Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen-and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-B (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-B, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-B was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-B. Analysis of individual subunits of NF-B revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IB␣, -␤, and -␥ were also observed. In transient-transfection experiments, constitutive activity of an NF-B-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-B in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-B-regulated genes. Furthermore, since recent data indicate that NF-B protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.Numerous experimental and clinical studies have established that estrogen plays a major role in the initiation and progression of breast cancers (37,44,49). Estrogen receptor (ER), which mediates the action of estrogen, is expressed in ϳ70% of breast cancers and is required for estrogen-dependent growth of breast cancers. As the cancer progresses, tumor cells acquire growth autonomy, no longer require estrogen, and become resistant to antiestrogens such as tamoxifen. Tumor heterogeneity and clonal selection of ER-negative cells, downregulation of ER expression, mutations of ER, altered regulation of ER-responsive genes, and generation of ER variants that function as dominant positive and estrogen-independent transcription factor are believed to be responsible for antiestrogen-resistant...

UK Efficacy and Mechanism Evaluation Programme and National Institutes of Health.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway elicits a survival signal against multiple apoptotic insults. In addition, phase II enzymes such as heme oxygenase-1 (HO-1) protect cells against diverse toxins and oxidative stress. In this work, we describe a link between these defense systems at the level of transcriptional regulation of the antioxidant enzyme HO-1. The herb-derived phenol carnosol induced HO-1 expression at both mRNA and protein levels. Luciferase reporter assays indicated that carnosol targeted the mouse ho1 promoter at two enhancer regions comprising the antioxidant response elements (AREs). Moreover, carnosol increased the nuclear levels of Nrf2, a transcription factor governing AREs. Electrophoretic mobility shift assays and luciferase reporter assays with a dominantnegative Nrf2 mutant indicated that carnosol increased the binding of Nrf2 to ARE and induced Nrf2-dependent activation of the ho1 promoter. While investigating the signaling pathways responsible for HO-1 induction, we observed that carnosol activated the ERK, p38, and JNK pathways as well as the survival pathway driven by PI3K. Inhibition of PI3K reduced the increase in Nrf2 protein levels and activation of the ho1 promoter. Expression of active PI3K-CAAX (where A is aliphatic amino acid) was sufficient to activate AREs. The use of dominant-negative mutants of protein kinase C and Akt1, two kinases downstream from PI3K, demonstrated a requirement for active Akt1, but not protein kinase C. Moreover, the long-term antioxidant effect of carnosol was partially blocked by PI3K or HO-1 inhibitors, further demonstrating that carnosol attenuates oxidative stress through a pathway that involves PI3K and HO-1.High levels of reactive oxygen species cause damage to cells and are involved in several human pathologies, including neurodegenerative disorders and cancer (1, 2). Therefore, the use of compounds with antioxidant properties may help prevent or alleviate diseases in which oxidative stress is a primary cause (3). Carnosol, a diterpene derived from the herb rosemary, is a representative member of a family of plant-derived phenols, which also include curcumin, carnosic acid, phenylethyl isothiocyanate, epigallocatechin gallate, and other green tea polyphenols. These bioactive phytochemicals exhibit Michael acceptor function and therefore behave as antioxidants (4). In addition, being themselves xenobiotic compounds, they activate a xenobiotic response in the target cells affecting the expression of phase II enzymes such as NAD(P)H:quinone oxidoreductase, aldoketoreductase, glutathione S-transferase, ␥-glutamylcysteine synthetase, glutathione synthetase, and heme oxygenase-1 (HO-1) 1 (5-7). Heme oxygenase isozymes (HO-1 and HO-2) catalyze the stepwise degradation of heme to release free iron and equimolar concentrations of carbon monoxide and the linear tetrapyrrole biliverdin, which is converted to bilirubin by the enzyme biliverdin reductase (8). The HO-1 isozyme is a phase II enzyme that is transcriptionally regulated by a large...

These experiments examined the influence of exercise intensity and duration on antioxidant enzyme activity in locomotor muscles differing in fiber type composition. Nine groups of female Sprague-Dawley rats (age 120 days) exercised 4 days/wk on a motor-driven treadmill for 10 wk. The impact of three levels of exercise intensity (low, moderate, and high: approximately 55, approximately 65, and approximately 75% of maximal oxygen consumption, respectively) and exercise duration (30, 60, and 90 min/day) was assessed. Sedentary animals served as controls. Oxidative capacity in the soleus and white and red gastrocnemius was assessed by measurement of citrate synthase (CS) activity, and antioxidant capacity was evaluated by assay of total superoxide dismutase, catalase, and total glutathione peroxidase (GPX) activities. In all muscles, CS activity increased as a function of exercise duration. Furthermore, in the soleus and white gastrocnemius, the magnitude of the training-induced increase in CS activity was directly related to exercise intensity. In contrast, the peak increase in CS activity in the red gastrocnemius was relatively independent of exercise intensity. Catalase activity was not increased (P > 0.05) in any muscle with training. Training-induced changes in superoxide dismutase and GPX activities were muscle specific; specifically, exercise training significantly (P < 0.05) increased superoxide dismutase activity in the soleus as a function of exercise duration up to 60 min/day. Conversely, training-induced significant (P < 0.05) increases in GPX activity occurred in red gastrocnemius only; the magnitude of the GPX increase was directly related to exercise duration but relatively independent of intensity. These data demonstrate that exercise training-induced changes in muscle antioxidant enzymes are muscle specific.

Acquisition of CCR7 expression is an important phenotype change during dendritic cell (DC) maturation that endows these cells with the capability to migrate to lymph nodes. We have analyzed the possible role of CCR7 on the regulation of the survival of DCs. Stimulation with CCR7 ligands CCL19 and CCL21 inhibits apoptotic hallmarks of serum-deprived DCs, including membrane phosphatidylserine exposure, loss of mitochondria membrane potential, increased membrane blebs, and nuclear changes. Both chemokines induced a rapid activation of phosphatidylinositol 3-kinase/Akt1 (PI3K/Akt1), with a prolonged and persistent activation of Akt1. Interference with PI3K, Gi, or G protein ␤␥ subunits abrogated the effects of the chemokines on Akt1 activation and on survival. In contrast, inhibition of extracellular signal-related kinase 1/2 (Erk1/2), p38, or c-Jun N-terminal kinase (JNK) was ineffective. Nuclear factor-B (NFB) was involved in the antiapoptotic effects of chemokines because inhibition of NFB blunted the effects of CCL19 and CCL21 on survival. Furthermore, chemokines induced down-regulation of the NFB inhibitor IB, an increase of NFB DNA-binding capability, and translocation of the NFB subunit p65 to the nucleus. In summary, in addition to its well-established role in chemotaxis, we show that CCR7 also induces antiapoptotic signaling in mature DCs. IntroductionApoptosis, or programmed cell death, is a physiologic process involved in the normal development and maintenance of tissue homeostasis. 1 The final stage of this process that leads to the demise of the cell is executed by proteases that degrade vital molecular components of the cell. 1 Hallmarks of cells undergoing apoptosis include disruption of mitochondria transmembrane potential, apparition of numerous blebs on the membrane, increased nuclear condensation, and increased appearance of phosphatidylserine (PS) in the outer leaflet of the cell membrane.Apoptosis is a programmed process that is regulated through a complex mechanism that involves multiple molecular intermediates. Surface receptors may inhibit apoptosis by relaying intracellular signals that either repress proapoptotic molecules and/or stimulate antiapoptotic ones. 1 Multiple pathways that inhibit apoptosis use as a common signaling intermediate phosphatidylinositol 3Ј-kinase (PI3K) and its downstream effector Akt1. 1-3 Akt1 phosphorylates and inhibits a variety of proapoptotic regulators and also regulates proteins that promote cell survival. [1][2][3] In this regard, it has been shown that Akt1 may activate IB kinase, which induces phosphorylation and subsequent degradation of IB, a molecule that binds and retains transcription factor nuclear factor-B (NFB) in the cytoplasm. 1-3 Upon IB degradation, NFB translocates to the nucleus and stimulates transcription from a variety of antiapoptotic genes. 2,4 Apart from PI3K/Akt1, in some cell settings, mitogen-activated protein kinase (MAPK) family members have also been shown to play an important role as regulators of apoptosis. [5][6][7] Dendritic ...