Antineoplastic and Cytotoxic Activities of Nickel(II) Complexes of Thiosemicarbazones

Nickel(II) complexes of thiosemicarbazons were observed to be potent cytotoxic agents in human and rodent tissue cultured tumor cells. Each compound demonstrated a slightly different profile in the various histological types of tumors. The nickel complex of Appip demonstrated the most potent in vivo activity in the Ehrlich ascites carcinoma. This agent selectively inhibited L1210 DNA and purine syntheses, and DNA polymerase α, PRPP-amido transferase, IMP-dehydrogenase, dihydrofolate reductase, TMP-kinase and thymidylate synthetase activities. L1210 DNA strand scission was evident and DNA viscosity was reduced after 24 hr incubation. The nickel complexes were not L1210 DNA topoisomerase II inhibitors.


Introduction
Copper, nickel and cobalt metal complexes of heterocyclic thiosemicarbazones, thioureas and 2substituted pyridines have previously been shown to be potent anti-neoplastic agents in the Ehrlich ascites carcinoma screen [1,2]. Cytotoxicity was demonstrated against L1210 and Tmolt leukemias, as well as human solid tumor growth, e.g. HeLa, KB nasopharynx, skin, bronchogenic lung carcinomas, bone osteosarcoma and glioma. A mode of action study with copper(II) complexes in L1210 leukemia cells showed that DNA synthesis followed by RNA synthesis was inhibited. The major enzymatic sites of inhibition by the agents include: IMP dehydrogenase, dihydrofolate reductase, DNA polymerase , ribonucleoside reductase, and nucleoside kinase [1,2]. More important was the observation that DNA strand scission occurred after 24 hr. incubation with the agents. There did not appear to be any evidence of cross linking of the DNA nor did the agents produce intercalation between nucleic bases of DNA. The copper complexes of the heterocyclic thiosemicarbazones, thioureas and 2-substituted pyridines were shown to be L1210 DNA topoisomerase II inhibitors much like the agent fostriecin in that they were competitive blockers of VP-|6's induction of DNA protein linked breaks [3][4][5]. The present investigation involves an in-depth study of the nickel complexes regarding their mode of action in L1210 leukemia cells.

Methods
All test compounds were previously synthesized and their chemical and physical characteristics reported [6,7] [20]. Carbamyl phosphate synthetase activity was determined with the method ofKalman et al. [21]; citrulline was determined colorimetrically [22]. Aspartate transcarbamylase activity was measured by the method of Kalman et a/. [21]; carbamyl aspartate was determined colorimetrically [23]. Thymidylate synthetase activity was analyzed by Kampf et al. 's method [24]. The 3H9O measured was proportional to the amount of TMP formed from 3H-dUMP. Dihydrofolate reductase activity was determined by the spectrophotometric method of Ho et a/. [25]. PRPP amidotransferase activity was determined by Spassova et al. 's method [26]; IMP dehydrogenase activity was analyzed with 8-14C-IMP (54 mCi/mmol) (Amersham, Arlington Heights, IL) atter separating XMP on PEI plates (Fisher Scientific) by TLC [27]. Protein content was determined for the enzymatic assays by the Lowry technique [28]. After deoxyribonucleoside triphosphates were extracted [29], d[NTP] pool levels were determined by the method of Hunting and Henderson [30] with calf thymus DNA, E. coli DNA polymerase I, non-limiting amounts of the three deoxyribonucleoside triphosphates not being assayed, and either 0.4 mCi of(3H-methyl)-dTTP or (5-3H)-dCTP.

DNA Studies
The effects of compound 1 on DNA strand scission was determined by the methods of Suzuki

Statistics
The mean and standard deviation are designated by "X + SD." The probable level of significance (p) between test and control samples was determined by the Student's "t" test with the raw data. The mode of action study with compound 1 in L1210 cells showed that DNA synthesis was reduced greater than 65% at 25 txM and 99% at 100 txM whereas RNA and protein syntheses were inhibited 37% and 34% respectively after 60 min at 100 tM of compound 1. DNA polymerase a activity was reduced 55% and m-RNA polymerase activity was reduced 33% at 100 tM but r-and t-RNA polymerase activities were not affected by compound 1. Ribonucleoside reductase activity was only reduced 20% but dihydrofolate reductase activity was inhibited 54% after 60 min at 100 lxM. De novo purine synthesis was inhibited in a concentration dependent manner with 63%,,reduction at 100 tM. The activities of both regulatory enzymes of the pathway were reduced PRPP amido transferase activity by 56% and IMP dehydrogenase activity 54% by compound I. De novo pyrimidine synthesis was not significantly affected by compound 1 nor were the early regulatory enzymes of the pathway affected. Thymidylate synthetase activity was reduced 44% and thymidine-monophosphate kinase activity was reduced 49% at 100 tM.
TDP kinase activity was elevated 185% by compound 1 DNA strand scission did occur with compound 1 after incubation for 24 hr. at 100 laM [Fig 2]. ct-DNA studies demonstrated that DNA viscosity was reduced from 323 see for the control to 283 see. for compound 1 indicating smaller fragments of DNA. The DNA thermal denaturation Tm value for the control was 87.5C and was 92C for compound 1. L1210 DNA topoisomerase activity was not affected by compound I at 100 tM.

Discussion
The nickel(II) complexes of thiosemicarbazones proved to be potent anti-neoplastic and cytotoxic agents. The complexes did demonstrate some specifi.city for select tumor growth inhibition. The two nickel compounds tested previously demonstrated significant activity in the ileum adenocarcinoma, and skin A431 screens and were more potent in the KB nasopharynx and osteosarcoma screens[ ], but the two new nickel complexes I and 2 were more potent in the L1210 screen. The mode of action in L1210 leukemia 178+7 cell for the nickel complex was Similar to that of the copper(Ii) c0mplexe"of hiosemicarbazones [2]. DNA synthesis was the major target of the nickel complexes with marginal effects on RNA and protein synthesis after 60 rain. Inhibition of DNA polymerase a activity by the compound was one of the reasons DNA synthesis was suppressed. This effect on this enzyme was similar to that observation with the copper(II) complexes as were the mixed effects on the RNA polymerase activities. The copper(II) complexes did have a higher magnitude of inhibition for ribonucleoside reductase activity[I,2] than the nickel compound; however, inhibition ofthis enzyme would be additive with the overall reduction of DNA synthesis since there would be a reduction in the deoxyribonucleosides and deoxyribonucleotides in the nucleus for their incorporation into DNA. A major site of inhibition of the nickel complex was in the purine pathway of L1210 cells. Both regulatory enzymes were markedly reduced in activity, similar to the effects afforded by 6-MP. This would lead to reduced deoxyribopurines as well as inhibition of DNA synthesis and cell death. There were not apparent blocks by the agent in the pyrimidine pathway, although some inhibition of thymidylate synthetase activity occurred. The elevation of TDP-kinase activity by the nickel complex may help explain the observed elevation of deoxyribopyrimidine pools. On the other hand, the d[NTP] pools would be elevated in the cell if DNA polymerase c was inhibited in that the deoxytriphosphate nucleotides were not incorporated into the new strand ofDNA. The nickel complex did cause fragmentation of DNA but did not cause alkylation of bases of DNA or intercalation between base pairs. The nickel complex did not inhibit L 1210 DNA topoisomerase II activity. The copper complexes of thiosemicarbazones were potent inhibitors of L1210 DNA topoisomerase II activity and were able to cause DNA protein linked breaks [2].