Polyamine content of Pneumocystis carinii and response to the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine.

Difluoromethylornithine (DFMO; eflornithine hydrochloride [Ornidyl]), a suicide inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase (ODC), is effective in treating Pneumocystis carinii pneumonia, a common opportunistic infection associated with AIDS. Despite DFMO's specificity for ODC, the reason for its selective toxicity against P. carinii is unknown since both host and parasite are dependent on the same enzyme for polyamine biosynthesis. A new high-performance liquid chromatography method was used with P. carinii cells isolated from infected rat lungs to measure polyamine content, to confirm the presence of ODC, and to examine the effect of DFMO on polyamine concentrations. Putrescine, spermidine, and spermine were found to be present at 2.00 +/- 0.54, 1.26 +/- 0.51, and 1.59 +/- 0.91 nmol (mg of protein)-1, respectively, neither unusually high nor low values. ODC's specific activity was 79 +/- 11 pmol (mg of protein)-1 h-1, again not a remarkable value. However, the rates of both DFMO-induced polyamine depletion and subsequent repletion upon DFMO removal were unusually high. A 3-h exposure to 1 mM DFMO in vitro caused the depletion of putrescine, spermidine, and spermine to levels 12, 29, and 16%, respectively, of that of control cells. After DFMO removal and incubation for 1 h in serum-free media, polyamine levels returned to 78, 88, and 64%, respectively, of that of the control cells not exposed to DFMO. Since such depletions and repletions usually occur over periods of days rather than hours, these rapid changes may provide a clue to the selective action of DFMO against P. carinii and may guide the development of new compounds and an optimal drug administration schedule for DFMO.     

Ornithine decarboxylase as an enzyme target for therapy.

Interest in ornithine decarboxylase (ODC) and the therapeutic effects of its inhibition with the consequent depletion of polyamine biosynthesis has been widespread since the late 1970s and 1980s. This review covers new information about the properties of ODC, recent findings with ODC inhibitors and a discussion of the mechanism of inactivation of ODC by eflornithine. Recent in vivo therapeutic approaches of ODC inhibition are also discussed including: cancer and cancer chemoprevention; autoimmune diseases; polyamines and the blood-brain barrier, ischemia and hyperplasia; the NMDA receptor and modulation by polyamines; hearing loss; African trypanosomiasis; Pneumocystis carinii pneumonia and Cryptosporidium in AIDS; and other infectious diseases/organisms.     

Deferoxamine and eflornithine (DL-alpha-difluoromethylornithine) in a rat model of Pneumocystis carinii pneumonia.

The iron chelator deferoxamine and the polyamine biosynthesis inhibitor eflornithine (DL-alpha-difluoromethylornithine) were examined for anti-Pneumocystis carinii activity in the rat model of P. carinii pneumonia. The activity of deferoxamine at 250, 500, and 1,000 mg/kg given intraperitoneally provides evidence that iron chelation is a promising novel approach to P. carinii chemotherapy. Results with eflornithine at 2, 3, and 4% in drinking water confirm and extend previously reported activity in the rat model.     

Effect of a bis-benzyl polyamine analogue on Pneumocystis carinii

Pneumocystis carinii is the causative agent of P. carinii pneumonia (PCP), an opportunistic infection associated with AIDS and other immunosuppressed conditions. Although polyamine metabolism of this fungus has been shown to be a chemotherapeutic target, this metabolism has not been thoroughly investigated. Reported here is the effect of one polyamine analogue, N, N'-bis[3-[(phenylmethyl)amino]propyl]-1,7-diaminoheptane (BBS), on P. carinii. BBS inhibits the growth of P. carinii in culture, but at concentrations higher than those required to inhibit the growth of other pathogens. However, BBS is at least as active in an animal model of PCP as in other models of diseases studied. BBS causes some reduction in P. carinii polyamine content and polyamine biosynthetic enzyme activities, but the effect is less than that observed with other pathogens and very much less than the effect of the polyamine biosynthesis inhibitor DL-alpha-difluoromethylornithine. BBS enters P. carinii cells via a polyamine transporter, unlike all other cells that have been studied. P. carinii cells do not remove the benzyl groups of BBS, as is reported for mammalian cells. The most likely mode of action is displacement of natural polyamines. Overall, the activity of BBS provides further evidence that polyamines and polyamine metabolism are rational targets for the development of drugs to treat PCP. Because the details of BBS-P. carinii interaction differ from those of other cells studied, polyamine analogues may provide a highly specific treatment for PCP.     

Inhibition of intestinal epithelial DNA synthesis and adaptive hyperplasia after jejunectomy in the rat by suppression of polyamine biosynthesis

Transient increases in the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, may be critical to initiation of cell growth. We previously reported such increases in ODC activity, and the polyamines, putrescine, and spermidine in rat ileal mucosa between days 1 and 4 after intestinal resection. During this time, there is initiation of mucosal cell hyperplasia, as measured morphologically and biochemically. Intestinal weight and mucosal thickness increase, as do mucosal DNA content and DNA synthesis. In the present study, we gave rats the specific irreversible ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), beginning 3 d before jejunectomy. DFMO completely suppressed the increases in ODC activity and polyamine content in the intestinal mucosa. The suppression in ODC activity was associated with an 87% suppression of DNA synthesis, and resulted in a complete abolition of intestinal adaptation, as manifested by the absence of intestinal weight gain, increase in mucosal thickness, or increase in crypt cell production. Our results indicate that the increases in ODC activity and polyamine biosynthesis are critical for adaptive postresectional crypt cell proliferation in vivo, and that the critical step mediated by polyamines in this adaptive process is the onset of new DNA synthesis.     

3-Aminooxy-1-aminopropane and derivatives have an antiproliferative effect on cultured Plasmodium falciparum by decreasing intracellular polyamine concentrations

The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with K(i) values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 microM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.     

Difluoromethylornithine enhances the uptake of methylglyoxal-bis(guanylhydrazone) prior to inhibiting leukemic cell proliferation

Difluoromethylornithine (DFMO) is a nonreversible inhibitor of ornithine decarboxylase (ODC), the initial rate-limiting enzyme in the polyamine biosynthetic pathway. When HL60 leukemic cells were incubated in the presence of concentrations of DFMO from 0.05 mM to 5 mM, there was a concentration-dependent inhibition of ODC activity apparent within 24 h. Likewise, cellular polyamine levels were reduced by the presence of DFMO in a concentration-dependent manner after 4 days. The growth of cells incubated with 0.5 mM or greater was inhibited after 3-4 cell doublings. When the concentration of DFMO was less than 0.5 mM, growth was not inhibited. Methylglyoxal-bis(guanylhydrazone) (MGBG) uptake was enhanced in cells treated with concentrations of 0.05-0.5 mM DFMO, but not enhanced in cells treated with DFMO concentrations of 1 mM or greater. DFMO-induced cellular polyamine depletion does enhance MGBG uptake into HL60 cells, but treatment with high concentrations of DFMO, which deplete polyamines to the extent that growth is inhibited, negate this effect.     

Method of testing the susceptibility of Pneumocystis carinii to antimicrobial agents in vitro.

Rat Pneumocystis carinii grown on lung-derived cell lines in tissue culture flasks and multiwell plates was tested for susceptibility to four antimicrobial agents currently being used in the treatment of human pneumocystosis. Standard criteria for organism quantitation, replication, viability, and inoculum size were established. Trimethoprim-sulfamethoxazole inhibited P. carinii growth at a concentration ratio of 1:19 microgram/ml, and pentamidine isethionate was active at 0.1 microgram/ml. alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, inhibited P. carinii at a concentration of 1 mM once erythrocytes (which are high in polyamine content) were removed from the inoculum; this effect could be overcome by the polyamine putrescine. Dapsone suppressed P. carinii replication at a dose of 0.1 microgram/ml, but this effect was lost after 72 h in culture. Overall, the reduction in P. carinii numbers with these drugs was relatively modest (45 to 84%), which is consistent with their lack of lethal effects on the organism in vivo. Thus, the system presented here should be helpful in developing new anti-P. carinii agents and in elucidating their mechanism of action.     

New transition state-based inhibitor for human ornithine decarboxylase inhibits growth of tumor cells

Pyridoxal 5'-phosphate (PLP)-dependent ornithine decarboxylase (ODC) is the key enzyme in polyamine synthesis. ODC is overexpressed in many tumor cells and thus a potential drug target. Here we show the design and synthesis of a coenzyme-substrate analogue as a novel precursor inhibitor of ODC. Structural analysis of the crystal structure of human ODC disclosed an additional hydrophobic pocket surrounding the epsilon-amino group of its substrate ornithine. Molecular modeling methods showed favorable interactions of the BOC-protected pyridoxyl-ornithine conjugate, termed POB, in the active site of human ODC. The synthesized and purified POB completely inhibited the activity of newly induced ODC activity at 100 micromol/L in glioma LN229 and COS7 cells. In correlation with the inhibition of ODC activity, a time-dependent inhibition of cell growth was observed in myeloma, glioma LN18 and LN229, Jurkat, COS7, and SW2 small-cell lung cancer cells if DNA synthesis and cell number were measured, but not in the nontumorigenic human aortic smooth muscle cells. POB strongly inhibited cell proliferation not only of low-grade glioma LN229 cells in a dose-dependent manner (IC(50) approximately 50 micromol/L) but also of high-grade glioblastoma multiforme cells. POB is much more efficient in inhibiting proliferation of several types of tumor cells than alpha-DL-difluoromethylornithine, the best known irreversible inhibitor of ODC.     

Effect of ethanol on polyamine synthesis during liver regeneration in rats.

Ethanol consumption retards the hepatic regenerative response to injury. This may contribute to the pathogenesis of liver injury in alcoholic individuals. The mechanisms responsible for ethanol-associated inhibition of liver regeneration are poorly understood. To determine if the antiregenerative effects of ethanol involve modulation of polyamine metabolism, parameters of polyamine synthesis were compared before and during surgically induced liver regeneration in ethanol-fed rats and isocalorically maintained controls. After partial hepatectomy, induction of the activity of ornithine decarboxylase (ODC), the rate limiting enzyme for polyamine synthesis, was delayed in rats that had been fed ethanol. This was correlated with reduced levels of putrescine, ODC's immediate product. Increases in hepatic spermidine and spermine were also inhibited. Differences in ODC activity between ethanol-fed and control rats could not be explained by differences in the expression of ODC mRNA or by differences in ODC apoenzyme concentrations, suggesting that chronic ethanol intake inactivates ODC posttranslationally. Supplemental putrescine, administered at partial hepatectomy and 4 and 8 h thereafter, increased hepatic putrescine concentrations and markedly improved DNA synthesis and liver regeneration in ethanol-fed rats. These data suggest that altered polyamine metabolism may contribute to the inhibition of liver regeneration that occurs after chronic exposure to ethanol.     

Continuous infusion of DL-alpha-difluoromethylornithine and improved efficacy against a rat model of Pneumocystis carinii pneumonia.

The rapid depletion of Pneumocystis carinii polyamines caused by in vitro exposure to DL-alpha-difluoromethylornithine (DFMO; also called eflornithine or Ornidyl) and the rapid repletion following removal of this drug suggested that the in vivo efficacy of DFMO against P. carinii pneumonia (PCP) may be limited by troughs in drug concentration resulting from the schedule of administration. This led to the prediction that, compared with the response to the standard animal protocol of administering DFMO in drinking water, the response of a rat model of PCP to DFMO would be lessened by bolus administration and improved by continuous infusion. These predictions were confirmed. Intraperitoneal bolus administration of up to 3 g of DFMO kg of body weight-1 was completely ineffective, although this dose has been shown to be effective when given in the drinking water. Conversely, continuous infusion improved the response against PCP seven- to ninefold over the response to drinking water administration. These findings suggest that, compared with the standard clinical investigational protocol for treatment of PCP with DFMO given in four divided daily doses, continuous infusion combined with monitoring of drug concentrations in plasma may improve efficacy and/or reduce the already low rate of adverse effects.     

Antileishmanial effect of 3-aminooxy-1-aminopropane is due to polyamine depletion

The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, catalyzes the conversion of ornithine to putrescine. As the polyamine biosynthetic pathway is essential for the growth and survival of Leishmania donovani, the causative agent of visceral leishmaniasis, inhibition of the pathway is an important leishmaniacidal strategy. In the present study, we examined for the first time the effects of 3-aminooxy-1-aminopropane (APA), an ODC inhibitor, on the growth of L. donovani. APA inhibited the growth of both promastigotes in vitro and amastigotes in the macrophage model, with the 50% inhibitory concentrations being 42 and 5 microM, respectively. However, concentrations of APA up to 200 microM did not affect the viability of macrophages. The effects of APA were completely abolished by the addition of putrescine or spermidine. APA induced a significant decrease in ODC activity and putrescine, spermidine, and trypanothione levels in L. donovani promastigotes. Parasites were transfected with an episomal ODC construct, and these ODC overexpressers exhibited significant resistance to APA and were concomitantly resistant to sodium antimony gluconate (Pentostam), indicating a role for ODC overexpression in antimonial drug resistance. Clinical isolates with sodium antimony gluconate resistance were also found to overexpress ODC and to have significant increases in putrescine and spermidine levels. However, no increase in trypanothione levels was observed. The ODC overexpression in these clinical isolates alleviated the antiproliferative effects of APA. Collectively, our results demonstrate that APA is a potent inhibitor of L. donovani growth and that its leishmaniacidal effect is due to inhibition of ODC.     

Leishmania donovani polyamine biosynthetic enzyme overproducers as tools to investigate the mode of action of cytotoxic polyamine analogs

A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.     

Ornithine decarboxylase activity in tumor cell lines correlates with sensitivity to cell death induced by histone deacetylase inhibitors

Inhibitors of histone deacetylases (HDAC) show significant promise as targeted anticancer agents against a variety of hematologic and solid tumors. HDAC inhibitors arrest the growth of primary cells, but they induce apoptosis or differentiation of tumor cells. Although the precise mechanism is unknown, differences in cell cycle checkpoints and chromatin structure may be responsible. Cellular polyamines regulate both cell cycle progression and chromatin structure. In tumors, polyamines are abundantly produced because of increased activity of the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase (ODC). To determine if polyamines contribute to the cellular response to HDAC inhibitors, we inhibited ODC activity with alpha-difluoromethylornithine. Polyamine depletion increased resistance to apoptosis induced by HDAC inhibitors. In addition, we found that ODC activity levels correlated with sensitivity to HDAC inhibitors in a panel of tumor cell lines. We conclude that polyamines participate in the cellular response to HDAC inhibitors and that ODC activity correlates with sensitivity to HDAC inhibitor-induced apoptosis. Thus, elevated polyamine levels might be a biomarker for tumor sensitivity to HDAC inhibitor-induced apoptosis. These findings warrant clinical evaluation of tumor samples to determine if high ODC activity levels predict sensitivity to HDAC inhibitors.     

Regulation of ornithine decarboxylase activity and polyamine transport by agmatine in rat pulmonary artery endothelial cells

Agmatine, a product of arginine decarboxylation in mammalian cells, is believed to govern cell polyamines by inducing antizyme, which in turn suppresses ornithine decarboxylase (ODC) activity and polyamine uptake. However, since agmatine is structurally similar to the polyamines, it is possible that it exerts antizyme-independent actions on polyamine regulatory pathways. The present study determined whether agmatine inhibited ODC activity and polyamine transport in rat pulmonary artery endothelial cells (PAECs) by an antizyme-dependent mechanism. Agmatine caused time-dependent reductions in ODC activity, which occurred before increases in antizyme. Interventions that suppressed proteasome function caused large increases in ODC activity but failed to attenuate inhibitory effects of agmatine. When agmatine was present in the culture medium, 14C-polyamine uptake was competitively inhibited as evidenced by substantial elevations in K(m) values. If PAECs were incubated with agmatine for periods sufficient to increase antizyme, there were modest decreases in V(max) for putrescine and spermidine but not for spermine. These effects of agmatine on polyamine transport were insensitive to protein synthesis inhibition. Collectively, our findings show that agmatine decreases ODC activity and polyamine transport in PAECs, but a causal role for antizyme in these actions of agmatine is difficult to establish. Nevertheless, these observations are consistent with a model in which PAECs express both antizyme-1 and -2, but only the latter contributes to agmatine-mediated suppression of ODC activity.     

Structure-activity and structure-selectivity studies on diaminoquinazolines and other inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Twenty-eight 2,4-diaminopteridines with alkyl and aralkyl groups at the 6- and 7-positions, five 1,3-diamino-7,8,9,10-tetrahydropyrimido [4,5-c]isoquinolines with an alkyl, alkylthio, or aryl group at the 6-position, and nine 4,6-diamino-1,2-dihydro-s-triazines with one or two alkyl groups at the 2-position and a substituted phenyl or naphthyl group at the 1-position were evaluated as inhibitors of dihydrofolate reductase enzymes from Pneumocystis carinii, Toxoplasma gondii, and rat liver. Halogen substitution at the 5- or 6-position of 2,4-diaminoquinazoline favored selective binding to the P. carinii enzyme but not the T. gondii enzyme. For example, the 50% inhibitory concentrations of 2,4-diamino-6-chloroquinazoline as an inhibitor of P. carinii, T. gondii, and rat liver dihydrofolate reductase were 3.6, 14 and 29 microM, respectively, corresponding to 12-fold selectivity for the P. carinii enzyme but only marginal selectivity for the T. gondii enzyme. Greater than fivefold selectivity for P. carinii but not T. gondii dihydrofolate reductase was also observed for the 2,4-diaminoquinazolines with 5-methyl, 5-fluoro, 5- and 6-bromo, 6-chloro, and 5-chloro-6-bromo substitution. In contrast, alkyl and aralkyl substitution at the 6- and 7-positions of 2,4-diaminopteridines was found to be a favorable feature for selective inhibition of the T. gondii enzyme and, in two cases, for both enzymes. Nine of the fifty-one compounds tested against P. carinii dihydrofolate reductase and four of the thirty compounds tested against T. gondii dihydrofolate reductase displayed fivefold or greater selectivity for the microbial enzyme versus the rat liver enzyme. The most selective against both enzymes was 2,4-diamino-6,7-bis(cyclohexylmethyl) pteridine, with a selectivity ratio 2 orders of magnitude greater than the value reported for trimetrexate and piritrexim. Since substitution at the 7-position is generally considered to be detrimental to the binding of 2,4-diaminop-teridines and related compounds to mammalian dihydrofolate reductase, the selectivity observed in this study with the 6,7-bis(cyclohexylmethyl) analog may represent a useful approach to enhancing selective inhibition of the enzyme from nonmammalian species.     

Inhibition of Pneumocystis carinii dihydropteroate synthetase by para-acetamidobenzoic acid: possible mechanism of action of isoprinosine in human immunodeficiency virus infection.

Isoprinosine has been reported to decrease progression to AIDS, primarily by preventing Pneumocystis carinii pneumonia (PCP), in human immunodeficiency virus-infected patients, but the mechanism of action is unknown. para-Acetamidobenzoic acid (PAcBA), one component of isoprinosine, is structurally related to para-aminobenzoic acid (PABA), a precursor of de novo folate synthesis. This pathway is known to be important for P. carinii because sulfonamides, which are effective anti-P. carinii agents, inhibit incorporation of PABA into folate precursors by the enzyme dihydropteroate synthetase (DHPS). Inhibition of P. carinii DHPS by PAcBA was investigated by using two assays. In short-term cultures of P. carinii from rats, [3H]PABA incorporation into reduced folates was inhibited by both isoprinosine (mean +/- standard error 50% inhibitory concentration [IC50], 20 +/- 8.4 microM) and PAcBA free acid (IC50, 240 +/- 100 microM); a soluble PAcBA salt was more potent than PAcBA free acid alone (IC50, 29 +/- 48 microM). The activity of PAcBA free acid was confirmed in a cell-free DHPS inhibition assay (IC50, 120 +/- 120 microM). Inosine and dimethylaminopropanol, two other components of isoprinosine, were poor inhibitors of PABA incorporation (IC50, > 1,000 microM). PAcBA free acid also showed activity in inhibiting the DHPS of Toxoplasma gondii, but was a poor inhibitor of the DHPSs of Escherichia coli and Saccharomyces cerevisiae. In a rat model of PCP, the PAcBA salt administered intraperitoneally demonstrated no activity against established PCP either alone or when used in combination with trimethoprim; the lack of efficacy in this model may be due to the rapid metabolism of the drug. Prevention of PCP by PaCBA through inhibition of P. carinii DHPS may explain the activity of isoprinosine in decreasing the progression to AIDS in human immunodeficiency virus-infected patients.     

Nonsteroidal anti‐inflammatory drugs stimulate spermidine/spermine acetyltransferase and deplete polyamine content in colon cancer cells

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colonic tumourigenesis and have an established usefulness in cancer prevention. Because polyamines are essential for neoplastic cell growth, the aim of this study was to evaluate the effect of NSAIDs (indomethacin, a nonselective COX-1 and COX-2 inhibitor) on polyamine metabolism in colon cancer cells. METHODS: Both cell counting and thymidine incorporation into cellular DNA were used to assess colon cancer cell growth. Activities of polyamine-metabolising enzymes, polyamine content (HPLC) and ODC and c-myc protein expression (Western blot) were measured in colon cancer cells treated with indomethacin during logarithmic phase of proliferation. RESULTS: Indomethacin impaired growth of human colon cancer cells (Caco-2 and HCT-116). As a result, ornithine decarboxylase activity and c-myc protein expression were decreased. Treatment with indomethacin induced intracellular oxidant formation in colon cancer cells significantly increased the spermidine/spermine-acetyltrasferase activity (SSAT) and enhanced polyamine acetylation and efflux from colon cancer cells. Impairment of cell growth by indomethacin could not be reversed by exogenous polyamines. CONCLUSION: Taken together, our results suggest that NSAIDs affect polyamine metabolism in colon cancer cells by inducing SSAT activity, and that polyamine depletion in NSAID-treated colon cancer cells is mainly due to enhanced polyamine acetylation and irreversible depletion of intracellular polyamine pools.     

Effects of atovaquone and diospyrin-based drugs on the cellular ATP of Pneumocystis carinii f. sp. carinii

Atovaquone (also called Mepron, or 566C80) is a napthoquinone used for the treatment of infections caused by pathogens such as Plasmodium spp. and Pneumocystis carinii. The mechanism of action against the malarial parasite is the inhibition of dihydroorotate dehydrogenase (DHOD), a consequence of blocking electron transport by the drug. As an analog of ubiquinone (coenzyme Q [CoQ]), atovaquone irreversibly binds to the mitochondrial cytochrome bc(1) complex; thus, electrons are not able to pass from dehydrogenase enzymes via CoQ to cytochrome c. Since DHOD is a critical enzyme in pyrimidine biosynthesis, and because the parasite cannot scavenge host pyrimidines, the drug is lethal to the organism. Oxygen consumption in P. carinii is inhibited by the drug; thus, electron transport has also been identified as the drug target in P. carinii. However, unlike Plasmodium DHOD, P. carinii DHOD is inhibited only at high atovaquone concentrations, suggesting that the organism may salvage host pyrimidines and that atovaquone exerts its primary effects on ATP biosynthesis. In the present study, the effect of atovaquone on ATP levels in P. carinii was measured directly from 1 to 6 h and then after 24, 48, and 72 h of exposure. The average 50% inhibitory concentration after 24 to 72 h of exposure was 1.5 microgram/ml (4.2 microM). The kinetics of ATP depletion were in contrast to those of another family of naphthoquinone compounds, diospyrin and two of its derivatives. Whereas atovaquone reduced ATP levels within 1 h of exposure, the diospyrins required at least 48 h. After 72 h, the diospyrins were able to decrease ATP levels of P. carinii at nanomolar concentrations. These data indicate that although naphthoquinones inhibit the electron transport chain, the molecular targets in a given organism are likely to be distinct among members of this class of compounds.