Norma E. López-Diazguerrero

Senescence associated secretory phenotype profile from primary lung mice fibroblasts depends on the senescence induction stimuli

Cellular senescence is a multifactorial phenomenon of growth arrest and distorted function, which has been recognized as an important feature during tumor suppression mechanisms and a contributor to aging. Senescent cells have an altered secretion pattern called Senescence-Associated Secretory Phenotype (SASP) that comprises a complex mix of factors including cytokines, growth factors, chemokines, and matrix metalloproteinases. SASP has been related with local inflammation that leads to cellular transformation and neurodegenerative diseases. Various pathways for senescence induction have been proposed; the most studied is replicative senescence due to telomere attrition called replicative senescence (RS). However, senescence can be prematurely achieved when cells are exposed to diverse stimuli such as oxidative stress (stress-induced premature senescence, SIPS) or proteasome inhibition (proteasome inhibition-induced premature senescence, PIIPS). SASP has been characterized in RS and SIPS but not in PIIPS. Hence, our aim was to determine SASP components in primary lung fibroblasts obtained from CD-1 mice induced to senescence by PIIPS and compare them to RS and SIPS. Our results showed important variations in the 62 cytokines analyzed, while SIPS and RS showed an increase in the secretion of most cytokines, and in PIIPS only 13 were incremented. Variations in glutathione-redox balance were also observed in SIPS and RS, and not in PIIPS. All senescence types SASP displayed a pro-inflammatory profile and increased proliferation in L929 mice fibroblasts exposed to SASP. However, the behavior observed was not exactly the same, suggesting that the senescence induction pathway might encompass dissimilar responses in adjacent cells and promote different outcomes.

New considerations on hormetic response against oxidative stress

In order to survive living organisms have developed multiple mechanisms to deal with tough environmental conditions. Hormesis is defined as a process in which exposure to a low dose of a chemical agent or environmental factor that is damaging at higher doses induces an adaptive beneficial effect on the cell or organism. In this paper, we examine several ideas that might be taken into consideration before using hormesis as a therapeutic tool to improve health and life span, and hopefully will open the discussion for new and interesting debates regard hormesis. The first one is to understand that the same stressor or inductor can activate different pathways in a parallel or dual response, which might lead to diverse outcomes. Another idea is related to the mechanisms involved in activating Nrf2, which might be different and have diverse hormetic effects.Last, we discuss mild oxidative stress in association to low-grade chronic inflammation as a stimulating avenue to be explored and the unexpected effects proposed by the obesity paradox theory. All the previous might help to clarify the reasons why centenarians are able to reach the extreme limits of human life span, which could probably be related to the way they deal with homeostasis maintenance, providing an opportunity for hormesis to intervene significantly.

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.

Biochemical Alterations during the Obese-Aging Process in Female and Male Monosodium Glutamate (MSG)-Treated Mice

Obesity, from children to the elderly, has increased in the world at an alarming rate over the past three decades, implying long-term detrimental consequences for individual’s health. Obesity and aging are known to be risk factors for metabolic disorder development, insulin resistance and inflammation, but their relationship is not fully understood. Prevention and appropriate therapies for metabolic disorders and physical disabilities in older adults have become a major public health challenge. Hence, the aim of this study was to evaluate inflammation markers, biochemical parameters and glucose homeostasis during the obese-aging process, to understand the relationship between obesity and health span during the lifetime. In order to do this, the monosodium glutamate (MSG) obesity mice model was used, and data were evaluated at 4, 8, 12, 16 and 20 months in both female and male mice. Our results showed that obesity was a major factor contributing to premature alterations in MSG-treated mice metabolism; however, at older ages, obesity effects were attenuated and MSG-mice became more similar to normal mice. At a younger age (four months old), the Lee index, triglycerides, total cholesterol, TNF-α and transaminases levels increased; while adiponectin decreased and glucose tolerance and insulin sensitivity levels were remarkably altered. However, from 16 months old-on, the Lee index and TNF-α levels diminished significantly, while adiponectin increased, and glucose and insulin homeostasis was recovered. In summary, MSG-treated obese mice showed metabolic changes and differential susceptibility by gender throughout life and during the aging process. Understanding metabolic differences between genders during the lifespan will allow the discovery of specific preventive treatment strategies for chronic diseases and functional decline.

Senescent phenotype achieved in vitro is indistinguishable, with the exception of Bcl-2 content, from that attained during the in vivo aging process

Senescent phenotype can be attained by diverse agents, thus suggesting that there might be molecular differences between the senescence achieved in vivo and the senescence‐like state attained in vitro under culture conditions. In this study we compare the senescent phenotype reached by cells derived from young animals when cultured in vitro with the one associated with the in vivo aging process. Several in vitro senescence parameters, including MTT reduction, proliferation rate, DNA synthesis, SA‐β‐gal staining, and both in vivo and in vitro Bcl‐2 content, were determined. Alterations in DNA electrophoretic mobility were evaluated to test differences in bulk chromatin structure. Our results indicate that although it is possible to achieve a senescent phenotype with cells derived from young animals aged in culture, this phenotype differs from the one observed in older animals, due to lack of in vivo damage inducers to which cells are being exposed during natural aging.

Msh2 promoter region hypermethylation as a marker of aging-related deterioration in old retired female breeder mice

Aging is a process where individuals decrease the performance of their physiological systems and cellular stress response, making them more susceptible to disease and death. The increase in DNA damage associated with age might be recognized as the accumulation of physiological and environmentally induced mutations accompanied with a decline in DNA repair. DNA mismatch repair (MMR) is the main postreplicative correction pathway, which is known to decrease with age. However, since infrequent occurrence of direct DNA damage contrasts with the extensive cell and tissue dysfunction seen in older individuals, the withdrawing of DNA-repairing systems might be also related to epigenetic changes, such as DNA methylation. It has been reported that the physiological stress related to breeding might accelerate the acquisition of aging-related markers; therefore, the aim of this work was to link age with epigenetic modifications in this animal population. Hence, the correlation of Msh2 gene silencing with the deterioration of breeding female mice associated to aging was determined. Combined bisulfite restriction analysis assay was used to compare methylation on DNA isolated from twelve-month-old retired breeders against nulliparous female mice aged-matched, and two-month-old young adults. Our experiments clearly reveal Msh2 promoter hypermethylation associated to the aging process. A higher degree methylation was additionally observed in breeding females DNA. Nevertheless, this additional methylation did not correlate with a further decrease Msh2 mRNA, suggesting that the increase in methylation in old retired breeder might account for further epigenetic changes that could additionally promote the aging process.

Telomerase activity in response to mild oxidative stress.

We have analysed telomerase activity to determine whether it can be modified when BCL‐2 is endogenously overexpressed in response to a mild oxidative stress treatment as part of a survival mechanism, in contrast with an exogenous bcl‐2 overexpression due to a retroviral infection. Endogenous bcl‐2 overexpression was induced after a low oxidative insult of H2O2 in mice primary lung fibroblasts and L929 cell, whereas bcl‐2 exogenous overexpression was performed using a retroviral infection in L929 cells. Telomerase activity was quantified in Bcl‐2 overexpressing cells by the TRAP assay. When the cells were treated with different H2O2 concentrations, only those exposed to 50 μM showed increased telomerase activity. This correlates with BCL‐2 expression as part of the endogenous response to mild oxidative stress. Oxidative stress generated during the toxic mechanism of chemotherapeutic drugs might induce BCL‐2 increment, enhancing telomerase activity and reactivating the oncogenic process. Clinical trials should take into consideration the possibility of telomerase activation following increased BCL‐2 expression when treating patients with ROS (reactive oxygen species) generation by anti‐cancer drugs.

Cell proliferation arrest and redox state status as part of different stages during senescence establishment in mouse fibroblasts

Senescence phenotype can be achieved by multiple pathways. Most of them involve the activation of negative cell cycle regulators as well as a shift to an oxidative status. However, the exact participation of these events in senescence establishment and maintenance is not completely understood. In this study we investigated the content of three final cell cycle regulators, as well as the redox state in some critical points during the pre-senescent and the full-senescent states. Our results highlight the existence of a critical pre-phase in senescent phenotype establishment, in which cell proliferation stops with the participation of the cell cycle inhibitors, and a second maintenance stage where the exacerbated pro-oxidant state inside the cell induces the physiological decline characteristic in senescent cells.

Relationship of inflammatory profile of elderly patients serum and senescence-associated secretory phenotype with human breast cancer cells proliferation: Role of IL6/IL8 ratio

HIGHLIGHTSOnly certain elderly patients serum were capable to induce MCF‐7 proliferation.The SASP from WI‐38‐senescent cells also induced MCF‐7 cellular proliferation.An inflammatory profile is required to induce MCF‐7 cellular proliferation.An IL‐6/IL 8 specific ratio is required within the inflammatory profile.Only EPS‐IP and IL‐6/IL‐8 ratio 3.0 treatment increased Cyclin D1 expression in MCF‐7 cells. ABSTRACT Aging is considered a systemic, chronic and low‐grade inflammatory state, called “inflammaging”, which has been contemplated as a risk factor for cancer development and progression in the elderly population. Cellular senescence is a multifactorial phenomenon of growth arrest and distorted function, which has been recognized as a contributor to aging. Senescent cells have an altered secretion pattern called Senescent Associated Secretory Phenotype (SASP), that comprise a complex mix of factors including cytokines, growth factors, chemokines and matrix metalloproteinases among others. The SASP secreted by accumulated senescent cells during old age has been related to local inflammation that leads to cellular transformation and therefore may be supporting the inflammaging process. Here, we evaluated if the pro‐inflammatory profile within the serum obtained from elderly patients (EPS) was able to induce cellular proliferation in the breast cancer transformed cell line (MCF‐7), in a similar way to the proliferation stimulated by the SASP obtained from WI‐38 primary cells prematurely induced to senescence by oxidative stress (SIPS). At the same time, the participation of IL‐6/IL‐8 ratio was determined. Our results showed that not all the EPS increased MCF‐7 proliferation. However, there was an interesting relationship between IL‐6 and IL‐8 concentrations, when the IL‐6 was higher than IL‐8. Similar results were found with SASP from SIPS‐WI‐38 on the MCF‐7 proliferation. Although it is known that those cytokines are fundamental factors to induce proliferation; the occurrence of other components in the cellular microenvironment is necessary to carry out this effect.

Cellular Senescence, Neurological Function, and Redox State

SIGNIFICANCE Cellular senescence, characterized by permanent cell cycle arrest, has been extensively studied in mitotic cells such as fibroblasts. However, senescent cells have also been observed in the brain. Even though it is recognized that cellular energetic metabolism and redox homeostasis are perturbed in the aged brain and neurodegenerative diseases (NDDs), it is still unknown which alterations in the overall physiology can stimulate cellular senescence induction and their relationship with the former events. Recent Advances: Recent findings have shown that during prolonged inflammatory and pathologic events, the blood-brain barrier could be compromised and immune cells might enter the brain; this fact along with the brains high oxygen dependence might result in oxidative damage to macromolecules and therefore senescence induction. Thus, cellular senescence in different brain cell types is revised here. CRITICAL ISSUES Most information related to cellular senescence in the brain has been obtained from research in glial cells since it has been assumed that the senescent phenotype is a feature exclusive to mitotic cells. Nevertheless, neurons with senescence hallmarks have been observed in old mouse brains. Therefore, although this is a controversial topic in the field, here we summarize and integrate the observations from several studies and propose that neurons indeed senesce. FUTURE DIRECTIONS It is still unknown which alterations in the overall metabolism can stimulate senescence induction in the aged brain, what are the mechanisms and signaling pathways, and what is their relationship to NDD development. The understanding of these processes will expose new targets to intervene age-associated pathologies.-Antioxid. Redox Signal. 28, 1704-1723.