Molecular correlates of the action of bis(ethyl)polyamines in breast cancer cell growth inhibition and apoptosis.

Polyamines are known to be involved in cell growth regulation in breast cancer. To evaluate the efficacy of bis(ethyl)polyamine analogs for breast cancer therapy and to understand their mechanism of action we measured the effects of a series of polyamine analogs on cell growth, activities of enzymes involved in polyamine metabolism, intracellular polyamine levels, and the uptake of putrescine and spermidine using MCF-7 breast cancer cells. The IC50 values for cell growth inhibition of three of the compounds, N1,N12-bis(ethyl)spermine, N1,N11-bis(ethyl)norspermine, and N1,N14-bis(ethyl)homospermine, were in the range of 1-2 microM. Another group of three compounds showed antiproliferative activity at about 5 microM level. These compounds are also capable of suppressing colony formation in soft agar assay and inducing apoptosis of MCF-7 cells. The highly effective growth inhibitory agents altered the activity of polyamine biosynthetic and catabolic enzymes and down-regulated the transport of natural polyamines, although each compound produced a unique pattern of alterations in these parameters. HPLC analysis showed that cellular uptake of bis(ethyl)polyamines was highest for bis(ethyl)spermine. We also analyzed polyamine analog conformations and their binding to DNA minor or major grooves by molecular modelling and molecular dynamics simulations. Results of these analyses indicate that tetramine analogs fit well in the minor groove of DNA whereas, larger compounds extend out of the minor groove. Although major groove binding was also possible for the short tetramine analogs, this interaction led to a predominantly bent conformation. Our studies show growth inhibitory activities of several potentially important analogs on breast cancer cells and indicate that multiple sites are involved in the mechanism of action of these analogs. While the activity of an analog may depend on the sum of these different effects, molecular modelling studies indicate a correlation between antiproliferative activity and stable interactions of the analogs with major or minor grooves of DNA.

Effects of epidermal growth factor on MDA-MB-468 breast cancer cells: alterations in polyamine biosynthesis and the expression of p21/CIP1/WAF1.

We examined the effects of epidermal growth factor (EGF) on MDA-MB-468 cells to understand its mechanism of action in an EGF receptor-rich breast cancer cell line. EGF inhibited the growth of MDA-MB-468 cells with an IC50 of 1.5 +/- 0.5 nM, as determined by measurements of DNA content of cells in culture over a period of 4 to 6 days. This growth inhibition included apoptosis 24 h after EGF addition, as detected by an enzyme-linked immunosorbent assay (ELISA) and Hoechst 33342 staining. In EGF-treated cells, peak activities of two key enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), were reduced by 57% and 83%, respectively. EGF treatment also caused a 30 to 50% decrease in cellular putrescine at all time points tested (12 to 48 h). EGF-induced inhibition of DNA synthesis was also partially reversed by the addition of putrescine or spermidine, but not by spermine. Western blot analysis of cell cycle regulatory proteins showed that EGF-mediated growth inhibition was associated with the induction of p21, an inhibitor of cyclin-dependent kinases. However, EGF had no significant effect on the expression of cyclin D1 or cyclin E. Furthermore, putrescine reversal of EGF effects was associated with the down-regulation of EGF-induced p21. These results suggest that the mechanism of growth inhibition by EGF in MDA-MB-468 cells include a down-regulation of polyamine biosynthesis and the induction of p21. Identification of growth regulatory pathways in breast cancer cells might be useful in the development of novel targets for therapeutic intervention.

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

Molecular characterization of two novel transporters from human and mouse kidney and from LLC-PK1 cells reveals a novel conserved family that is homologous to bacterial and Aspergillus nucleobase transporters.

Nucleobase transport is important for the metabolism of nucleic acids and antiviral and antineoplastic drugs. This transport has been functionally described in several mammalian cells but has not been well characterized molecularly. We report the cloning of two novel transporters. YSPL2 encodes a 650-residue protein and has an ubiquitous 8 kb transcript. The human and pig homologs are 95% similar. YSPL3 encodes a 598-residue protein with a 3 kb transcript that is expressed only in kidney and liver. Human YSPL2 and YSPL3 are 60% similar at the amino acid level and both show 31% similarity to the first nucleobase permease gene described in vertebrates, YSPL1. These proteins appear to be members of a new family of possible nucleobase transporters with significant sequence similarities with bacterial and Aspergillus nucleobase transporters. Further functional studies will be needed to unveil the role of these transporters in nucleic acid metabolism in normal and in disease states.

Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans.

We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegans K-Cl cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share approximately 45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains 10 exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.

Induction of p21 (CIP1/WAF1/SID1) by estradiol in a breast epithelial cell line transfected with the recombinant estrogen receptor gene: a possible mechanism for a negative regulatory role of estradiol.

Estrogens stimulate the growth of a majority of estrogen receptor (ER)-positive breast cancer cells. In contrast, estradiol exerted a 75% inhibition of DNA synthesis in the MCF-10AE(wt5) cell line, obtained by the transfection of the ER gene into a normal breast epithelial cell line, MCF-10A. The estradiol-mediated growth inhibitory effect was reversed by ICI 164384, a pure anti-estrogen. Analysis of cell cycle by flow cytometry showed a significant increase of G1 cells by estradiol treatment compared to controls. To understand the mechanism of action of estradiol on MCF-10AE(wt5) cells, we examined the level of a cyclin dependent kinase inhibitor (CKI), p21, by Western blot analysis. Our results showed a 5- to 10-fold increase in the level of p21 in estradiol-treated MCF-10AE(wt5) cells compared to controls. ICI 164384 reversed estradiol-mediated induction of p21. Northern blot analysis of p21 mRNA indicated that estradiol stimulated its message in MCF-10AE(wt5) cells. Analysis of a panel of 6 breast cancer cell lines showed the absence of p21 protein, whereas it was present at a very low level in MCF-10A cells. Comparison of p21 in MCF-10A and MCF-10AE(wt5) cells showed an abundance of p21 in the ER-transfected cells. However, this p21 appears to be inactive in the absence of estradiol. These results suggest a p21-mediated pathway as a possible mechanism for the growth inhibitory effects of estradiol on at least a subset of ER-transfected cell lines.

Mechanism of action of a tyrphostin, 3,4-dihydroxy-alpha-cyanothiocinnamamide, in breast cancer cell growth inhibition involves the suppression of cyclin B1 and the functional activity of cyclin B1/p34cdc2 complex.

Tyrphostins are a group of compounds specifically targeted for the inhibition of tyrosine phosphorylation in signal transduction pathways. We studied the effects of a tyrphostin, 3,4-dihydroxy-alpha-cyanothiocinnamamide (tyrphostin-47), on hormone-responsive MCF-7 and hormone-unresponsive MCF-7-5C cell growth by DNA analysis for a period of 10 days. The growth of both cell lines was inhibited by this drug at 50 and 100 microM concentrations. Flow cytometric analysis showed that tyrphostin treatment caused a significant delay in the progression of MCF-7 cells through G1 and S phases of the cell cycle. The level of cyclin B1, a component of the mitosis promoting factor (MPF), was reduced by 90% in the presence of 100 microM tyrphostin. The other component of MPF, p34cdc2 kinase, was not affected; however, its functional activity was dramatically reduced, as determined by histone H1 phosphorylation assay. In contrast, G1 cyclins (D1 and E) and tyrosine kinase activity were not markedly affected by tyrphostin-47, as determined by Western immunoblot detection with specific antibodies. Our results suggest that a possible mechanism of tyrphostin action in breast cancer cells might involve the suppression of cyclin B1 and inhibition of the functional activity of cyclin B1/p34cdc2 complex. Our data indicate that the cell cycle machinery might be a target for developing novel drugs for breast cancer.

Effects of a bis(benzyl)spermine analog on MCF-7 breast cancer cells in culture and nude mice xenografts.

We studied the effects of a polyamine analog, N,N'-bis{3-[(phenylmethyl)amino]propyl}-1,7-diaminoheptane (MDL 27695) on MCF-7 cells, as part of an attempt to develop new drugs for breast cancer treatment. Using [H-3]-thymidine incorporation assay and long-term growth curves, we found that MDL 27695 inhibited the growth of MCF-7 cells in a dose-dependent manner in the low mu M range. G1 synchronized cells progressing in cell cycle showed delayed and inefficient entry into S phase in the presence of 4 mu M MDL 27695. Consistent with a G1 arrest, MDL 27695 significantly reduced estradiol-mediated increase in the expression of cyclin D1. HPLC analysis showed that treatment of MCF-7 cells with MDL 27695 reduced the accumulation of natural polyamines, putrescine, spermidine, and spermine, by 43, 38, and 45%, respectively, at 8 h after the initiation of cell cycle. This decrease in polyamine levels was not associated with a decrease in the activity of polyamine biosynthetic (ornithine decarboxylase, ODC; s-adenosylmethionine decarboxylase, SAMDC) or catabolizing (spermidine/spermine acetyltransferase, SSAT) enzymes. However, there was a 40% decrease in the uptake of putrescine and spermidine, in cells treated with MDL 27695. Our studies also showed that MDL 27695, at a dose of 20 mg/kg, caused a significant inhibition of tumor growth in nude mice harboring MCF-7 cell derived tumors, without overt symptoms of toxicity. These data indicate that the polyamine analog MDL 27695 is an efficient inhibitor of MCF-7 breast cancer cell growth in vitro and in vivo. Our results suggest that polyamines are critical factors in cell cycle regulation of breast cancer cells and potential targets for therapy.

Estrogenic regulation of a novel 34 kDa protein associated with cyclin B1 in MCF-7 breast cancer cells.

Recent studies have revealed altered regulation of cyclins in breast cancer cells. To understand the role of aberrant cyclin B1 expression in the proliferation of breast cancer cells, we examined cyclin B1-associated proteins in estrogen-responsive MCF-7 cells in a cell cycle-dependent manner. Immunoprecipitation of cell lysate with a monoclonal anti-human cyclin B1 antibody, followed by Western blot probing with an anti-human cdc2 (PSTAIR) antibody revealed the presence of a 34 kDa protein in estradiol-treated cells at 16 h after initiation of cell cycle progression. Flow cytometry and [H-3]-thymidine (Thd) incorporation experiments showed a dramatic increase in the percentage of S phase cells at this time point. This protein was suppressed by an antiestrogen, 4-hydroxytamoxifen. It was not found in MCF-1OA, a normal breast epithelial cell line. The 34 kDa protein was not reactive with antibodies raised against other cyclin dependent kinases (CDKs), including p34(cdc2(Carboxy terminal)). This protein was functionally active as determined by histone H1 kinase activity. These data suggest that the induction of a cyclin B1-associated 34 kDa protein during the G1 --> S transition might be a positive regulator of cell cycle progression in estrogen-responsive breast cancer cells.

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

SAMDC 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 Ciba-Geigy, 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 microM concentration. In contrast, CGP48664 and CGP39937, containing 2 aromatic rings that delocalize the pi electron system of the backbone of MGBG, were potent inhibitors with 50% growth inhibition at 0.5 microM 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 hormone-responsive breast cancer cell line and suggest potential application of SAMDC inhibitors as therapeutic agents.

Suppression of c-myc oncogene expression by a polyamine-complexed triplex forming oligonucleotide in MCF-7 breast cancer cells.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.

Inhibition of epidermal growth factor-stimulated EGF receptor tyrosine kinase activity in A431 human epidermoid carcinoma cells by polyamines.

Polyamines--putrescine, spermidine, and spermine--are ubiquitous cellular components that play an important role in cell growth and differentiation. Using A431 cells, a cell line that overexpresses the epidermal growth factor (EGF) receptor, we found that polyamines modulate EGF-mediated growth inhibition. The natural polyamine, putrescine, was the most effective, followed by diamines containing lower and higher methylene bridging between the amino groups. To understand the mechanism, we examined the effects of polyamines on EGF-mediated signal transduction in A431 cells. All three polyamines partially inhibited EGF-receptor tyrosine kinase activity in a dose-dependent manner. The maximal inhibition was 75% with spermidine. Polyamine effects were exerted 12-16 h after treatment, although HPLC analysis revealed uptake of polyamines within 1 h. Homologues of putrescine had no significant effect on tyrosine kinase activity, indicating structural specificity of naturally occurring polyamines in this process. Amine oxidase inhibitors did not alter spermidine and spermine-mediated effects, suggesting that the inhibition of tyrosine kinase activity was not a consequence of the oxidative metabolism of polyamines. Difluoromethylornithine, a specific inhibitor of polyamine biosynthesis, did not affect EGF receptor tyrosine kinase activity. Polyamines also had no effect on EGF receptor levels or EGF-EGF receptor high-affinity binding, indicating that they are not competitive inhibitors of the EGF receptor tyrosine kinase. Our results suggest that polyamine action in A431 cells involves modulation of EGF receptor signal transduction pathways.

Rapid uptake of tyrphostin into A431 human epidermoid cells is followed by delayed inhibition of epidermal growth factor (EGF)-stimulated EGF receptor tyrosine kinase activity.

Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C.

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.