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Nitric oxide is a pleiotropic ancestral molecule, which elicits beneficial effect in many physiological settings but is also tenaciously expressed in numerous pathological conditions, particularly breast tumors. Nitric oxide is particularly harmful in adipogenic milieu of the breast, where it initiates and promotes tumorigenesis. Epidemiological studies have associated populations at a greater risk for developing breast cancer, predominantly estrogen receptor positive tumors, to express specific polymorphic forms of endothelial nitric oxide synthase, that produce sustained low levels of nitric oxide. Low sustained nitric oxide generates oxidative stress and inflammatory conditions at susceptible sites in the heterogeneous microenvironment of the breast, where it promotes cancer related events in specific cell types. Inflammatory conditions also stimulate inducible nitric oxide synthase expression, which dependent on the microenvironment, could promote or inhibit mammary tumors. In this review we re-examine the mechanisms by which nitric oxide promotes initiation and progression of breast cancer and address some of the controversies in the field.

Nitric oxide has varied effects on human breast cancer cells. At low concentration (micromolar range) it increases proliferation by increasing synthesis of some cells cycle protein and in higher concentration (nanomolar range) it leads to cytostasis or apoptosis by decreasing the translation of some cell cycle proteins.

Protein synthesis is a tightly controlled process, and its deregulation plays an important role in tumorigenesis. Protein synthesis remains poorly understood with very few well-identified validated targets for therapeutic purposes. In this study, we use nitric oxide (NO), which suppresses protein synthesis by inactivating eukaryotic initiation factor 2-alpha (eIF2-alpha), to examine the mechanism by which low and high oxidative stress inhibits protein synthesis. In breast cancer cells, low NO stress induced heme-regulated inhibitor (HRI) activation, which facilitated gradual decline in short half-life proteins. High NO stress induced HRI and protein kinase R (PKR) activation, leading to a sharp decline in protein synthesis as accessed by a decline in short and long half-life proteins and dramatic morphologic changes. In contrast, human mammary epithelial (HME) and Ras transfected untransformed HME (MCF-10A1 neo N) cells were less susceptible to NO-induced inhibition of protein synthesis and cytostasis. Our results suggest that NO-induced cytostasis in breast cancer cells was due to PKR activation and increased phosphorylation of eIF2-alpha, whereas the reduced susceptibility of normal mammary epithelial cells to NO could be due to the inaccessibility of PKR, which is bound to inhibitor p58.

Nitric oxide (NO) in nanomolar (nmol/L) concentrations is consistently detected in tumor microenvironment and has been found to promote tumorigenesis. The mechanism by which NO enhances tumor progression is largely unknown. In this study, we investigated the possible mechanisms and identified cellular targets by which NO increases proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7. DETA-NONOate, a long acting NO donor, with a half-life of 20 h, was used. We found that NO (nmol/L) dramatically increased total protein synthesis in MDA-MB-231 and MCF-7 and also increased cell proliferation. NO specifically increased the translation of cyclin D1 and ornithine decarboxylase (ODC) without altering their mRNA levels or half-lives. Critical components in the translational machinery, such as phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets, phosphorylated eukaryotic translation initiation factor and p70 S6 kinase, were up-regulated following NO treatment, and inhibition of mTOR with rapamycin attenuated NO induced increase of cyclin D1 and ODC. Activation of translational machinery was mediated by NO-induced up-regulation of the Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK (Raf/MEK/ERK) and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt signaling pathways. Up-regulation of the Raf/MEK/ERK and PI-3 kinase/Akt pathways by NO was found to be mediated by activation of Ras, which was cyclic guanosine 3',5'-monophosphate independent. Furthermore, inactivation of Ras by farnesyl transferase inhibitor or K-Ras small interfering RNA attenuated NO-induced increase in proliferation signaling and cyclin D1 and ODC translation, further confirming the involvement of Ras activation during NO-induced cell proliferation.

Apoptosis is regulated by a series of biochemical events that commits a cell to death. We are interested in understanding and have been investigating the mechanisms by which nitric oxide (NO) induces apoptosis in human breast cancer cell lines. In this study, we investigated the possible interplay of extracellular signal-regulated kinase (ERK) and Akt pathways in NO-induced apoptosis. MKP-1 transcripts were induced in these cells as early as 4 h, peaking at 8 h leading to inactivation of ERK1/2 at 16-24 h after exposure to NO. We also found 50% decrease in the levels pAkt at 24 h of DETA-NONOate treatment. The inactivation of ERK1/2 preceded the dephosphorylation of Akt and apoptosis. NO was not able to inactivate ERK1/2 or Akt or to induce apoptosis in the presence of a phosphatase inhibitor, sodium orthovanadate, or antisense oligonucleotides, suggesting a cross-talk between the two pathways. NO also up-regulated MKP-1 in another breast cancer cell line, ZR 75-30, which led to inactivation of ERK1/2 and induced apoptosis. In MDA-MB-231, NO did not induce MKP-1, and there was no ERK inactivation or apoptosis. Our results indicate that expression of MKP-1 by NO leading to dephosphorylation of ERK1/2 is the initial essential event that commits the cells to the apoptotic pathway in breast cancer cells.

BackgroundBreast cancer is a complex heterogeneous disease where many distinct subtypes are found. Younger African American (AA) women often present themselves with aggressive form of breast cancer with unique biology which is very difficult to treat. Better understanding the biology of AA breast tumors could lead to development of effective treatment strategies. Our previous studies indicate that AA but not Caucasian (CA) triple negative (TN) breast cancer cells were sensitive to nitrosative stress-induced cell death. In this study, we elucidate possible mechanisms that contribute to nitric oxide (NO)-induced apoptosis in AA TN breast cancer cells.MethodsBreast cancer cells were treated with various concentrations of long-acting NO donor, DETA-NONOate and cell viability was determined by trypan blue exclusion assay. Apoptosis was determined by TUNEL and caspase 3 activity as well as changes in mitochondrial membrane potential. Caspase 3 and Bax cleavage, levels of Cu/Zn superoxide dismutase (SOD) and Mn SOD was assessed by immunoblot analysis. Inhibition of Bax cleavage by Calpain inhibitor, and levels of reactive oxygen species (ROS) as well as SOD activity was measured in NO-induced apoptosis. In vitro and in vivo effect of NO treatment on mammary cancer stem cells (MCSCs) was assessed.Results and discussionNO induced mitocondria-mediated apoptosis in all AA but not in CA TN breast cancer cells. We found significant TUNEL-positive cells, cleavage of Bax and caspase-3 activation as well as depolarization mitochondrial membrane potential only in AA TN breast cancer cells exposed to NO. Inhibition of Bax cleavage and quenching of ROS partially inhibited NO-induced apoptosis in AA TN cells. Increase in ROS coincided with reduction in SOD activity in AA TN breast cancer cells. Furthermore, NO treatment of AA TN breast cancer cells dramatically reduced aldehyde dehydrogenase1 (ALDH1) expressing MCSCs and xenograft formation but not in breast cancer cells from CA origin.ConclusionsEthnic differences in breast tumors dictate a need for tailoring treatment options more suited to the unique biology of the disease.

Vitamin D signaling in mammary cancer stem cells (MCSCs), which are implicated in the initiation and progression of breast cancer, is poorly understood. In this study, we examined vitamin D signaling in mammospheres which are enriched in MCSCs from established breast cancer cell lines. Breast cancer cells positive for aldehyde dehydrogenase (ALDH(+)) had increased ability to form mammospheres compared to ALDH(-) cells. These mammospheres expressed MCSC-specific markers and generated transplantable xenografts in nude mice. Vitamin D receptor (VDR) was significantly down-regulated in mammospheres, as well as in ALDH(+) breast cancer cells. TN aggressive human breast tumors as well as transplantable xenografts obtained from SKBR3 expressed significantly lower levels of VDR but higher levels of CD44 expression. Snail was up-regulated in mammospheres isolated from breast cancer cells. Inhibition of VDR expression by siRNA led to a significant change in key EMT-specific transcription factors and increased the ability of these cells to form mammospheres. On the other hand, over-expression of VDR led to a down-regulation of Snail but increased expression of E-cad and significantly compromised the ability of cells to form mammospheres. Mammospheres were relatively insensitive to treatment with 1,25-dihydroxyvitamin D (1,25D), the active form of vitamin D, compared to more differentiated cancer cells grown in presence of serum. Treatment of H-Ras transformed HMLE(HRas) cells with DETA NONOate, a nitric oxide (NO)-donor led to induction of MAP-kinase phosphatase -1 (MKP-1) and dephosphorylation of ERK1/2 in the mammospheres. Combined treatment of these cells with 1,25D and a low-concentration of DETA NONOate led to a significant decrease in the overall size of mammospheres and reduced tumor volume in nude mice. Our findings therefore, suggest that combination therapy using 1,25D with drugs specifically targeting key survival pathways in MCSCs warrant testing in prospective clinical trial for treatment of aggressive breast cancer.

Abstract: Nitric oxide has varied effects on human breast cancer cells. At low concentration (micromolar range) it increases proliferation by increasing synthesis of some cells cycle protein and in higher concentration (nanomolar range) it leads to cytostasis or apoptosis by decreasing the translation of some cell cycle proteins. [Copyright &y& Elsevier]

Studies the nitric oxide (NO)-induced cytostasis and cell cycle arrest of a human breast cancer cell line, MDA-MB-231). Role of the down-regulation of cyclin D1 protein by DETA-NONOate, an NO donor, in the cytostasis and arrest of arrest of tumor cells in the G1 phase of the cell cycle; Measurement of rate of NO release by DETA-NONOate.

Determines whether induction of nitric oxide synthase (NOS) by lipopolysaccharide and cytokines is accompanied by sufficient arginase induction to render arginine concentration rate limiting for high-output NO production. Conditions for presence of arginase and NOS in unactivated and activated endothelial cells; Influence of arginine concentration.