Sera from women with different metabolic and menopause states differentially regulate cell viability and Akt activation in a breast cancer in-vitro model.

Obesity is associated with an increased incidence and aggressiveness of breast cancer and is estimated to increment the development of this tumor by 50 to 86%. These associations are driven, in part, by changes in the serum molecules. Epidemiological studies have reported that Metformin reduces the incidence of obesity-associated cancer, probably by regulating the metabolic state. In this study, we evaluated in a breast cancer in-vitro model the activation of the IR-ß/Akt/p70S6K pathway by exposure to human sera with different metabolic and hormonal characteristics. Furthermore, we evaluated the effect of brief Metformin treatment on sera of obese postmenopausal women and its impact on Akt and NF-κB activation. We demonstrated that MCF-7 cells represent a robust cellular model to differentiate Akt pathway activation influenced by the stimulation with sera from obese women, resulting in increased cell viability rates compared to cells stimulated with sera from normal-weight women. In particular, stimulation with sera from postmenopausal obese women showed an increase in the phosphorylation of IR-ß and Akt proteins. These effects were reversed after exposure of MCF-7 cells to sera from postmenopausal obese women with insulin resistance with Metformin treatment. Whereas sera from women without insulin resistance affected NF-κB regulation. We further demonstrated that sera from post-Metformin obese women induced an increase in p38 phosphorylation, independent of insulin resistance. Our results suggest a possible mechanism in which obesity-mediated serum molecules could enhance the development of luminal A-breast cancer by increasing Akt activation. Further, we provided evidence that the phenomenon was reversed by Metformin treatment in a subgroup of women.

Glucocorticoid-dependent expression of IAP participates in the protection against TNF-mediated cytotoxicity in MCF7 cells.

BACKGROUND: Glucocorticoid receptor (GR) activation has been associated with breast cancer cell survival in vitro. Glucocorticoid (GC)-dependent protection against tumor necrosis factor (TNF)-induced cell death has been well characterized in MCF7 luminal A breast cancer cells. The GR activates a variety of protective mechanisms, such as inhibitors of apoptosis proteins (IAPs). However, the relative contribution of the GR-dependent expression of IAPs in the protection of cell death has not, to our knowledge, been evaluated. METHODS: MCF7 cells were used for all experiments. GR was activated with cortisol (CORT) or dexamethasone (DEX) and inhibited with mifepristone (RU486). Cell viability was determined in real-time with the xCELLigence™ RTCA System and at specific endpoints using crystal violet stain. The mRNA levels of the eight members of the IAP family were measured by qRT-PCR. The protein levels of GR, PR, ERα, HER2, PARP1, c-IAP1 and XIAP were evaluated by Western blot analysis. The knockdown of c-IAP1 and XIAP was accomplished via transient transfection with specific siRNAs. GR activation was verified by a gene reporter assay. Via the cBioportal interphase we queried the mRNA levels of GR and IAPs in breast cancer tumors. RESULTS: RU486 significantly inhibited the anti-cytotoxic effect of both GCs. PARP1 processing was diminished in the presence of both GCs. The combined treatments of GCs + TNF increased the relative mRNA levels of Survivin>c-IAP1 > NAIP>Apollon>XIAP>Ts-IAP > ML-IAP > c-IAP2. Additionally, GR mRNA content increased with the combined treatments of GCs + TNF. Sustained levels of the proteins c-IAP1 and XIAP were observed after 48 h of the combined treatments with GCs + TNF. With c-IAP1 and XIAP gene silencing, the GC-mediated protection was diminished. In the breast tumor samples, the GR mRNA was coexpressed with Apollon and XIAP with a Pearson coefficient greater than 0.3. CONCLUSIONS: The effect of GCs against TNF-mediated cytotoxicity involves increased mRNA expression and sustained protein levels of c-IAP1 and XIAP. The antagonist effects of RU486 and the qRT-PCR results also suggest the role of the GR in this process. This finding may have clinical implications because the GR and IAPs are expressed in breast tumor samples.

Primary cultured astrocytes from old rats are capable to activate the Nrf2 response against MPP+ toxicity after tBHQ pretreatment.

Astrocytes are key players for brain physiology, protecting neurons by releasing antioxidant enzymes; however, they are also susceptible to damage by neurotoxins. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a central regulator of the antioxidant response, and therefore, pharmacologic inducers are often used to activate this transcription factor to induce cellular protection. To date, it still remains unknown if cells from aged animals are capable of developing this response. Therefore, the purpose of this work was to determine if cortical astrocytes derived from old rats are able to respond to tertbuthyl-hydroquinene (tBHQ) pretreatment and stimulate the Nrf2-antioxidant response pathway to induce an antioxidant strategy against MPP+ toxicity, one of the most used molecules to model Parkinson's disease. Our results show that, although astrocytes from adult and old rats were more susceptible to MPP+ toxicity than astrocytes from newborn rats, when pretreated with tertbuthyl-hydroquinene, they were able to transactivate Nrf2, increasing antioxidant enzymes and developing cellular protection. These results are discussed in terms of the doses used to create protective responses.

HSP-72 accelerated expression in mononuclear cells induced in vivo by acetyl salicylic acid can be reproduced in vitro when combined with H2O2.

BACKGROUND: Among NSAIDs acetyl salicylic acid remains as a valuable tool because of the variety of benefic prophylactic and therapeutic effects. Nevertheless, the molecular bases for these responses have not been complete understood. We explored the effect of acetyl salicylic acid on the heat shock response. RESULTS: Peripheral blood mononuclear cells from rats challenged with acetyl salicylic acid presented a faster kinetics of expression of HSP-72 messenger RNA and protein in response to in vitro heat shock. This effect reaches its maximum 2 h after treatment and disappeared after 5 h. On isolated peripheral blood mononuclear cells from untreated rats, incubation with acetyl salicylic acid was ineffective to produce priming, but this effect was mimicked when the cells were incubated with the combination of H2O2+ ASA. CONCLUSIONS: Administration of acetyl salicylic acid to rats alters HSP-72 expression mechanism in a way that it becomes more efficient in response to in vitro heat shock. The fact that in vitro acetyl salicylic acid alone did not induce this priming effect implies that in vivo other signals are required. Priming could be reproduces in vitro with the combination of acetyl salicylic acid+H2O2.

A noncanonical NF-κB pathway through the p50 subunit regulates Bcl-2 overexpression during an oxidative-conditioning hormesis response.

Cells can respond to damage and stress by activating various repair and survival pathways. One of these responses can be induced by preconditioning the cells with sublethal stress to provoke a prosurvival response that will prevent damage and death, and which is known as hormesis. Bcl-2, an antiapoptotic protein recognized by its antioxidant and prosurvival functions, has been documented to play an important role during oxidative-conditioning hormesis. Using an oxidative-hormetic model, which was previously established in the L929 cell line by subjecting the cells to a mild oxidative stress of 50 µM H2O2 for 9 h, we identified two different transductional mechanisms that participate in the regulation of Bcl-2 expression during the hormetic response. These mechanisms converge in activating the nuclear transcription factor NF-κB. Interestingly, the noncanonical p50 subunit of the NF-κB family is apparently the subunit that participates during the oxidative-hormetic response.

Glycine suppresses TNF-α-induced activation of NF-κB in differentiated 3T3-L1 adipocytes.

Glycine strongly reduces the serum levels of pro-inflammatory cytokines and increases the levels of anti-inflammatory cytokines. Recently, glycine has been shown to decrease the expression and secretion of pro-inflammatory adipokines in monosodium glutamate-induced obese (MSG/Ob) mice. It has been postulated that these effects may be explained by a reduction in nuclear factor kappa B (NF-κB) activation. NF-κB is a transcription factor, which is crucial to the inflammatory response. Hasegawa et al. (2011 and 2012) recently reported a glycine-dependent reduction in NF-κB levels. Here, we have investigated the role of glycine in the regulation of NF-κB in differentiated 3T3-L1 adipocytes. The results revealed that pretreatment with glycine interfered with the activation of NF-κB, which has been shown to be stimulated by tumor necrosis factor-alpha (TNF-α). Glycine alone stimulated NF-κB activation in an unusual way such that the inhibitor κB-ß (IκB-ß) degradation was more significant than that of the inhibitor κB-α (IκB-α) and led to NF-κB complexes comprised of p50 and p65 subunits; IκB-ε degradation did not affect by glycine. These findings suggest that glycine could be used as an alternative treatment for chronic inflammation, which is a hallmark of obesity and other comorbidities, and is characterized by an elevated production of pro-inflammatory cytokines.

Bcl-2 overexpression in hepatic stellate cell line CFSC-2G, induces a pro-fibrotic state.

BACKGROUND AND AIM: Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro- and anti-apoptotic molecules. Since Bcl-2 overexpression preserves viability against OS, our objective was to address the effect of Bcl-2 overexpression in the hepatic stellate cells (HSC) cell-line CFSC-2G under acetaldehyde and H(2)O(2) challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. METHODS: To induce Bcl-2 overexpression, HSC cell line CFSC-2G was transfected by lipofection technique. Green fluorescent protein-only CFSC-2G cells were used as a control. Cell survival after H(2)O(2) treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation-rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue-inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a-actin (alpha-SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor-beta (TGF-beta) mRNA. RESULTS: Cells overexpressing Bcl-2 survived approximately 20% more than control cells when exposed to H(2)O(2) and approximately 35% proteins were protected from oxidation, but Bcl-2 did not slow proliferation or induced senescence. Bcl-2 overexpression did not change alpha-SMA levels, but it increased TIMP-1 (55%), tissue transglutaminases (tTG) (25%) and TGF-beta mRNA (49%), when exposed to acetaldehyde, while MMP-13 content decreased (47%). CONCLUSIONS: Bcl-2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP-1, tTG and TGF-beta mRNA levels and decreased MMP-13 content, suggesting that Bcl-2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases.

Bcl-2 sustains hormetic response by inducing Nrf-2 nuclear translocation in L929 mouse fibroblasts.

Hormesis is the process whereby exposure to a low dose of a chemical agent induces an adaptive effect on the cell or organism. This response evokes the expression of cytoprotective and antioxidant proteins, allowing pro-oxidants to emerge as important hormetic agents. The antiapoptotic protein Bcl-2 is known to protect cells against death induced by oxidants; it has been suggested that Bcl-2 might also modulate steady-state reactive oxygen species levels. The aim of this work was to find out if Bcl-2 might play a role during the hormetic response and in Nrf-2 activation. We have established a model to study the oxidative conditioning hormesis response (OCH) by conditioning the cell line L929 with 50muM H(2)O(2) for 9h. This condition did not induce oxidative damage nor oxidative imbalance, and OCH cells maintained a 70-80% survival rate after severe H(2)O(2) treatment compared to nonconditioned cells. When cells were pretreated with the Bcl-2 inhibitor HA14-1 or were silenced with Bcl-2-siRNA, both the hormetic effect and the Nrf-2 nuclear translocation previously observed were abrogated. Our results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and gammaGCS.

Locus on chromosome 6p linked to elevated HDL cholesterol serum levels and to protection against premature atherosclerosis in a kindred with familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (FH) is a highly atherogenic genetic disorder leading to premature coronary heart disease (CHD), usually before 60 years of age. We studied an extended multigenerational kindred with FH linked to chromosome 1p32 in which atherosclerotic complications were either delayed or prevented in individuals with elevated HDL cholesterol (HDL-C) levels or hyperalphalipoproteinemia (HA). Premature CHD was observed in FH individuals without HA. The study of this family established that the HA trait in the family also followed an autosomal dominant mode of inheritance with a pattern of segregation independent from FH. We identified a locus on chromosome 6 linked to elevated HDL-C levels (HA) in this family. Haplotype analysis refined the localization to a 7.32-cM interval (73 to 80 cM from pter) flanked by markers D6S1280 and D6S1275. Parametric 2-point and multipoint analyses yielded maximum LOD scores of 3.05 and 3.17, respectively. This finding was confirmed with a nonparametric multipoint score of 3.78 (P=0.0009). We propose that this locus, linked to elevated HDL-C levels, confers protection against premature CHD within an FH context.