Stress-induced Senescence Research Articles

Inhibition of Sumoylation Alleviates Oxidative Stress-induced Retinal Pigment Epithelial Cell Senescence and Represses Proinflammatory Gene Expression.

Advanced age is the largest risk factor for age-related macular degeneration (AMD). Sumoylation is a reversible post-translational modification that conjugates small peptide, small ubiquitin-like modifier (SUMO), to a target protein. Dysregulation of sumoylation is recently found to be critically involved in several age-related disorders. However, the effects of sumoylation during retina senescence and aging remains elusive. This study is aimed to investigate the function and regulation of sumoylation pathway in the aging retina and premature senescent retinal pigment epithelial (RPE) cells. 1.5- and 10-month C57/B6 mice were used for comparative aging study. Both ARPE primary cultures and ARPE-19 cells were used for assay systems. The qRT-PCR was used for analysis of mRNA expression. Western blot and immunofluorescence were used to analyze the protein expression. Cell flow cytometry was used for cell cycle progression analysis. RPE barrier function and senescent-associated β-galactosidase (SA β-gal) activity were analyzed to measure cellular senescence. We show that the expression of SUMO enzymes and global protein sumoylation were downregulated in the aging mouse retina, and in the oxidative stress (OS) -induced premature senescent RPE cells. Dramatical altered distribution of SUMO E1, E2 and E3 enzymes were observed during RPE senescence. Inhibition of sumoylation alleviated OS-induced cell senescence in RPE cells, as indicated by decreased p21 and p53 expression and decreased percentage of cell cycle arrest at G0/G1 phase. Intriguingly, inhibition of SUMO E1 repressed the expression of proinflammatory cytokine and chemokine in the premature senescent RPE cells. However, inhibition of sumoylation did not prevent DNA damage during the OS-induced RPE senescence process. Our data indicate sumoylation critically regulates retina and RPE aging and that targeting sumoylation process may provide potential therapeutic strategy for AMD treatment.

Length doesn't matter-telomere damage triggers cellular senescence in the ageing heart.

Telomere shortening induces cellular senescence in proliferative cells. Yet, it is presently unclear how it is triggered in post‐mitotic cells such as cardiac myocytes. A new study by Anderson et al (2019) reports that during ageing of the heart, cellular senescence develops independently of telomere length, but is evoked by DNA damage, which preferentially accumulates at the telomere. Removal of senescent cells using senolytic drugs ameliorated cardiac hypertrophy and fibrosis and may inform novel approaches to improve the conditions for the ageing heart.

Leaf Senescence can be Induced by Inhibition of Root Respiration

Constituting the last stage of leaf development, leaf senescence is a complicated process that involves many senescence-associated genes, and numerous factors can induce leaf senescence. To elucidate the relationship between root respiration and leaf senescence, we treated the roots of Alhagi sparsifolia with nitrogen (N2) with the purpose of inhibiting root respiration (denoted as the N2 group). The results showed that compared with the control group, N2 treatment decreased the root respiration rate, chlorophyll (Chl) a, Chl b and carotenoid (Car) contents, the Chl/Car ratio, stomatal conductance (Gs), photosynthesis rate (Pn), maximum photochemical efficiency (φPo), and performance index on absorption basis (PIabs). In contrast, it increased leaf proline (Pro), malonaldehyde (MDA), and abscisic acid (ABA) contents. Moreover, no significant decline of Chl a/b was found in the N2 group. The results of the present work implied that leaf senescence can be induced by root respiration inhibition. Root respiration inhibition may result in ABA accumulation in leaves and thus induce leaf senescence. Another mechanism may be that root respiration inhibition resulted in the decrease of root water uptake, which subsequently led to water stress-induced leaf senescence. Root respiration inhibition-induced leaf senescence is a highly regulated process that is similar to natural senescence. In this process, no significant decline of Chl a/b was found. Root respiration inhibition-induced senescence is a “mild” process, in which most of the function of the photosynthetic apparatus performed well. Carotenoids play a key photoprotective role in leaf senescence. Overall, root respiration inhibition accelerated leaf senescence. In both types of senescence (root respiration induced senescence and natural senescence), the photosynthetic apparatus maintains a good performance until the last stage of senescence.

DCR2, a Cellular Senescent Molecule, Is a Novel Marker for Assessing Tubulointerstitial Fibrosis in Patients with Immunoglobulin A Nephropathy

Background/Aims: Stress-induced cell senescence, which contributes to cell cycle arrest and is independent of age, plays an important role in chronic kidney disease (CKD) progression. DcR2, as a senescent marker, exclusively expressed in senescent tubular epithelia. The objective of this study was to examine whether urinary DcR2 (uDcR2) could be a potential biomarker for tubulointerstitial fibrosis (TIF) in patients with immunoglobulin A nephropathy (IgAN). Methods: This study included 210 IgAN patients and 80 healthy volunteers, with uDcR2 levels measured using enzyme-linked immunosorbent assay. We examined the relationship among uDcR2/Cr levels, renal function, and pathological parameters, using regression analysis to identify risk factors for TIF and the area under the curve (AUC) approach to predict TIF. Renal DcR2 expression was quantified by immunohistochemistry. Co-expression of DcR2 with fibrotic markers (α-smooth muscle actin [α-SMA], collagen III) was analyzed by confocal microscopy. Results: Levels of uDcR2/Cr were significantly higher in IgAN patients and in those with more severe TIF, compared with healthy controls. Serum DcR2 levels were similar across groups. The proportion of IgAN patients with stages 1–2 CKD and T0 was highest among those with uDcR2/Cr <130 ng/g. In contrast, the majority of those with uDcR2/Cr >201 ng/g had stages 4–5 CKD and T2. Levels of uDcR2/Cr were positively associated with urinary albumin to creatinine ratio (ACR), urinary N-acetyl-β-D-glucosaminidase (uNAG)/Cr, and TIF scores and negatively associated with estimated glomerular filtration rate (eGFR). uDcR2/Cr, uNAG, ACR, and eGFR were independent predictors for TIF, with AUC of 0.907 for uDcR2/Cr. This AUC value was higher than that observed for eGFR, uNAG/Cr, or ACR. The sensitivity and specificity of uDcR2/Cr in predicting TIF were 87.0 and 80.5%, respectively. Moreover, uDcR2/Cr levels were positively associated with the percentage of renal DcR2 expression. Renal DcR2 co-localized with α-SMA and collagen III in the kidneys of IgAN patients. Conclusions: Levels of uDcR2/Cr were closely associated with the severity of TIF and renal function parameters. uDcR2/Cr represents a potential biomarker for predicting TIF in IgAN patients.

Application of ex-vivo spheroid model system for the analysis of senescence and senolytic phenotypes in uterine leiomyoma

Cellular senecence is an important biologic endpoint. Naturally occuring (aging) senescence is common in uterine leiomyoma (ULM). AKT is one of major pathways in promoting ULM growth and survival. Inactivation of AKT by MK2206 in ULM resulted in stress-induced senescence in vitro. Study of the senescent phenotypes and molecular changes in ULM may greatly facilitate the understanding of the tumor biology and potential clinical therapy for this common disease associated with high morbidity. To study senescence in a model system that closely resembles primary ULM in vivo, we applied an ex vivo model of three-dimensional (3D) spheroid culture system which maintained the molecular and cellular characteristics of primary ULM and matched myometrium as seen in vivo. Gene expression profiling done on ULM induced to undergo replication (passaging) or stress-induced (MK2206) senescence revealed that ROS and hypoxic-related genes were upregulated in the two types of senescences. Overexpression of two selected genes, WIPI1 and SLITKR4, induced cellular senescence in ULM spheroids. Additionally, administration of ABT263 (a BH3 mimetic) effectively reduced the senescent cells induced in ULM spheroids. This study identified novel genes associated with senescence in ULM and demonstrated a BH3 mimetic to act as a senolytic to remove senescent cells.Cellular senescence induced by replication and an AKT inhibitor in ex-vivo spheroid leiomyomas was examined in this work. Several dysregulated genes in the ROS, hypoxic and AKT pathways were identified, including WIPI1 and SLITKR4. Induced senescence in spheroids can be reversed by ABT263, a BH3 mimetic, which may be therapeutic modality for treatment of leiomyoma.

Genotoxic Stress-Induced Senescence.

A cell's genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply. If the DNA repair machinery fails to fix the damaged site during a temporary cell-cycle arrest, or if massive genotoxic stress overwhelmed the repair capacity, cellular failsafe programs such as apoptosis or senescence will be triggered to limit aberrant propagation of these damaged and potentially harmful cells. After decades of scientific focusing on apoptosis, cellular senescence is increasingly recognized as an equally important but biologically andfundamentally different type of ultimate cell-cycle exit program, because of its lastingly persistent nature and cell-intrinsic and extrinsic roles within the tissue and tumor microenvironment. We established primary apoptosis-compromised, Bcl2-expressing Eμ-myc transgenic mouse lymphomas as a versatile and clinically relevant model system to study therapy-induced senescence (TIS). Given the lack of a single specific senescence-defining marker, we previously exploited co-staining of senescence-associated β-galactosidase (SA-β-gal) activity with immunohistochemical detection of trimethylated histone H3 lysine 9 (H3K9me3), an established S-phase gene expression-controlling, repressive chromatin mark, and the proliferation marker Ki67. This biomarker panel is instrumental to characterize cells as senescent via their high SA-β-gal activity, strong nuclear H3K9me3 expression and Ki67-negative profile. In this chapter, we demonstrate the detection of viable senescent cells by novel methods based on a fluorescent version of the SA-β-gal (fSA-β-gal) assay, combined with immuno-fluoroscencestaining of H3K9me3 or Ki67, or analysis of the DNA replication status by incorporating 5-ethynyl-2'-deoxyuridine (EdU) detection into the protocol. Notably, while most senescence markers, irrespective of their specificity and sensitivity, may only be assessed in endpoint assays, we wouldlike to emphasize here the strength of viablefSA-β-gal to track single-cell fate in senescent populations over time.

Melatonin protects chronic kidney disease mesenchymal stem cells against senescence via PrPC‐dependent enhancement of the mitochondrial function

Although mesenchymal stem cell (MSC)-based therapy is a treatment strategy for ischemic diseases associated with chronic kidney disease (CKD), MSCs of CKD patients undergo accelerated senescence, with decreased viability and proliferation upon uremic toxin exposure, inhibiting their utility as a potent stem cell source for transplantation therapy. We investigated the effects of melatonin administration in protecting against cell senescence and decreased viability induced by pathophysiological conditions near the engraftment site. MSCs harvested from CKD mouse models were treated with H2 O2 to induce oxidative stress. CKD-derived MSCs exhibited greater oxidative stress-induced senescence than normal-mMSCs, while melatonin protected CKD-mMSCs from H2 O2 and associated excessive senescence. The latter was mediated by PrPC -dependent mitochondrial functional enhancement; melatonin upregulated PrPC , which bound PINK1, thus promoting mitochondrial dynamics and metabolism. In vivo, melatonin-treated CKD-mMSCs survived longer, with increased secretion of angiogenic cytokines in ischemic disease engraftment sites. CKD-mMSCs are more susceptible to H2 O2 -induced senescence than normal-mMSCs, and melatonin administration protects CKD-mMSCs from excessive senescence by upregulating PrPC and enhancing mitochondrial function. Melatonin showed favorable therapeutic effects by successfully protecting CKD-mMSCs from related ischemic conditions, thereby enhancing angiogenesis and survival. These results elucidate the mechanism underlying senescence inhibition by melatonin in stem cell-based therapies using mouse-derived CKD-mMSCs.

C-terminus of Hsc70-interacting protein (CHIP) enhances stemness properties of human Wharton’s jelly mesenchymal stem cell

ABSTRACT Mesenchymal stem cells are an attractive source of multipotent cells in part because they are easy to obtain. Several E3 ligases regulate the stability and functions of various factors in different adult stem cells through the ubiquitylation pathway. We investigated the C-terminus of Hsc70-interacting protein (CHIP) E3 ligase that regulates pluripotency of human Wharton’s jelly mesenchymal stem cells (hWJMSC). We found that CHIP increases protein kinase B (Akt) phosphorylation by decreased expression of phosphatase and tensin homolog (PTEN), which suggests improvement of the survival pathway by CHIP over-expression. We also found that increased CHIP expression induced Sox2 and NANOG, which can promote stem cell self-renewal and prevent oxidative stress-induced senescence of hWJMSC by decreased p21. We found that CHIP could be used to enhance the multiple functions of hWJMSC.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

New Urea Derivatives Are Effective Anti-senescence Compounds Acting Most Likely via a Cytokinin-Independent Mechanism.

Stress-induced senescence is a global agro-economic problem. Cytokinins are considered to be key plant anti-senescence hormones, but despite this practical function their use in agriculture is limited because cytokinins also inhibit root growth and development. We explored new cytokinin analogs by synthesizing a series of 1,2,3-thiadiazol-5-yl urea derivatives. The most potent compound, 1-(2-methoxy-ethyl)-3-1,2,3-thiadiazol-5-yl urea (ASES - Anti-Senescence Substance), strongly inhibited dark-induced senescence in leaves of wheat (Triticum aestivum L.) and Arabidopsis thaliana. The inhibitory effect of ASES on wheat leaf senescence was, to the best of our knowledge, the strongest of any known natural or synthetic compound. In vivo, ASES also improved the salt tolerance of young wheat plants. Interestingly, ASES did not affect root development of wheat and Arabidopsis, and molecular and classical cytokinin bioassays demonstrated that ASES exhibits very low cytokinin activity. A proteomic analysis of the ASES-treated leaves further revealed that the senescence-induced degradation of photosystem II had been very effectively blocked. Taken together, our results including data from cytokinin content analysis demonstrate that ASES delays leaf senescence by mechanism(s) different from those of cytokinins and, more effectively. No such substance has yet been described in the literature, which makes ASES an interesting tool for research of photosynthesis regulation. Its simple synthesis and high efficiency predetermine ASES to become also a potent plant stress protectant in biotechnology and agricultural industries.

Abstract B19: Modulation of cytotoxic effects of vemurafenib by chloroquine in malignant melanoma cells G-361: Role of dermcidin

Abstract Dermcidin (DCD) is a candidate oncogene localized at 12q13.1 and co-amplified with multiple oncogenes in many tumor cells, including breast carcinomas and melanomas. DCD is one of the 100 gene signature for melanomas. To further understand the role of DCD on growth, survival and progression of melanoma cells, we selected G-361 melanoma cell clones expressing shRNA to DCD and established in vitro and in vivo models. In this study we evaluated the pharmacologic effects of vemurafenib (BRAFV600E inhibitor) and chloroquine (autophagy inhibitor) in cell viability and tumor growth of two melanoma cell lines identified as G-361 pLKO, which expresses DCD and BRAFV600E, and G-361 IBC I which express shRNA to silence DCD constitutively. Vemurafenib induces features of stress-induced senescence in addition to apoptosis. G-361 melanoma cells responded to vemurafenib (1-2 μM) alone or combined with chloroquine (50-100 μM) which increased apoptosis rates, while decreasing senescent cells expressing β-galactosidase enzyme. Vemurafenib (50 mg/kg / 21 days) inhibited melanoma growth in immunodeficient mice dependent on DCD expression. Chloroquine (30 mg/kg) in combination with vemurafenib, accelerated (given at 24-hour interval), and reduced (given at 72 hours interval), melanoma growth. Tumor cells resistant to vemurafenib and chloroquine displayed atypical cell morphology and nuclear histological patterns and diferential expression of melanocytic differentiation biomarkers S100, HMB-45, Melan-A and pancytokeratins. This work confirms the efficacy of vemurafenib and suggests potential adjuvant effects of chloroquine. It also confirms the role of dermcidin as growth factor and oncogene for melanoma cells. Citation Format: Jennifer Montoya Neyra, Jose Belizario. Modulation of cytotoxic effects of vemurafenib by chloroquine in malignant melanoma cells G-361: Role of dermcidin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B19.

PGC-1α Protects RPE Cells of the Aging Retina against Oxidative Stress-Induced Degeneration through the Regulation of Senescence and Mitochondrial Quality Control. The Significance for AMD Pathogenesis.

PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is a transcriptional coactivator of many genes involved in energy management and mitochondrial biogenesis. PGC-1α expression is associated with cellular senescence, organismal aging, and many age-related diseases, including AMD (age-related macular degeneration), an important global issue concerning vision loss. We and others have developed a model of AMD pathogenesis, in which stress-induced senescence of retinal pigment epithelium (RPE) cells leads to AMD-related pathological changes. PGC-1α can decrease oxidative stress, a key factor of AMD pathogenesis related to senescence, through upregulation of antioxidant enzymes and DNA damage response. PGC-1α is an important regulator of VEGF (vascular endothelial growth factor), which is targeted in the therapy of wet AMD, the most devastating form of AMD. Dysfunction of mitochondria induces cellular senescence associated with AMD pathogenesis. PGC-1α can improve mitochondrial biogenesis and negatively regulate senescence, although this function of PGC-1α in AMD needs further studies. Post-translational modifications of PGC-1α by AMPK (AMP kinase) and SIRT1 (sirtuin 1) are crucial for its activation and important in AMD pathogenesis.

Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling.

Aim: Premature senescence of vascular endothelial cells is a leading cause of various cardiovascular diseases. Therapies targeting endothelial senescence would have important clinical implications. The present study was aimed to evaluate the potential of heme oxygenase-1 (HO-1) as a therapeutic target for endothelial senescence.Methods and Results: Upregulation of HO-1 by Hemin or adenovirus infection reversed H2O2-induced senescence in human umbilical vein endothelial cells (HUVECs); whereas depletion of HO-1 by siRNA or HO-1 inhibitor protoporphyrin IX zinc (II) (ZnPP) triggered HUVEC senescence. Mechanistically, overexpression of HO-1 enhanced the interaction between HO-1 and endothelial nitric oxide synthase (eNOS), and promoted the interaction between eNOS and its upstream kinase Akt, thus resulting in an enhancement of eNOS phosphorylation at Ser1177 and a subsequent increase of nitric oxide (NO) production. Moreover, HO-1 induction prevented the decrease of eNOS dimer/monomer ratio stimulated by H2O2 via its antioxidant properties. Contrarily, HO-1 silencing impaired eNOS phosphorylation and accelerated eNOS uncoupling. In vivo, Hemin treatment alleviated senescence of endothelial cells of the aorta from spontaneously hypertensive rats, through upregulating eNOS phosphorylation at Ser1177.Conclusions: HO-1 ameliorated endothelial senescence through enhancing eNOS activation and defending eNOS uncoupling, suggesting that HO-1 is a potential target for treating endothelial senescence.

Water- and Nitrogen-Use Efficiencies of Hemp (Cannabis sativa L.) Based on Whole-Canopy Measurements and Modeling.

Interest in hemp (Cannabis sativa L.) as a crop for the biobased economy is growing worldwide because hemp produces a high and valuable biomass while requiring low inputs. To understand the physiological basis of hemp's resource-use efficiency, canopy gas exchange was assessed using a chamber technique on canopies exposed to a range of nitrogen (N) and water levels. Since canopy transpiration and carbon assimilation were very sensitive to variations in microclimate among canopy chambers, observations were adjusted for microclimatic differences using a physiological canopy model, with leaf-level parameters estimated for hemp from our previous study. Canopy photosynthetic water-use efficiency (PWUEc), defined as the ratio of gross canopy photosynthesis to canopy transpiration, ranged from 4.0 mmol CO2 (mol H2O)−1 to 7.5 mmol CO2 (mol H2O)−1. Canopy photosynthetic nitrogen-use efficiency (PNUEc), the ratio of the gross canopy photosynthesis to canopy leaf-N content, ranged from 0.3 mol CO2 d−1 (g N)−1 to 0.7 mol CO2 d−1 (g N)−1. The effect of N-input levels on PWUEc and PNUEc was largely determined by the N effect on canopy size or leaf area index (LAI), whereas the effect of water-input levels differed between short- and long-term stresses. The effect of short-term water stress was reflected by stomatal regulation. The long-term stress increased leaf senescence, decreased LAI but retained total canopy N content; however, the increased average leaf-N could not compensate for the lost LAI, leading to a decreased PNUEc. Although hemp is known as a resource-use efficient crop, its final biomass yield and nitrogen use efficiency may be restricted by water limitation during growth. Our results also suggest that crop models should take stress-induced senescence into account in addition to stomatal effects if crops experience a prolonged water stress during growth.

Evidence of oxidative stress-induced senescence in mature, post-mature and pathological human placentas

IntroductionPremature ageing has been implicated in placental dysfunction. Senescence can be activated by oxidative stress, a key intermediary in the pathophysiology of pre-eclampsia. We examined senescence markers across normal gestation, and in pathological and post-mature pregnancies. Inducers of oxidative stress were used to mimic senescence changes in term explants. MethodsPlacental samples were collected with ethical approval and informed consent: first and second trimester samples from surgical terminations; term and pre-term controls, and early-onset pre-eclampsia samples from caesarean deliveries. Paraffin and EM blocks of post-mature placentas were from an archival collection. Term explants were subjected to hypoxia-reoxygenation (HR) or hydrogen peroxide (H2O2). Resultsp21 was increased significantly in term homogenates compared to first and second trimester samples, and was significantly higher in PE compared to term controls. Immunostaining revealed nuclear localisation of p21 and phosphorylated histone γH2AX in syncytiotrophoblast, with abundant foci in pathological and post-mature placentas. Abnormal nuclear appearances were observed in post-mature placentas. Sudan-Black-B staining demonstrated abundant lipofuscin, an aggregate of oxidised proteins, lipids and metals, in post-mature and pathological placentas. The percentage of nuclei positive for 8-hydroxy-2′-deoxy-guanosine, a marker of oxidised DNA/RNA, was increased in pathological placentas compared to age-matched controls. These changes could be mimicked by challenge with HR or H2O2. DiscussionSenescence markers increase in normal placentas with gestational age, and are exaggerated in post-mature and pathological cases. Oxidative stress triggers equivalent changes in explants, and may precipitate senescence in vivo. The consequent pro-inflammatory senescence-associated secretory phenotype may contribute to the pathophysiology of pre-eclampsia.

PIM1-catalyzed CBX8 phosphorylation promotes the oncogene-induced senescence of human diploid fibroblast

The proto-oncogene PIM1 encodes Ser/Thr kinase and regulates cell growth, differentiation and apoptosis. However, more and more studies including ours have found that PIM1 can induce senescence in normal human diploid fibroblasts and behave as a tumor suppressor. But the relevant molecular mechanism of this process is not yet clear. It has been reported that Chromobox homolog 8 (CBX8) binds directly to INK4A as a transcriptional repressor, thereby suppressing stress-induced senescence. Here we report that PIM1 can phosphorylate CBX8 to promote its degradation, thereby up-regulating p16, during PIM1-induced cell senescence. Overexpression of CBX8 can inhibit PIM1-induced cell senescence. These data suggest that to promote CBX8 degradation may be an important molecular mechanism of PIM1-induced cell senescence.

P-273 - Stress-induced premature senescence program activated in the mesenchymal stem cells by antioxidant treatments

It is now well established that intracellular reactive oxygen species (ROS) mediate the regulation of many signaling cascades that define cell fate. The delicate balance between the production and elimination of ROS is essential for maintaining normal cell functions. Disruption of this balance caused by the antioxidant treatment can disturb various intracellular processes controlled by the systemic signaling. In the present study, we investigate the response of the mesenchymal stem cells (MSCs) to the sub-cytotoxic treatment with various antioxidants (tempol, NAC, resveratrol, DPI). We show that exposure of the proliferating MSC cultures to the antioxidants at concentrations which are widely used in the cell studies to protect cells from the oxidative stress, can cause replication stress and initiate the program of stress-induced preliminary senescence. We demonstrate that these effects can be explained by the disturbance of the ROS-depended degradation of the key proteins regulating DNA replication, e.g. cyclin A, Emi1, geminin. Our data show that enhanced intracellular elimination of ROS resulted from the pharmacologic manipulations can cause the antioxidant-induced damage of cells.

1122 Nicotinamide prevents oxidative stress-induced senescence and differentiation in 2D and 3D human skin models

1122 Nicotinamide prevents oxidative stress-induced senescence and differentiation in 2D and 3D human skin models

Inhibitory effect of phytol on cellular senescence

BackgroundPhytol is a component of chlorophyll with demonstrated anticancer and immune-enhancing effects. In particular, it has been reported that phytol enhances the activity of natural killer cells that detect and remove cancer cells and promotes macrophage functions in immunity. In this study, based on the recently published precedent articles, we examined whether phytol can also inhibit cellular senescence to provide evidence for its possible use as a base material for cosmetics.MethodsHaCaT keratinocytes were pretreated with phytol for 24 h and then treated with oxidative stress-inducing hydrogen peroxide (H2O2) for 6 h before the analysis; anti-inflammation analysis, cell cycle analysis, cytoprotection analysis, caspase 3 (CASP3) activity analysis, and senescence-associated β-galactosidase (SA-β-gal) assay were performed to examine the cell potency of phytol.ResultsIn this study, HaCaT keratinocytes, treated with 750 μM H2O2, were tested with a phytol concentration below 10 μM to obtain the following results. First, phytol pretreatment suppressed H2O2-induced inflammation in a concentration-dependent manner, as indicated by the reduced expression of the mRNA levels of TNF-α (tumor necrosis factor-alpha), IL6 (interleukin 6), IL8 (interleukin 8), and COX2 (cyclooxygenase 2). Second, the quantitative analysis of cell cycle, NRF2 (nuclear factor erythroid 2-related factor 2), HO-1 (heme oxygenase 1), 14-3-3σ (Stratifin), cyclin B mRNA, and CASP3 was performed to examine the cytoprotective effects of phytol, as evidenced by increased mRNA expression of the cytoprotective genes NRF2 and HO-1 and decreased mRNA expression of the G2 cell cycle arrest-inducing genes 14-3-3σ and Cyclin B. CASP3, a protein that induces apoptosis, was reduced in a concentration-dependent manner, confirming that phytol protected cells from apoptosis. Third, the SA-β-gal assay revealed that phytol reduced H2O2-induced senescence in a concentration-dependent manner, and the cellular senescence was delayed on treatment using phytol.ConclusionsThis study verifies that phytol can inhibit oxidative stress-induced senescence of HaCaT keratinocytes. The results suggest the use of phytol as a base material for cosmetics to inhibit cellular senescence.

Radical scavenging activity of sulfated Bupleurum chinense polysaccharides and their effects against oxidative stress-induced senescence

In this study, BCPS-1, a polysaccharide previously isolated and characterized from Bupleurum chinense was chemically modified to yield two sulfated derivatives, which we designated as S-BCP1-4 and S-BCP1-8. The physicochemical properties of these sulfated derivatives were then determined by high performance liquid chromatography (HPLC), Fourier transform infrared spectrometry (FT-IR), and gas chromatography (GC), and then compared with those of BCPS-1. Furthermore, the antioxidant activities of all three polysaccharides were also evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay, superoxide radical assay and hydroxyl radical assay, while their effects against H2O2-induced cellular senescence were determined using senescence-associated β-galactosidase (SA-β-gal) staining, cell cycle assay and immunoblotting in H2O2-induced mouse lung endothelial cells (MLECs). Compared to BCPS-1, S-BCP1-4 and S-BCP1-8 exhibited remarkable antioxidant effect, and in a concentration-dependent manner. They also provided stronger protection against H2O2-induced cellular senescence in MLECs. These results indicated that the sulfate group in the modified B. chinense polysaccharides might play an important role in radical scavenging and resistance to H2O2-induced senescence. These sulfated polysaccharides could be considered as novel pharmaceutical products with potential antioxidant and anti-senescence effects.