Senescence In Normal Fibroblasts Research Articles

Doxorubicin-induced senescence in normal fibroblasts promotes in vitro tumour cell growth and invasiveness: The role of Quercetin in modulating these processes

Ageing is a complex biological phenomenon representing the major risk factor for developing age-related diseases, such as cardiovascular pathologies, neurodegenerative diseases, and cancer. Geroscience, the new vision of gerontology, identifies cellular senescence as an interconnected biological process that characterises ageing and age-related diseases. Therefore, many strategies have been employed in the last years to reduce the harmful effects of senescence, and among these, the most intriguing ones use nutraceutical compounds. Here we show that a pre-treatment with Quercetin, a bioactive flavonoid present in many fruits and vegetables, increasing cellular antioxidant defence, can alleviate Doxorubicin (Doxo)-induced cellular senescence in human normal WI-38 fibroblasts.Furthermore, our work demonstrates that Quercetin pre-treatment, reducing the number of senescent cells and the production of the senescence-associated secretory phenotype (SASP) factors, can decrease the pro-tumour effects of conditioned medium from Doxo-induced senescent fibroblasts on osteosarcoma cells. Overall, our findings are consistent with the hypothesis that targeting senescent cells can be an emerging strategy for cancer treatment, especially in elderly patients, in which senescent cells are already abundant in several tissues and organs.

Enhanced Activity of Exportin-1/CRM1 in Neurons Contributes to Autophagy Dysfunction and Senescent Features in Old Mouse Brain.

Brain aging is characterized by dysfunctional autophagy and cellular senescence, among other features. While autophagy can either promote or suppress cellular senescence in proliferating cells, in postmitotic cells, such as neurons, autophagy impairment promotes cellular senescence. CRM1 (exportin-1/XPO1) exports hundreds of nuclear proteins into the cytoplasm, including the transcription factors TFEB (the main inducer of autophagy and lysosomal biogenesis genes) and STAT3, another autophagy modulator. It appears that CRM1 is a modulator of aging-associated senescence and autophagy, because pharmacological inhibition of CRM1 improved autophagic degradation in flies, by increasing nuclear TFEB levels, and because enhanced CRM1 activity is mechanistically linked to senescence in fibroblasts from Hutchinson–Gilford progeria syndrome patients and old healthy individuals; furthermore, the exogenous overexpression of CRM1 induced senescence in normal fibroblasts. In this work, we tested the hypothesis that impaired autophagic flux during brain aging occurs due to CRM1 accumulation in the brain. We found that CRM1 levels and activity increased in the hippocampus and cortex during physiological aging, which resulted in a decrease of nuclear TFEB and STAT3. Consistent with an autophagic flux impairment, we observed accumulation of the autophagic receptor p62/SQSTM1 in neurons of old mice, which correlated with increased neuronal senescence. Using an in vitro model of neuronal senescence, we demonstrate that CRM1 inhibition improved autophagy flux and reduced SA-β-gal activity by restoring TFEB nuclear localization. Collectively, our data suggest that enhanced CRM1-mediated export of proteins during brain aging perturbs neuronal homeostasis, contributing to autophagy impairment, and neuronal senescence.

Abstract 458: Relationship between autophagy, activation and senescence in normal and cancer-associated human oral fibroblasts

Abstract Cancer-associated fibroblasts (CAFs) isolated from oral squamous cell carcinoma (OSCC) display an activated phenotype in that they express α-smooth muscle actin (αSMA) and are frequently prematurely senescent. OSCC cell lines can induce senescence in normal fibroblasts in vitro by a mechanism that involves TGF-β and reactive oxygen species. Another phenotype closely associated with senescence is autophagy, but the relationship between autophagy, activation and senescence in CAFs has not been examined in detail. We first examined autophagosome accumulation in normal human oral fibroblasts (NHOFs; n=3) and in a panel of CAFs (n=8) that exhibited different degrees of senescence. Autophagosome accumulation was observed in senescent CAFs, as shown by the increased expression of the LC3B-II protein and by the number of LC3B cytoplasmic puncta, which were quantified using immunofluorescence. The number of LC3B puncta was significantly greater (p<0.01) in senescent CAFs compared to NHOFs, and senescent CAFs contained a higher fraction of autophagic fibroblasts than NHOFs, as indicated by increased number of LC3B puncta per cell. Further, the number of LC3B puncta correlated positively with the degree of senescence, as demonstrated by senescence-associated β-galactosidase (SA β-Gal) activity. To investigate whether autophagy, activation and senescence develop simultaneously when fibroblasts are stimulated to undergo senescence, we examined the temporal induction of these phenotypes in NHOFs in response to TGF-β1. NHOFs were treated with TGF-β1 (4 ng/ml) for up to 10 days to induce autophagy, αSMA expression and SA β-Gal activity. The results showed that autophagic flux preceded fibroblast activation, which in turn was followed by senescence. In summary, our results demonstrate that CAFs from OSCC concomitantly display autophagic, activated and senescent phenotypes and that the induction of autophagy occurs before fibroblast activation and sensecence. Taken together, these data suggest that autophagy, activation and sensecence in fibroblasts may be part of a unified programme leading to the development of CAFs. Citation Format: May Leng Tan, Eric K. Parkinson, Lee Fah Yap, Ian C. Paterson. Relationship between autophagy, activation and senescence in normal and cancer-associated human oral fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 458.

Regulation of p53 during senescence in normal human keratinocytes.

p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene-induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age-dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16INK4A, induced G2 cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells.

Hypoxia Enhances the Senescence Effect of Bortezomib—The Proteasome Inhibitor—On Human Skin Fibroblasts

The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in some cancer cells. However, the nature of its selectivity remains unknown. The study presented here provides novel information on cellular effects of bortezomib in normal fibroblasts. We have found that in normoxic conditions the percent of apoptotic cells did not change significantly, independently on incubation time and examined concentration of bortezomib (25 nmol/L or 50 nmol/L). In hypoxic conditions we did not observe any effect of bortezomib on apoptosis of fibroblasts incubated for 24 h and 48 h in comparison to control. Only in the case of fibroblasts incubated for 12 hours in hypoxia significant increase in apoptosis, dependent on concentration of bortezomib, was observed. Our study has shown that bortezomib causes a time-dependent increase in senescence of normal fibroblasts, especially of these incubated in hypoxic conditions. Moreover, we demonstrated that oxygen regulated protein 150 (ORP150) expression was induced in fibroblasts in hypoxia conditions only, suggesting that this protein may play an important role in the cytoprotective response to environmental stress.

Abstract 4507: Small molecule inhibitor of MDM2 induces p53-dependent HMGB1 secretion followed by apoptosis in cancer cells and incomplete senescence in normal fibroblasts

Abstract An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63 (Ascenta Therapeutics). Both can rapidly increase intracellular p53wt levels and induce apoptosis or a senescence growth arrest, depending on the neoplastic status of the target cell. A critical question in the clinical development of such MDM2 inhibitors is their potential for normal tissue toxicity. In the absence of MDM2, p53 activation can cause fatal normal tissue pathology; and MDM2 inhibition can induce p53-dependent cell senescence in mouse fibroblasts. To assess the effects of MI-63 effects in human cells, we compared treatment (2.5-10 uM, 1-7 d) responses in p53wt malignant human mammary epithelial cells (MCF7) with those of p53wt normal human foreskin fibroblasts (HCA2). As expected, MI-63 had little effect on p53mut human cancer cells (MDA-231). However, against p53wt cancer cells and fibroblasts, MI-63 produced a dose-dependent increase in both nuclear p53 levels and reporter gene (p21) expression within 24 h. MCF7 cells responded with a dose- and time-dependent increase in apoptosis after 2 d exposure (vital dye uptake, cleaved PARP). By contrast, HCA2 cells responded with partially reversible growth arrest and a senescent-like phenotype (increased ROS, SOD2, and SA-βgal) within 3 days. A common early (24 h) MI-63 response by both the malignant and normal cells was nuclear loss of high-mobility group box-1 protein (HMGB1) associated with its active extracellular secretion. Acetylation of this damage-reporting cytokine, thought to be required for its secretion, was detected by western and mass spectrometry analyses. Although HMGB1 is generally pro-inflammatory and may be a harbinger of the senescence-associated secretory phenotype (SASP), MI-63 treated fibroblasts failed to exhibit induction of DNA damage foci or other features characteristic of SASP including IL-6 secretion. Surprisingly, MI-63 treatment of HCA2 beginning 24 or 48 h after a senescence-inducing dose of ionizing radiation (10 Gy) prevented enlarged cell morphology and produced a >80% reduction in SASP associated IL-6 secretion. These findings indicate that upregulation of p53wt in proliferating fibroblasts by MI-63, in the absence of DNA damage or other cell stresses, induces an incomplete senescence like growth arrest without a SASP response, consistent with our finding that p53 restrains SASP. In contrast, MI-63 upregulation of p53wt in malignant epithelial cells caused apoptotic cell death. The early release of HMGB1 from these dying cancer cells may modulate host innate immune response and/or recruit tumor infiltrating inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2011-4507

Overexpression of the Pituitary Tumor Transforming Gene Induces p53-dependent Senescence through Activating DNA Damage Response Pathway in Normal Human Fibroblasts

Pituitary tumor transforming gene (PTTG1, securin) is involved in cell-cycle control through inhibition of sister-chromatid separation. Elevated levels of PTTG1 were found to be associated with many different tumor types that might be involved in late stage tumor progression. However, the role of PTTG1 in early stage of tumorigenesis is unclear. Here we utilized the adenovirus expression system to deliver PTTG1 into normal human fibroblasts to evaluate the role of PTTG1 in tumorigenesis. Expressing PTTG1 in normal human fibroblasts inhibited cell proliferation. Several senescence-associated (SA) phenotypes including increased SA-beta-galactosidase activities, decreased bromodeoxyuridine incorporation, and increased SA-heterochromatin foci formation were also observed in PTTG1-expressing cells, indicating that PTTG1 overexpression induced a senescent phenotype in normal cells. Significantly, the PTTG1-induced senescence is p53-dependent and telomerase-independent, which is distinctively different from that of replicative senescence. The mechanism of PTTG1-induced senescence was also analyzed. Consistent with its role in regulating sister-chromatid separation, overexpression of PTTG1 inhibited the activation of separase. Consequently, the numbers of cells with abnormal nuclei morphologies and chromosome separations were increased, which resulted in activation of the DNA damage response. Thus, we concluded that PTTG1 overexpression in normal human fibroblasts caused chromosome instability, which subsequently induced p53-dependent senescence through activation of DNA-damage response pathway.

Abstract 4999: Glutamate receptors in cancer: Stable expression of GluR6 induces growth arrest and senescence in hepatocellular carcinoma HCC 7703 cells

Abstract Applying a functional strategy, we identified a gene, SEN6A, located at 6q21, which restores senescence in breast tumor cells. Human genome database search identified SEN6A as GluR6, a kainate type - ionotropic glutamate receptor gene. Kainate receptors constitute a family of ionotropic glutamate receptors that plays a role in the rapid synaptic transmission in the central nervous system. Glutamate receptors have been implicated in several pathophysiological conditions including Epilepsy, Schizophrenia and Parkinson's disease. We cloned the GluR6 gene by RT-PCR and investigated the structure, splicing pattern and its role in the cell senescence and cancer. We show that GluR6A, a splice variant is specifically expressed in brain, and four other variants; namely, GluR6B, GluR6C, GluR6D and GluR6E are expressed in other tissues and fibroblast cell lines. Genomic analysis and cloning of the sequence preceding the transcribed region led to the identification of two tissue specific promoters designated as neuronal promoter Pn and non-neuronal promoter Pnn. GluR6 is expressed in most tissues and its expression is elevated in late passage cells, relative to young human cells. The gene is either turned off or expressed at a reduced level in a large number of tumor cell lines. The chromosomal location of GluR6 at 6q21, a LOH hot spot in multiple cancers, points to its potential role in the etiology of cancer. The analysis for mutations in 22 cancer cell lines revealed a point mutation in exon 15 and deletion of exon 6 in two hepatoma cell lines. Open reading frame of GluR6 was cloned in a retroviral expression vector, in frame with EGFP tag, where EGFP is added after the signal peptide. In this vector GluR6-EGFP gene is expressed as a single transcript, from a CMV promoter, along with puromycin gene for selection. Ecotopic expression of GluR6 (variants A-E) induces growth arrest and senescence in hepatoma HCC7703 cells, and induce premature senescence in normal fibroblasts. Clones of GluR6 transfer cells display doubling time of 72-96 hours that gradually increases to 3-4 days ending in complete growth arrest and senescence. In contrast the transfer of empty vector or a construct carrying truncated GluR6 gene did not affect the growth of HCC7703 or normal cells. These result show that GluR6 is involved in the cascade of signaling circuits that govern cell growth and differentiation. Future studies are designed to investigate signaling pathways responsive to the expression of GluR6 gene. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4999.

Differential Oncogenic Ras Signaling and Senescence in Tumor Cells

Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.