To understand the mechanisms behind platinum drug/DENSPM-induced inhibition of cancer cell growth, we compared the effects of oxaliplatin and cisplatin when combined with DENSPM on the induction of SSAT mRNA, activity, polyamines and cell growth in A2780 human ovarian carcinoma cells and their oxaliplatin- and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively.

We have previously showed that platinum drugs up-regulate SSAT and SMO and down-regulate ODC and SAMDC in the polyamine pathway. Several studies including our own established that platinum drugs combined with polyamine analog DENSPM produces synergistic increase in SSAT activity with polyamine depletion. Since polyamine pathway is an important therapeutic target, we investigated whether agents containing both platinum and polyamines have similar effects on the polyamine pathway. Two complexes i) Pt-spermine with two cisplatin molecules linked to a spermine in the center and ii) Pd-spermine with similar structure i, but Pd (II) substituted for Pt (II) were analyzed with respect to their effect on the expression of genes in polyamine pathway, SSAT and SMO protein expression, SSAT activity and polyamine pools. Pt-, Pd-spermine complexes induced significant down-regulation of SMO, arginase 2 and NRF-2, with no change in SSAT, while cisplatin as a single agent or in combination with DENSPM induced significant up-regulation of SSAT and SMO. The SSAT activity was not induced by either Pt- or Pd-spermine in A2780 cells; SMO protein levels were significantly elevated compared to the no-drug control and to a similar extent as cisplatin/DENSPM. The Pd-spm treatment induced a fall in putrescine levels to 33%, spermidine to 62% and spermine to 72% while Pt-spm did not induce such a decline. Comparative cytotoxicity studies in A2780 cells indicated the potency to be cisplatin> Pd-Spm>Pt-Spm. Although both complexes exhibit a lower potency, the degree of resistance itself is much lower for Pt-spermine and Pd-spermine in that order (2.5 and 7.5, respectively) compared to cisplatin ( approximately 12) as tested in cisplatin resistant A2780/CP cells. These studies suggest that Pd (II)-polyamine complexes may constitute a promising group of inorganic compounds for further studies in the development of novel chemotherapy/adjuvant chemotherapy strategies.

The expression of catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) increases after ischemic reperfusion injury. We hypothesized that polyamine catabolism is upregulated and that this increase in catabolic response contributes to tissue damage in endotoxin-induced acute kidney injury (AKI). SSAT mRNA expression peaked at threefold 24 h following LPS injection and returned to background levels by 48 h. The activity of SSAT correlated with its mRNA levels. The expression of SMO also increased in the kidney after LPS administration. Serum creatinine levels increased significantly at approximately 15 h, peaking by 24 h, and returned to background levels by 72 h. To test the role of SSAT in endotoxin-induced AKI, we injected wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice with LPS. Compared with SSAT-wt mice, the SSAT-ko mice subjected to endotoxic-AKI had less severe kidney damage as indicated by better preservation of kidney function. The role of polyamine oxidation in the mediation of kidney injury was examined by comparing the severity of renal damage in SSAT-wt mice treated with MDL72527, an inhibitor of both polyamine oxidase and SMO. Animals treated with MDL72527 showed significant protection against endotoxin-induced AKI. We conclude that increased polyamine catabolism through generation of by-products of polyamine oxidation contributes to kidney damage and that modulation of polyamine catabolism may be a viable approach for the treatment of endotoxin-induced AKI.

Purpose Our previous studies showed that combined treatment of oxaliplatin and N 1 , N 11 diethyl-norspermine (DENSPM) results in massive induction of spermidine/spermine N 1 -acetyltransferase (SSAT) mRNA and activity. Since oxaliplatin and 5-fluorouracil (5FU) are used clinically in treatment of colorectal cancers, this study examines the effect of adding DENSPM to oxaliplatin/5FU combination on SSAT and spermine oxidase (SMO) in HCT-116 cells. Methods HCT-116 cells were treated with clinically relevant concentrations of drugs for 20 h followed by 24 h in drug free medium. SSAT and SMO mRNA and protein were assayed by QRT-PCR and Westerns respectively; polyamine pools were measured by HPLC. SSAT and SMO mRNA in tumor biopsies from patients with rectal cancer receiving oxaliplatin, capecitabine and radiation were measured by QRT-PCR. Results Oxaliplatin + 5FU + DENSPM produced significantly higher levels of SSAT and SMO mRNA, protein and activity than those seen with oxaliplatin+5FU with a significant depletion of cellular spermine and spermidine pools. Oxaliplatin/DENSPM was superior to 5FU/DENSPM in SSAT induction but similar for SMO. Oxaliplatin + DENSPM revealed synergistic growth inhibition at >IC 50 concentrations and antagonism at <IC 50 . SMO and SSAT induction occurred in 60 and 30% of the patient samples examined. Conclusions These studies demonstrated that combining DENSPM with oxaliplatin + 5FU provides an added benefit by aiming at the clinically relevant therapeutic target, the polyamine catabolism. Further, we show for the first time, that SMO and SSAT induction could be measured in tumor biopsies in patients receiving chemo-radiation. Optimization of treatment conditions in vivo should facilitate a clinical evaluation of the three drug combination.

Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N1-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 μm 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by ∼5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail.

Numerous studies have correlated elevated polyamine levels with abnormal or rapid cell growth. One therapeutic strategy to treat diseases with increased cellular proliferation rates, most obviously cancer, has been to identify compounds which lower cellular polyamine levels. An ideal target for this strategy is the protein antizyme-a negative regulator of polyamine biosynthesis and import, and a positive regulator of polyamine export. In this study, we have optimized two tissue-culture assays in 96-well format, to allow the rapid screening of a 750-member polyamine analog library for compounds which induce antizyme frameshifting and fail to substitute for the natural polyamines in growth. Five analogs (MQTPA1-5) containing xylene (1,4-dimethyl benzene) were found to be equal to or better than spermidine at stimulating antizyme frameshifting and were inefficient at rescuing cell growth following polyamine depletion. These compounds were further characterized for effects on natural polyamine levels and enzymes involved in polyamine metabolism. Finally, direct measurements of antizyme induction in cells treated with two of the lead compounds revealed an 8- to 15-fold increase in antizyme protein over untreated cells. The impact of the xylene moiety and the distance between the positively charged amino groups on antizyme frameshifting and cell growth are discussed.

Biochemical modulation of methylation processes can be accomplished by agents which either reduce pools of 5-adenosylmethionine (AdoMet), the principal methyl donor, or alternatively, which raise levels of ,Sadenosylhomocysteine (AdoHcy), a potent product inhibitor of methyltransferase reactions. Both strategies have apparent limitations arising from their direct interference with only one determinant of the intracellular AdoHcy/AdoMet ratio, a parameter proposed to be indicative of methylation inhibition. The biological consequences of maximally altering this ratio have been examined by the combined use of an inhibitor of AdoMet synthetase, L-2-amino-4-methoxy-cii-but-3-enoic acid (LcisAMB), with inhibitors of AdoHcy hydrolase, 9-(trans-2\trans-3 -dihydroxycyclopent-4'-enyI)adenine (DHCA) and neplanocin A. At concen trations which inhibited growth of LI 210 cells by 50% at 48 h, I cisAMB alone rapidly depleted AdoMet pools, while neplanocin A or DHCA alone led to an accumulation of AdoHcy. When L-cisAMB was combined with either neplanocin A or DHCA, AdoHcy increased and, concomitantly, AdoMet pools decreased. The resultant AdoHcy/AdoMet ratios for up to 48 h ranged from 2.2 to 3.6—a value 4-fold greater than those achieved with neplanocin A or DHCA alone. This elevation in the AdoHcy/AdoMet ratio was accompanied by marked and sustained inter ference with DNA and RNA methylation and with a near-total inhibition of cell growth for a period of 24 to 96 h. Thus, the combined treatment with these two types of mechanistically different methylation inhibitors resulted in significantly enhanced interference with nucleic acid methyl ation and cell growth, both of which correlated directly with unprece dented increases in the AdoHcy/AdoMet ratio. This approach may have therapeutic implications in antiviral and/or antitumor strategies targeting methylation.

The cellular effects of a-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC). and Arl,N8-bis(ethyl)spermidine (BES), an apparent regulator of the enzyme were compared in cultured LI210 cells. Unlike DFMO, BES has no direct inhibitory effect on OIK activity. Rather the polyamine analogue is believed, from previous studies, to behave similarly to exogenous spermidine in its ability to suppress intracellular OIK activity but not in its ability to perform functions required for cell growth. The kinetics and extent of growth inhibition by 30 /IM or 100 MMBES and 1 HIMDFMO were nearly identical as were their effects on macromolecular precursor incorporation with leucine being the first and most significantly affected. By flow cytometry, neither BES nor DFMO induced obvious perturbations in the cell cycle. Both compounds effectively eliminated OIK activity in treated cells and depleted putrescine and spermidine pools with very similar kinetics of decline. These close similarities in drug effects between BES and DFMO, an established polyamine inhibitor, support previous indications that BES induces growth inhibition by depletion of cellular polyamines. BES differed distinctly from the OIK inhibitor by decreasing speratine pools, and by not increasing 5-adenosyl-methionine decarboxylase activity, ,Vadenosylmethionine pools, or stimulating cellular uptake of polyamines. The data suggest that enzyme regulation by polyamine analogues such as BES represents a viable alternative to enzyme inhibition as an antiproliferative strategy directed at polyamine biosynthesis.

We have previously shown that the clinically relevant polyamine analog N1,N11-diethylnorspermine (DENSPM) causes rapid apoptosis in human melanoma SK-MEL-28 cells via a series of events that include mitochondrial release of cytochrome c and activation of the caspase cascade. Upstream to these events, DENSPM downregulates polyamine biosynthesis and potently upregulates polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT). In searching for downstream effectors that either contribute to or abrogate the apoptotic response, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs). Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP protein (but not XIAP) levels by 18 h via an apparently Smac/Diablo-independent pathway. Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis, suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome. We also observed that structural analogs of DENSPM which differentially induced SSAT and apoptosis lowered survivin and ML-IAP levels in a manner that correlated with enzyme activity. The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interfering RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment. Among the melanoma cell lines (SK-MEL-28, MALME-3M, A375 and LOX), survivin degradation correlated temporally with the onset of DENSPM induced apoptosis or growth inhibition. By contrast, ML-IAP degradation occurred only during rapid apoptosis seen in SK-MEL-28 cells. These data suggest a sequence of events whereby DENSPM induction of SSAT leads to loss of IAP proteins and a more fulminate apoptotic response. The findings implicate survivin and ML-IAP as important determinants of polyamine analog drug action in melanoma cells.

The mechanistic effectiveness of various polyamine analogs and enzyme inhibitors is typically determined by their ability to deplete intracellular polyamine pools. In this study, we describe an assay that may prove useful in augmenting this relatively static assessment of drug action. The assay relies upon the substitution of 4-fluoro-L-ornithine (Fl-Orn) for ornithine as a polyamine precursor to provide a means to measure metabolic flux through polyamine pools. At concentrations up to 500 microM, the analog did not inhibit the growth of L1210 murine leukemia cells during incubations of up to 72 hr. Using HPLC, the analog was processed metabolically over time to what was deduced to be 2-fluoroputrescine, 6-fluorospermidine and 6-fluorospermine. The relative proportion of fluorinated polyamine analog to the natural polyamine increased with time and Fl-Orn concentration. The sum of the two was found to be nearly identical to the respective polyamine pool of control cells exposed instead to 500 microM ornithine. This indicates that Fl-Orn was recognized and utilized as a precursor at a rate very similar to that of ornithine itself. Using L1210 cells at different stages of cell growth, it was determined that the metabolic flux through the pools, as indicated by the rate of appearance of individual fluorinated polyamine species, reflected the proliferation status of the cells--non-growing cells failed to incorporate the analog. Likewise, in cell types with varying polyamine pool profiles, such as polyamine enzyme overproducers or those with constitutively different spermidine of spermine ratios, the incorporation of the fluorinated analogs into pools was found to be proportional to the size to the natural polyamine pool. In cells treated with inhibitors of S-adenosylmethionine decarboxylase, Fl-Orn incorporation indicated a total blockade of polyamine synthesis at that enzyme site. Overall, the Fl-Orn assay has demonstrated that polyamine pool profiles generally reflect the rate of flux through the pathway in proliferating cells, suggesting that most intracellular polyamines are freely exchangeable with those undergoing metabolic flux.

A Chinese hamster ovary cell subline (CHO/664) > 1000-fold resistant to the S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, CGP-48664 (4-(aminoiminomethyl)-2,3-dihydro-1H-inden-1-one diaminomethylenehydrazone), has been developed and characterized. The cells were also cross-resistant to the highly specific nucleoside analog inhibitor of AdoMetDC, MDL-73811. These unique cells stably overexpress AdoMetDC due to a 10-16-fold amplification of the AdoMetDC gene, which resulted in a similar increase in AdoMetDC transcript levels. In the presence of 100 microM CGP-48664, the CHO/664 cells displayed AdoMetDC activities similar to the parental line. Following removal of the inhibitor, AdoMetDC activity increased steadily over 20 days to 10-12 times that found in parental CHO cells. Decarboxylated (dc) AdoMet pools accumulated rapidly from < 5 pmol/10(6) cells to approximately 1000-1500 pmol/10(6) cells at 3 days due to diffusion away of intracellular inhibitor and to the depletion of putrescine and spermidine as aminopropyl acceptors in dcAdoMet-mediated synthase reactions. Polyamine pools shifted as putrescine, and spermidine pools were processed forward to spermine. During the period from 3 days to 20 days, dcAdoMet pools fell steadily and eventually stabilized at 100-200 pmol/10(6) cells. Providing excess putrescine at this time as an aminopropyl acceptor rapidly lowered dcAdoMet pools and led to a near normalization of polyamine pools, indicating that both dcAdoMet and putrescine are essential in maintaining steady-state polyamine pool profiles. As with cell line variants that overproduce ornithine decarboxylase, polyamine transport was found to be increased in CHO/664 cells due to an apparent inability of the system to down-regulate polyamine transport in response to polyamine excess. Given the unique metabolic disturbances seen in these cells, we anticipate that in addition to providing a useful system for evaluating the specificity of newly developed AdoMetDC inhibitors, they will undoubtedly prove valuable for investigating the various regulatory interrelationships involved in polyamine homeostasis and possibly other aspects of purine metabolism.

Inhibitors of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC), derived from methylglyoxal-bis(guanylhydrazone) (MGBG), have been shown to have significant antitumor activity in several human solid tumor systems (U. Regenass et al., Cancer Res., 52:4712-4718, 1992). From an ongoing effort to synthesize derivatives with increased enzyme specificity and potency and improved antitumor efficacy, we have now identified CGP 48664, a 4-amidinoindan-1-one 2'-amidinohydrazone (J. Stanek et al., J. Med. Chem., 36:2168-2171, 1993). The compound displays potent inhibition of SAMDC (50% inhibitory concentration, 5 nM), modest inhibition of diamine oxidase (50% inhibitory concentration, 4 microM), and no detectable inhibition of ornithine decarboxylase. CGP 48664 inhibits the growth of a panel of human and mouse tumor cell lines, including one which expresses the multidrug resistance phenotype, with 50% inhibitory concentrations ranging between 0.3 and 3 microM. CGP 48664 does not seem to utilize the polyamine transport carrier system since it competes poorly with spermidine for uptake into L1210 cells (Ki 161 microM) and inhibits the growth of polyamine transport-deficient Chinese hamster ovary cells. Relative to MGBG or previously described MGBG analogues, CGP 48664 accumulates to much lower intracellular concentrations. Treatment of the L1210 cell for 48 h with 3 microM CGP 48664 decreases SAMDC activity to < 10% of control and initiates a compensatory 3-fold rise in ornithine decarboxylase. Consistent with SAMDC inhibition, putrescine pools increase 10-fold, whereas spermidine and spermine pools fall to < 10% of control. In contrast to MGBG, CGP 48664 displays attenuated antimitochondrial activity as indicated by a lack of effect on pyruvate oxidation and mitochondrial DNA levels under treatment conditions which inhibit cell proliferation. Specificity of drug action was indicated further by prevention of L1210 cell growth inhibition by exogenous spermidine or spermine. More convincingly, Chinese hamster ovary cells made approximately 1000-fold resistant by chronic exposure to the analogue were found to selectively overexpress SAMDC mRNA due to gene amplification. The new SAMDC inhibitor showed potent antitumor activity against syngeneic tumors (B16 melanoma and Lewis lung carcinoma) and nude mouse human tumor xenografts (T-24 bladder carcinoma, SK MEL-24 melanoma, and MALME-3M melanoma). On the basis of its novel structure, its apparent specificity of action, and its potent antitumor activity, CGP 48664 is the candidate drug for further preclinical development.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and antiproliferative effects of novel 5'-fluorinated analogs of 5'-deoxy-5'-(methylthio)adenosine [Erratum to document cited in CA110(21):193314b]Janice R. Sufrin, Arthur J. Spiess, Debora L. Kramer, Paul R. Libby, and Carl W. PorterCite this: J. Med. Chem. 1990, 33, 4, 1270Publication Date (Print):April 1, 1990Publication History Published online1 May 2002Published inissue 1 April 1990https://pubs.acs.org/doi/10.1021/jm00166a030https://doi.org/10.1021/jm00166a030research-articleACS PublicationsRequest reuse permissionsArticle Views31Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

The essentiality of S-adenosylmethionine (AdoMet) in transmethylation reactions and polyamine biosynthesis provides a rationale for the development of methionine (Met) analog inhibitors of AdoMet synthesis as potential antitumor agents. Recent studies indicating that “Met dependent” tumors have defects in Met and AdoMet metabolism suggest that certain tumors may be particularly susceptible to growth inhibition by Met analogs (Hoffman, 1985). Systematic approaches to the design of such analogs have been facilitated by the earlier studies on Met analog inhibitors of microbial and mammalian isofunctional methionine adenosyl transferases (MAT; EC 2.5.1.6) reported by Talalay and coworkers (Chou et. al., 1977), in which certain critical features of the enzyme bound conformation of L-Met and of the complementary topography at its L-Met binding site were defined.

A number of studies have demonstrated that pretreatment of tumor-bearing animals with the inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), potentiates the antitumor activity of methylglyoxal bis(guanylhydrazone) (MGBG). The present study examines whether this phenomenon is related to a DFMO-mediated increase in the selectivity of MGBG for tumor tissue. Specifically, the effect of DFMO pretreatment on the tissue distribution and content of MGBG was investigated in mice bearing ascites L1210 leukemia. At 3 and 18 h following a single i.v. injection of [14C]MGBG (50 mg/kg), L1210 cells and seven tissues from nonpretreated (control) and DFMO-pretreated (3% by drinking water for 3 days) animals were compared for their [14C]MGBG content. In control mice, the greatest amount of drug was found in L1210 cells, small intestine, and kidney (in decreasing order of magnitude) at both 3 and 18 h. This distribution was not altered following DFMO pretreatment, but the relative MGBG content of other tissues was shifted. On an average, DFMO pretreatment increased the accumulation of MGBG by 30% in normal tissues and 32% in tumor tissues at 3 h and 56% and 69%, respectively, at 18 h. Thus, pretreatment of leukemic mice with DFMO fails to improve the selectivity of MGBG for L1210 cells. It is possible that other tumor systems might demonstrate sufficient DFMO-mediated increases in MGBG uptake to enhance drug selectivity but not without significantly increasing MGBG uptake (and hence toxicity) in normal tissues.

Mitochondria of cultured L1210 leukemia cells undergo extensive swelling and structural disruption when exposed to the anticancer agent, methylglyoxal-bis(guanylhydrazone) (MGBG). Similar damage has now been observed in ascites L1210 cells treated in vivo with a single dose (>50 mg/kg) of either MGBG or ethidium bromide (EB). After 24 hr, the mitochondria swell significantly, lose their inner structure, and, in the case of EB treatment, develop numerous electron densities within the matrix. Analysis of the ribonucleotide pools of these cells by high-pressure liquid chromatography reveals that treatment with either MGBG or EB depletes the adenosine triphosphate pools and markedly reduces the overall adenylate energy charge of the cell. Thus, in addition to being structurally damaged, the mitochondria are functionally impaired by both drugs. When MGBG-treated cells are harvested, washed, and placed in untreated mice, all cells recover near to normal ultrastructure after 48 hr, whereas EB-treated cells do not, even after 96 hr. Analysis of the ribonucleotide pools of the MGBG-treated cells indicates that the mitochondira have recovered their functional capabilities as well. The adenylate energy charge for these cells is essentially the same as that for untreated cells. If allowed to remain in the untreated mice, the cells previously treated with MGBG display unaltered leukemogenicity, killing the animals at nearly the same average time as untreated cells. The significance of the contribution of MGBG-induced mitochondrial damage to the antiproliferative action of the drug and its relationship to inhibition of spermidine and spermine biosynthesis by the drug remains to be established.