Novel lysine-spermine conjugate inhibits polyamine transport and inhibits cell growth when given with DFMO

Polyamines are ubiquitous molecules with multiple intracellular functions. Cells tightly regulate their levels through feedback mechanisms affecting synthesis, intracellular conversion, and transport. Because polyamines have an important role in regulating cell growth, they are a target for cancer therapeutic development. However, to effectively inhibit cell growth through polyamine depletion one needs to inhibit both polyamine synthesis and import. Although the mammalian polyamine transporter has not been cloned, we have identified ORI 1202, an N(1)-spermine-L-lysinyl amide, as an effective polyamine transport inhibitor. ORI 1202 prevents the cellular accumulation of [(3)H]spermidine over a 20-h test period. ORI 1202 (30-100 microM) effectively inhibits cell growth when used in conjunction with the polyamine synthesis inhibitor alpha-difluoromethylornithine (DFMO; > or =230 microM). Human breast, prostate, and bladder carcinoma cell lines and melanoma cell lines show ORI 1202 EC(50) values in the low micromolar range when tested in conjunction with DFMO. This cytostatic effect correlates with a reduction in the intracellular levels of putrescine and spermidine. When ORI 1202 (45 mg/kg, i.p., tidx5) and DFMO (1% in drinking water) were delivered over 14 days, MDA-MB-231 breast tumor xenografts in nude mice showed 50% growth inhibition. Polyamine depletion therapy provides a cytostatic therapy that could be useful against cancer and other diseases resulting from uncontrolled cell growth.     

Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism

OBJECTIVE: Iron loading has been observed to have a hyperproliferative effect on hepatocytes in vitro and on tumour cells in vivo; removal of this iron being required to induce antitumour activity. MATERIAL AND METHODS: Antiproliferative effects of orally active tridentate iron chelator ICL670 (deferasirox) and bidentate iron chelator CP20 (deferiprone), mediated through the chelation of intracellular iron, were compared in rat hepatoma cell line FAO and human hepatoma cell line HUH7. RESULTS: In FAO cell cultures, we have shown that ICL670 decreased cell viability and DNA replication and induced apoptosis more efficiently than an iron-binding equivalent concentration of CP20. Moreover, ICL670 decreased significantly the number of the cells in G(2)-M phase. In the HUH7 cell cultures, ICL670 and a four-time higher iron-binding equivalent concentration of CP20, decreased cell viability and DNA replication in the same range. CP20 increased the number of the cells in G(2)-M phase. However, ICL670 inhibited polyamine biosynthesis by decreasing ornithine decarboxylase mRNA level; in contrast, CP20 increased polyamine biosynthesis, particularly putrescine level, by stimulating spermidine-spermine N(1)-acetyl transferase activity that could activate the polyamine retro-conversion pathway. By mass spectrometry, we observed that ICL670 cellular uptake was six times higher than CP20. CONCLUSIONS: These results suggest that ICL670 has a powerful antitumoural effect and blocks cell proliferation in neoplastic cells by a pathway different from that of CP20 and may constitute a potential adjuvant drug for anticancer therapy.     

Comparison of the effects of treatment with the polyamine analogue N1,N8 bis(ethyl)spermidine (BESpd) or difluoromethylornithine (DFMO) on the Topoisomerase II mediated formation of 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) induced cleavable complex in the human lung carcinoma line NCI H157

The positively charged polyamines putrescine, spermidine, and spermine are thought to be important in the maintenance of chromosomal structure. Polyamine depletion by the ornithine decarboxylase inhibitor, 2-difluoromethyl-ornithine (DFMO) is known to alter the effect of several DNA active agents, presumably resulting from the altered conformation of the polyamine depleted DNA. Here we compare the polyamine depletion effects of DFMO and the spermidine analogue N1,N8 bis(ethyl)spermidine (BESpd) on the formation of Topoisomerase II mediated, 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) induced cleavable complex formation in human large cell undifferentiated lung carcinoma NCI H157 cells. This human cell line responds in the normal cytostatic manner to DFMO, whereas it responds in an unusual cytotoxic manner to treatment with BESpd. Here we report that neither DFMO nor BESpd alone affects the formation of cleavable complex. However, both compounds significantly enhance the m-AMSA induced formation of cleavable complex, each by approximately 1.6 fold. These results indicate that both DFMO and BESpd lead to a similar depletion of nuclear polyamines. Additionally, although BESpd closely resembles the natural polyamine spermidine, it appears that it cannot substitute for Spd at the level of DNA.     

Antiproliferative and iron chelating efficiency of the new bis-8-hydroxyquinoline benzylamine chelator S1 in hepatocyte cultures

If a new generation of iron chelators specifically devoted for cancer chemotherapy emerged these last years, any of them has not yet been approved at this time. Accordingly, there is a need to optimize new chelating molecules for iron chelation therapy and cancer treatment. So, the objective of the present investigation was to characterize the antiproliferative activity and the iron chelating capacity of the iron chelator S1 [bis-N-(8-hydroxyquinoline-5-ylmethyl)benzylamine]. Its effects were compared to O-trensox which binds ferric iron with a very high affinity (pFe(3+)=29.5). For this purpose, primary rat hepatocyte stimulated by EGF and human hepatoma HepaRG cell cultures were used. In these models, the anti-proliferative effect, the inhibition of DNA synthesis and the iron-chelating efficiency of increasing concentrations of S1 and O-trensox (0 up to 200 μM) were investigated. In the two cell culture models, we observed that S1 was about 100 times more efficient than O-trensox and the antiproliferative effect of S1 in HepaRG cells appeared at concentrations as low as 0.1 μM without cytotoxicity. Moreover, the stoichiometry of S1 for iron seemed to be in the range S1/Fe(3+)=1. Using the calcein fluorescence assay, we demonstrated that the affinity of S1 for iron was better than that of O-trensox since it was at least two times more effective to restore the fluorescence of calcein previously quenched by iron. So, the iron chelating efficiency of S1 could explain at least partially its higher anti-proliferative effect compared to O-trensox. Finally, these results suggest that molecules such as S1 may constitute a promising starting point to improve cancer treatment.     

多胺生物合成的抑制对转化细胞c－Ha－ras癌基因表达的调控

抗多胺代谢剂──二氟甲基鸟氨酸（ＤＦＭＯ）作用于经含点突变的Ｈａ－ｒａｓ基因片段转染的转化细胞（ＨＲ－１细胞）引起细胞生长的抑制,其抑制率随ＤＦＭＯ浓度的增加而增大,此时细胞多停滞于Ｇ_１期;多胺合成的关键酶鸟氨酸脱羧酶（ＯＤＣ）活性显著下降;Ｈａ－ｒａｓ癌基因ｍＲＮＡ及ｒａｓＰ~（２１）蛋白的表达受到抑制;而外源性腐胺与ＤＦＭＯ的同时加入可防止上述一系列改变的发生,说明ＤＦＭＯ使ＨＲ－１细胞某些表型向亲本细胞逆转的作用是与细胞多胺生物合成的抑制直接相关。     

Nebivolol inhibits vascular smooth muscle cell proliferation by mechanisms involving nitric oxide but not cyclic GMP.

The objective of this study was to elucidate the mechanisms by which nebivolol, a cardio-selective beta-adrenergic receptor antagonist, inhibits rat aortic smooth muscle cell (RASMC) proliferation. Nebivolol was compared with DETA-NO and S-nitroso-N-acetylpenicillamine (SNAP), two nitric oxide (NO) donor agents, and alpha-difluoromethylornithine (DFMO), a known inhibitor of ornithine decarboxylase (ODC). All four test agents inhibited RASMC proliferation in a concentration-dependent manner, with nebivolol being the most potent (IC(50) = 4.5 microM), whereas atenolol, another relatively selective beta(1)-blocker, was inactive. DFMO, nebivolol, and DETA-NO interfered with cell proliferation in a cell-density-dependent manner, the lower the cell density the greater the inhibition of cell proliferation. The cytostatic effects of nebivolol and DETA-NO were completely independent of cyclic GMP, as neither ODQ (cytosolic guanylyl cyclase inhibitor) nor zaprinast (cyclic GMP phosphodiesterase inhibitor) affected the antiproliferative action of nebivolol or DETA-NO. The cytostatic effects of nebivolol, SNAP, and DFMO were largely prevented by the addition of excess putrescine, but not ornithine, to cell cultures. Moreover, nebivolol caused a marked reduction in the intracellular levels of putrescine, spermidine, and spermine. Like DFMO, nebivolol and DETA-NO interfered with the G(1)-phase to S-phase cell cycle transition in RASMC. These observations confirm previous findings that DFMO and NO interfere with RASMC proliferation by inhibiting ODC and polyamine production and provide evidence that nebivolol works by the same mechanism.     

Effects of alpha-difluoromethylornithine-induced polyamine depletion on the radiosensitivity of a human colon carcinoma cell line

The effect of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) on the in vitro radiation response of Clone A human colon adenocarcinoma cells was investigated. Analysis of intracellular polyamine levels showed that exposure of Clone A cells to 1 mM DFMO for 96 h reduced putrescine and spermidine to nondetectable levels, while spermine was decreased by approximately 50%. This DFMO treatment protocol enhanced the radiosensitivity of Clone A cells, which was reflected by a decrease in both the Do and Dq. The addition of putrescine (1 mM) for the final 48 h of DFMO exposure restored polyamine levels and returned clone A radiosensitivity to that of control cells. These results indicate that polyamine depletion by DFMO sensitizes Clone A tumor cells to ionizing radiation.     

RhoA stimulates IEC-6 cell proliferation by increasing polyamine-dependent Cdk2 activity

Although RhoA plays an important role in cell proliferation and in Ras transformation in fibroblasts and mammary epithelial cells, its role in intestinal epithelial cells (IEC) is unknown. In a previous study (Ray RM, Zimmerman BJ, McCormack SA, Patel TB, and Johnson LR. Am J Physiol Cell Physiol 276: C684-C691, 1999), we showed that polyamine depletion [dl-alpha-difluoromethylornithine (DFMO) treatment] strongly inhibits the proliferation of IEC. In this report, we examined the effect of RhoA on IEC-6 cell proliferation and whether polyamine depletion inhibits cell proliferation in the presence of constitutively active RhoA. Constitutively active RhoA and vector-transfected IEC-6 cell lines were grown in the presence or absence of DFMO, which causes polyamine depletion by inhibiting ornithine decarboxylase, the first rate-limiting step in polyamine synthesis. Constitutively active RhoA significantly increased the rate of cell proliferation. These cells also lost contact inhibition and formed conspicuous foci when they were fully confluent. Decreased p21Waf1/Cip1 expression and increased cyclin-dependent kinase (Cdk2) mRNA levels and activity accompanied the increased proliferation. The inhibition of p21Waf1/Cip1 was independent of p53. There was no activation of the Ras-Raf-MEK-ERK pathway in the RhoA-transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on IEC-6 cell proliferation, focus formation, and Cdk2 expression. The stability of mRNA and protein for Cdk2 and p21Waf1/Cip1 in V14-RhoA cells was not significantly different from that of vector-transfected cells. In conclusion, RhoA activation decreased p21Waf1/Cip1 expression and increased basal and serum-induced ornithine decarboxylase activity, Cdk2 expression, Cdk2 protein, and Cdk2 activity, leading to the stimulation of IEC proliferation and transformation. Polyamine depletion totally prevented RhoA's effect on proliferation by decreasing Cdk2 expression and activity.     

p53 independent G(1) arrest induced by DL-alpha-difluoromethylornithine

Ornithine decarboxylase (ODC), which catalyzes polyamine biosynthesis, plays an essential role in cell growth. DL-alpha-Difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, inhibits cell growth. However, the exact mechanism by which polyamine depletion by DFMO results in growth inhibition remains to be elucidated. We clarified the mechanisms by which DFMO inhibits human gastric cancer cell (MKN45) growth. DFMO induced MKN45 cell G(1) phase arrest after 48 h, and the percentage of G(1) arrest cells continued to increase until 72 h. Expression of p21 and phosphorylation of Stat1 were significantly induced by DFMO at 24 h. Luciferase assay and gel shift assay showed specific binding of Stat1 to the p21 promoter, and promoter activity was activated at 24 h. In dominant negative p53 expressing cells, DFMO significantly induced p21 expression, arrested cells at G(1) phase, and suppressed cell growth effectively. These results suggest that DFMO induced MKN45 cell arrest at G(1) phase in a p53 independent manner, and Stat1 is, at least in part, involved in G(1) arrest.     

Effects of agmatine accumulation in human colon carcinoma cells on polyamine metabolism, DNA synthesis and the cell cycle

Putrescine, spermidine and spermine are low molecular polycations that play important roles in cell growth and cell cycle progression of normal and malignant cells. Agmatine (1-amino-4-guanidobutane), another polyamine formed through arginine decarboxylation, has been reported to act as an antiproliferative agent in several non-intestinal mammalian cell models. Using the human colon adenocarcinoma HT-29 Glc(-/+) cell line, we demonstrate that agmatine, which markedly accumulated inside the cells without being metabolised, exerted a strong cytostatic effect with an IC50 close to 2 mM. Agmatine decreased the rate of L-ornithine decarboxylation and induced a 70% down-regulation of ornithine decarboxylase (ODC) expression. Agmatine caused a marked decrease in putrescine and spermidine cell contents, an increase in the N1-acetylspermidine level without altering the spermine pool. We show that agmatine induced the accumulation of cells in the S and G2/M phases, reduced the rate of DNA synthesis and decreased cyclin A and B1 expression. We conclude that the anti-metabolic action of agmatine on HT-29 cells is mediated by a reduction in polyamine biosynthesis and induction in polyamine degradation. The decrease in intracellular polyamine contents, the reduced rate of DNA synthesis and the cell accumulation in the S phase are discussed from a causal perspective.     

Differential effect of αdifluoromethyl-ornithine on the proliferation of balb 3T3 and chemically transformed 3T3 cells

In order to study the effect of polyamine depletion on growth and proliferation of untransformed and chemically transformed cells, α-difluoromethyl-ornithine (DFMO) was added to cultures of 3T3 cells and their benzo[a]pyrene derivative BP-3T3. Both types of cells stopped their proliferation after 72 hr of treatment with the inhibitor. When DFMO was removed and cells were cultivated afterwards in fresh medium without the drug, untransformed cells resumed growth after a lag period, whereas transformed cells were unable to proliferate unless exogenous polyamine was added. These alterations showed a strict correlation with intracellular polyamine pools, since after removal of DFMO from the culture medium, polyamine concentrations increased to almost normal values in 3T3 cells, but remained at low levels or decreased even more in the transformed cells BP-3T3. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled proteins of both cell extracts has indicated that the described control of cell proliferation by intracellular polyamine levels might be related to the synthesis of at least two proteins with molecular weights of about 36,000 and 55,000 daltons.     

Polyamines affect histamine synthesis during early stages of IL‐3‐induced bone marrow cell differentiation

Mast cells synthesize and store histamine, a key immunomodulatory mediator. Polyamines are essential for every living cell. Previously, we detected an antagonistic relationship between the metabolisms of these amines in established mast cell and basophilic cell lines. Here, we used the IL‐3‐driven mouse bone marrow‐derived mast cell (BMMC) culture system to further investigate this antagonism in a mast cell model of deeper physiological significance. Polyamines and histamine levels followed opposite profiles along the bone marrow cell cultures leading to BMMCs. α‐Difluoromethylornithine (DFMO)‐induced polyamine depletion resulted in an upregulation of histidine decarboxylase (HDC, the histamine‐synthesizing enzyme) expression and activity, accompanied by increased histamine levels, specifically during early stages of these cell cultures, where an active histamine synthesis process occurs. In contrast, DFMO did not induce any effect in either HDC activity or histamine levels of differentiated BMMCs or C57.1 mast cells, that exhibit a nearly inactive histamine synthesis rate. Sequence‐specific DNA methylation analysis revealed that the DFMO‐induced HDC mRNA upregulation observed in early bone marrow cell cultures is not attributable to a demethylation of the gene promoter caused by the pharmacological polyamine depletion. Taken together, the results support an inverse relationship between histamine and polyamine metabolisms during the bone marrow cell cultures leading to BMMCs and, moreover, suggest that the regulation of the histamine synthesis occurring during the early stages of these cultures depends on the concentrations of polyamines. J. Cell. Biochem. 108: 261–271, 2009. © 2009 Wiley‐Liss, Inc.     

Haematological cell proliferation and differentiation responses to perturbations of polyamine biosynthesis

Combined administration of methylglyoxal-bis-guanylhydrazone (MGBG) (25 mg/kg) with difluoromethylornithine (DFMO), or MGBG alone at a higher dose (50 mg/kg), to mice resulted in a decreased white cell count (WBC) in the peripheral blood while DFMO or MGBG alone at a lower dose (25 mg/kg) had no effect. As expected, DFMO alone increased the number of colony forming units spleen (CFU-s), colony forming units diffusion chamber granulocyte (CFU-dg) and colony forming units culture (CFU-c) in the bone marrow. MGBG treatment led to an increase in CFU-dg alone. Combined treatment seemingly had no effect on marrow stem cells. Total tibial and differential counts were not affected by any of the treatments. Cell proliferation in diffusion chamber cultures, as judged by CFU-dg colony formation, was impaired by MGBG alone or in combination with DFMO, at dose levels which had no effect or increased the precursor cell number in the bone marrow. This effect was partially reversed with either putrescine or spermidine. Determination of intracellular polyamine concentrations, demonstrated decreased putrescine and spermidine levels after DFMO administration. As expected, MGBG treatment resulted in decreased spermidine and spermine levels, concomitant with an increase in putrescine. In mice which received both agents, rather than only MGBG, after 3 days higher intracellular polyamine concentrations were observed. After 11 days, however, there was no significant difference between the two groups.     

Modulation of cell proliferation in rat liver cell cultures by new calix[4]arenes

Cell cycle progression is dependent on intracellular iron level and chelators lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of new synthetic calix[4]arene podands bearing two aspartic/glutamic acid, ornithine groups or hydrazide function at the lower rim, designed as potential iron chelators. The synthesis only afforded calix[4]arenes in the cone conformation. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the rat hepatoma cell line Fao by measuring mitochondrial succinate dehydrogenase activity. Their cytotoxicity was evaluated by extracellular LDH activity. Preliminary results indicated that among all tested compounds, monohydrazidocalix[4]arene 2 which is not cytotoxic in Fao cells exhibits interesting antiproliferative activity. This effect, independent on iron depletion, remains to be further explored. Moreover, it also shows that new substituted calix[4]arenes could open the way to new valuable medicinal chemistry scaffolding.     

Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

Thiol depletion induces lethal cell injury in cultured cardiomyocytes.

Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or alpha-difluoromethylornithine

We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and alpha-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 microM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G1 and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 x 10(5)), medium (5 x 10(5)), or high (2 x 10(6)) cell densities in a 25 cm2 flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.