Noemi Prajda

Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas.

Xanthine oxidase was decreased 2- to 10-fold in all examined rat hepatomas irrespective of the malignancy; growth rate and degrees of histological differentiation of the neoplasms. The affinity to substrate (KM=6-8 muM) and the pH optimum (8.0) of the liver and hepatoma enzymes were the same. The reprogramming of gene expression, as manifested in the decreased activity of this key purine metabolizing enzyme, appears to be specific to neoplastic transformation. Since glutamine PRPP amidotransferase activity was increased but the opposing enzyme, xanthine oxidase, was decreased in all the hepatomas, the reprogramming of gene expression results in an imbalance that favors synthesis against catabolism. This enzymatic imbalance should confer selective advantages to the cancer cells.

Molecular mechanisms in the antiproliferative action of quercetin

A single treatment with quercetin (5.5 microM), a plant flavonoid, activated both apoptosis and differentiation programs in K562 human leukemia cells. K562 cells expressed commitment to apoptosis after 1 h exposure, however, at least 12 h of drug exposure was needed to induce differentiation. Early (1 h) down-regulation of the c-myc and Ki-ras oncogenes and rapid reduction of inositol-1,4,5-trisphosphate (IP3) concentration (IC50 = 9 microM, 1 h incubation) are part of the antiproliferative action of quercetin and appear to relate to induction of differentiation and/or apoptotic program of K562 leukemia cells treated with quercetin.

Key enzymes of IMP metabolism: transformation and proliferation-linked alterations in gene expression.

Abstract The purpose of this investigation was to test the concept that key enzymes of IMP synthesis, degradation and utilization may show characteristic patterns in proliferative conditions. In the synthetic pathway of IMP the first enzyme committed to de novo purine biosynthesis, glutamine PRPP amidotransferase, was measured. In the pathway of IMP catabolism the rate-limiting enzyme, xanthine oxidase, and uricase, the final enzyme, were examined. In the synthetic utilization of IMP, IMP dehydrogenase, the enzyme involved in channeling IMP into the production of XMP and adenylosuccinate synthetase, the enzyme channeling IMP into SAMP biosynthesis, were studied. Investigations were also carried out on GMP synthetase and adenylosuccinase. The enzymes of IMP metabolism were examined in normal and neoplastic liver proliferation, such as in differentiating and regenerating liver and in a spectrum of hepatomas of different growth rates. The following main observations were made. 1. 1.|In differentiating liver the specific activities of IMP dehydrogenase and amidotransferase were high after birth and decreased during development to the levels observed in the adult rat liver. In contrast, the adenylosuccinate synthetase and xanthine oxidase activities were low after birth and rose to the adult levels during development. 2. 2.|In a spectrum of hepatomas of different growth rates the specific activity of IMP dehydrogenase was 2- to 3-fold increased in the slow growing hepatomas and it increased in parallel with tumor growth rates, reaching 12- to 13-fold increases in the most rapidly growing neoplasms. The behavior of IMP dehydrogenase belongs to Class 1 as grouped by the molecular correlation concept. 3. 3.|The activities of adenylosuccinate synthetase and glutamine PRPP amidotransferase were increased in all hepatomas to approximately 2- to 4-fold, and those of the adenylosuccinase 1.2- to 2-fold of the levels observed in the corresponding normal rat livers. In contrast, the activities of the catabolic enzymes, xanthine oxidase and uricase, were decreased in all hepatomas to 2- to 10-fold of the activity of the relevant control rat livers. The ratios of amidotransferase/xanthine oxidase were markedly increased in all hepatomas and they reached 20- to 27-fold increases in those of rapid growth rate. The behavior of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase belongs to Class 2 of the molecular correlation concept where enzymes are grouped that exhibit alterations that occur in all hepatomas irrespective of growth rate and degree of differentiation. 4. 4.|Since the alterations in the activities of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase are expressed in all the hepatomas, even in the slowest growing, most liver-like tumors, this reprogramming of gene expression appears to be linked with the malignant transformation per se . However, these enzyme activities do not signal a linkage with the degrees in the expression of malignancy and growth rate. In contrast, the activity of IMP dehydrogenase is an indicator both of malignant transformation and growth rate. 5. 5.|In the regenerating liver IMP dehydrogenase specific activity rapidly increased to 500% in 6 to 18 hours; it slowly returned towards normal range in 96 hours after operation. Amidotransferase specific activity reached a peak of 165% of the sham-operated value at 48 hours after operation and then returned to normal range by 96 hours. The activities of SAMP synthetase, adenylosuccinase, xanthine oxidase and uricase did not change in regeneration. Thus, IMP dehydrogenase appears to be the earliest enzyme to rise in the regenerating liver, whereas the elevation of amidotransferase is a delayed and a minor one. The increase in activities of these two enzymes in the regenerating liver is in the range of the activity observed in the newborn rat liver. However, the activities are markedly lower than those observed in the very malignant hepatomas that exhibit the same growth rate as the regenerating liver. 6. 6.|The discriminating power of the biochemical pattern of enzymes of purine metabolism indicates that the imbalance in the pattern of alteration of the key enzymes of IMP synthesis, degradation and utilization is specific to malignancy. The pattern of these enzyme activities also provides quantitative and qualitative discriminants for the identification of regenerating and differentiating liver and for distinguishing the enzyme patterns from those observed in normal and rapidly growing neoplastic liver. 7. 7.|These experiments indicate the operation of an antagonistic behavior for the key synthetic enzymes, glutamine PRPP amidotransferase and IMP dehydrogenase, that are high in differentiation, regeneration and neoplasia as compared to the rate-limiting catabolic enzyme, xanthine oxidase, that is low in differentiation and in neoplasia and is unaltered in the regenerating liver. These observations are in line with earlier studies that demonstrated an antagonistic behavior for the opposing key enzymes of gluconeogenesis and glycolysis and of the synthetic and catabolic pathways of the de novo and salvage pathways of pyrimidine metabolism.

Regulation of the signal transduction program by drugs

The purpose of this paper was to clarify critical aspects of the behavior of signal transduction activity in normal and cancer cells. 1. Signal transduction activity in the conversion of phosphatidylinositol through PI and PIP kinases and PLC to IP3 is regulated at multiple sites. In liver, hepatomas and human carcinomas PIP kinase is the rate limiting enzyme and PLC activity is present in great excess. 2. The steady-state signal transduction activity as measured by the three enzyme activities and IP3 concentration was markedly up-regulated in rat hepatomas of different growth rates. The steady-state specific activities of the three signal transduction enzymes were elevated in ovarian carcinomas as compared to normal ovary. Increased enzyme activities were also observed in human breast carcinoma cells as compared to normal human breast parenchymal cells. In breast, ovarian and rat hepatoma cells as they go through lag, log and plateau phases, IP3 concentration in the early lag phase increased 4.5- to 20-fold and PI and PIP kinase activities peaked in mid-log phase. These events returned to baseline levels in the plateau phase. PLC activity did not change. 3. The bone marrow PI and PIP kinase activities in 3-day starvation were decreased to 13% and IP3 concentration was reduced to 24%; at 1-day refeeding they returned to normal. PLC activity changed little. These alterations are in line with the rapid t1/2 degradation rates (12 min) of PI and PIP kinases observed in studies with cycloheximide. By contrast, PLC has a long half-life. 4. The molecular action of tiazofurin entails inhibition of IMP DH activity, decrease in GTP and IP3 concentrations, reduction of ras and myc oncogene expression, and signal transduction enzyme activities. These events are followed by induced differentiation and apoptosis. There are also decreases in enzyme activities which have rapid turnover, including TdR kinase, dTMP synthase, and GPRT. In vitro studies indicated that these events are abrogated by addition of guanine which restores GTP concentrations. Therefore, most or all these events were brought about by the reduced GTP concentration in the tiazofurin target cells. 5. Quercetin and genistein are able to inhibit PI and PIP kinase activities and reduce IP3 concentration in vivo and in tissue culture systems. These flavonoids are also inhibitors of cell proliferation and clonogenic ability in rat hepatoma 3924A and in human OVCAR-5 and MDA-MB-435 cells. Quercetin down-regulated the expression of c-myc and Ki-ras oncogenes and led to induced differentiation and apoptosis in K562 cells. Genistein reduced IP3 concentration in vivo and in the tissue culture system. Genistein is antiproliferative and has cytototoxicity in human carcinoma cells. All three drugs, tiazofurin, quercetin and genistein, act, in part at least, through depression of cellular IP3 concentration although the mechanisms may not be identical. 6. Quercetin and genistein, which attack different targets and different phases of the cell cycle, proved to be synergistic in OVCAR-5 cells. The impact of tiazofurin, genistein and quercetin is of interest because the drugs crucially inhibit the display of the neoplastic program of cells and lead to induced differentiation and apoptosis.

Multi-enzyme-targeted chemotherapy by acivicin and actinomycin

On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.

Linkage of reduction in 1-phosphatidylinositol 4-kinase activity and inositol 1,4,5-trisphosphate concentration in human ovarian carcinoma cells treated with quercetin.

Quercetin inhibited 1-phosphatidylinositol (Pl) 4-kinase, EC 2.7.1.67 (Pl kinase) activity in a concentration-dependent manner (IC50 = 4 microM) in particulate extracts from human ovarian carcinoma. In OVCAR-5 cells quercetin produced growth inhibition (IC50 = 63 microM) and cytotoxicity (LC50 = 17 microM). The growth inhibition by quercetin was accompanied by an 80% decrease in Pl kinase activity and a 65% decrease in the concentration of the second messenger, inositol 1,4,5-trisphosphate (IP3). When human OVCAR-5 cells were plated and expressed their neoplastic proliferative program in the log phase, Pl kinase and Pl 4-phosphate 5-kinase, EC 2.7.1.68 (PIP kinase) activities coordinately increased to a peak of 5.8- and 4.5-fold, respectively. The results demonstrate for the first time inhibition by quercetin of the enhanced capacity for operation of signal transduction in human ovarian carcinoma cells, thus providing a novel target in cancer cells.

Regulation of signal transduction

1. A systematic study is reported on the control of 1-phosphatidylinositol 4-kinase (PI kinase) and PI 4-phosphate 5-kinase (PIP kinase), enzymes of the phosphatidylinositol phosphorylation pathway which leads to the production of second messengers. IP3 and DAG. In liver of normal male, adult, fed Wistar rats the steady state activity of PI kinase was 0.5 +/- 0.01 and that of PIP kinase was 0.046 +/- 0.003 nmol/hr/mg protein. The concentration of IP3 was 1.8 +/- 0.1 pmol/mg protein. 2. That the two kinases have short half-lives was observed in starvation. where in the rat liver or bone marrow activities rapidly decreased and on refeeding were restored in a day. Injection to rats of the protein synthetic inhibitor, cycloheximide, yielded t1/2 = 80 min for the two enzymes in bone marrow and t1/2 = 80 min in liver. 3. Linkage of the signal transduction enzymes with proliferation was shown by the high activities as compared to liver of these enzymes in rat organs of high cell renewal capacity, e.g., thymus, bone marrow, spleen and testes. 4. Linkage with malignant proliferation was indicated by the observation that in rat hepatomas the enzyme activities increased 5- to 9-fold and were highest in rapidly growing hepatoma 3924A (29- and 45-fold). 5. In human primary ovarian carcinoma PI and PIP kinase activities were elevated 4.4 and 2.9-fold, respectively, and in OVCAR-5 cells, 32- and 11-fold, respectively. Similar increases were observed in MDA-MB-435 human breast carcinoma cells in comparison with normal breast parenchymal cells. 6. The linkage of signal transduction enzyme activities with malignant proliferation was also observed in experiments when human breast carcinoma cells were plated in flasks and expressed their proliferative capacity in the log phase. PI and PIP kinase activities steadily and coordinately increased to a peak 11-fold rise in mid-log phase. In late log and plateau phases the kinase activities gradually declined to the starting level. Similar observations were made for the two enzymes in human ovarian carcinoma OVCAR-5 cells and in rat hepatoma 3924A cells in tissue culture. 7. In animals injected with cycloheximide the bone marrow PI and PIP kinase activities exhibited t1/2 = 0.12 hr, the shortest decay rate in comparison with 8 enzymes of purine and pyrimidine biosynthesis with t1/2 = 0.6 to 4.3 hr. 8. Injection of tiazofurin decreased PI and PIP kinase activities in the bone marrow with t1/2 = 82 and 78 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzyme targets of antiglutamine agents in cancer chemotherapy.

The modes of action of azaserine and acivicin were compared. The results were evaluated by assessing the impact of these drugs on primary targets, the activities of key enzymes, and on secondary and tertiary targets, the concentrations of pools of ribonucleotides and deoxyribonucleotides. It was observed that both drugs act as competitive inhibitors for glutamine-utilizing enzymes involved in the biosynthesis of purines and pyrimidines, but in addition acivicin exerts a direct inactivating effect (probably by alkylation) on the enzymes. The different tissues examined displayed varying sensitivity to the drugs which may be attributed in part at least to the tissue glutamine content. Acivicin markedly depleted the CTP pools, but ATP and UTP were unaffected. It also decreased the concentration of all 4 deoxynucleoside triphosphates. These biochemical targets serve as indicators of acivicin action in cancer cells and should also be helpful in the design of combination chemotherapy. On the basis of the biochemical action of acivicin, actinomycin and dipyridamole were selected for testing in combination chemotherapy. Both drugs acted synergistically with acivicin.

Targeted and non-targeted actions of anti-cancer drugs

(1) The currently used clinical anti-metabolites are targeted against-key enzymes of de novo purine and pyrimidine biosynthesis. However, the activities of salvage enzymes in each of the biosynthetic segments are markedly higher than those of the rate-limiting enzymes of de novo biosynthesis. Enzyme-pattern-targeted chemotherapy has been suggested to overcome the circumvention activity of salvage. Combination of inhibition of de novo and salvage pathways does provide a synergistic impact. Examples that enzyme-pattern-targeted drug treatment yields synergism include the following: tiazofurin (against IMP DH) and allopurinol (by raising serum hypoxanthine levels it inhibits GPRT); methotrexate or 5-FU lead to inhibition of the dTMP synthase reaction and AZT (a competitive inhibitor of thymidine kinase) or dipyridamole (a nucleoside transport inhibitor); acivicin, an inhibitor and inactivator of glutamine-utilizing enzymes in the de novo pathways of purine and pyrimidine biosynthesis, and dipyridamole. (2) Administration of MTX, 5-FU, tiazofurin or acivicin causes inhibition and/or inactivation of target enzymes. That these drugs are effective in spite of the presence of highly active salvage enzymes is now accounted for, at least in part, by new observations showing that these drugs markedly reduce (but do not eliminate) the activities (amounts) of CdR and TdR kinases, dTMP synthase and GPRT. This action is attributed to the rapid decay rate of these enzymes. (3) Studies on the bone marrow enzymic programs indicate that there is a window of opportunity for strengthening therapy and for the protection of bone marrow by administering salvage metabolites when the salvage enzymes are still present in high enough activities, i.e., 2-6 hr after administration of the blockers of de novo enzyme activities. (4) These results are a strong argument for discovering and utilizing inhibitors of purine and pyrimidine salvage enzymes to achieve more successful enzyme-pattern-targeted chemotherapy and to avoid development of resistant clones of cancer cells. (5) These approaches provide greater explanatory coherence than the previous accounts because recognition of (a) the importance of salvage and (b) rapid decay of key and salvage enzymes reveals a paradigm shift. The problem-solving process in chemotherapy should now be not only data-driven but also explanation-driven.

Current issues in the regulation of signal transduction

(1) In all examined rat and human tissues and cells, PIP kinase activity was rate-limiting and PLC activity was present in great excess. (2) The steady-state activities of the signal transduction enzymes, PI kinase, PIP kinase and PLC, and the concentration of the end product, IP3, were determined in rat liver and hepatomas of different malignancies. The activities of all three enzymes were elevated in the hepatomas in a non-random fashion. A generalization emerged that the enzyme with the lowest activity in liver, PIP kinase, increased to the highest extent and the enzyme with the highest activity in liver, PLC, increased to the smallest extent in rapidly growing hepatomas. The IP3 concentration in the hepatomas was elevated in a progression-linked fashion. (3) The three signal transduction enzyme activities were elevated in human ovarian carcinoma samples and in human breast carcinoma cells. (4) When human breast carcinoma MDA-MB-435 cells were allowed to go through lag, log and plateau phases, the IP3 concentration reached a 20-fold peak at 12 hr after plating. The elevation in IP3 concentration preceded the rise in PI and PIP kinase activities which increased 11-fold in the log phase. The IP3 concentration and PI and PIP kinase activities returned to their baseline levels when the plateau phase was reached. The PLC activity did not change significantly during the whole period. (5) Administration of cycloheximide i.p. in rats revealed short half-lives in the bone marrow for the two kinases (8 min) and a long half-life for PLC (> 6 hr). In a group of 10 enzymes, the half-lives of the kinases were the shortest. In cycloheximide-injected rats, the bone marrow IP3 concentration was reduced to about 50% in 30 min. The reduction of IP3 concentration is attributed to the decline to 15 and 12%, respectively, in PI and PIP kinase activities since PLC activity did not change. (6) In 3-day starved rats, the bone marrow PI and PIP kinase were reduced to activities (13%) that were markedly lower than the decrease in the protein concentration (to 55%). By contrast, the PLC activity was preferentially maintained (to 78%) over the protein level. Under starvation, the IP3 concentration decreased (to 24%), indicating that starvation can markedly disrupt IP3 homeostasis. Refeeding returned the enzymic activities and the IP3 concentration to the normal level in bone marrow in 24 hr. (7) Comparison of the absolute activities of PI and PIP kinases and PLC showed that PLC is present in an excess; therefore, it does not appear to have a rate-limiting action in cycloheximide treated rats or in starvation. (8) Whereas PI and PIP kinases have short half-lives and apparently rapid synthetic rates, PLC has high activity, a long half-life and responds to starvation with only a small decrease. (9) The gain in function manifested in the over-expressed capacity for signal transduction confers growth advantages to cancer cells. These increased activities, particularly those of PI and PIP kinases, should be sensitive targets for chemotherapy.