Sharlene Washington

The Impact of Fish Oil on the Chemopreventive Efficacy of Tamoxifen against Development of N-Methyl-N-Nitrosourea- Induced Rat Mammary Carcinogenesis

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-γ (PPARγ), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARγ 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 PPARγ 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 Prev Res; 3(3); 322–30

Metastasis of hormone-independent breast cancer to lung and bone is decreased by α-difluoromethylornithine treatment

IntroductionPolyamines 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.α-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.MethodsMDA-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.ResultsDFMO 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).ConclusionTaken 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.

Interaction between Polyamines and the Mitogen-Activated Protein Kinase Pathway in the Regulation of Cell Cycle Variables in Breast Cancer Cells

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.

Effects of α-difluoromethylornithine on local recurrence and pulmonary metastasis from MDA-MB-435 breast cancer xenografts in nude mice

We have recently shown that administration of α-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.

Cellular mechanisms mediating the anti-invasive properties of the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO) in human breast cancer cells

We have shown that inhibition of polyamine biosynthesis with α-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.

Effects of fish oil and Tamoxifen on preneoplastic lesion development and biomarkers of oxidative stress in the early stages of N-methyl-N-nitrosourea-induced rat mammary carcinogenesis

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.

Effect of α-difluoromethyl-ornithine on the expression and function of the epidermal growth factor receptor in human breast epithelial cells in culture

We have previously shown that ornithine decarboxylase (ODC) overexpression enhances the transforming effects of HER-2neu and epidermal growth factor (EGF) in normal MCF-10A human breast epithelial cells. Our data suggest that such potentiation may be mediated by activation of the mitogen-activated protein kinase (MAPK) pathway and, possibly, STAT signalling. To further explore the interaction between the polyamine pathway and EGF/HER-2neu signalling in this system, we inhibited endogenous ODC activity with α-difluoromethylornithine (DFMO) and assessed the effects of this blockade on the expression of EGF receptors (EGFR) and HER-2neu as well as activation of downstream EGF target genes. We found that DFMO administration to MCF-10A cells increased EGF-R mRNA and protein levels in a dose-response fashion, while HER-2neu expression was not affected. The effect of DFMO was mediated through polyamine depletion since it could be reversed by exogenous putrescine administration. Our results also indicated that the increase in EGFR induced by DFMO was not a non-specific consequence of inhibition of cell proliferation. The upregulated EGFRs were functional since they could be phosphorylated by EGF and they were able to promote phosphorylation of downstream signalling molecules including ERK, STAT-3, and STAT-5. We propose that physiologic levels of ODC activity may be critical for regulation of a yet undefined signalling pathway, whose blockade by DFMO leads to a compensatory increase in functional EGFR.

Influence of omega-3 fatty acids on Tamoxifen-induced suppression of rat mammary carcinogenesis

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague‐Dawley rats were injected ip with 1‐methyl‐1‐nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO‐rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n‐3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n‐3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n‐3FA.

Immunohistochemical detection of ornithine-decarboxylase in primary and metastatic human breast cancer specimens

Increased ornithine decarboxylase (ODC) activity in human breast cancer specimens has recently been shown to be an independent adverse prognostic factor for recurrence and death. Biochemical measurement of ODC, however, is not practical for routine clinical use. Furthermore, it does not take into account the heterogeneous composition of human breast cancers which contain variable proportions of epithelial and stromal elements. Therefore, we developed an immunohistochemical method for ODC determination which can be applied to formalin-fixed, paraffin-embedded tissue sections. We report here our results in a series of 30 human breast cancer samples. ODC expression was detected most consistently in the malignant epithelial component of the tumors. Twenty-seven of 30 samples stained positive with intensities ranging from 1+ to 3+. The fraction of malignant epithelial cells expressing ODC varied among specimens between 10% and >90%. When quantitated by H-SCORE, ODC expression was significantly higher in the malignant epithelial component than in normal appearing epithelial cells and stroma admixed within the tumor. Normal mammary tissue adjacent to the cancer was available for analysis in six cases. ODC expression was absent in two (while both cancers were positive) but present in four to a degree which was overall comparable to that observed in the corresponding tumors. We believe that this technique will be useful for future studies aimed at expanding our knowledge of the role of ODC and polyamines (PA) in breast cancer biology.

Effects of polyamine depletion by α-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anti-cancer therapy. Polyamine depletion by 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 STAT3, JNK, and ERK, 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 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 anti-proliferative effect at the metastatic sites.