Inhibition of polyamine synthesis suppresses human lymphocyte proliferation without decreasing cytokine production or interleukin 2 receptor expression

Difluoromethylornithine (DFMO) irreversibly inhibits ornithine decarboxylase (ODC), a crucial enzyme in polyamine synthesis, and impairs mitogen-induced lymphocyte proliferation. To examine the mechanism of action of DFMO, we studied the effect of this ODC inhibitor on lymphokine production and interleukin 2 (IL 2) receptor expression. DFMO decreased thymidine uptake of peripheral blood mononuclear cells stimulated by the mitogens phytohemagglutinin, concanavalin A, phorbol myristate acetate and ionomycin 60-70% compared with untreated cells, and the inhibition could be completely reversed by 10 mM spermidine. DFMO had no effect on IL 1 production by monocytes exposed to silica particles. Concentrations of IL 2 increased 7-fold in DFMO-treated, PHA-stimulated PBMC cultures, compared with untreated cells; whereas IL 2 receptor expression as measured by the anti-Tac monoclonal antibody was not affected by the inhibition of ODC. Mixing experiments using cells cultured with or without DFMO indicated that the inhibition by DFMO was not mediated by suppressor cells. Our results strongly support the concept that polyamines are required for a relatively late event in lymphocyte activation occurring after the interaction of IL 2 and its receptor.     

Suppression of lymphocyte proliferation by hexamethylene diamine

The antiproliferative potential of hexamethylene diamine (HMDA) for mitogen-stimulated splenic lymphocytes was evaluated in vitro at final concentrations of 0.1-16 mM. Addition at the start of culture or after 24 or 48 h of culture decreased the proliferative response to T and B cell mitogens. However, the concentration of HMDA required to cause suppression increased with incubation time. Removal of diamine after 24 h allowed cells to proliferate normally upon reculture with mitogen. Mitogenic responses of cultures containing the potent ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were also inhibited in a time and dose dependent fashion. ODC activity, which was much greater in cultures stimulated with Con A than LPS, was markedly decreased by inclusion of diamine or DFMO in the culture medium. Addition of putrescine to cultures did not reverse the suppressive effects of diamine on proliferation but did restore DFMO-containing cultures to control levels of activity. These results indicate that HMDA does suppress lymphocyte proliferation in vitro by alteration of ODC and polyamine activity. However, comparison of results obtained with DFMO and HMDA suggests that HMDA may act via multiple pathways, only one of which involves inhibition of ODC activity.     

Inhibition of Polyamine Formation Antagonizes Vascular Smooth Muscle Cell Proliferation and Preserves the Contractile Phenotype

The polyamines putrescine, spermidine and spermine play essential roles in cell proliferation and migration, two processes involved in the development of vascular disease. Thus, intervention with polyamine formation may represent a way to inhibit unwanted vascular smooth muscle cell (VSMC) proliferation. The aim of the present study was to assess the importance of polyamines for VSMC proliferation and vascular contractility. The rate‐limiting step in polyamine biosynthesis is catalysed by ornithine decarboxylase (ODC). Treatment with α‐difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, reduced DNA synthesis in primary rat VSMCs in a concentration‐dependent manner with an IC₅₀ value of 100 μM. Moreover, DFMO reduced VSMC migration assessed in a scratch assay. The DFMO‐induced attenuation of VSMC proliferation was associated with lowered cellular amount of polyamines. The antiproliferative effect of DFMO was specific because supplementation with polyamines reversed the effect of DFMO on proliferation and normalized cellular polyamine levels. Isometric force recordings in cultured rat tail artery rings showed that DFMO counteracts the decrease in contractility caused by culture with foetal bovine serum as growth stimulant. We conclude that inhibition of polyamine synthesis by DFMO may limit the first wave of cell proliferation and migration, which occurs in the acute phase after vascular injury. Besides its antiproliferative effect, DFMO may prevent loss of the smooth muscle contractile phenotype in vascular injury.     

Cyclosporine and α-difluoromethylornithine exhibit differential effects on colon and pancreatic cancer in vitro

-Difluoromethylornithine (DFMO) is a known irreversible inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Cyclosporine (CsA) has been reported to inhibit ODC activity in vitro. In the present study, we compared the effects of DFMO and CsA on growth, survival, and polyamine levels in mouse colon cancer (MC-26) and hamster pancreatic cancer (H2T) cells in vitro. The growth and survival of MC-26 and H2T cells were inhibited by both DFMO and CsA. However, H2T cells were observed to be significantly more sensitive than MC-26 cells to both CsA and DFMO. The inhibitory effects of CsA were blocked by the addition of the polyamine, putrescine, in both MC-26 and H2T cells. Polyamine levels were altered significantly in both MC-26 and H2T cells treated with CsA and DFMO. However, the profile of these alterations differed between MC-26 and H2T cell lines. Putrescine and spermidine levels in MC-26 cells were more sensitive to DFMO inhibition than were H2T cells. Spermine levels were consistently elevated in MC-26 cells exposed to CsA or DFMO, while the level of spermine in H2T cells decreased significantly in response to the same drugs. These results suggest that CsA and DFMO exhibit different effects on colon and pancreatic cancer growth in vitro. In addition, the differences in the sensitivity of pancreatic and colon cancer to CsA and DFMO indicate potentially important differences in polyamine metabolism between the two cell lines.     

Marked and prolonged inhibition of mammalian ornithine decarboxylase in vivo by esters of (E)-2-(fluoromethyl)dehydroornithine

(E)-2-(fluoromethyl)dehydroornithine, a new enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) is no more effective than alpha-difluoromethylornithine (DFMO) at inhibiting polyamine biosynthesis in rat hepatoma tissue culture (HTC) cells and in rat organs even though its potency is over 15 times higher than that of DFMO in vitro. The methyl, ethyl, octyl and benzyl esters of (E)-2-(fluoromethyl)dehydroornithine were synthesized as potential prodrugs of the amino acid. When tested at concentration equivalent to the Ki value of the amino acid, they are devoid of ODC-inhibitory property. When measured 6 hr after its addition to the HTC cell culture medium, the absorption of methyl ester was 20 times higher than that of the parent amino acid or that of DFMO, and was accompanied by a more marked intracellular accumulation of (E)-2-(fluoromethyl)dehydroornithine than that achieved by the addition of the parent amino acid. The methyl ester used at 10 times lower concentrations is as effective as its parent amino acid or as DFMO at inhibiting polyamine biosynthesis in HTC cells. Similarly, the methyl and the ethyl esters of (E)-2-(fluoromethyl)dehydroornithine used at 10 times lower doses are as effective as the parent amino acid and as DFMO at inhibiting ODC in the ventral prostate of rat, 6 hr after oral administration. All the esters of (E)-2-(fluoromethyl)dehydroornithine produce a particularly long duration of ODC inhibition in the ventral prostate and in the testes. Repeated administration (25 mg/kg given once a day by gavage) of the methyl ester of (E)-2-(fluoromethyl)dehydroornithine for 8 days to rats results in a constant 80% inhibition of ODC over a 24-hr period, accompanied by a 90% decrease of putrescine and spermidine concentrations in the ventral prostate.     

Treatment with polyamine synthesis inhibitors reduces the positive inotropic effect of ouabain, noradrenaline and calcium

Polyamines (putrescine, spermidine and spermine) are considered to act as intracellular second messengers by increasing Ca++ influx and mobilizing intracellular calcium. On the other hand, intracellular Ca++ increase is the common final step of the mechanism of action of many inotropic agents. To discover whether the functional integrity of the cardiac ornithine decarboxylase (ODC)/polyamine system is necessary to cope with a stimulated inotropism, we studied the effect of ouabain, noradrenaline, and calcium on ventricle strips obtained from rats treated with polyamine synthesis inhibitors. The combined administration of methylglioxal bis (guanylhydrazone) (MGBG) (single i.p. injection of 50 mgkg-1) and of alpha-di fluoromethylornithine (DFMO) (100 mgkg-1 every 12 h for 7 consecutive days) caused a 62.5% inhibition of ventricular ODC activity, and a significant decrease of the ventricular content of putrescine and spermidine (-59.5%, and -40.1%, respectively). While the basal isometric tension developed by ventricle strips obtained from rats treated with MGBG+DFMO was similar to that developed by ventricle strips from controls, the response to ouabain (1 microM), noradrenaline (10 microM), or Ca++ (3.6 mM) was significantly reduced. It cannot be excluded that effects of MGBG unrelated to the inhibition of polyamine synthesis may have also concurred in part to influence the effect of ouabain, Ca++ and noradrenaline adversely. However, the present results seem to indicate that the heart response to inotropic agents requires an efficient ODC/polyamine system, polyamines probably being involved in calcium ion movements or affecting the Ca++ sensitivity of contractile proteins.     

Schedule dependent potentiation of antitumor drug effects by α-difluoromethylornithine in human gastric carcinoma cells in vitro

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with -difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line.     

Inhibition of ornithine decarboxylase activity decreases polyamines and suppresses DNA synthesis in human colonic lamina propria lymphocytes

Ornithine decarboxylase (ODC) and the polyamines are essential for cell proliferation in a variety of cells including lymphocytes. In this study, we investigated the potential role of ODC and polyamines in human colonic lamina propria lymphocytes (LPL) compared to peripheral blood lymphocytes (PBL). Our results show that con A stimulation of LPL and PBL was associated with marked increases in ODC and polyamines. The specific inhibitor of ODC, alpha-difluoromethylornithine (DFMO), resulted in a complete inhibition of ODC activity and depletion of putrescine, spermidine and spermine levels. DFMO also suppressed DNA synthesis of LPL and PBL by up to 48% and 62% respectively. This antiproliferative effect was reversed by adding back the polyamines putrescine (1 mM), spermidine (10 μM) or spermine (10 μM) to the culture medium. We conclude that ODC and the polyamines are important for human LPL proliferation, and hence may play a role in human mucosal immune function.     

The methionine salvage pathway compound 4-methylthio-2-oxobutanate causes apoptosis independent of down-regulation of ornithine decarboxylase

4-Methylthio-2-oxobutanoic acid (MTOB) is the final compound of the methionine salvage pathway that converts the polyamine byproduct methylthioadenosine to adenine and methionine. Here we find that MTOB inhibits growth of several human cell lines in a dose-dependent manner. Growth inhibition was specific for MTOB as we did not observe any inhibition with other chemically related compounds. MTOB treatment causes apoptosis and reduction of ornithine decarboxylase (ODC) activity but not ODC mRNA. To determine if MTOB exerts its effects primarily via ODC inhibition, we compared the effects of MTOB with the ODC-specific inhibitor difluoromethylornithine (DFMO). We found that MTOB was a more potent inducer of apoptosis than DFMO, lacked activation of caspase 3/7, and was able to induce apoptosis in cells lacking p53. Our results show that MTOB-induced growth inhibition and apoptosis is not simply secondary due to ODC inhibition and implies that MTOB activates apoptosis via other mechanisms.     

Apoptosis inhibition and ornithine decarboxylase superinduction as early epigenetic events in morphological transformation of Syrian hamster embryo cells exposed to 2-methoxyacetaldehyde, a metabolite of 2-methoxyethanol

We have conducted a study to determine the carcinogenic potential of ethylene glycol monomethyl ether (EGME), a member of the glycol ether family, as compared to its reactive metabolite 2-methoxy-acetaldehyde (MALD). Since disruption of equilibrium between cell proliferation and cell death is thought to play a key role in multistage carcinogenesis, we investigated, in Syrian hamster embryo (SHE) cells exposed to various doses of EGME and MALD, impairment in apoptosis rate and in ornithine decarboxylase (ODC) metabolism. The activity of this rate-limiting enzyme of polyamine biosynthesis is closely related to cell proliferation and cell transformation. At the end-point, comparative action of the two products on SHE cell morphological transformation frequency was evaluated. One-stage exposure of SHE cells to 2 mM EGME and 200 microM MALD for 5 h did not change basal apoptotic level, whereas 0.16 microM phorbol ester (TPA) decreased it. Using two-stage exposure protocol (1 h xenobiotic followed by 5 h TPA), MALD strongly inhibited apoptosis more than did TPA alone; the parent compound EGME did not have any effect on TPA inhibiting action. Western blotting analysis showed that sequential treatment (MALD/TPA) increased Bcl-2 oncoprotein expression, whereas Bcl-XL and Bax proteins were not changed. The same staged exposure of SHE cells to MALD/TPA strongly induced ODC activity, and the rate was higher than that obtained with TPA alone: this was accompanied by an increase of ODC protein level. This ODC superinduction was not observed with EGME/TPA treatment. In long-term SHE-cell morphological transformation assay, staged exposure to MALD (800 microM or 1 mM for 24 h) followed by TPA applications increased the number of transformed colonies at the seventh day. Such early cooperative events as apoptosis inhibition and ODC superinduction, followed by the increase of SHE-cell transformation frequency, are highly indicative of a carcinogenic potential for the metabolite, MALD.     

Molecular basis for the antiproliferative effect of agmatine in tumor cells of colonic, hepatic, and neuronal origin

The aim of the present study was to challenge potential mechanisms of action underlying the inhibition of tumor cell proliferation by agmatine. Agmatine inhibited proliferation of the human hepatoma cells HepG2, the human adenocarcinoma cells HT29, the rat hepatoma cells McRH7777, and the rat pheochromocytoma cells PC-12. Inhibition of proliferation of HepG2 cells was associated with an abolition of expression of ornithine decarboxylase (ODC) protein and a doubling of mRNA content encoding ODC. In HepG2 cells, silencing of ODC-antizyme-1, but not of antizyme inhibitor, by RNA interference resulted in an increase of agmatine's antiproliferative effect. Thus, the distinct decrease in intracellular polyamine content by agmatine was due to a reduced translation of the synthesizing protein ODC but was not essentially mediated by induction of ODC-antizyme or blockade of antizyme inhibitor. In interaction experiments 1 mM L-arginine, 1 mM D-arginine, 1 mM citrulline, 100 microM N(omega)-nitro-L-arginine methyl ester, 1 and 10 microM sodium nitroprusside, and 1 microM N1-guanyl-1,7-diaminoheptane failed to alter agmatine's antiproliferative effect. Hence, the antiproliferative effect of agmatine in HT29 and HepG2 cells is due to an interaction with neither the NO synthases, the hypusination of eIF5A, nor an agmatine-induced reduction in availability of intracellular L-arginine. L-Arginine and citrulline, but not d-arginine, inhibited tumor cell proliferation by themselves. Their inhibitory effect was abolished after silencing of arginine decarboxylase (ADC) expression by RNA interference indicating the conversion to agmatine by ADC. Finally, in the four cell lines under study, agmatine-induced inhibition of cell proliferation was paralleled by an increase in intracellular caspase-3 activity, indicating a promotion of apoptosis.     

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

1. We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by α-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages.
2. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48±5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production.
3. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10  mM) to potentiate LPS-induced NO synthesis.
4. Metabolism of L-[³H]arginine to L-[³H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1–10 mM) or by putrescine (0.001–1 mM), spermidine (0.001–1 mM) or spermine (0.001–1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70±0.07%) by L-NMMA (100 μM).
5. Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (∼2 fold) iNOS protein expression.
6. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.

     

多胺合成抑制剂DFMO抗人膀胱癌EJ细胞增殖的实验观察

目的 :探讨多胺生物合成抑制剂二氟甲基鸟氨酸 (α- difluoeom ethyl- ornithine,DFMO)抗人膀胱癌 EJ细胞增殖的作用。方法 :活细胞显微摄影、伊红染色细胞计数、绘制细胞生长曲线方法。结果 :4m mol～ 8mmol DFMO可以抑制人膀胱癌 EJ细胞的增殖 ,并具有剂量依赖性 ;外源性腐胺可以阻止 DFMO上述作用。结论 :DFMO抗人膀胱癌 EJ细胞增殖的作用是通过抑制多胺生物合成而实现的 ;抑制多胺生物合成可能是治疗膀胱瘤的一种有效途径。     

Suppression of allograft immunity by 3,4,5,3',4',5'-hexachlorobiphenyl. II. Effects of exposure on mixed lymphocyte reactivity and induction of suppressor cell activity in vitro

Previous studies in our laboratory have established the sensitivity of the in vivo allogeneic cytotoxic T lymphocyte (CTL) response to suppression by 3,4,5,3',4',5'-hexachlorobiphenyl[(345)2-HxCB], a toxic, Ah receptor-binding polychlorinated biphenyl isomer. The present studies have examined possible cellular mechanisms for this suppression. A modest dose-dependent suppression of the proliferative response to alloantigen in mixed lymphocyte culture (MLC) was observed with lymphocytes from B6 mice exposed to 10 or 100 mg/kg (345)2-HxCB while the CTL response generated in MLC was significantly suppressed only following exposure to 100 mg/kg (345)2-HxCB. The amount of time between treatment with (345)2-HxCB and sacrifice, which ranged from 2 to 23 days, did not appear to influence the degree of immunosuppression produced by (345)2-HxCB exposure. Mitomycin C-treated lymphocytes from B6 mice treated with (345)2-HxCB were not suppressive when added as third party cells to an independent MLC. However, if the mice were alloimmune, lymphocyte-mediated suppression of the MLC response was observed and directly correlated with the magnitude of the CTL response present in the same population. Thus, (345)2-HxCB-treated mice which had less CTL activity as compared to vehicle-treated mice also had less suppressor activity. Further analysis indicated that stimulator cell lysis by the CTL was likely to be responsible for the inhibitory activity of the alloimmune lymphocytes rather than suppressor cells per se. Avoidance of stimulator cell lysis by using H-2-incompatible MLC stimulator cells revealed the existence of antigen-nonspecific suppressor activity that was greater with lymphocytes from vehicle-treated than from (345)2-HxCB-treated mice, suggesting that both CTL and suppressor cell activities were suppressed by (345)2-HxCB exposure. Direct addition of (345)2-HxCB to lymphocyte cultures in vitro indicated a lack of direct toxicity of (345)2-HxCB on lymphoproliferative responses to mitogen or alloantigen at concentrations equal to or less than 1 x 10(-6) M. Thus, the in-vitro functional integrity of lymphocytes obtained from (345)2-HxCB-treated mice coupled with the lack of a direct lymphotoxic effect of (345)2-HxCB in vitro suggest an indirect mechanism of action for (345)2-HxCB-mediated suppression of CTL activity in vivo. Previous reports implicating suppressor cell induction and/or activation by Ah-receptor-binding halogenated aromatic hydrocarbons that mediate the inhibition of CTL generation were not confirmed in these studies.     

Polyamines and nonmelanoma skin cancer

Elevated levels of polyamines have long been associated with skin tumorigenesis. Tightly regulated metabolism of polyamines is critical for cell survival and normal skin homeostasis, and these controls are dysregulated in skin tumorigenesis. A key enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC) is upregulated in skin tumors compared to normal skin. Use of transgenic mouse models has demonstrated that polyamines play an essential role in the early promotional phase of skin tumorigenesis. The formation of skin tumors in these transgenic mice is dependent upon polyamine biosynthesis, especially putrescine, since treatment with inhibitors of ODC activity blocks the formation of skin tumors and causes the rapid regression of existing tumors. Although the mechanism by which polyamines promote skin tumorigenesis are not well understood, elevated levels of polyamines have been shown to stimulate epidermal proliferation, alter keratinocyte differentiation status, increase neovascularization, and increase synthesis of extracellular matrix proteins in a manner similar to that seen in wound healing. It is becoming increasingly apparent that elevated polyamine levels activate not only epidermal cells but also underlying stromal cells in the skin to promote the development and progression of skin tumors. The inhibition of polyamine biosynthesis has potential to be an effective chemoprevention strategy for nonmelanoma skin cancer.     

Suppression of polyamine biosynthesis prevents monocrotaline-induced pulmonary edema and arterial medial thickening

Previous work in our laboratory has shown that the continuous administration of alpha-difluoromethylornithine (DFMO), a highly specific irreversible inhibitor of ornithine decarboxylase (ODC), which is the rate-limiting enzyme in polyamine biosynthesis, prevented the development of pulmonary hypertension and right ventricular hypertrophy induced in rats 21 days after a single injection of monocrotaline (MCT). We now report that DFMO treatment did not influence the proposed first step of MCT pneumotoxicity, that is, the hepatic metabolism of MCT to toxic pyrrolic metabolites. In contrast, DFMO treatment blunted the development of lung perivascular edema at Day 7, inhibited the respective four- and twofold increases in lung putrescine and spermidine contents at Day 21 without significantly altering spermine content, and prevented the arterial medial thickening at Day 21. It thus appears that increased lung polyamine biosynthesis may be essential for the expression of MCT-induced perivascular edema as well as the development of the medial thickening stage of MCT-induced hypertensive pulmonary vascular disease.     

The effect of alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, on mitogen-induced interleukin 2 production

The objective of the present investigation was to examine the effect of DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, on mitogen-induced interleukin 2 production. Treatment with DFMO reduced nylon wool T cell polyamine levels. In contrast, DFMO treatment enhanced, greater than two-fold, detectable levels of concanavalin A-induced interleukin 2 activity. This observed augmentation was not limited to in vitro DFMO treatment, since oral administration of DFMO to C57BL/6 mice also enhanced concanavalin A-induced interleukin 2 levels in vitro. Treatment with exogenous putrescine reversed the effect of DFMO on interleukin 2 levels. These results suggest that the effect of DFMO on interleukin 2 levels is mediated through polyamines. Therefore, polyamine biosynthesis may play a role in the intracellular regulation of interleukin 2 production.     

Cell cycle kinetics responses of human stomach cancer cells to reduction in polyamine levels by treatment with αDifluoromethylornithine in vitro

Treatment of human gastric cancer clones in vitro with low doses of DFMO (5 mM) produced elongation of the cell population doubling times and lowering of the saturation densities. By contrast, DFMO treatment of normal human skin fibroblasts altered only the saturation density. The lack of an effect of 5 mM DFMO on the doubling time of normal fibroblasts may be directly related to baseline intracellular putrescine levels, which were about 2.5 times higher than in the cancer cells. The same dose of DFMO caused a rapid decrease in intracellular polyamine levels in the tumor clones. The effects on the doubling time and saturation density were almost totally abolished by the addition of 50 M putrescine to the growth medium during the first 24 h of treatment with DFMO. Exposure to 5 mM DFMO for 24 h caused the human gastric cancer cells to become blocked in G₁ phase only, and this led to a reduction in the fraction of cells in S phase. The G₁ block was reversible and this cohort of cells eventually passed through S phase and then through G₂ and M.A higher 100 mM dose of DFMO and longer exposure times for both doses produced cell cycle changes and death of more than 90% of the cell population. These data suggest that cell kinetics changes observed under these experimental conditions may reflect polyamine-related alterations in the biochemical events of cell cycle progression kinetics; but may also be the result of DFMO-induced loss of cell viability.Abbreviations DFMO -Difluoromethylornithine - ODC ornithine decarboxylase - ARA C cytosine arabinosidede - MGBG methylglyoxal bis(guanylhydrazone)     

多胺类似物CPENSpm通过干扰多胺代谢抑制肺癌细胞的增殖

目的研究多胺类似物CPENSpm对肺癌细胞株A549增殖和细胞凋亡的影响,以探讨CPENSpm抗肿瘤的作用机制。方法MTS法分析细胞的增殖速度,化学分析法测定多胺代谢酶的活性,HPLC法分析细胞内的多胺含量,亚凋亡峰测定法和DNA片段化分析法鉴定细胞程序性凋亡。结果CPENSpm处理A549肺癌细胞可导致:①癌细胞生长抑制并激发细胞凋亡;②抑制多胺合成关键酶ODC的活性,活化多胺降解代谢关键酶SSAT和SMO的活性;③耗竭细胞内多胺含量。SMO抑制剂MDL72527可拮抗CPENSpm对A549细胞的生长抑制作用。结论CPENSpm通过干扰A549细胞的多胺代谢途径,耗竭肿瘤快速生长必需的多胺成分,诱导产生活性氧H2O2从而抑制癌细胞生长并激发细胞程序性凋亡。     

2-deoxy-D-glucose inhibits the antitumor effects of α-difluoromethylornithine on the growth of colon cancer in vivo

Summary The glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), has been shown to inhibit the growth of certain cancers. -Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the ratelimiting enzyme in polyamine biosynthesis. DFMO has been shown to inhibit cancer growth in a number of models. The present study was designed to investigate the effects of 2-DG alone and combined with DFMO on MC-26 mouse colon adenocarcinoma tumors growing in vivo. Twenty-eight male Balb/c mice were inoculated with 250,000 MC-26 cells, and then randomized into four groups of 7 each: group I served as control; group II received DFMO (3% in drinking water); group III received 2-DG (500 mg/kg/d IP); group IV received a combination of 2-DG and DFMO. Treatment began 5 days after tumor cell inoculation. MC-26 tumor area was reduced 73% by DFMO compared to a 24% reduction caused by 2-DG. The tumor weight was reduced 80% by DFMO and 52% by 2-DG. The tumor contents of DNA, RNA, and protein were significantly reduced by DFMO but not 2-DG. The tumor concentration of the polyamines putrescine and spermidine were reduced by DFMO alone or combined with 2-DG while spermine levels remained unchanged. 2-DG alone did not alter polyamine levels. These results indicate that both 2-DG and DFMO, when added as single agents, inhibit tumor growth. However, the addition of 2-DG to the DFMO regimen inhibited the antitumor effects of DFMO. Survival studies performed on MC-26 cells in vitro corroborated the antagonisms between DFMO and 2-DG that were shown in vivo.Dr. Upp was awarded a fellowship grant from the American Cancer Society Texas Division.