210104b0Nature2105031196604021041050028-0836196610.1038/210104b0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v210/n5031issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue210104b0Synergistic Interaction of Kethoxal bis(Thiosemicarbazone) and Cupric Ions in Sarcoma 180
AU  - BOOTH, BARBARA A.
AU  - SARTORELLI, ALAN C.Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut.2-KETO-3-ethoxybutyraldehyde bis (thiosemicarbazone) [kethoxal bis(thiosemicarbazone); KTS], an agent that chelates metals of the first, second and third transition series1, has been reported to be an inhibitor of the growth of several transplanted rodent neoplasms2-5. The anti-neoplastic potency of KTS was enhanced by the presence of either cupric or zinc ions6; however, the preformed cupric chelate was found to be the most potent of several different metal chelates of KTS1,5,7. To investigate the relationship of copper ions to the antineoplastic activity of KTS, the effects of combinations of cupric ions (as cupric chloride) and KTS, both on growth and on the synthesis of nucleic acids and proteins, were measured in sarcoma 180 ascites cells.The tumour-inhibitory effects of such combinations on neoplastic growth were measured by determining the survival time of adult HajICR Swiss mice inoculated with approximately 4 x 106 sarcoma 180 ascites tumour cells by intraperitoneal injection as previously described8. Therapy was initiated 24 h after implantation of the neoplastic cells and was continued for six consecutive days. The results obtained are shown in Table 1. Neither KTS in daily doses up to 60 mg/kg, nor cupric chloride in daily doses up to 10 mg/kg, increased the average survival time of tumour-bearing mice. In combination, however, significant prolongation of life was produced at low levels of each drug. No concomitant toxicity to the host was evident; thus, no weight loss from onset to termination of therapy was observed.
To probe for metabolic alterations associated with the growth-inhibitory potency of combinations of cuprie chloride and KTS, the effect of a single dose of these agents on the synthesis of DNA, KNA and proteins was monitored using thymidine-methyl-3H, uridine-3H and DL-leucine-l-14C, respectively (Table 2). Little or no inhibition of these biosynthetic pathways was produced by either cupric chloride or KTS alone; however, combination of these agents caused about 82 per cent inhibition of the formation of DNA, 54 per cent inhibition of the synthesis of UNA, and a 62 per cent depression in the rate of formation of proteins. Although the magnitude of the effects of the combination on DNA were comparable with those produced by administration of the preformed cupric chelate of KTS, [Cu(II)KTS]9, the degree of inhibition of the synthesis of proteins by the mixture of copper chloride and KTS could not be explained by the formation of Cu(II)KTS in vivo. Furthermore, the magnitude of inhibition achieved by the mixture of cupric chloride and KTS could not be duplicated by appropriate quantities of Cu(II)KTS in combination with either KTS or cupric chloride10. Thus it would appear that cupric chloride, KTS and possibly some resultant Cu(II)KTS produce distinct alterations that result in synergistic inhibition of the synthesis of proteins.
Table 1. EFFECT OF COMBINATIONS OF CUPRIC CHLORIDE AND KTS ON THE SURVIVAL TIME OF MICE BEARING SARCOMA 180 ASCITES CELLS
Daily dosage[ast]	(nag/kg)	Av. A wt. t	Av. survival!
Cupric chloride	KTS	(%)	(days)
0	0	+ 22-3	12-8 + 0-5
0-5	0	+ 20-0	13-8 [plusmn]0-8
1-0	0	+ 20-8	12-1 + 0-8
0	2-5	+ 22-4	13-4 [plusmn]0-6
0	5-0	+ 26-8	11-2 + 0-9
0-5	2-5	+ 5-0	21-6 + 1-3
1-0	5-0	+ 0-8	23-3 + 1-7
[ast] Drugs were administered intraperitoneally once daily for 6 consecutive days, beginning 24 h after implantation of tumour cells, with combinations given simultaneously.
t Average weight change from onset to termination of drug treatment.
% Each value represents the average survival time ([plusmn] standard error) obtained with 9-20 mice.
Table 2. EFFECT OF CUPRIC CHLORIDE, KTS OR THEIR COMBINATION ON THE SYNTHESIS OF DNA, UNA AND PROTEINS IN SARCOMA 180 ASCITES CELLS
Treatment None
Cupric chloride KTS Cupric chloride + KTS
Specific activity DNA[ast] UNAt Protein
136-8 [plusmn]10-6 13-05 [plusmn]1-10 3,720 [plusmn]147
109-9 [plusmn]13-5 10-45 [plusmn]1-35 3,380 [plusmn]274
132-9 [plusmn]11-8 8-55 [plusmn]0-90 3,700 [plusmn]147
25-2 [plusmn] 6-4 6-00 [plusmn]1-05 1,420 [plusmn]503
Six days after implantation of tumour cells, each mouse received a single intraperitoneal injection of either 2-5 mg of cupric chloride/kg body-weight, 50 mg of KTS/kg, or a combination of the two drugs administered simultaneously. Twelve h later an isotopic tracer was administered intraperitoneally to each mouse, and 1 h was allowed for incorporation. Isolations and analyses were performed as previously described (ref. 13).
[ast] Determined as c.p.m./^mole of thymine x 10~2 after administration of 200 MS of thymidine-methyl-8H (5-7 x 103 c.p.m.//ug) to each mouse. Each number represents the mean value ([plusmn] standard error) obtained with 8-12 mice.
t Determined as c.p.m./m^mole of ribose after administration of 200 jug of uridine-3H (7-1 x 103 c.p.m./yug) to each mouse. Each number represents the mean value ([plusmn] standard error) obtained with four mice.
% Determined as c.p.m./mg of residual protein after administration of 125 fig of DL-leucine-l-14C (1-45 x 104 c.p.m./^g) to each mouse. Each number represents the mean value ([plusmn] standard error) obtained with eight mice.
Table 3. COPPER CONTENT OF SARCOMA 180 ASCITES CELLS AFTER EXPOSURE TO CUPRIC CHLORIDE, KTS OR THEIR COMBINATION
Treatment m^moles Copper/g packed cells
None 9-2 [plusmn] 3-9
Cupric chloride 38-0 [plusmn]4-4
KTS 10-2 [plusmn] 3-3
Cupric chloride + KTS 144-2 [plusmn]29-1
Six days after implantation of tumour cells each mouse received a single intraperitoneal injection of either 2-5 mg of cupric chloride/kg body-weight, 50 mg of KTS/kg, or a combination of the two drugs administered simultaneously. Twelve h later ascites cells were wet-ashed and analysed for ocopper content as described by Stoner and Dasler (ref. 14). Each point represents the mean value ([plusmn] standard error) obtained with four mice.
Since Cu(II)KTS, formed from the mixture of cupric chloride and KTS, is a relatively lipid-soluble form of copper and, therefore, would penetrate cellular membranes with comparatively greater ease than would copper ions derived from cupric chloride, the quantity of copper associated with the tumour cells after a single exposure to the drugs alone or in combination was determined (Table 3). The amount of copper/g of wet weight of cells was four times greater in sarcoma 180 cells removed from mice treated with cupric chloride than in untreated controls, whereas KTS did not significantly alter the copper content. Exposure of the neoplastic cells to the drug combination, however, resulted in a copper content 15-5 times that of untreated control cells. Although the large copper content of cells may be responsible for some of the observed biochemical effects, no direct correlation can be made between the quantity of copper found in the neoplastic cells and the degree of metabolic blockade10.
The enhanced inhibitory effects observed in the present study with copper chloride and KTS are reminiscent of the synergistic chromosomal aberrations produced by combinations of ethyl methane sulphonate and copper ions in Viciafaba11[ast]12. Investigations are now in progress to define precisely the enzymatic sites involved in the metabolic lesions produced by cupric chloride, KTS and Cu(II)KTS.
This research was supported by a U.S. Public Health Service Research grant from the National Cancer Institute. We thank Dr. H. G. Petering of the Upjohn Company, Kalamazoo, Michigan, for generously donating KTS and Cu(II)KTS, and Miss Sheila J. Feld for her assistance in performing these experiments.Van Giessen, , G. J., and Petering, , H. G., Abst. Amer. Chem. Soc. Meeting, 13N (1965).Petering, , H. G., and Buskirk, , H. H., Fed. Proc., 21, 163 (1962).ISIMihich, , E., and Nichol, , C. A., Proc. Amer. Assoc. Cancer Res., 4, 44 (1963).Petering, , H. G., Buskirk, , H. H., and Underwood, , G. E., Cancer Res., 24, 367 (1964).PubMedISIChemPortMihich, , E., and Mulhern, , A. I., Fed. Proc., 24, 454 (1965).ISIPetering, , H. G., Buskirk, , H. H., Crim, , J. A., and Van Giessen, , G. J., Pharmacologist, 5, 271 (1963).Petering, , H. G., Buskirk, , H. H., and Kupiecki, , F. P., Fed. Proc., 24, 454 (1965).ISIBooth, , B. A., and Sartorelli, , A. C., Cancer Res., 23, 1762 (1963).PubMedISIChemPortSartorelli, , A. C., and Booth, , B. A., Fed. Proc., 24, 454 (1965).ISIBooth, , B. A., and Sartorelli, , A. C. (unpublished observations).Moutschen-Dahmen, , J., and Moutschen-Dahmen, , M., Experientia, 19, 144 (1963).PubMedChemPortMoutschen, , J., Moes, , A., and Gilot, , J., Experientia, 20, 494 (1964).PubMedChemPortBooth, , B. A., Creasey, , W. A., and Sartorelli, , A. C., Proc. U.S. Nat. Acad. Sci., 52, 1396 (1964).ChemPortStoner, , R. E., and Dasler, , W., Clin. Chem., 10, 845 (1964).PubMedISIChemPort