Thresia Thomas

Polyamine metabolism and cancer

Polyamines are aliphatic cations present in all cells. In normal cells, polyamine levels are intricately controlled by biosynthetic and catabolic enzymes. The biosynthetic enzymes are ornithine decarboxylase, S‐adenosylmethionine decarboxylase, spermidine synthase, and spermine synthase. The catabolic enzymes include spermidine/spermine acetyltransferase, flavin containing polyamine oxidase, copper containing diamine oxidase, and possibly other amine oxidases. Multiple abnormalities in the control of polyamine metabolism and uptake might be responsible for increased levels of polyamines in cancer cells as compared to that of normal cells. This review is designed to look at the current research in polyamine biosynthesis, catabolism, and transport pathways, enumerate the functions of polyamines, and assess the potential for using polyamine metabolism or function as targets for cancer therapy.

Regulation of Estrogenic Effects by Beclin 1 in Breast Cancer Cells

Beclin 1 is an essential mediator of autophagy and a regulator of cell growth and cell death. We examined the effect of Beclin 1 overexpression on the action of estradiol (E(2)) and two antiestrogens, raloxifene and 4-hydroxytamoxifen, in estrogen receptor alpha (ERalpha)-positive MCF-7 breast cancer cells. [(3)H]-thymidine incorporation studies showed that Beclin 1-overexpressing cells (MCF-7 x beclin) had a lower proliferative response to E(2) compared with cells transfected with vector control (MCF-7 x control). There was only a 35% increase in [(3)H]-thymidine incorporation, after 24 hours of E(2) treatment of MCF-7 x beclin cells compared with untreated cells, whereas this increase was 2-fold for MCF-7 x control cells. E(2)-induced changes in the expression of early-response genes were examined by real-time quantitiative PCR. There were significant differences in the pattern of expression of E(2)-induced genes c-myc, c-fos, Erg-1, and Nur77 between MCF-7 x beclin and MCF-7 x control cells two hours after treatment. Although E(2)-induced growth of MCF-7 x control cells was completely inhibited by 500 nmol/L raloxifene or 500 nmol/L 4-hydroxytamoxifen, these concentrations of antiestrogens had no significant effect on the growth of MCF-7 x beclin cells. Confocal microscopic and coimmunoprecipitation studies showed evidence for colocalization and association of Beclin 1 and ERalpha. In addition, E(2) caused a decrease in Akt phosphorylation in MCF-7 x beclin cells, compared with a 3-fold increase in MCF-7 cells, five minutes after treatment. These results indicate that Beclin 1 can down-regulate estrogenic signaling and growth response, and contribute to the development of antiestrogen resistance. This observation might be useful to define and overcome antiestrogen resistance of breast cancer.

Activation of cyclin D1 by estradiol and spermine in MCF-7 breast cancer cells: a mechanism involving the p38 MAP kinase and phosphorylation of ATF-2.

Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.

Regulation of estrogenic and nuclear factor κB functions by polyamines and their role in polyamine analog-induced apoptosis of breast cancer cells

The natural polyamines -putrescine, spermidine, and spermine- are essential for cell growth and differentiation. Polyamines are involved in several gene regulatory functions, although their mechanism(s) of action has not been elucidated. We investigated the role of polyamines in the function of NF-κB and estrogen receptor-α (ERα), two transcription factors implicated in breast cancer cell proliferation and cell survival, using MCF-7 breast cancer cells. We found that spermine facilitated the binding of ERα and NF-κB to estrogen response element (ERE)- and NF-κB response element (NRE), respectively, and enhanced ERα-mediated transcriptional activation in transient transfection experiments. We also found that the association of the co-regulatory protein CBP/p300 with ERα and NF-κB was increased by spermine treatment of MCF-7 cells. Spermine also increased the nuclear translocation of NF-κB compared to the control. In contrast, treatment of MCF-7 cells with polyamine analogs, BE-3-4-3 and BE-3-3-3, resulted in transcriptional inhibition of both ERE- and NRE-driven reporter plasmids. In addition, polyamine analogs inhibited the association of ERα and NF-κB with CBP/p300 and were unable to facilitate nuclear translocation of NF-κB. APO-BRDU assay demonstrated that polyamine analogs induced apoptosis, with a loss of the anti-apoptotic protein Bcl-2. These data show a gene regulatory function of polyamines involving transcriptional activation of ERα and NF-κB, potentially leading to the up-regulation of genes involved in breast cancer cell proliferation. Our results with BE-3-4-3 and BE-3-3-3 suggest that down-regulation of ERα- and NF-κB-regulated genes is a possible mechanism for the action of polyamine analogs in inducing apoptosis of breast cancer cells.

Estradiol control of ornithine decarboxylase mRNA, enzyme activity, and polyamine levels in MCF-7 breast cancer cells: therapeutic implications

Previous studies have shown that natural polyamines - putrescine, spermidine, and spermine - play a key role in the mechanism of action of estrogens in breast cancer. Ornithine decarboxylase (ODC) is the first enzyme of the polyamine biosynthetic pathway. To examine estrogenic regulation of polyamine biosynthesis in breast cancer, we measured ODC mRNA, ODC activity, and polyamine levels in G1 synchronized MCF-7 cells. ODC mRNA and activity increased four-fold over that of cells in G1 phase between 8 to 16 h after the addition of estradiol. Polyamine levels showed a sharp increase by 8 h after the addition of estradiol and decreased by 12 h. We further examined whether synthetic homologs of putrescine or spermidine could replace natural polyamines in supporting MCF-7 cell growth. Treatment of MCF-7 cells with 1 mM difluoromethylornithine (DFMO), an inhibitor of ODC, suppressed putrescine, spermidine, and spermine levels by 74, 78, and 10%, respectively, within 48 h. Cells treated with DFMO for 48 h were supplemented with either putrescine or its homologs or spermidine or its homologs. Diaminopropane, diaminobutane (putrescine), and diaminopentane were capable of fully or partially reversing the growth inhibitory effects of DFMO, whereas diaminoethane had no significant effect. Among a series of triamines, H2N(CH2)nNH(CH2)3NH2 (where n = 2 to 8; abbreviated as APn n = 4 for spermidine, or AP4), spermidine was most effective in reversing the effects of DFMO, whereas compounds with shorter or longer methylene bridging regions were less effective. AP8 was ineffective in reversing the growth inhibitory effects of DFMO. At 10 µM concentration, AP8 also inhibited DNA synthesis by 66%, as measured by [3H]-thymidine incorporation. These data show that MCF-7 cells have a strong requirement for polyamines for their growth and that estradiol stimulates the polyamine cascade by inducing the ODC mRNA level. Our results also suggest that polyamine homologs such as AP8 might be potentially useful in breast cancer therapy.

Estrogen Receptors as Targets for Drug Development for Breast Cancer, Osteoporosis and Cardiovascular Diseases

Estrogen receptors (ERs) are proteins that mediate the action of estradiol and a series of natural and synthetic chemicals that mimic the estradiol structure. Estrogenic action was initially attributed to a single type of ER, now known as ERalpha, but ERbeta was discovered in 1995. Tissue specific distribution and the intensity of expression of these proteins determine the first response of tissues to estrogenic compounds. Estrogens and ERs play a major role in the origin and progression of breast cancer, and antiestrogens that block ER function are useful for breast cancer prevention and treatment. Estrogen mimetics, however, do not fall into distinct categories of agonists and antagonists, since their action is regulated by tissue-specific expression of a number of auxiliary proteins called coactivators or corepressors. In addition, small molecules such as polyamines, fattyacids, and thioredoxin may modulate ER function. Estrogenic functions encompass multiple organ systems, including the reproductive, skeletal, cardiovascular, and nervous system. Estrogens are critical for bone remodeling and mineralization so that estrogen replacement therapy is proven to strengthen bone health in post-menopausal women. Ideally, selective blockade of ER function in breast epithelial cells should be accompanied by growth support on bone and cardiovascular systems. The details of estrogenic function in different organs are to be fully realized, in order to better utilize selective estrogen receptor modulators (SERMs) to fight not only breast cancer but also osteoporosis and cardiovascular diseases. Current research on SERMs points toward accomplishing this goal by exploiting ER as a versatile target against multiple diseases.

Polyamine-mediated conformational perturbations in DNA alter the binding of estrogen receptor to poly(dG-m5dC).poly(dG-m5dC) and a plasmid containing the estrogen response element

The binding estrogen receptor (ER) to the upstream regions of estrogen-responsive genes, the estrogen response elements (ERE), is of fundamental importance in the regulation of gene expression by estradiol. Multiple cell-specific factors affect ER-ERE binding and modulate the responses of estradiol. We studied the role of polyamines in the recognition of ER, a ligand-activated transcription factor, with a left-handed Z-DNA forming polynucleotide as well as with a plasmid containing ERE. Polyamines are cellular organic cations with multiple functions in cell growth and differentiation. Polyamines induce Z-DNA conformation in alternating purine-pyrimidine sequences. To understand the role of polyamine-induced DNA conformational transition in ER-DNA interaction, we studied the binding of partially purified rabbit uterine ER to poly(dG-m5dC).poly(dG-m5dC). The induction of Z-DNA in the polynucleotide was monitored by circular dichroism and ultraviolet spectroscopic measurements. Binding of ER to poly(dG-m5dC).poly(dG-m5dC) increased from 15% to approx. 50-60% in the presence of 7.5 mM putrescine, 0.5 mM spermidine or 0.25 mM spermine. Maximal binding of ER to the polynucleotide was observed near the midpoint of the B-DNA to Z-DNA transition of the polynucleotide. N1-acetyl spermidine and N1-acetyl spermine facilitated the B-DNA to Z-DNA transition and the binding of ER although they were less effective than the unacetylated analog. Co(NH3)6(3+), a trivalent inorganic cation, also provoked the B-DNA to Z-DNA transition of the polynucleotide and increased its binding to ER. At higher polyamine concentrations, there was an inhibition of ER binding to the polynucleotide. In the presence of polyamines, the binding of ER to a plasmid containing ERE was 2-3-fold higher than that to a control plasmid devoid of ERE. Polyamine-induced facilitation of ER-ERE binding was also confirmed by gel mobility shift assay. Our data indicate that conformational perturbations, similar to that of the early stages of B-DNA to Z-DNA transition, are important in the recognition of ER and ERE.

Direct Measurement of the Association Constant of HER2/ neu Antisense Oligonucleotide to Its Target RNA Sequence Using a Molecular Beacon

A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4 degrees C +/- 1 degree C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka, deltaG, and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.

Structure-activity relations of S-adenosylmethionine decarboxylase inhibitors on the growth of MCF-7 breast cancer cells.

SummarySAMDC is a key enzyme in the biosynthesis of spermidine and spermine, 2 polyamines that are essential for cell proliferation. Inhibition of polyamine biosynthesis is often targeted as a therapeutic strategy to suppress cancer cell growth as these cells contain elevated levels of polyamines. We examined the effect of a new group of SAMDC inhibitors, CGP33829, CGP35753, CGP36958, CGP39937, and CGP48664, (obtained from CibaGeigy, Basel, Switzerland), and their parent compound, MGBG, on the proliferation of MCF-7 breast cancer cells. MGBG had minimal effects on the proliferation of MCF-7 cells up to 6 µM concentration. In contrast, CGP48664 and CGP39937, containing 2 aromatic rings that delocalize the π electron system of the backbone of MGBG, were potent inhibitors with 50% growth inhibition at 0.5 µM concentration. Other CGP compounds were less effective in inhibiting cell growth. The ability of CGP48664 to inhibit MCF-7 cell proliferation was related to its ability to inhibit SAMDC and to consequently deplete spermidine and spermine levels in the cell. Exogenous spermidine and spermine could reverse the growth inhibitory effects of this compound. CGP compounds also increased the activity of ODC, another enzyme involved in polyamine biosynthesis. Northern blot analysis of mRNA from MCF-7 cells progressing in cell cycle after G1 synchronization did not show an increase in ODC mRNA level by CGP48664. These data demonstrate structure-activity relationships of a series of MGBG derivatives on cell growth, enzyme activities, and polyamine biosynthesis in a hormoneresponsive breast cancer cell line and suggest potential application of SAMDC inhibitors as therapeutic agents.

Liquid Crystalline Phase Behavior of High Molecular Weight DNA: A Comparative Study of the Influence of Metal Ions of Different Size, Charge and Binding Mode

The ability of Li(+), Na(+), K(+), Rb(+), Cs(+), Mg(2+), Ca(2+), Sr(2+), Ba(2+), Cu(2+), Cd(2+), Al(3+), V(4+), Hg(2+), Pd(2+), Au(3+), and Pt(4+) to provoke liquid crystalline (LC) phases in high molecular weight DNA was investigated. The alkali and alkaline earth metal ions provoked typical cholesteric/columnar structures, whereas transition metal ions precipitated DNA into solid/translucent gel-like aggregates. Heavy metal ions reduced viscosity of DNA solution, disrupting rigid, rod-like DNA structure necessary for LC textures. Three-layer quantum mechanical-molecular mechanical (QM/MM) studies of Li(+), Na(+), K(+), Mg(2+), and Ca(2+) binding DNA fragment suggested several possible binding modes of these ions to the phosphate groups. The dianion mode of metal binding, involving the phosphate groups of both strands of DNA, allowed for higher DNA binding affinity of the alkaline earth metal ions. These results have implications in understanding the biological role of metal ions and developing DNA-based sensors and nanoelectronic devices.