Polyamine Catabolic Pathway Research Articles

Cumulative effect of AOC1 gene variants on symptoms and pathological conditions in adult women with fibromyalgia: a pilot study.

Introduction: The amine oxidase copper-containing 1 (AOC1) gene encodes for the diamine oxidase (DAO) enzyme. DAO is an enzyme that catabolizes some molecules, including histamine, and is the degradative enzyme in the polyamine catabolic pathway that is active in intestinal mucosal cells. Variants of AOC1 are associated with reduced DAO activity, resulting in accumulation of high levels of histamine and causing a wide range of neurological, gastrointestinal, and epidermal disorders, which are present in people with fibromyalgia. This study aimed to evaluate the impact of four AOC1 gene variants, namely, rs10156191, rs1049742, rs1049793, and rs2052129, on fibromyalgia symptoms measured by the Fibromyalgia Impact Questionnaire (FIQ), such as sleep disorders, atopic dermatitis, migraine, gastrointestinal (GI) disorders, allergies, and intolerances, in adult women with fibromyalgia. Methods: The sample consisted of 100 unrelated women with fibromyalgia between 33 and 60years of age (48.48years ±7.35), whose were diagnosed by a rheumatologist based on symptoms such as pain, stiffness, and fatigue. Single-nucleotide polymorphisms (SNPs) of AOC1 were identified using oral mucosa samples collected following a standard hygiene protocol. DNA was extracted, and gene variants of interest were analyzed using multiplex single-nucleotide primer extension (SNPE). Clinical data were collected using the FIQ and a series of variables that quantified the intensity and frequency of the symptoms. Results: The minor allele frequencies of rs10156191, rs1049742, rs1049793, and rs2052129 were 31.5, 10, 32.5, and 27%, respectively. Each variant was found to be in Hardy-Weinberg equilibrium, but partial linkage disequilibrium between AOC1 SNPs is suspected. The results show that fibromyalgia symptoms measured using the FIQ tend to increase with the number of risk alleles and that the intensity of dry skin and low stool consistency may be associated with an increase in the number of these alleles. Conclusion: This study constitutes the first step in investigating associations between fibromyalgia symptoms and candidate variants of the AOC1 gene in DAO enzyme activity. Identification of reduced DAO activity may improve the quality of life and treatment of symptoms in fibromyalgia patients.

Abstract 5905: Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression

Abstract Background: With more than 2.3 million cases worldwide, breast cancer persists as a major health burden and warrants a greater understanding of the molecular triggers underlying this disease. Focusing on microbial dysbiosis, our lab discovered the pivotal role of the enterotoxigenic Bacteroides fragilis (ETBF) in promoting breast tumorigenesis through activation of key oncogenic pathways. A gap in knowledge exists regarding how microbes/microbial toxins modulate the oncogenic pathways in cancer cells. Polyamines are imperative for various physiological processes, including cell growth, survival, differentiation and apoptosis and are strongly associated with breast cancer. We questioned the involvement of polyamine pathway in mediating the oncogenic effects of microbial dysbiosis on breast cancer. Methods: Human Her2 expressing spontaneous breast cancer model, MMTV.f.HuHer2 mice, were infected with ETBF and tumor incidence and progression were monitored for 18 months. We used qRT-PCR, western blotting, immunocytochemistry and immunohistochemistry to examine expression of key nodes of polyamine pathway. Various growth, migration and invasion assays were performed to assess carcinogenic properties in breast cancer cells. We employed a colorimetric-based assay to analyze enzymatic activities of key polyamine metabolic members. ROS generation was examined via DCFA staining. Results: Our in vivo data showed that ETBF colonization in mice led to a considerable increase in the level of spermine oxidase (SMOX) in breast tumors, hinting at a correlation between ETBF and polyamine metabolism. Moreover, a marked rise in expression of γH2AX foci was observed in these tumors and in breast cancer cells, suggesting a critical role of ETBF in SMOX-related DNA damage. In agreement, we found SMOX upregulation in breast cancer cells. Our findings displayed a substantial increase in SMOX enzymatic activity in the presence of ETBF toxin, BFT, in breast tumor cells. BFT caused a marked rise in ROS generation of breast tumor cells, suggesting a plausible association of ETBF in SMOX-mediated oxidative stress. Interestingly, we noted a significant induction of ornithine decarboxylase (ODC) expression and activity in response to ETBF or BFT in breast cancer cells. Treatment of BFT-exposed breast cancer cells with SMOX inhibitors led to a sharp inhibition of neoplastic transformations, like cellular migration and γH2AX induction. Conclusion: Our collated evidences suggest the existence of an important cross-talk between key polyamine metabolic enzymes and ETBF that aids in exacerbating breast tumorigenesis. Of importance, therapeutic inhibition of SMOX dramatically impeded BFT-related breast tumorigenesis. Collectively, our results demonstrated the attractive potential of intervening the polyamine catabolic pathway for the management of breast cancer. Citation Format: Deeptashree Nandi, Sheetal Parida, Sowjanya Thatikonda, Jackson Foley, Tracy Stewart, Cynthia L. Sears, Robert A. Casero, Dipali Sharma. Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5905.

Structural Insights into the Substrate Range of a Bacterial Monoamine Oxidase.

Monoamine oxidases (MAOs) play a key role in the breakdown of primary and secondary amines. In eukaryotic organisms, these enzymes are vital to the regulation of monoamine neurotransmitters and the degradation of dietary monoamines. MAOs have also been identified in prokaryotic species, although their role in these organisms is not well understood. Here, we report the biophysical and structural properties of a promiscuous, bacterial MAO from Corynebacterium ammoniagenes (caMAO). caMAO catalyzes the oxidation of a number of monoamine substrates including dopamine and norepinephrine, as well as exhibiting some activity with polyamine substrates such as cadaverine. The X-ray crystal structures of Michaelis complexes with seven substrates show that conserved hydrophobic interactions and hydrogen-bonding pattern (for polar substrates) allow the broad specificity range. The structure of caMAO identifies an unusual cysteine (Cys424) residue in the so-called "aromatic cage", which flanks the flavin isoalloxazine ring in the active site. Site-directed mutagenesis, steady-state kinetics in air-saturated buffer, and UV-vis spectroscopy revealed that Cys424 plays a role in the pH dependence and modulation of electrostatics within the caMAO active site. Notably, bioinformatic analysis shows a propensity for variation at this site within the "aromatic cage" of the flavin amine oxidase (FAO) superfamily. Structural analysis also identified the conservation of a secondary substrate inhibition site, present in a homologous member of the superfamily. Finally, genome neighborhood diagram analysis of caMAO in the context of the FAO superfamily allows us to propose potential roles for these bacterial MAOs in monoamine and polyamine degradation and catabolic pathways related to scavenging of nitrogen.

The Pivotal Role of PAO in CDK Inhibitors (Roscovitine and Purvalanol)- Triggered Apoptosis in PUMA Null HCT116 Colon Cancer Cells

Background: Cycline-dependent kinase inhibitors (CDKi); roscovitine and purvalanol, are promising anti-cancer drugs due to their strong anti-proliferative effectiveness due to activation of PA catabolism. Besides transforming acetylated spermine and spermidine into spermidine and putrescine, respectively, polyamine oxidase (PAO) also generates hydrogen peroxide in high concentrations as a by-product. PAO was assumed as a pivotal key molecule during drug-induced apoptosis in cancer cells. Our aim is to reveal the role of PAO action in CDKi-triggered apoptosis in Puma knock-out HCT116 colon cancer cells. Methods: HCT116 wt and HCT116 Puma-/- cells were treated with Roscovitine and Purvalanol and cell viability and apoptosis were determined. Protein was isolated from treated and untreated cells and key molecules of cell cycle control and polyamine pathways were investigated at translational level. Polyamine content was determined by HPLC for all conditions. MDL-72527 was used as a PAO inhibitor and apoptotic cell death was analysed. Results: Roscovitine and purvalanol induced apoptosis and increased the cytotoxic responses in HCT116 wt and HCT116 Puma-/- colon carcinoma cell lines by modulating CDK1, 4, cyclin-B1, D3. Both, CDKi altered intrinsic apoptotic pathways in HCT116 wt. Whereas, drug-induced apoptosis occurred caspase-independent in Puma-/- colon cancer cells. Roscovitine and purvalanol up-regulated polyamine catabolic enzymes, whereas CDK inhibitors decreased the polyamine levels in HCT116 wt and HCT116 Puma-/- colon cancer cells. In addition, PAO inhibitor MDL72527 prevented drug-induced apoptosis. Conclusion: PAO expression profile might be a critical target in CDK inhibitors-triggered apoptosis in HCT116 colorectal cancer cells. Thus, MAPK signaling pathway relations with cell cycle and polyamine catabolic pathway investigations are in progress.

Polyamine Analogues of Propanediamine Series Inhibit Prostate Tumor Cell Growth and Activate the Polyamine Catabolic Pathway.

Polyamines are important for the growth of eukaryotic cells. At high levels, they promote proliferation, invasion and migration of tumour cells. Polyamine metabolism is an important new target for anticancer therapy. Some polyamine analogues can have an inhibitory effect on tumour cells. The aim of this study was to explore the potential of certain butylated derivatives of propanediamine for prostate cancer chemotherapy. Human prostate cancer cells, LNCaP, were used for the evaluation of the antiproliferative activity of polyamine analogs and their influence on spermine oxidase. Tetrabutyl propanediamine and two new polyamine analogues inhibited the growth of LNCaP cells. At the same time, a strong activation of spermine oxidase was observed. The investigated compounds demonstrated their potential value in the therapy of human prostate cancer. Their effect might be attributed to the activation of the polyamine catabolic pathway.

Bioprospecting Reveals Class III ω-Transaminases Converting Bulky Ketones and Environmentally Relevant Polyamines.

Amination of bulky ketones, particularly in (R) configuration, is an attractive chemical conversion; however, known ω-transaminases (ω-TAs) show insufficient levels of performance. By applying two screening methods, we discovered 10 amine transaminases from the class III ω-TA family that were 38% to 76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (S) selectivity. We also report representatives from the class III ω-TAs capable of converting (R) and (S) amines and bulky ketones and one that can convert amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (R) amines. This configuration was also found to favor the utilization of putrescine as an amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 also contributes to the preference for amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained ≥50% of their activity in water-soluble solvents (up to 50% [vol/vol]). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (R) amines for the production of high-value products and to screen for those converting putrescine.IMPORTANCE Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

Exploring the activity of polyamine analogues on polyamine and spermine oxidase: methoctramine, a potent and selective inhibitor of polyamine oxidase

Fourteen polyamine analogues, asymmetric or symmetric substituted spermine (1–9) or methoctramine (10–14) analogues, were evaluated as potential inhibitors or substrates of two enzymes of the polyamine catabolic pathway, spermine oxidase (SMOX) and acetylpolyamine oxidase (PAOX). Compound 2 turned out to be the best substrate for PAOX, having the highest affinity and catalytic efficiency with respect to its physiological substrates. Methoctramine (10), a well-known muscarinic M2 receptor antagonist, emerged as the most potent competitive PAOX inhibitor known so far (Ki = 10 nM), endowed with very good selectivity compared with SMOX (Ki=1.2 μM vs SMOX). The efficacy of methoctramine in inhibiting PAOX activity was confirmed in the HT22 cell line. Methoctramine is a very promising tool in the design of drugs targeting the polyamine catabolism pathway, both to understand the physio-pathological role of PAOX vs SMOX and for pharmacological applications, being the polyamine pathway involved in various pathologies.

Curcumin inhibits autocrine growth hormone-mediated invasion and metastasis by targeting NF-κB signaling and polyamine metabolism in breast cancer cells.

Curcumin is assumed to be a plant-derived therapeutic drug that triggers apoptotic cell death in vitro and in vivo by affecting different molecular targets such as NF-κB. Phase I/II trial of curcumin alone or with chemotherapeutic drugs has been accomplished in pancreatic, colon, prostate and breast cancer cases. Recently, autocrine growth hormone (GH) signaling-induced cell growth, metastasis and drug resistance have been demonstrated in breast cancer. In this study, our aim was to investigate the potential therapeutic effect of curcumin by evaluating the molecular machinery of curcumin-triggered apoptotic cell death via focusing on NF-κB signaling and polyamine (PA) metabolism in autocrine GH-expressing MCF-7, MDA-MB-453 and MDA-MB-231 breast cancer cells. For this purpose, a pcDNA3.1 (+) vector with a GH gene insert was transfected by a liposomal agent in all breast cancer cells and then selection was conducted in neomycin (G418) included media. Autocrine GH-induced curcumin resistance was overcome in a dose-dependent manner and curcumin inhibited cell proliferation, invasion-metastasis and phosphorylation of p65 (Ser536), and thereby partly prevented its DNA binding activity in breast cancer cells. Moreover, curcumin induced caspase-mediated apoptotic cell death by activating the PA catabolic enzyme expressions, which led to generation of toxic by-products such as H2O2 in MCF-7, MDA-MB-453 and MDA-MB-231 GH+ breast cancer cells. In addition, transient silencing of SSAT prevented curcumin-induced cell viability loss and apoptotic cell death in each breast cancer cells. In conclusion, curcumin could overcome the GH-mediated resistant phenotype via modulating cell survival, death-related signaling routes and activating PA catabolic pathway.

DENSpm overcame Bcl-2 mediated resistance against Paclitaxel treatment in MCF-7 breast cancer cells via activating polyamine catabolic machinery.

The Bcl-2 mediated resistance is one of the most critical obstacle in cancer therapy. Conventional chemotherapeutics such as Paclitaxel, a commonly used in the treatment of metastatic breast cancer, is not sufficient to overcome Bcl-2 mediated drug resistance mechanism. Thus, combinational drug regimes are favored by researchers to overcome resistance phenotype against drugs. N1,N11-diethylnorspermine (DENSpm), a polyamine analogue, which is a promising drug candidate induced-cell cycle arrest and apoptosis in various cancer cells such as prostate, melanoma, colon and breast cancer cells via activated polyamine catabolism and reactive oxygen generation. Recent studies indicated the potential therapeutic role of DENSpm in phase I and II trials in breast cancer cases. Although the molecular targets of Paclitaxel in apoptotic cell death mechanism is well documented, the therapeutic effect of DENSpm and Paclitaxel in breast cancer cells has not been investigated yet. In this study, our aim was to determine the time dependent effect of DENSpm and Paclitaxel on apoptotic cell death via determination of polyamine metabolism related targets in wt and Bcl-2 overexpressing MCF-7 breast cancer cells. In our experimental study, Paclitaxel decreased cell viability in dose-dependent manner within 24h. Co-treatment of Paclitaxel (30nM) with DENSpm (20μM) further increased the cytoxicity of Paclitaxel (30nM) compared to alone Paclitaxel (30nM) treatment in MCF-7 Bcl-2+ breast cancer cells. In addition, we determined that resistance against Paclitaxel-induced apoptotic cell death in Bcl-2 overexpressed MCF-7 cells was overcome due to activation of polyamine catabolic pathway, which caused depletion of polyamines. DENSpm combinational treatment might increase the effect of low cytotoxic paclitaxel in drug-resistant breast cancer cases.

Polyamine analog TBP inhibits proliferation of human K562 chronic myelogenous leukemia cells by induced apoptosis.

The aim of the present study was to investigate the effects of the novel polyamine analog tetrabutyl propanediamine (TBP) on the growth of K562 chronic myelogenous leukemia (CML) cells and the underlying mechanism of these effects. MTT was used for the analysis of cell proliferation and flow cytometry was performed to analyze cell cycle distribution. DNA fragmentation analysis and Annexin V/propidium iodide double staining were used to identify apoptotic cells. The activity of the key enzymes in polyamine catabolism was detected using chemiluminescence. TBP can induce apoptosis and significantly inhibit K562 cell proliferation in a time- and dose-dependent manner. TBP treatment significantly induced the enzyme activity of spermine oxidase and acetylpolyamine oxidase in K562 cells, and also enhanced the inhibitory effect of the antitumor drug doxorubicin on K562 cell proliferation. As a novel polyamine analog, TBP significantly inhibited proliferation and induced apoptosis in K562 cells by upregulating the activity of the key enzymes in the polyamine catabolic pathways. TBP also increased the sensitivity of the K562 cells to the antitumor drug doxorubicin. These data indicate an important potential value of TBP for clinical therapy of human CML.

Abstract 1326: Regulation of AKT phosphorylation by a CDK inhibitor purvalanol A in breast cancer cells

Abstract Akt is a serine/threonine kinase and an essential component in the PI3K-Akt-mTOR axis. Multiple cell signals stimulate the activation of Akt through Akt activating kinases. The Akt is activated through the phosphorylation in serine and threonine residues. The activated Akt increases in cancer cells and plays promoting functions in proliferation, survival, invasion, and drug/radiation resistance. Several Akt inhibitors have been tested for cancer treatment. Purvalanol A is such a CDK inhibitor. In synchronized cells, purvalanol A induced a reversible cell cycle arrest in the G1 and G2 phases. Purvalanol A treated MCF7 cells induced apoptosis via activating the polyamine catabolic pathway. In the study of the cell cycle and radiation resistance of breast cancer cells, purvalanol A was found to inhibit phosphorylated Akt. MCF7 cells were treated with 20µM of purvalanol A one hour before the radiation. The protein was extracted at 15, 60, and 300 minutes after the radiation at 6Gy. Western analyses were performed with antibody against phosph-Akt(Ser473). Compared to control cells, purvalanol A treated cells showed reduced phosph-Akt(Ser473) by 59 to 88% depending on the specific time point. To clarify if the reduction of phosphorylated Akt in purvalanol A treated cells is a result of radiation, the effect of purvalanol A on the expression of phosphorylated Akt was examined in cells without radiation. Two breast cancer cell lines, MCF7 and MDA-MB231 cells, and one prostate cancer cell line, PC3 cells were treated with purvalanol A for 2 hours at doses of 0.5,1,5,10, and 20µM. Expression of phosph-Akt(Ser473) and phosph-Akt(Thr308) was investigated by Western blot analyses. The result from three cell lines showed that purvalanol A regulated the expression of phosph-Akt(Ser473) and phosph-Akt(Thr308) in a dose-dependent way. At a low dose of 1µM, purvalanol A increased the phosph-Akt(Ser473) in MCF7 cells by 34% and phosph-Akt(Thr308) by 50%, respectively. In MDA-MB231 cells treated with 1µM of purvalanol A, phosph-Akt(Ser473) increased by 35% and phosph-Akt(Thr308) increased by 1.6 fold. In PC3 cells, treatment with 1µM of purvalanol A led to an 87% increase in phosph-Akt(Ser473). However, the same treatment also reduced phosph-Akt(Thr308) by 13%. At a high dose of 20µM, purvalanol A treatment significantly reduced phosphorylated Akt. The phosph-Akt(Ser473) was reduced by 80% in MDA-MB231 and by 84% in MCF7 cells, respectively. The phosph-Akt(Thr308) was reduced by 91% in MDA-MB231 and by 97% in MCF7 cells, respectively. In PC3 cells treatment with 20µM of purvalanol A reduced phosph-Akt(Ser473) by 50% and phosph-Akt(Thr308) by 90%. The results suggest that purvalanol A could be a regulator of Akt activation and a multiple targeting effector in cancer treatment. Citation Format: Hong Yin. Regulation of AKT phosphorylation by a CDK inhibitor purvalanol A in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1326. doi:10.1158/1538-7445.AM2014-1326

Epibrassinolide-induced apoptosis regardless of p53 expression via activating polyamine catabolic machinery, a common target for androgen sensitive and insensitive prostate cancer cells.

Epibrassinolide (EBR), is a member of the brassinosteroids (BR), has been shown as an apoptotic inducer in different cancer cell lines. We previously showed that EBR induced apoptosis by activating polyamine catabolic pathway, which lead to the accumulation of cytotoxic compounds such as hydrogen peroxide and aldehydes in LNCaP and DU 145 prostate cancer cells. However, we found that LNCaP prostate cancer cells expressing functional androgen receptor (AR) was found more sensitive to EBR than those with non-functional AR (DU 145 cells). To better understand the apoptotic effect of EBR, we aimed to investigate the cellular responses in p53 null, PC3 prostate cancer cells. We showed that EBR induced mitochondria-mediated and caspase-dependent apoptosis in wt and p53 stable transfected PC3 cells, which suggesting that EBR-induced apoptosis regardless of p53 expression. In addition, inhibition of p53 by pifithrin-α orthe activation of Mdm2 by Nutlin-3 co-treatment did not alter EBR induced PARP cleavage. Furthermore, EBR treatment was also induced apoptosis in both LNCaP(wt p53) and DU 145 (mt p53)cells, respectively. These all findings verified that EBR-induced apoptosis regardless of p53 expression. The PA catabolic pathway was also altered in PC3 cells causing the generation of reactive oxygen species (ROS) and intracellular PA pool decrease. However, the silencing of spermidine-spermineacetyltransferase (SSAT), a key enzyme at polyamine catabolic machinery prevented the EBR-induced apoptosis. Therefore, we concluded that EBR-induced apoptosis was mainly related with PA catabolic pathway and independent from p53 expression.

CDK Inhibitors Induce Mitochondria-mediated Apoptosis Through the Activation of Polyamine Catabolic Pathway in LNCaP, DU145 and PC3 Prostate Cancer Cells

Androgen signaling is critical in prostate cancer development and progression. The co-existence of hormone responsive and irresponsive cells due to functional androgen receptor (AR) in prostate gland is the major obstacle in prostate cancer therapy models. Targeting aberrant cell cycle by novel cell cycle blocking agents is a promising strategy to treat various types of malignancies. Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors able to activate apoptotic cell death by inducing cell cycle arrest at G1/S and G2/M phases in cancer cells. Polyamines are unique cationic amine derivatives involved in the regulation of cell proliferation. Although the elevated intracellular level of polyamines (putrescine, spermidine and spermine) is typical for prostate gland, abnormal regulation of polyamine metabolism might result in rapid cell proliferation and, thus in prostate cancer progression. Therefore, treatment with drug-induced depletion of intracellular polyamine levels through the activated polyamine catabolism is critical to achieve successful strategies for prostate cancer. In this study we aimed to investigate the apoptotic efficiency of CDK inhibitors in three prostate cancer cell lines (LNCaP, DU145 and PC3), showing different AR expression profile. We found that both purvalanol and roscovitine were able to induce apoptosis at moderate cytotoxic concentrations by decreasing mitochondria membrane potential. The apoptotic effect of both CDK inhibitors was due to activation of caspases by modulating Bcl-2 family members. The efficiency of drugs was quite similar on the three prostate cell lines used in this study. However, DU145 cells were found the least sensitive against CDK inhibitors while purvalanol was more potent than roscovitine. Similarly to classical chemotherapeutic agents, both drugs could up-regulate polyamine catabolic enzymes (SSAT, SMO and PAO) in cell type dependent manner. Transient silencing of SSAT and/or inhibition of PAO/ SMO with MDL72527 prevented CDK inhibitors- induced apoptotic cell death in DU145 and PC3 cells. Although roscovitine was less effective in DU145 cells, pre-treatment with α-difluoromethylornithine (DFMO), an inhibitor of ODC, enhanced the roscovitine-induced apoptotic cell death through the cleavage of caspase-9 and caspase-3. Therefore, we conclude that polyamine catabolism might have essential role in the cellular responses against CDK inhibitors in different androgen-responsive or irresponsive prostate cancer cells.

UORF, a regulatory mechanism of the Arabidopsis polyamine oxidase 2

The translational efficiency of an mRNA can be modulated by elements located in the 5'-untranslated region. The flavin-containing polyamine oxidases catabolize oxidative deamination of spermidine and spermine, thus contributing to polyamine homeostasis as well as diverse biological processes through their reaction products. In this study, we characterized the uORF of AtPAO2 gene using the GUS reporter gene. Transgenic lines harboring the native AtPAO2 promoter or the constitutive CaMV 35S promoter show that the uORF negatively affects GUS expression. Exogenous applications of PAs positively modulate GUS expression, thus alleviating the negative effect of AtPAO2 uORF, while treatments with MGBG inhibitor show an opposite effect. Our data suggest that AtPAO2 uORF regulatory mechanism is modulated by polyamines. In addition, we present a comparative in silico study of the uORFs identified in several plant transcripts encoding polyamine oxidases in both mono- and dicotyledonous plants as well as in the Bryophyte Physcomitrella patens. The polyamine oxidase uORF-encoded peptides are conserved among families and share conserved features such as their position, length, and amino acid sequence. Our findings provide new insights into the regulatory mechanism of polyamine oxidase genes and encourage further exploration to assess the biological significance of uORFs in the polyamine catabolic pathway.

Purvalanol A is a strong apoptotic inducer via activating polyamine catabolic pathway in MCF-7 estrogen receptor positive breast cancer cells

Purvalanol A is a specific CDK inhibitor which triggers apoptosis by causing cell cycle arrest in cancer cells. Although it has strong apoptotic potential, the mechanistic action of Purvalanol A on significant cell signaling targets has not been clarified yet. Polyamines are crucial metabolic regulators affected by CDK inhibition because of their role in cell cycle progress as well. In addition, malignant cells possess impaired polyamine homeostasis with high level of intracellular polyamines. Especially induction of polyamine catabolic enzymes spermidine/spermine N1-acetyltransferase (SSAT), polyamine oxidase (PAO) and spermine oxidase (SMO) induced toxic by-products in correlation with the induction of apoptosis in cancer cells. In this study, we showed that Purvalanol A induced apoptosis in caspase- dependent manner in MCF-7 ER(+) cells, while MDA-MB-231 (ER-) cells were less sensitive against drug. In addition Bcl-2 is a critical target for Purvalanol A, since Bcl-2 overexpressed cells are more resistant to Purvalanol A-mediated apoptosis. Furthermore, exposure of MCF-7 cells to Purvalanol A triggered SSAT and PAO upregulation and the presence of PAO/SMO inhibitor, MDL 72,527 prevented Purvalanol A-induced apoptosis.

Histone Deacetylase Inhibition Overcomes Drug Resistance through a miRNA-Dependent Mechanism

The treatment of specific tumor cell lines with poly- and oligoamine analogs results in a superinduction of polyamine catabolism that is associated with cytotoxicity; however, other tumor cells show resistance to analog treatment. Recent data indicate that some of these analogs also have direct epigenetic effects. We, therefore, sought to determine the effects of combining specific analogs with an epigenetic targeting agent in phenotypically resistant human lung cancer cell lines. We show that the histone deacetylase inhibitor MS-275, when combined with (N(1), N(11))-bisethylnorspermine (BENSpm) or (N(1), N(12))-bis(ethyl)-cis-6,7-dehydrospermine tetrahydrochloride (PG-11047), synergistically induces the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT), a major determinant of sensitivity to the antitumor analogs. Evidence indicates that the mechanism of this synergy includes reactivation of miR-200a, which targets and destabilizes kelch-like ECH-associated protein 1 (KEAP1) mRNA, resulting in the translocation and binding of nuclear factor (erythroid-derived 2)-like 2 (NRF2) to the polyamine-responsive element of the SSAT promoter. This transcriptional stimulation, combined with positive regulation of SSAT mRNA and protein by the analogs, results in decreased intracellular concentrations of natural polyamines and growth inhibition. The finding that an epigenetic targeting agent is capable of inducing a rate-limiting step in polyamine catabolism to overcome resistance to the antitumor analogs represents a completely novel chemotherapeutic approach. In addition, this is the first demonstration of miRNA-mediated regulation of the polyamine catabolic pathway. Furthermore, the individual agents used in this study have been investigated clinically; therefore, translation of these combinations into the clinical setting holds promise.

Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines

Epibrassinolide (EBR) is a biologically active compound of the brassinosteroids, steroid-derived plant growth regulator family. Generally, brassinosteroids are known for their cell expansion and cell division-promoting roles. Recently, EBR was shown as a potential apoptotic inducer in various cancer cells without affecting the non-tumor cell growth. Androgen signaling controls cell proliferation through the interaction with the androgen receptor (AR) in the prostate gland. Initially, the development of prostate cancer is driven by androgens. However, in later stages, a progress to the androgen-independent stage is observed, resulting in metastatic prostate cancer. The androgen-responsive or -irresponsive cells are responsible for tumor heterogeneity, which is an obstacle to effective anti-cancer therapy. Polyamines are amine-derived organic compounds, known for their role in abnormal cell proliferation as well as during malignant transformation. Polyamine catabolism-targeting agents are being investigated against human cancers. Many chemotherapeutic agents including polyamine analogs have been demonstrated to induce polyamine catabolism that depletes polyamine levels and causes apoptosis in tumor models. In our study, we aimed to investigate the mechanism of apoptotic cell death induced by EBR, related with polyamine biosynthetic and catabolic pathways in LNCaP (AR+), DU145 (AR-) prostate cancer cell lines and PNT1a normal prostate epithelial cell line. Induction of apoptotic cell death was observed in prostate cancer cell lines after EBR treatment. In addition, EBR induced the decrease of intracellular polyamine levels, accompanied by a significant ornithine decarboxylase (ODC) down-regulation in each prostate cancer cell and also modulated ODC antizyme and antizyme inhibitor expression levels only in LNCaP cells. Catabolic enzymes SSAT and PAO expression levels were up-regulated in both cell lines; however, the specific SSAT and PAO siRNA treatments prevented the EBR-induced apoptosis only in LNCaP (AR+) cells. In a similar way, MDL 72,527, the specific PAO and SMO inhibitor, co-treatment with EBR during 24 h, reduced the formation of cleaved fragments of PARP in LNCaP (AR+) cells.

Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention.

Polyamines, including spermine, spermidine, and the precursor diamine, putrescine, are naturally occurring polycationic alkylamines that are required for eukaryotic cell growth, differentiation, and survival. This absolute requirement for polyamines and the need to maintain intracellular levels within specific ranges require a highly regulated metabolic pathway primed for rapid changes in response to cellular growth signals, environmental changes, and stress. Although the polyamine metabolic pathway is strictly regulated in normal cells, dysregulation of polyamine metabolism is a frequent event in cancer. Recent studies suggest that the polyamine catabolic pathway may be involved in the etiology of some epithelial cancers. The catabolism of spermine to spermidine utilizes either the one-step enzymatic reaction of spermine oxidase (SMO) or the two-step process of spermidine/spermine N (1)-acetyltransferase (SSAT) coupled with the peroxisomal enzyme N (1)-acetylpolyamine oxidase. Both catabolic pathways produce hydrogen peroxide and a reactive aldehyde that are capable of damaging DNA and other critical cellular components. The catabolic pathway also depletes the intracellular concentrations of spermidine and spermine, which are free radical scavengers. Consequently, the polyamine catabolic pathway in general and specifically SMO and SSAT provide exciting new targets for chemoprevention and/or chemotherapy.

Putrescine catabolism is a metabolic response to several stresses in Escherichia coli

Genes whose products degrade arginine and ornithine, precursors of putrescine synthesis, are activated by either regulators of the nitrogen-regulated (Ntr) response or σ(S) -RNA polymerase. To determine if dual control regulates a complete putrescine catabolic pathway, we examined expression of patA and patD, which specify the first two enzymes of one putrescine catabolic pathway. Assays of PatA (putrescine transaminase) activity and β-galactosidase from cells with patA-lacZ transcriptional and translational fusions indicate dual control of patA transcription and putrescine-stimulated patA translation. Similar assays for PatD indicate that patD transcription required σ(S) -RNA polymerase, and Nac, an Ntr regulator, enhanced the σ(S) -dependent transcription. Since Nac activation via σ(S) -RNA polymerase is without precedent, transcription with purified components was examined and the results confirmed this conclusion. This result indicates that the Ntr regulon can intrude into the σ(S) regulon. Strains lacking both polyamine catabolic pathways have defective responses to oxidative stress, high temperature and a sublethal concentration of an antibiotic. These defects and the σ(S) -dependent expression indicate that polyamine catabolism is a core metabolic response to stress.

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N1-acetylspermidine (N1AcSpd), N8-acetylspermidine (N8AcSpd), N1-acetylspermine, N1,N8-diacetylspermidine, and N1,N12-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N1AcSpd and N8AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with 13C2-N1AcSpd and 13C2-N8AcSpd which have the 13C2-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N1-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N1-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-15N3]-N1-acetylspermine and [1,4,8-15N3]spermidine (15N3-Spd), respectively; for SMO, [1,4,8,12-15N4]spermine and 15N3-Spd, respectively; and for SSAT, 15N3-Spd and [1,4,8-15N3]-N1-acetylspermidine, respectively.