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Lucas Tadeu Bidinotto,
Ricardo López de Cicco,
Johana Erika Vanegas,
Julia Santucci-Pereira,
John Patrick Vanden Heuvel,
Sharlene Washington,
Cesar Aliaga,
Haifang Xu,
Irma H Russo, Andrea Manni,
Karam El-Bayoumy,
Jose Russo
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ABSTRACT: We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publisher's online edition of Nutrition and Cancer to view the supplemental file.
Nutrition and Cancer 10/2012; 64(7):991-9. · 2.78 Impact Factor
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ABSTRACT: An increased ratio of dietary n-3 relative to n-6 fatty acids has been shown to inhibit the development of mammary cancer in animal models. However, the molecular mechanisms by which n-3 fatty acids affect tumor growth remain unknown. Here, we investigated the effects of varying dietary ratios of n-3:n-6 fatty acids on cell signaling in a rat model of chemically induced mammary carcinoma. Cell proliferation was reduced by 60% in carcinomas from the high n-3:n-6 treatment group compared with the low n-3:n-6 treatment group. These changes were associated with decreased cyclin-D1 and phospho-retinoblastoma protein expression and increased levels of cyclin-dependent kinase inhibitors, CIP1 (p21) and KIP1 (p27). In addition, the apoptotic index was increased in carcinomas from the high n-3:n-6 group and was associated with elevated apoptotic protease-activating factor 1 and a higher ratio of Bax/Bcl-2. Interestingly, changes in protein expression were consistent with reduced inflammation and suppressed mTOR activity, and the molecular signature associated with high n-3:n-6 treatment revealed changes in PPARγ activation and suppression of lipid synthesis. Together, our findings indicate that the molecular effects of high dietary n-3 to n-6 ratios are heterogeneous in nature but point to consistent changes in lipid metabolism pathways, which may serve as potential therapeutic targets for cancer prevention and control. This study identifies the pathways modulated by dietary fatty acid ratios in a rat model of breast cancer, with implications for cancer prevention.
Cancer Research 05/2012; 72(15):3795-806. · 7.86 Impact Factor
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Andrea Manni,
John P Richie,
Haifang Xu,
Sharlene Washington,
Cesar Aliaga,
Timothy K Cooper,
Richard Bruggeman,
Arunangshu Das,
Bogdan Prokopczyk,
Ana Calcagnotto,
Neil Trushin,
Rebecca Dickinson,
Jason Liao,
Michael F Verderame,
Karam El-Bayoumy
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ABSTRACT: Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p<0.0001) in FO-fed rats, possibly as a result of FO-induced depletion of arachidonic acid in the mammary gland. These results suggest that the protective effect of n:3 in this experimental system is not mediated by changes in the levels of oxidative stress but may result from suppression of arachidonic acid-specific pathways.
International Journal of Oncology 11/2011; 39(5):1153-64. · 2.40 Impact Factor
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ABSTRACT: Despite the perception that omega-3 fatty acids (n-3 FA) protect against breast cancer, epidemiologic studies have yielded inconsistent results. Although preclinical data have been, in general, more supportive of a protective effect of n-3 FA on breast cancer, inconsistencies still remain, which preclude definite conclusions or in-depth mechanistic investigations despite 30 years of research in this area. In this review, we discuss key variables that may account for inconsistencies of results across preclinical studies and provide recommendations for future experiments testing the chemopreventive effect of n-3 FAs in breast cancer, as part of a multiagent approach under rigorously controlled conditions.
Cancer Research 09/2011; 71(19):6091-6. · 7.86 Impact Factor
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ABSTRACT: This study investigated the effect of a broad range of dietary ratios of n-3:n-6 fatty acids on mammary gland density and mammary cancer risk. Cancer was induced in female rats by N-methyl-N-nitrosourea. Purified diet that provided 30% of dietary kilocalories from fat was formulated to contain ratios of n-3:n-6 fatty acids from 25:1 to 1:25. Mammary gland density was determined by digital analysis, fatty acids by gas chromatography/flame ionization detection, and other plasma analytes via ELISA. Mammary gland density was reduced dose dependently at n-3:n-6 ratios from 1:1 to 25:1 (r = -0.477, P = 0.038), with a 20.3% decrease of mammary gland density between n-3:n-6 of 1:1 versus 25:1, P < 0.001. Mammary carcinogenesis was inhibited in the absence or presence of tamoxifen (1 mg/kg diet) in a manner predicted by mammary gland density. Plasma n-3 fatty acid concentrations failed to increase above an n-3:n-6 ratio of 5:1, and changes in specific plasma n-3 or n-6 fatty acids were not predictive of mammary gland density or cancer inhibitory activity. A strong reciprocal effect of the n-3:n-6 ratio on plasma leptin (decreased, P = 0.005) and adiponectin (increased, P < 0.001) was observed indicating adipose tissue function was modulated. However, neither cytokine was predictive of mammary gland density. Plasma insulin-like growth factor I (IGF-I) decreased with increasing dietary n-3:n-6 ratio (P = 0.004) and was predictive of the changes in mammary gland density (r = 0.362, P < 0.005). These findings indicate that (i) mammary gland density predicted the carcinogenic response, (ii) the n-3:n-6 ratio exerts effects in the presence or absence of hormonal regulation of carcinogenesis, and (iii) signaling pathways regulated by IGF-I are potential targets for further mechanistic investigation.
Cancer Prevention Research 08/2011; 4(10):1675-85. · 4.91 Impact Factor
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Andrea Manni,
Haifang Xu,
Sharlene Washington,
Cesar Aliaga,
Arunangshu Das,
Timothy Cooper,
John P Richie,
Bogdan Prokopczyk,
Ana Calcagnotto,
Neil Trushin,
John P Van den Heuvel,
Christopher Hamilton,
Laurence M Demers,
Jason Liao,
Michael F Verderame,
Karam El-Bayoumy
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ABSTRACT: In these experiments, we tested the hypothesis that inhibition of the estrogen receptor (ER) with Tamoxifen and activation of PPARγ with fish oil (FO) rich in omega-3 (n-3; known PPAR agonists) inhibit the development of hormone-independent breast cancer in view of the known crosstalk between the ER and PPARγ pathways. We selected the polyoma middle T transgenic mouse model, since in this system the development of ER- tumors is preceded by ER positive preneoplastic lesions. Tamoxifen admixed with a 20% corn oil (CO) modified AIN-76A diet delayed mammary carcinogenesis and inhibited tumor multiplicity, volume, and weight in a dose-dependent (1, 10, and 100 ppm) fashion. Administration of increasing concentrations of FO in the diet (5%, 10%, and 17%) did not affect any of the tumor parameters. Combined administration of different doses of Tamoxifen and FO delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as Tamoxifen alone. Mice fed 10% FO exhibited the expected increase in n-3/n-6 ratio in plasma and tumor based on diet analysis. Further increase in the n-3/n-6 ratio was not observed in mice fed the 17% FO diet. FO reduced tissue levels of arachidonic acid and its metabolite PGF-2α. Our results support the role of ER expression by preneoplastic lesions in the development of hormone-independent tumors and consequently the importance of including ER targeting in combination with mechanistically based novel chemopreventive agents.
Hormones and Cancer 08/2011; 2(4):249-59.
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ABSTRACT: To discuss the implications of a young age at diagnosis in a family member with hyperparathyroidism-jaw tumor syndrome, the youngest published case to date, due to a mutation of the CDC73 gene (formerly known as HRPT2); to review this family with regard to modifications of guidelines for surveillance of hyperparathyroidism and other associated features in affected and at-risk relatives; and to discuss surgical recommendations in this syndrome.
A review of English-language publications in PubMed and a review of GeneReviews were conducted pertaining to the subject of familial hyperparathyroidism. A case is described, and the family pedigree is discussed.
Review of the literature revealed that CDC73-related disorder has not previously been reported in patients younger than 10 years. This finding has been the basis for the recommendation for initiation of surveillance for disease manifestations at that age. Review of the family history of our current patient revealed a 7-year-old nephew with hypercalcemia attributable to primary hyperparathyroidism.
Surveillance of hyperparathyroidism in affected persons and genetic testing of relatives at risk are currently recommended to start at 10 years of age. We recommend that these be conducted at a younger age, preferably 5 to 10 years before the earliest diagnosis of hyperparathyroidism within the family, and potentially at birth in families with a known mutation of the CDC73 gene, in light of the malignant potential of the disease.
Endocrine Practice 02/2011; 17(4):602-9. · 2.49 Impact Factor
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Andrea Manni,
Haifang Xu,
Sharlene Washington,
Cesar Aliaga,
Timothy Cooper,
John P Richie,
Richard Bruggeman,
Bogdan Prokopczyk,
Ana Calcagnotto,
Neil Trushin,
David Mauger,
Michael F Verderame,
Karam El-Bayoumy
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ABSTRACT: The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-gamma (PPARgamma), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARgamma receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea-induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARgamma is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA-rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies.
Cancer Prevention Research 02/2010; 3(3):322-30. · 4.91 Impact Factor
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ABSTRACT: This article presents an overview of medical management of persistent or recurrent differentiated thyroid cancer, in particular focusing on monitoring strategy and treatment plans. Most patients with differentiated thyroid carcinoma can be successfully rendered to be free of disease with initial treatments, and those with persistent or recurrent disease can still expect long-term survival when they are monitored properly and treated accordingly. Along with serum thyroglobulin, neck ultrasonography, and radioactive iodine whole-body scans, the use of cross-sectional imaging studies and (18F) fluoro-2-deoxy-D-glucose-positron emission tomography have facilitated the effort in localizing lesions and traditional treatments can be implemented effectively. For disease resistant to conventional therapies, there are new treatment modalities emerging and being tested, including several agents targeting specific signaling pathways, each of which may offer the potential remedy.
Otolaryngologic Clinics of North America 01/2009; 41(6):1241-60, xi-xii. · 1.65 Impact Factor
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ABSTRACT: Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anticancer therapy. Polyamine depletion by alpha-difluoromethylornithine (DFMO) has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of signal transducers and activator of transcription 3, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or the pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of antiproliferative effect at the metastatic sites.
Breast Cancer Research and Treatment 02/2008; 107(1):33-40. · 4.43 Impact Factor
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ABSTRACT: We have shown that alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, the first and rate-limiting enzyme in polyamine synthesis, has significant antiproliferative and antiinvasive effects in breast cancer cells. We have also reported that these antitumor effects are associated with activation of multiple signaling pathways, including STAT-3, STAT-1, Jun-N-Terminal kinase (JNK), and Mitogen activated protein kinase (MAPK), the latter being found to mediate its antiinvasive action in MDA-MB-435 cells. The present experiments were designed to test the effect of DFMO on the protein kinase A (PKA) pathway and determine its biological significance. We found that DFMO administration (1 mM) to MDA-MB-435 breast cancer cells significantly increased cAMP response element (CRE)-binding protein (CREB) phosphorylation as well as the transactivation of pCRE-luc, a CREB-dependent promoter activated by PKA. To determine the significance of this biochemical effect of DFMO, we used the PKA inhibitor H89 which, as expected, suppressed in a dose-dependent manner (1 and 10 microM) basal and DFMO-induced CREB phosphorylation in our system. Administration of H89 alone was able to suppress proliferation of MDA-MB-435 cells when used at a concentration (10 microM) shown to completely block basal CREB phosphorylation. At concentrations of 0.5 and 1 muM, H89 treatment, while having no antiproliferative effect of its own, potentiated in a dose-dependent fashion the growth inhibitory action of a suboptimal concentration of DFMO (0.01 mM). Ten micromoles of H89 reduced invasiveness of MDA-MB-435 cells in matrigel by approximately 40% (an effect similar to that of 1 mM DFMO). The combination treatment further reduced invasiveness by approximately 80% (P < 0.01 versus the individual treatments). H89 treatment (10 microM) partially reduced DFMO-induced phosphorylation of STAT-3 but not that of STAT-1, Extracellular regulated kinase (ERK), and JNK. In conclusion, our results indicate that PKA signaling exerts proproliferative and proinvasive effects in our experimental system. Therefore, its activation by DFMO represents a compensatory mechanism which should be blocked in order to maximize the antitumor action of the drug. Our data are also consistent with the notion that STAT-3 activation by DFMO is at least in part mediated through the PKA pathway.
Breast Cancer Research and Treatment 01/2008; 107(1):63-70. · 4.43 Impact Factor
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ABSTRACT: We have shown that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) biosynthesis, reduces the invasive and metastatic properties of MDA-MB-435 breast cancer cells while activating multiple signal transduction pathways, including MAPK, Stat3, Stat1, and JNK. Since the activity of these signaling mechanisms is frequently regulated by upstream tyrosine kinases (TKs), we tested whether non-receptor and receptor TKs may be involved in the signaling and biological effects of DFMO in MDA-MB-435 cells. Treatment with DFMO (1 mM for 48 h) did not affect Src phosphorylation (Tyr 416). Administration of the Src-family members inhibitor PP-1 (1 microM), blocked Src phosphorylation in the absence and in the presence of DFMO, but did not block the signaling effects of DFMO (increased phosphorylation of Stat3, Stat1, ERK and JNK). PP-1 treatment, on the other hand, inhibited the invasiveness of MDA-MB-435 cells in matrigel and potentiated the anti-invasive effect of DFMO. Next, we focused on the role of receptor TK. Western analysis of cell lysates from MDA-MB-435 cells failed to show the presence of EGF-R and HER-2neu but demonstrated the expression of c-Met, the receptor for hepatocyte growth factor (HGF). Therefore, we tested the effect of DFMO on the HGF/c-Met pathway which is strongly implicated in the progression of human breast cancer. We found that DFMO treatment blocked HGF-induced c-Met phosphorylation in MDA-MB-435 cells, suggesting that its anti-invasion action may be mediated, at least in part, by blocking c-Met signaling. Next, we showed that 1 mM DFMO suppressed HGF induced invasiveness of MDA-MB-435 cells in matrigel. Combination administration of DFMO with suboptimal doses of PHA-665752, a specific c-Met inhibitor, reduced invasiveness to an even greater extent than the individual treatment. These findings indicate that Src-family members, while not involved in DFMO action, promote invasiveness of breast cancer cells and their inhibition may enhance the antitumor effect of PA depletion. Our data also point to inhibition of HGF/c-Met pathway as a possible novel approach to enhancing the antitumor action of DFMO.
Breast Cancer Research and Treatment 01/2008; 112(2):255-61. · 4.43 Impact Factor
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ABSTRACT: We have previously observed that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) upregulates production of thrombospondin-1 (TSP-1), an extracellular matrix protein with potent anti-angiogenic and antimetastatic properties, by MDA-MB-435 human breast cancer cells in culture. The present experiments were designed to investigate the mechanisms by which DFMO regulates TSP-1 production in this system. 35S-methionine pulse chase experiments indicated that DFMO administration increased TSP-1 synthesis by approximately 6-fold, while it slightly but significantly decreased protein half-life from 35 to 28 min. DFMO treatment increased steady state TSP-1 mRNA levels by 2-fold in MDA-MB-435 cells. TSP-1 promoter reporter studies indicated that this increase was largely due to activation of transcription. Analysis of distribution of TSP-1 mRNA levels between non-polysomal, subpolysomal and polysomal fractions in control and DFMO-treated cells suggested a major stimulatory effect of the drug on TSP-1 translation. A similar increase in TSP-1 transcription and translation in response to DFMO treatment was also observed in vivo in MDA-MB-435 breast cancer xenografts. Surprisingly however, we failed to detect an increase in TSP-1 protein as assessed by Western blot analysis. The reason for this unexpected finding is unknown but may be due to DFMO-induced stimulation of TSP-1 secretion into the systemic circulation, thus preventing its accumulation within the tumor.
International Journal of Oncology 12/2007; 31(5):1187-91. · 2.40 Impact Factor
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ABSTRACT: Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) has been shown to inhibit proliferation of breast cancer cells although its mechanism of action has not been fully elucidated. To address this issue, we tested the effects of DFMO on cell cycle variables of MDA-MB-435 human breast cancer cells in culture. We also focused on the possible mediatory role of the mitogen-activated protein kinase (MAPK) pathway on the cell cycle effects of DFMO because this compound has been shown to activate MAPK signaling. We found that DFMO caused a p53-independent increase in p21 and its association with cyclin-dependent kinase (cdk)-2 and decreased cdk-2 protein as well as its phosphorylation on Thr160. In addition, DFMO markedly suppressed the expression of the full-length and low molecular weight forms of cyclin E. These effects of DFMO were reversible with exogenous putrescine, thus indicating that they are specifically mediated through polyamine depletion. Cdk-2 activity was drastically reduced in DFMO-treated breast cancer cells which exhibited a reduction in retinoblastoma (Rb) phosphorylation and protein. As a predictable consequence of these effects, DFMO caused a G1-S block. In addition, DFMO inhibited G2-M transition, most likely as a result of its induction of p21 expression. Inhibition of the MAPK pathway with PD98059 or U0126 blocked the DFMO-induced induction of p21 and the reduction of cdk-2 protein. PD98059 reversed the G2-M block induced by DFMO (probably as a result of suppression of p21) but not the G1-S arrest. MDA-MB-435 cells treated with PD98059 or U0126 in the presence and absence of DFMO exhibited a marked increase in the expression of p27 and its association with cdk-2, a decrease in phosphorylation of cdk-2 on Thr160, and a decrease in cyclin E expression. As predicted, PD98059 treatment reduced cdk-2 activity and Rb phosphorylation while reversing the decrease in Rb protein induced by DFMO. Neither DFMO nor PD98059, either alone or in combination, reduced cdk-4 activity despite a marked induction in p15 expression caused by DFMO. Our results indicate that activation of the MAPK pathway accounts for some of the effects of DFMO on cell cycle events of breast cancer cells. Inhibition of the MAPK pathway, however, does not reverse the cell cycle arrest induced by DFMO because of activation of alternative mechanisms leading to suppression of cdk-2 activity.
Cancer Research 01/2006; 65(23):11026-33. · 7.86 Impact Factor
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ABSTRACT: 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.
Clinical and Experimental Metastasis 02/2005; 22(3):255-63. · 3.52 Impact Factor
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ABSTRACT: 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.
Clinical and Experimental Metastasis 02/2005; 22(4):331-9. · 3.52 Impact Factor
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ABSTRACT: 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.
International Journal of Oncology 01/2005; 25(6):1831-8. · 2.40 Impact Factor
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ABSTRACT: Polyamines affect proliferation, differentiation, migration and apoptosis of cells, indicating their potential as a target for cancer chemotherapy. Ornithine decarboxylase converts ornithine to putrescine and is the rate-limiting step in polyamine synthesis.alpha-Difluoromethylornithine (DFMO) irreversibly inhibits ornithine decarboxylase and MDA-MB-435 human breast cancer metastasis to the lung without blocking orthotopic tumor growth. This study tested the effects of DFMO on orthotopic tumor growth and lung colonization of another breast cancer cell line (MDA-MB-231) and the effects on bone metastasis of MDA-MB-435 cells.
MDA-MB-231 cells were injected into the mammary fat pad of athymic mice. DFMO treatment (2% per orally) began at the day of tumor cell injection or 21 days post injection. Tumor growth was measured weekly. MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment began 7 days prior to injection, or 7 or 14 days post injection. The number and incidence of lung metastases were determined. Green fluorescent protein-tagged MDA-MB-435 cells were injected into the left cardiac ventricle in order to assess the incidence and extent of metastasis to the femur. DFMO treatment began 7 days prior to injection.
DFMO treatment delayed MDA-MB-231 orthotopic tumor growth to a greater extent than growth of MDA-MB-435 tumors. The most substantial effect on lung colonization by MDA-MB-231 cells occurred when DFMO treatment began 7 days before intravenous injection of tumor cells (incidence decreased 28% and number of metastases per lung decreased 35-40%). When DFMO treatment began 7 days post injection, the incidence and number of metastases decreased less than 10%. Surprisingly, treatment initiated 14 days after tumor cell inoculation resulted in a nearly 50% reduction in the number of lung metastases without diminishing the incidence. After intracardiac injection, DFMO treatment decreased the incidence of bone metastases (55% vs 87%) and the area occupied by the tumor (1.66 mm2 vs 4.51 mm2, P < 0.05).
Taken together, these data demonstrate that DFMO exerts an anti-metastatic effect in more than one hormone-independent breast cancer, for which no standard form of biologically-based treatment exists. Importantly, the data show that DFMO is effective against metastasis to multiple sites and that treatment is generally more effective when administered early.
Breast cancer research: BCR 01/2005; 7(5):R819-27. · 5.24 Impact Factor
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ABSTRACT: We have shown that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) reduces in vitro invasiveness and metastatic capacity of MDA-MB-435 breast cancer cells. These experiments investigated the mechanisms mediating the anti-invasive properties of DFMO. DFMO did not affect phosphorylation of FAK or Akt, but increased ERK phosphorylation by approximately threefold. To test the biologic significance of this finding, we tested the effect of the MEK inhibitor PD98059 on in vitro invasiveness of MDA-MB-435 breast cancer cells, both in the absence and in the presence of the proinvasive peptide hepatocyte growth factor (HGF) as a chemoattractant. We observed that PD98059 treatment reversed the anti-invasive effect of DFMO under both experimental conditions. Next, we tested the influence of DFMO on the production of the prometastatic peptide osteopontin (OPN) and the anti-metastatic protein thrombospondin-1 (TSP-1). DFMO treatment, while not affecting OPN production, markedly increased the TSP-1 level in the conditioned media. This effect was abolished by putrescine administration, thus indicating the specificity of the DFMO action through the polyamine pathway. PD98059 completely blocked the stimulatory effect of DFMO on TSP-1 production, which supports a mediatory role for activation of the MAPK pathway in the upregulation of this anti-metastatic peptide by DFMO. In summary, our results show that the increase in ERK phosphorylation induced by DFMO plays a critical role in the anti-invasive action of the drug and in its ability to upregulate TSP-1 production.
Clinical and Experimental Metastasis 02/2004; 21(5):461-7. · 3.52 Impact Factor
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Andrea Manni,
Sharlene Washington,
Laura Craig,
Michael Cloud,
James W Griffith,
Michael F Verderame,
Lindsay J Texter,
David Mauger,
Laurence M Demers,
John F Harms,
Danny R Welch
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ABSTRACT: We have recently shown that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) biosynthesis reduces pulmonary metastasis from MDA-MB-435 breast cancer xenografts in nude mice. The present experiments were designed to further explore PA involvement in breast cancer metastasis, using GFP-tagged MDA-MB-435 cells that can be tracked at the single cell level. Administration of DFMO significantly reduced the number of mice with pulmonary metastasis as well as the number of metastases per mouse. Both single-cell and multicellular metastatic deposits were similarly suppressed, thus suggesting that DFMO was inhibiting lung colonization by tumor cells rather than preventing progression of single-cell deposits to overt metastasis. DFMO administration also significantly reduced local recurrences following removal of the primary tumor. Prolongation of DFMO treatment to 14 weeks did not yield a superior antimetastatic effect beyond that provided by a 10-week course of therapy. Discontinuation of DFMO, on the other hand, was associated with local regrowth of the tumors and, possibly, recurrence of pulmonary metastasis. These data provide a rationale for testing the efficacy of anti-PA treatment within the context of adjuvant therapy of breast cancer.
Clinical and Experimental Metastasis 02/2003; 20(4):321-5. · 3.52 Impact Factor
