cgp-48664 and Breast-Neoplasms

We have previously reported that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) reduces pulmonary metastasis from MDA-MB-435 human breast cancer xenografts without affecting the volume of the primary tumors (Manni et al. Clin Exp Mets 20:321, 2003). In these experiments, we show that DFMO treatment (2% in drinking H(2)O) reduced the growth fraction of the primary tumors by 60%. However, this effect was counter-balanced by a similar reduction in non-apoptotic necrosis, thus accounting for the preservation of tumor volume in DFMO-treated mice. DFMO treatment caused a 4-fold increase in cytoplasmic staining for cleaved caspase-3 (as opposed to the nuclear staining observed in control tonsil tissue) in the absence of histologic evidence of apoptosis. DFMO treatment reduced the number of mice with pulmonary metastasis by approximately 80% and the number of metastasis per mouse by >90% in association with a reduction in invasiveness of the primary tumor in the surrounding dermis and muscle by approximately 30%. DFMO treatment increased ERK phosphorylation in the tumors, an effect that has been found by us in vitro to be causally linked to the anti-invasive effect of the drug (Manni et al. Clin Exp Metast 2004; 21: 461]. DFMO also increased tyrosine phosphorylation of STAT-3 and expression of STAT-1 and JNK proteins. Administration of SAM486A (1 mg/kg/i.p. daily), an inhibitor of S-adenosylmethionine decarboxylase, either individually or in combination with DFMO, was not found to exert any biological or biochemical effects, most likely as a result of its failure to suppress tissue polyamine levels under these experimental conditions.

Topics: Amidines; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Death; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Eflornithine; Female; Humans; Indans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Polyamines; STAT1 Transcription Factor; STAT3 Transcription Factor

Inhibition of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by the irreversible inhibitor alpha-difluoromethylornithine (DFMO) has been shown to decrease the invasiveness of metastatic human breast cancer cell lines. However, the mechanism by which DFMO acts to reduce invasiveness is unclear. Using the human breast cancer cell line MDA-MB-435, the effect of DFMO on metalloprotease gene expression was investigated. DFMO treatment decreases the expression of the metalloprotease meprin alpha, while concurrent treatment with DFMO and the polyamine putrescine partially restored meprin alpha expression levels. Expression of MMP-7 mRNA was reduced by DFMO, while MMPs-1, -2, -3, -14, and meprin beta were unaffected. Treatment of cells with a second inhibitor of polyamine biosynthesis, the S-adenosylmethionine decarboxylase (SAMDC) inhibitor SAM486A, also resulted in a dosage dependent decrease in meprin alpha and MMP-7 mRNA. In addition, DFMO treatment decreased meprin alpha at the protein level by 2 days of treatment, and MMP-7 protein levels at 4 and 6 days. Previous studies have shown that DFMO treatment increases ERK phosphorylation and signaling through the MAP kinase pathway. The decrease in meprin alpha expression was reversed with the MEK inhibitor PD98059, demonstrating that MAP kinase signaling mediates the effect of DFMO and SAM486A. MDA-MB-435 cells treated with the meprin alpha inhibitor actinonin (5 nM) were less invasive in vitro, indicating that meprin alpha is mechanistically involved in invasion. The decrease in meprin alpha expression in DFMO and SAM486A-treated cells indicates a means by which these compounds can decrease the invasiveness of metastatic breast cancer cells.

Topics: Amidines; Antineoplastic Agents; Breast Neoplasms; Down-Regulation; Eflornithine; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression; Humans; Hydroxamic Acids; Indans; Matrix Metalloproteinase 7; Metalloendopeptidases; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Ornithine Decarboxylase Inhibitors; Phosphorylation; Polyamines; RNA, Messenger

The antitumor activity of the S-adenosylmethionine decarboxylase (SAMDC) inhibitor SAM486A in human breast cancer cells was investigated. Our in vitro study focused on testing the effects of SAM486A on the proliferation, clonogenicity, invasiveness, cell signaling and PA levels of hormone-independent MDA-MB-435 human breast cancer cells. We also investigated the antitumor action, effects on polyamine pools and tolerability of SAM486A administered to nude mice carrying MDA-MB-435 xenografts. SAM486A suppressed anchorage-independent and -dependent growth and invasiveness of breast cancer cells and the inhibition of cell growth was associated with suppression of spermine synthesis. Combined administration of SAM486A and alpha-difluoromethylornithine (DFMO), a selective inhibitor of ornithine decarboxylase (ODC), exerted greater antiproliferative and anti-invasive effects and induced an overall greater suppression of cellular PA levels than the individual treatments. Both SAM486A and DFMO increased phosphorylation of STAT-1, -3, ERK1/2 and p38, thus indicating activation of both STAT signaling and the MAPK pathway. SAM486A (1 mg/kg) significantly suppressed the growth and spermine levels of established MDA-MB435 breast tumors in nude mice. SAM486A exerts a potent antitumor action in MDA-MB-435 breast cancer cells both in vitro and in vivo. Inhibition of cellular spermine is consistently observed with SAM486A treatment and may mediate its antitumor action. Combination treatment with DFMO may allow the use of lower and, hence, less toxic doses of each compound with preservation of optimal therapeutic effect. The role of activation of STAT signaling and the MAPK pathway in the antitumor action of SAM486A remains to be determined.

Topics: Adenosylmethionine Decarboxylase; Amidines; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Female; Humans; Indans; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Signal Transduction; Transplantation, Heterologous; Tumor Cells, Cultured

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.

Topics: Amidines; Animals; Biogenic Polyamines; Breast Neoplasms; Cell Division; Eflornithine; Estradiol; Female; Gene Expression; Humans; Indans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Recombinant Fusion Proteins; Spermidine; Tumor Cells, Cultured

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.

Topics: Acetyltransferases; Adenosylmethionine Decarboxylase; Amidines; Antineoplastic Agents; Biogenic Polyamines; Breast Neoplasms; Cell Division; Enzyme Inhibitors; Estradiol; Female; Humans; Indans; Mitoguazone; Ornithine Decarboxylase; RNA, Messenger; Structure-Activity Relationship; Tumor Cells, Cultured

CGP 48664 [4-aminoindanon-1-(2'-amidino)hydrazone dihydrochloride monohydrate] is a newly introduced inhibitor of S-adenosylmethionine decarboxylase (SAMDC) with increased selectivity of action and reduced toxicity. We analyzed the biochemical and antiproliferative effects of this compound in a panel of hormone-dependent (3 clones of MCF-7, T47D) and -independent (MDA-MB-231, BT-20) human breast cancer cell lines in culture. For comparison, we also tested its effects in the spontaneously immortalized human breast epithelial cell line MCF-10A. All cell lines were highly sensitive to the growth-inhibitor effect of CGP 48664 with an IC50 between 0.1 and 0.5 microM. A dose-dependent bell-shaped increase in SAMDC was observed in normal and malignant breast cells resulting from enzyme stabilization by the inhibitor as supported by Western blot analysis. While ornithine decarboxylase (ODC) activity consistently increased, the effect of CGP 48664 on spermidine/spermine N'acetyltransferase (SSAT) was variable in the breast cancer cell lines. In contrast, the inhibitor consistently reduced SSAT activity level in the MCF-10A cell line and its derivative partially transformed by a mutated ras oncogene. As expected cellular putrescine levels were markedly increased by CGP 48664 administration, whereas spermidine and spermine contents were reduced. However, the degree of reduction was usually only moderate. Furthermore, exogenous polyamine administration was relatively ineffective in rescuing the antiproliferative effect of CGP 48664 in MCF-7 cells, while exerting a more complete rescue in the MDA-MB-231 cell line. We conclude that CGP 48664 exerts a potent growth-inhibitory effect on mammary cells in culture. However, its action may not always be entirely mediated through the polyamine pathway.

Topics: Acetyltransferases; Adenosylmethionine Decarboxylase; Amidines; Breast; Breast Neoplasms; Cell Division; Cell Line, Transformed; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Indans; Neoplasms, Hormone-Dependent; Ornithine Decarboxylase; Spermidine; Spermine; Tumor Cells, Cultured