Robert A. Casero

Properties of purified recombinant human polyamine oxidase, PAOh1/SMO.

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.

Polyamines in normal and cancer cells

Based on available evidence, it appears that polyamines are critical for proliferation of both normal and transformed cells. Although the requirement of polyamines for DNA replication and cell proliferation is established, the molecular events in which the polyamines are essential are yet unknown. Furthermore, transformed and cancer cells, possibly because of their higher proliferative rate, appear to be more dependent on polyamine metabolism than their normal counterparts. This has been shown by the in vivo response of tumor models and human tumor xenografts in nude mice to polyamine depletion by DFMO. Although there has been associated toxicity to the host, the inhibition of cell proliferation has been higher in the implanted tumors than in the host. DFMO, a specific irreversible inhibitor of ODC, has been used extensively in studies which have shed light on the role of polyamines in cell proliferation and differentiation. DFMO has shown interesting anti-tumor effect in a number of experimental tumor models. Currently, DFMO clinical trials are being completed, and it will be of interest to see whether this polyamine inhibitor, or other newer polyamine analogs and inhibitors, will find a place in the treatment of neoplastic disorders.

Modulation of growth gene expression by selective alteration of polyamines in human colon carcinoma cells

The biosynthesis of the polyamines, putrescine, spermidine and spermine, is temporally linked with expression of many growth related genes. Our previous studies have shown that generalized polyamine depletion of the human colon cancer cell line COLO 320 by 2-difluoromethylornithine is associated with decreased transcription of the c-myc, c-fos, and ornithine decarboxylase (ODC) genes. In the current study, the role of individual polyamines was further defined by the use of a specific inhibitor of spermidine synthase, S-adenosyl-1,8, diamino-3-thio-octane (AdoDATO), and a spermine analogue, N1,N12 bis(ethyl)spermine. Our data demonstrate that depletion of spermidine results in a 60-90% decrease in c-myc mRNA steady state levels. In contrast, c-fos mRNA levels are decreased only when both spermidine and spermine are diminished. Furthermore, ODC mRNA levels are increased when all polyamines are decreased by DFMO, but are unaffected by a selective reduction in intracellular spermidine levels by AdoDATO. These studies suggest that individual polyamines may have a selective role in the expression of specific growth related genes.

Chronic lithium treatment prevents the dexamethasone-induced increase of brain polymine metabolizing enzymes

The paper describes the effects of various regimens of lithium chloride treatment on dexamethasone-induced increases in brain polyamine metabolizing enzymes. In contrast to peripheral tissues where acute lithium treatment suppresses the increase in ornithine decarboxylase activity, in the brain only chronic treatment was effective in preventing this increase and also the increases in the activities of S-adenosylmethionine decarboxylase and spermidine/spermine N1-acetyltransferase. This findings indicate a novel brain target for lithiums action and in turn provide new avenues for exploring polyamine function in the brain.

Self-immolative nanoparticles for simultaneous delivery of microRNA and targeting of polyamine metabolism in combination cancer therapy

Abstract Combination of anticancer drugs with therapeutic microRNA (miRNA) has emerged as a promising anticancer strategy. However, the promise is hampered by a lack of desirable delivery systems. We report on the development of self‐immolative nanoparticles capable of simultaneously delivering miR‐34a mimic and targeting dysregulated polyamine metabolism in cancer. The nanoparticles were prepared from a biodegradable polycationic prodrug, named DSS‐BEN, which was synthesized from a polyamine analog N1,N11‐bisethylnorspermine (BENSpm). The nanoparticles were selectively disassembled in the cytoplasm where they released miRNA. Glutathione (GSH)‐induced degradation of self‐immolative linkers released BENSpm from the DSS‐BEN polymers. MiR‐34a mimic was effectively delivered to cancer cells as evidenced by upregulation of intracellular miR‐34a and downregulation of Bcl‐2 as one of the downstream targets of miR‐34a. Intracellular BENSpm generated from the degraded nanoparticles induced the expression of rate‐limiting enzymes in polyamine catabolism (SMOX, SSAT) and depleted cellular natural polyamines. Simultaneous regulation of polyamine metabolism and miR‐34a expression by DSS‐BEN/miR‐34a not only enhanced cancer cell killing in cultured human colon cancer cells, but also improved antitumor activity in vivo. The reported findings validate the self‐immolative nanoparticles as delivery vectors of therapeutic miRNA capable of simultaneously targeting dysregulated polyamine metabolism in cancer, thereby providing an elegant and efficient approach to combination nanomedicines. Graphical abstract Figure. No Caption available.

Role of p53/p21Waf1/Cip1 in the regulation of polyamine analogue-induced growth inhibition and cell death in human breast cancer cells

Intracellular polyamines are absolutely required for cell proliferation and many tumors have abnormal requirements for polyamines. Therefore, the polyamine metabolic pathway represents a rational target for antineoplastic intervention. A number of polyamine analogues act as potent modulators of cellular polyamine metabolism and exhibit encouraging effects against tumor growth in both cell culture and animal studies. In this study we demonstrate that specific polyamine analogues exhibit differential inhibitory action against the growth of human breast cancer MCF-7 cells. Treatment of MCF-7 cells with oligoamine analogues and the symmetrically substituted bis(alkyl)- substituted analogue, BENSpm, produced a G1 cell cycle arrest, while the unsymmetrically substituted bis(alkyl)-substituted analogue, CHENSpm, induced a G2/M cell cycle arrest. All four compounds significantly up-regulated p53 and p21 expression in MCF-7 cells. Stable transfection of small interfering RNA (siRNA) targeting p53 blocked the expression of p21 induced by the polyamine analogues and significantly reduced polyamine analogue-induced growth inhibition and apoptosis, suggesting that polyamine analogue-induced p21 expression occurs through p53-dependent mechanisms. The effects of analogue exposure on cyclins and cyclin dependent kinases varied with the specific agent used. Expression of p53 siRNA reversed only BENSpm modulated the cell cycle arrest, suggesting that regulation of cell cycle arrest by p53/p21 induced by polyamine analogues occurs through agent-specific mechanisms. Understanding the mechanism of p53-mediated cellular responses to polyamine analogue may help to improve the therapeutic efficacy of polyamine analogues in human breast cancer.

Human lung tumor sensitivity to difluoromethylornithine as related to ornithine decarboxylase messenger RNA levels

The differential response to polyamine depletion has been studied in two types of human lung tumor cells. Small cell lung carcinoma cells die following polyamine depletion by difluoromethylornithine treatment while non-small cell lines demonstrate a typical cytostatic response. We now report that a small cell line, NCI H82, has a lower apparent capacity for polyamine biosynthesis than does a representative non-small cell, NCI H157. In subconfluent cultures, the ornithine decarboxylase activity is 25 times lower in the small cell than the non-small cell and by comparison, the polyamines in the small cell line are markedly reduced. Most significantly, levels of mRNA coding for ornithine decarboxylase are approximately 100-fold lower in the small cell than the non-small cell line, and this difference does not appear to be a result of gene rearrangement. These results suggest that differential sensitivity to polyamine depletion is related to different steady-state levels of ornithine decarboxylase mRNA.

Lithium exerts a time-dependent and tissue-selective attenuation of the dexamethasone-induced polyamine response in rat brain and liver

It has previously been shown that chronic, but not acute, lithium treatment indirectly prevents the dexamethasone-induced increase in brain polyamine-metabolizing enzymes. In the present study we determined the effects of lithium treatment on changes in cellular polyamines, 6 h after dexamethasone challenge (3 mg/kg intraperitoneally). The findings demonstrate that chronic lithium (daily intraperitoneal 2.5 mmol/kg injections for 2 weeks) treatment completely prevents the accumulation of putrescine, in parallel to its prevention of the dexamethasone-induced increase in ornithine decarboxylase activity. A partial attenuation of this polyamine response was also observed in the liver. Only minor and inconsistent changes were observed in the concentrations of the polyamines, spermidine and spermine. Acute lithium treatment (a single injection at times ranging from 1 to 24 h prior to dexamethasone challenge) did not attenuate the dexamethasone-induced increases in brain putrescine concentration nor in ornithine decarboxylase activity. It is suggested that prevention of the stress-induced polyamine response in the brain may be an important mechanism through which prophylactic lithium may exert its beneficial effect in manic-depressive illness.

N1-Nonyl-1,4-diaminobutane ameliorates brain infarction size in photochemically induced thrombosis model mice

Inhibitors for polyamine oxidizing enzymes, spermine oxidase (SMOX) and N1-acetylpolyamine oxidase (PAOX), were designed and evaluated for their effectiveness in a photochemically induced thrombosis (PIT) mouse model. N1-Nonyl-1,4-diaminobutane (C9-4) and N1-tridecyl-1,4-diaminobutane (C13-4) competitively inhibited the activity of PAOX and SMOX in a manner comparable to N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527), an irreversible inhibitor of both enzymes. The two compounds were then tested for their effects in the PIT model. Both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of C9-4 decreased infarct volumes significantly. By contrast, C13-4 reduced the volume of brain infarction by i.c.v. administration, but no reduction was observed after i.p. administration. C9-4 administered by i.p. injection reduced the volume of brain infarction significantly at doses of more than 3 mg/kg, and the dosage of 5 mg/kg or 10 mg/kg demonstrated the most potent effect and were more effective than equivalent doses of the other inhibitors such as MDL72527 and N-benzylhydroxylamine. I.P. injection of 5 mg/kg of C9-4 provided a therapeutic time window of longer than 12 h. This report demonstrates that C9-4 is a potent inhibitor of the polyamine oxidizing enzymes and is useful lead compound for candidate drugs with a long therapeutic time window, to be used in the treatment of ischemic stroke.

Polymeric Prodrugs Targeting Polyamine Metabolism Inhibit Zika Virus Replication

The Zika virus (ZIKV) is primarily transmitted via an infected mosquito bite, during sexual intercourse, or in utero mother to child transmission. When a fetus is infected, both neurological malformations and deficits in brain development are frequently manifested. As such, there is a need for vaccines or drugs that may be used to cure ZIKV infections. Metabolic pathways play a crucial role in cell differentiation and development. More importantly, polyamines play a key role in replication and translation of several RNA viruses, including ZIKV, Dengue virus, and Chikungunya virus. Here, we present polyamine analogues (BENSpm and PG11047) and their corresponding polymer prodrug derivatives for inhibiting ZIKV infection by intersecting with polyamine catabolism pathways. We tested the compounds against ZIKV African (MR766) and Asian (PRVABC59) strains in human kidney epithelial (Vero) and glioblastoma derived (SNB-19) cell lines. Our results demonstrate potent inhibition of ZIKV viral replication in both cell lines tested. This antiviral effect was mediated by the upregulation of two polyamine catabolic enzymes, spermine oxidase, and spermidine (SMOX)/spermine N1-acetyltransferase (SAT1) as apparent reduction of the ZIKV infection following heterologous expression of SMOX and SAT1. On the basis of these observations, we infer potential use of these polyamine analogues to treat ZIKV infections.