经典多胺的
类似物，胍丁胺对细胞增殖、分化的作用已引起了我们对其抗肿瘤研究的兴趣。
本研究旨在整体和体外培养两种实验模型上观察胍丁胺对肿瘤组织和肿瘤细胞增
殖的抑制作用，探讨胍丁胺可能的抗肿瘤作用机制，为深入研究胍丁胺的抗肿瘤
作用提供药效学依据。
皿丁胺抗肿瘤作用的药效学评价与机制探讨
中文摘要
二、肌丁胺抗肿瘤作用的机制研究
1、咪哇琳受体拮抗剂咪哇克生本身不影响体外培养MCF细胞的增殖，亦不
能阻断肌丁胺抑制MCF细胞增殖，提示在本实验模型上肌丁胺抑制MCF细胞的
增殖并非通过激活咪哇琳受体实现的。
2、肌丁胺(1一looopM)对MCF细胞培养介质中的乳酸脱氢酶活性没有显
著影响，1000 pM浓度以下的肌丁胺对ovca、Kse:和Kat。一111细胞无抑制增殖作用，
提示肌丁胺无细胞毒作用，在本实验模型上肌丁胺抑制MCF细胞的增殖与细胞毒
性无关。
3、在生物体内的多胺包括腐胺、精胺和亚精胺。在0.01一1000 pM浓度范围
内腐胺对MCF细胞增殖无影响，腐胺(12.5一loop玛能浓度依赖性地拮抗l000p
M肌丁胺对体外培养MCF细胞增殖抑制作用。在0.01一1 pM浓度范围内，精胺和
亚精胺对MCF细胞增殖无影响，但其浓度达到1一1 00 pM时则具有明确的抑制
MCF细胞增殖的作用，且在此浓度范围内与肌丁胺合用能增强肌丁胺抑制肿瘤细
胞增殖的作用。这些实验结果提示肌丁胺的抗肿瘤作用可能与其抑制多胺合成有
关。
三、结论:
1.肌丁胺有明确的抑制肿瘤细胞增殖的作用，且其抑制肿瘤细胞增殖的作用
具有瘤细胞株特异性。
2.肌丁胺抑制肿瘤增殖的作用与激活咪哇琳受体和细胞毒作用无关。
3.肌丁胺抑制体外培养MCF细胞增殖的作用与其影响多胺合成有关。
Agmatine, the analog of polyamine, which was formed by the decarboxylation of L-Arginine by the enzyme L-arginine decarboxylase, has been postulated to be an endogenous ligand for imidazoline receptors (I-R). Many biological functions of agmatine have been found such as neuron protection, influence on the release of hormones and neurotransmitters, antidepression and modulation on opioid functions. The recent discovery for the effect of agmatine on cell proliferation and differentiation has caused our interest in the effects of agmatine on tumor. However, there is no report about whether agmatine can inhibit the proliferation of tumor by now. In this experiment, we have studied the inhibitory effect of agmatine on the proliferation of tumor tissues in mice in vivo and tumor cells in culture in vitro for the first times. In addition, the possible mechanisms for the anti-proliferative effect of agmatine were studied at the same time.
Section A: Pharmacological effects of agmatine on tumor cell lines in vitro
1 A weak inhibitory effect of agmatine was found on the proliferation of ovaries cancer cell line (Ovca), gastric carcinoma cell line ( K562 ) and human erythroleukemia cell line (Kato-III) in the current experiments. However, in MCF cell line, agmatine inhibited the proliferation of the tumor cells in MTT assay and 3H-thymidine incorporation assay. These results inferred that agmatine had anti-proliferative effect on tumor cells, but this effect was specific for cell lines. Agmatine could inhibit the proliferation of MCF cell in the range of 1~1000u M in a concentration-dependent manner both in the MTT assay and 3H-thymidine incorporation test. The highest inhibitory rate was over 50%. The anti-proliferative effect of agmatine 1000 u M on MCF cell lines exhibited a time-dependent characteristic during 48 hours.
2 In mice transplanted with S180 sarcoma and B16 melanoma tumor cell lines, the tumor weight was significantly reduced after the mice were treated with agmatine 5-40 mg.kg-1, and these efFects exhibited a dose-dependent manner.
Section B: Possible mechanisms for the antiproliferation of agmatine on tumor cells
1 Idazoxan, an antagonist of I-R, had no influence on the proliferation of MCF cell
itself and had no action on the inhibitory effects of agmatine on the proliferation of MCF cells. This result indicated that the inhibitory effect of agmatine didn't due to the activation of I-R.
2 As to MCF cells, agmatine (1-1000 u M) had no influence on the activity of LDH, which suggest that agmatine has no direct cell toxicity in vitro and the inhibitory effect of agmatine on the proliferation of MCF cells had nothing to do with the cell toxicity.
3 Polyamines include putrescine, spermidine and spermine. Putrescine at the concentration of 0.01-1000 u M, had no inhibitory and facilitory effects on the MCF proliferation. When co-administrated with agmatine, putrescine (12.5-100 u M) reversed agmatine's inhibitory effect on MCF. Spermine and spermidine, at the concentration of 0.01-1 u M, had no inhibitory and facilitory effect on the proliferation of tumor cells. However, at the concentration of 1-100 u M, spermidine and spermine significantly inhibited the proliferation of tumor cells and enhanced the anti-proliferative effect of agmatine. These results implicated that agmatine had anti-proliferative effect on tumor cells and the possible mechanism might relate to interfere with the synthesis of polyamines.
Section C: Conclusion
1 Agmatine inhibited the proliferation of tumor cells, and this effect was specific for
tumor cell lines.
2 The anti-proliferative effect of agmatine on tumor cells didn't due to the activation of I-R and its cell toxicity.
3 The possible mechanism for the anti-proliferative effect of agmatine on MCF cells might be relate to its influence on the synthesis of polyamines.
是一种新的神经递质和／或神经调质，是咪唑啉受体的内源性配体。国内外研究
已证实胍丁胺具有多种生物学功能，主要包括神经元保护、影响激素或递质的释
放、降低血糖浓度、促进肾脏水钠排泄、减少心肌细胞坏死、抑制血管内皮细胞
过度增生、刺激胃酸分泌、抗炎、抗抑郁、调节阿片功能等等。作为经典多胺的
类似物，胍丁胺对细胞增殖、分化的作用已引起了我们对其抗肿瘤研究的兴趣。
本研究旨在整体和体外培养两种实验模型上观察胍丁胺对肿瘤组织和肿瘤细胞增
殖的抑制作用，探讨胍丁胺可能的抗肿瘤作用机制，为深入研究胍丁胺的抗肿瘤
作用提供药效学依据。
皿丁胺抗肿瘤作用的药效学评价与机制探讨
中文摘要
二、肌丁胺抗肿瘤作用的机制研究
1、咪哇琳受体拮抗剂咪哇克生本身不影响体外培养MCF细胞的增殖，亦不
能阻断肌丁胺抑制MCF细胞增殖，提示在本实验模型上肌丁胺抑制MCF细胞的
增殖并非通过激活咪哇琳受体实现的。
2、肌丁胺(1一looopM)对MCF细胞培养介质中的乳酸脱氢酶活性没有显
著影响，1000 pM浓度以下的肌丁胺对ovca、Kse:和Kat。一111细胞无抑制增殖作用，
提示肌丁胺无细胞毒作用，在本实验模型上肌丁胺抑制MCF细胞的增殖与细胞毒
性无关。
3、在生物体内的多胺包括腐胺、精胺和亚精胺。在0.01一1000 pM浓度范围
内腐胺对MCF细胞增殖无影响，腐胺(12.5一loop玛能浓度依赖性地拮抗l000p
M肌丁胺对体外培养MCF细胞增殖抑制作用。在0.01一1 pM浓度范围内，精胺和
亚精胺对MCF细胞增殖无影响，但其浓度达到1一1 00 pM时则具有明确的抑制
MCF细胞增殖的作用，且在此浓度范围内与肌丁胺合用能增强肌丁胺抑制肿瘤细
胞增殖的作用。这些实验结果提示肌丁胺的抗肿瘤作用可能与其抑制多胺合成有
关。
三、结论:
1.肌丁胺有明确的抑制肿瘤细胞增殖的作用，且其抑制肿瘤细胞增殖的作用
具有瘤细胞株特异性。
2.肌丁胺抑制肿瘤增殖的作用与激活咪哇琳受体和细胞毒作用无关。
3.肌丁胺抑制体外培养MCF细胞增殖的作用与其影响多胺合成有关。
Agmatine, the analog of polyamine, which was formed by the decarboxylation of L-Arginine by the enzyme L-arginine decarboxylase, has been postulated to be an endogenous ligand for imidazoline receptors (I-R). Many biological functions of agmatine have been found such as neuron protection, influence on the release of hormones and neurotransmitters, antidepression and modulation on opioid functions. The recent discovery for the effect of agmatine on cell proliferation and differentiation has caused our interest in the effects of agmatine on tumor. However, there is no report about whether agmatine can inhibit the proliferation of tumor by now. In this experiment, we have studied the inhibitory effect of agmatine on the proliferation of tumor tissues in mice in vivo and tumor cells in culture in vitro for the first times. In addition, the possible mechanisms for the anti-proliferative effect of agmatine were studied at the same time.
Section A: Pharmacological effects of agmatine on tumor cell lines in vitro
1 A weak inhibitory effect of agmatine was found on the proliferation of ovaries cancer cell line (Ovca), gastric carcinoma cell line ( K562 ) and human erythroleukemia cell line (Kato-III) in the current experiments. However, in MCF cell line, agmatine inhibited the proliferation of the tumor cells in MTT assay and 3H-thymidine incorporation assay. These results inferred that agmatine had anti-proliferative effect on tumor cells, but this effect was specific for cell lines. Agmatine could inhibit the proliferation of MCF cell in the range of 1~1000u M in a concentration-dependent manner both in the MTT assay and 3H-thymidine incorporation test. The highest inhibitory rate was over 50%. The anti-proliferative effect of agmatine 1000 u M on MCF cell lines exhibited a time-dependent characteristic during 48 hours.
2 In mice transplanted with S180 sarcoma and B16 melanoma tumor cell lines, the tumor weight was significantly reduced after the mice were treated with agmatine 5-40 mg.kg-1, and these efFects exhibited a dose-dependent manner.
Section B: Possible mechanisms for the antiproliferation of agmatine on tumor cells
1 Idazoxan, an antagonist of I-R, had no influence on the proliferation of MCF cell
itself and had no action on the inhibitory effects of agmatine on the proliferation of MCF cells. This result indicated that the inhibitory effect of agmatine didn't due to the activation of I-R.
2 As to MCF cells, agmatine (1-1000 u M) had no influence on the activity of LDH, which suggest that agmatine has no direct cell toxicity in vitro and the inhibitory effect of agmatine on the proliferation of MCF cells had nothing to do with the cell toxicity.
3 Polyamines include putrescine, spermidine and spermine. Putrescine at the concentration of 0.01-1000 u M, had no inhibitory and facilitory effects on the MCF proliferation. When co-administrated with agmatine, putrescine (12.5-100 u M) reversed agmatine's inhibitory effect on MCF. Spermine and spermidine, at the concentration of 0.01-1 u M, had no inhibitory and facilitory effect on the proliferation of tumor cells. However, at the concentration of 1-100 u M, spermidine and spermine significantly inhibited the proliferation of tumor cells and enhanced the anti-proliferative effect of agmatine. These results implicated that agmatine had anti-proliferative effect on tumor cells and the possible mechanism might relate to interfere with the synthesis of polyamines.
Section C: Conclusion
1 Agmatine inhibited the proliferation of tumor cells, and this effect was specific for
tumor cell lines.
2 The anti-proliferative effect of agmatine on tumor cells didn't due to the activation of I-R and its cell toxicity.
3 The possible mechanism for the anti-proliferative effect of agmatine on MCF cells might be relate to its influence on the synthesis of polyamines.
引文
1． Thomas T, Thomas TJ. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci 2001; 58:244-58．
2． Tabib A and Bachrach U. Polyamines induce malignanttransforamation in cultured NIH 3T3 fibroblasts, Int. J.Biochem. Cell Biol. 1998; 30 :135-46．
3． Nesrin Gundogus-Ozcanli,Cafer Sayilirt and Wayne E. Effects of Polyamines, Polyamine Synthesis Inhibitors, and Polyaine Analogs on Casein Kinase 2 Using Myc Oncoprotein as Substrate. Biochemical Pharmacology, 1999; 58:251-4．
4． Su RB, Li J, Qin BY. Biphasic opioid function modulator: agmatine. Acta Pharmacol Sin 2003; 24:631-6．
5． Trujillo KA, Akil H. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801． Science 1991;251(4989) :85-7．
6． Verma V, Mediratta PK, Sharma KK. Potentiation of analgesia and reversal of tolerance to morphine by calcium channel blockers. Indian. J Exp Biol. 2001; 39(7) :636-42．
7． Michaluk J, Karolewicz B, Antkiewicz-Michaluk L, Vetulani J. Effects of various Ca2+ channel antagonists on morphine analgesia, tolerance and dependence, and on blood pressure in the rat. Eur J Pharmacol. 1998; 352(2-3) : 189-97．
8． Li J, Li X, Pei G, Qin BY. Effects of agmatine on tolerance to and substance dependence on morphine in mice. Acta Pharmacol Sin, 1999;20(3) :232-8．
9． Li J. Opioid function modulator. Information of the Chinese pharmacological society, 2002;19(1) :14-5．
10． Nester EJ. Molecular mechanisms of drug addiction. J Neurosci, 1992; 12(7) : 2439-50．
11． Bhargava HA. Diversity of agents that modify opioid tolerance, physical dependence, abstinence syndrome, and self-administrative behavior. Pharmacol Rev, 1994;46(3) :293-324．
12． Coderre TJ. Potent analgesia induced in rats by combined action at PCP and polyamine recognition sites of the NMDA receptor complex. Eur J Neurosci 1993; 5(4) :390-3．
13． Li, G., Regunathan, S., Barrow, CJ., Eshraghi, J., Cooper, R., Reis, DJ.. Agmatine: an endogenous clonidine-displacing substance in the brain. Science 1994; 263(18) : 966-9．
14． Reis DJ. Regunathan S. Agmatine: an endogenous ligand at imidazoline receptors is a novel neurotransmitter. Ann N Y Acad Sci 1999; 881: 65-80．
15． Kolesnikov, Y, Jain, S., Pasternak, GW. Modulation of opioid analgesia by agmatine. Eur J Pharmacol. 1996; 296: 17-22．
16． Li, J., Li, X., Pei, G., Qin, BY. Analgesic effect of agmatine and its enhancement on morphine analgesia in mice and rats. Acta Pharmacol Sin 1999; 20: 81-5．
17． Li, J., Li, X., Pei, G., Qin, BY. Effects of agmatine on tolerance to and substance dependence on morphine in mice. Acta Pharmacol Sin 1999; 20: 232-8．
18． Li J, Li X, Pei G, Qin BY. Correlation between inhibitions of morphine withdrawal and nitric oxide synthase by agmatine. Acta Pharmacologica Sinica 1999, 20(4) : 375-80．
19． Su, RB., Li, J., Gao, K., Pei, G., Qin, BY. Influence of idazoxan on analgesia, tolerance, and physical dependence of morphine in mice and rats in vivo. Acta Pharmacol Sin. 2000; 21: 1011-5．
20． Eick AJ van. A change in the response of the mouse in the "hot plate" analgesia-test,owing to a central action of atrophine and related compounds. Acta physiol Pharmacol Neerl ,1967, 14(4) : 499-500．
21． Kossel A. Zeischrift fur Physiologiche Chemie 1910:257-61．
22． Reis, DJ. and Regunathan, S. Is agmatine a novel neurotransmitter in brain? Trends Pharmacol Sci. 2000; 21(5) : 187-93．
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41． Moldering GJ, Menzel S, Gothert M. Imidazoline derivatives and agmatine induce histamine release from the rat stomach. Naunyn Schmiedebergs Arch Pharmacol, 1999, 360(6) :711-4．
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45． Fairbanks CA, Schreiber KL, Brewer KL. Agmatine reverses pain induced by inflammation, neuropathy, and spinal cord injury. Proc Natl Acad Sci U S A 2000; 97(19) :10584-9．
46． Y esilyurt O, Uzbay IT. Agmatine potentiates the analgesic effect of morphine by an alpha(2) -adrenoceptor-mediated mechanism in mice. Neuropsychopharmacology 2001;25(1) :98-103．
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50． Mauro Sparapani, Ottavio Gandolfi, Elisabetta Ciani, Neurotoxicity of Polyamines and Pharmacological Neuroprotection in Cultures of Rat Cerebellar Granule Cells.Experimental Neurology 1997;148; 157-66 .
2． Tabib A and Bachrach U. Polyamines induce malignanttransforamation in cultured NIH 3T3 fibroblasts, Int. J.Biochem. Cell Biol. 1998; 30 :135-46．
3． Nesrin Gundogus-Ozcanli,Cafer Sayilirt and Wayne E. Effects of Polyamines, Polyamine Synthesis Inhibitors, and Polyaine Analogs on Casein Kinase 2 Using Myc Oncoprotein as Substrate. Biochemical Pharmacology, 1999; 58:251-4．
4． Su RB, Li J, Qin BY. Biphasic opioid function modulator: agmatine. Acta Pharmacol Sin 2003; 24:631-6．
5． Trujillo KA, Akil H. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801． Science 1991;251(4989) :85-7．
6． Verma V, Mediratta PK, Sharma KK. Potentiation of analgesia and reversal of tolerance to morphine by calcium channel blockers. Indian. J Exp Biol. 2001; 39(7) :636-42．
7． Michaluk J, Karolewicz B, Antkiewicz-Michaluk L, Vetulani J. Effects of various Ca2+ channel antagonists on morphine analgesia, tolerance and dependence, and on blood pressure in the rat. Eur J Pharmacol. 1998; 352(2-3) : 189-97．
8． Li J, Li X, Pei G, Qin BY. Effects of agmatine on tolerance to and substance dependence on morphine in mice. Acta Pharmacol Sin, 1999;20(3) :232-8．
9． Li J. Opioid function modulator. Information of the Chinese pharmacological society, 2002;19(1) :14-5．
10． Nester EJ. Molecular mechanisms of drug addiction. J Neurosci, 1992; 12(7) : 2439-50．
11． Bhargava HA. Diversity of agents that modify opioid tolerance, physical dependence, abstinence syndrome, and self-administrative behavior. Pharmacol Rev, 1994;46(3) :293-324．
12． Coderre TJ. Potent analgesia induced in rats by combined action at PCP and polyamine recognition sites of the NMDA receptor complex. Eur J Neurosci 1993; 5(4) :390-3．
13． Li, G., Regunathan, S., Barrow, CJ., Eshraghi, J., Cooper, R., Reis, DJ.. Agmatine: an endogenous clonidine-displacing substance in the brain. Science 1994; 263(18) : 966-9．
14． Reis DJ. Regunathan S. Agmatine: an endogenous ligand at imidazoline receptors is a novel neurotransmitter. Ann N Y Acad Sci 1999; 881: 65-80．
15． Kolesnikov, Y, Jain, S., Pasternak, GW. Modulation of opioid analgesia by agmatine. Eur J Pharmacol. 1996; 296: 17-22．
16． Li, J., Li, X., Pei, G., Qin, BY. Analgesic effect of agmatine and its enhancement on morphine analgesia in mice and rats. Acta Pharmacol Sin 1999; 20: 81-5．
17． Li, J., Li, X., Pei, G., Qin, BY. Effects of agmatine on tolerance to and substance dependence on morphine in mice. Acta Pharmacol Sin 1999; 20: 232-8．
18． Li J, Li X, Pei G, Qin BY. Correlation between inhibitions of morphine withdrawal and nitric oxide synthase by agmatine. Acta Pharmacologica Sinica 1999, 20(4) : 375-80．
19． Su, RB., Li, J., Gao, K., Pei, G., Qin, BY. Influence of idazoxan on analgesia, tolerance, and physical dependence of morphine in mice and rats in vivo. Acta Pharmacol Sin. 2000; 21: 1011-5．
20． Eick AJ van. A change in the response of the mouse in the "hot plate" analgesia-test,owing to a central action of atrophine and related compounds. Acta physiol Pharmacol Neerl ,1967, 14(4) : 499-500．
21． Kossel A. Zeischrift fur Physiologiche Chemie 1910:257-61．
22． Reis, DJ. and Regunathan, S. Is agmatine a novel neurotransmitter in brain? Trends Pharmacol Sci. 2000; 21(5) : 187-93．
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