Cellular and molecular mechanisms of xenobiotics-induced premature senescence

Abstract

Premature senescence, which share common features with replicative senescence such as morphology, senescence-associated galactosidase (SA-β-gal) activity, cell cycle regulation, and gene expression, can be triggered by the exposure of various xenobiotics including environmental pollutant, peroxides, and anticancer drugs. The exact mechanisms underlying the senescence onset and stabilization are still obscure. In this review, we summarized the possible cellular and molecular mechanisms of xenobiotics-induced premature senescence, including induction of reactive oxygen species (ROS), tumor suppressors, and DNA damage; disequilibrium of calcium homeostasis; activation of transforming growth factor-β (TGF-β); and blockage of aryl hydrocarbon receptor (AHR) pathway. The deeper understanding of the molecular mechanisms underlying xenobiotics-induced senescence may shed light on new therapeutic strategies for age-related pathologies and extend healthy lifespan.

Graphical Abstract

Graphical Abstract.

Introduction

Senescence, first introduced by Hayflick and Moorhead in 1961 [1], is characterized by irreversible cell cycle arrest. Later, this particular type of senescence (replicative senescence) was known as the phenomenon by which normal diploid cells lose the ability to divide with telomere shortening, normally after about 60 cell generations in vitro [2]. Senescence was believed to guard against unrestricted growth of damaged cells. Senescent cells, which exert flatted and enlarged appearance with increased senescence-associated galactosidase (SA-β-gal) activity, are different from apoptotic cells [3]. Premature senescence is another type of senescence that occurs after only few replications cycles and is morphologically and physiologically similar to replicative senescence [4]. Premature senescence can be triggered by oncogene activation, stress, or xenobiotics exposure. The expression of oncogenes such as RAS and C-Myc can unlock oncogene-induced senescence (OIS), which showed flattened cellular morphology, a large nucleus with a prominent nucleolus, and the activation of the p53 and p16 pathways [5, 6]. OIS is recognized as a potent antitumor barrier in vivo against aberrant oncogenic signaling and cancer development [7]. Various stresses including ionizing radiation can stir up stress-induced premature senescence (SIPS), with the characteristic of p53-dependent irreversible G1 arrest [8]. Xenobiotics are the chemicals which are found in organisms but which are not normally produced or expected to be present in them. They also cover substances which are present in high enough concentrations. It is reported that all kinds of xenobiotics [9], such as anticancer drugs, peroxides such as hydrogen peroxide (H₂O₂) [10], and the pollutants such as hexavalent chromium [Cr(VI)] [11], may lead to premature senescence.

In order to better understand premature senescence, researchers identified biomarkers by analyzing senescent pathways and the related mechanisms. These biomarkers include (i) SA-β-gal: SA-β-gal activity is typically determined by in situ staining with the chromogenic substrate X-gal [12]. It remains as the most widely used reliable biomarker for the detection of senescent cells even its expression is not required for senescence. (ii) Apolipoprotein J (Apo J): Apo J, also known as clusterin, is an 80 kDa secreted glycoprotein that upregulated after the exposure of the senescence inducers [11]. (iii) Connective tissue growth factor (CTGF): CTGF, an age-related protein that regulated by transforming growth factor- β (TGF-β) pathway, is known to be a useful marker for identifying senescence [13]. (iv) Fibronectin: Fibronectin, a senescence-related protein, is a high molecular weight glycoprotein of the extracellular matrix. The significant increase of Fibronectin expression during senescence has been found to correlate closely with the increasing size of senescent cells [14]. (v) Senescence marker protein-30 (SMP30): SMP30, which plays an important role in intracellular Ca²⁺ homeostasis, is found to be decreased in senescent cells [15].

In the daily life, people cannot keep off from being exposed to various xenobiotics. If induced by anticancer drugs, premature senescence in cancer cells means chemotherapy, while in normal cells it indicates drug side effects, and when the cancer cells bypass senescent state (escapers) they may become drug resistant. If induced by environmental pollutant, premature senescence means toxicity, and the escapers may have the tendency of malignant transformation [16, 17]. However, the exact mechanisms underlying the senescence onset and stabilization are still obscure. In this review, we summarized the possible cellular and molecular mechanisms of xenobiotics-induced premature senescence.

Induction of reactive oxygen species

Reactive oxygen species (ROS), a family of highly reactive oxygen molecules which includes free radicals that contain unpaired electron, such as superoxide (O₂^•−), hydroxyl radicals (OH^•), and non-radical molecules like H₂O₂, are defined as oxygen-containing chemical species with reactive chemical properties [18]. Oxidative phosphorylation (OXPHOS) is a metabolic pathway that generates 70–80% of the ATP demand of cells using energy released by the oxidation of nutrients [19]. During OXPHOS, the electrons are removed from nicotinamide adenine dinucleotide (NADH) and passed to oxygen through a series of enzymes consisting of respiratory chain complexes I- IV (complex I: NADH coenzyme Q reductase; complex II: succinate dehydrogenase; complex III: cytochrome bc1 complex; complex IV: cytochrome c oxidase) and F1F0-ATP synthase (complex V) to synthesize ATP [20]. ROS could generate mainly from mitochondrial electron transport system such as complex I and III [21], or from a family of membrane-bound enzymes such as NADH phosphate (NAD(P)H) oxidases [22], and also from xanthine oxidase or nitric oxide synthase [23]. Although ROS have long been regarded as unwanted byproducts and being invariably harmful, it is clear that ROS are essential, health-engendering signaling molecules that play a role in promoting health and lifespan [24]. The significant increase of ROS level has been confirmed to be the critical event in the induction and maintenance of cell senescence [25].

While suppressing the proliferation in cancer cells, anticancer drugs also induce premature senescence in normal cells, which may account for the occurrence of drug side effect [26]. Busulfan (BU) is a commonly used chemotherapeutic drug in the treatment of chronic myelogenous leukemia. The therapeutic efficacy of BU can be compromised by normal tissue damage caused by the induction of the premature senescence. Probin et al. [27] reported that the induction of premature senescence by BU is initiated by the transient depletion of intracellular glutathione (GSH) and followed by the continuous accumulation of ROS via NADPH oxidase, which lead to the activation of extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK). The antioxidant N-acetylcysteine (NAC) abrogated the burst generation of ROS, ameliorated Erk and p38 MAPK activation, and further attenuated BU-induced senescence.

Environmental pollutants could trigger premature senescence in normal cells to exert toxicity, which may account for the occurrence of occupational disease. Cr(VI), which can be found of various uses in industries such as metallurgy and textile dying, is a recognized human carcinogen [28]. Continuous treatment with low concentration of Cr(VI) (0.3 μg/l) for 10 consecutive weeks in adult skin fibroblast cultures resulted in the induction of premature senescence, which is characterized by the induction of various morphological and molecular biomarkers, such as positive SA-β-gal staining and overexpression of Apo J [29]. Cr(VI) treatment can increase the production of cellular ROS, such as hydroxyl and superoxide radicals [30]. Zhang et al. [31] revealed that Cr(VI) targeted and inhibited mitochondrial complex I to enhance ROS production, which may account for the induction of premature senescence. By applying antioxidant Trolox and a tetracycline-inducible lentiviral expression system containing shRNA to p53, the study demonstrated that Cr(VI) has a role in premature senescence by promoting ROS-dependent p53 activation in L-02 hepatocytes. Under normal conditions, mitochondrial oxidation and phosphorylation are coupled. OXPHOS can be uncoupled by various chemical uncouplers. The potential causal connection between impaired mitochondrial coupling and premature senescence has also been demonstrated [32]. Chronic exposure of chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused the uncoupling of OXPHOS and the burst generation of ROS, resulting in a significant proportion of the cells entering premature senescence. Thus, besides the well-established influence of ROS on proliferation and senescence, the dysfunction of OXPHOS is causally related to reduced cell proliferation and the induction of premature senescence.

Some xenobiotics could suppress premature senescence by modulating cellular oxidative status to exert their antiaging effect. Salidroside, the phenylpropanoid glycoside isolated from medicinal plant Rhodiola rosea, has been reported to have antiaging, anticancer, and anti-inflammatory functions [33]. Salidroside considerably reversed the H₂O₂-induced premature senescence phenotypes, including alterations of morphology, cell cycle, SA-β-gal staining, and the related molecules expressions of senescence pathways. The protection effect occurred in a dose-dependent manner and was achieved by modulating both ROS-relevant and ROS-irrelevant oxidative status. This finding demonstrates salidroside as an attractive and bio-safe agent with the potential to retard aging process and attenuate age-related diseases [34]. Hayashi et al. [35] reported that nitric oxide (NO) could delay endothelial cell senescence by suppression of NADPH oxidase-dependent ROS production. This finding may shed new light on the regulatory role of NO in anti-senescence therapy for age-related human vascular disorders.

Induction of tumor suppressors and cell cycle arrest

Cellular senescence is a state of irreversible and permanent cell cycle arrest that has been considered to be an intrinsic barrier against tumorigenesis [36]. Cell cycle arrest contributes to senescence but cell cycle arrest is not yet senescence [37]. Signaling that induces senescence mainly contains two major tumor suppressor cascades, the Rb/p16 pathway and the p53/p21 pathway [38]. During the induction of senescence, p53 transactivates the p21 gene, which is the only known mediator of pro-senescent function, and the p21protein binds to and inhibits G1-regulatory cyclin-dependent kinase 2 (CDK2), which leads cells to undergo G1 cell cycle arrest and senescence [39]. Rb could be sustained through CDK inhibition as a consequence of p53-dependent upregulation of p21, as well as the independent upregulation of p16 [40]. However, in addition to Rb/p16 and the p53/p21 pathway, the PTEN tumor suppressor is also involved in cellular senescence. It is reported that complete loss of PTEN in mouse prostate triggers premature senescence, which evidently limits the ability of PTEN loss to induce prostate cancer [41].

Methotrexate (MTX) is known as a classic antifolate used in cancer chemotherapy for many decades. Senescent growth arrest of human colorectal adenocarcinoma C85 cells treated for 48 h with 1 μM MTX was found to occur at the G1 and S phases of the cell cycle, and this is executed due to the activation of p53 and its downstream effector p21 [42]. Continuous treatment of ganoderiol F (GolF), a triterpene isolate, was found to arrest cell growth and induce senescent phenotypes such as the enlarged and flattened cell morphology, and SA-β-gal positive staining in hepatoma HepG2 cells [43]. Upregulation of CDK inhibitor p16 was found in early stages of GolF treatment and was presumed to block cell cycle progression, maintain cell cycle arrest, and further trigger premature senescence. The chronic hydroxyurea treatment leads to premature senescence only in association with the induction of p53 and p21 in a dose- and time-dependent manner [44]. Similar to hydroxyurea, only p53 pathway was involved in interferon-γ (IFN-γ) induced cellular senescence, because the study demonstrated that IFN-γ treatment increased SA-β-gal staining in p16-knockout cells but not in p53-knockout cells [45]. Tert-butyl hydroperoxide (t-BHP), an analog of hydroperoxide, could induce characteristic phenotypes of premature senescence in human diploid fibroblasts. Chen et al. [46] demonstrated that ginsenoside Rg1, the active ingredient of ginseng, markedly inhibited senescent morphological changes induced by t-BHP, and mechanism study revealed that this effect was achieved by regulating cell cycle-related proteins and enhancing mitochondrial functioning. It has been confirmed that the decrease of cell viability of human osteoblast MG-63 after exposure to Cr (VI) is related to cell cycle arrest and DNA damage [47].

DNA damage

DNA damages include telomeric DNA damage, genome instability, and mitochondrial DNA (mtDNA) damage [48]. A telomere is a region of repetitive DNA sequences that protects the end of the chromosome from deterioration. Telomere dysfunction induces the increased secretion of DNA damage response (DDR) factors such as Rad17, ataxia telangiectasia mutated (ATM), and phosphorylated histone 2A family member X (γ-H2AX) to further trigger senescence [49, 50]. Genomic instability, which has been implicated as a major causal factor in growth arrest, is broadly classified into microsatellite instability that associated with DNA level instability, and chromosome instability recognized by chromosomal abnormalities [51]. Human mtDNA, the naked compact DNA molecule without protection by histones, exhibits inefficient DNA repair mechanisms and is prone to damage [52]. The increased damage of mtDNA caused by various stress may lead to the impaired electron transport, overproduction of ROS, and ATP depletion, which may accelerate the progress of premature senescence [53]. The intracellular ROS level is also recognized as an important determinant of genomic integrity and cellular response to DNA damage.

By measuring the mean length of telomere restriction fragment (TRF) in human diploid fibroblasts, Duan et al. [54] demonstrated that low dosage of H₂O₂ treatment significantly accelerated the process of telomere shortening, and this may further act as the trigger of premature senescence through DDR-ATM-p53-dependent pathway. Treatment of Nevirapine, an effective specific inhibitor of the endogenous non-telomeric reverse transcriptase, induced premature senescence that is characterized by growth arrest and morphological changes. Mechanism study revealed that Nevirapine could possibly destabilize the genome, rendering it prone to damage and consequently activation of the DDR [55]. Resveratrol, a phytoalexin that naturally occurs in many species of plants and assists in the response against pathogen infections, has cancer-preventive and anticancer properties. However, it could also promote genomic instability. Rusin et al. [56] reported that Resveratrol induces DNA instability, which can be exemplified in telomeric DNA of osteosarcoma cells. Consequently, Resveratrol activates DDR, which can be inferred from histone H2AX phosphorylation at serine 139, upregulation and phosphorylation of p53 at serines 15 and 37, loss of nucleolar accumulation of werner syndrome protein (WRN) as well as S-phase arrest. Axitinib is a potent and selective inhibitor of vascular endothelial growth factor receptor (VEGFR) for the treatment of patients with metastatic renal cell carcinoma (RCC). Axitinib treatment induces premature senescence via DDR, which initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and later by p21 upregulation in human kidney cancer (Caki-2 and A-498) cell lines [57]. Wise et al. [58] found that DNA repair disorder is the key process in the carcinogenic process of Cr(VI), and chromosome instability (CIN), repair defects and centrosome amplification are important mechanisms of malignant transformation and permanent inheritance.

Disequilibrium of calcium homeostasis

Although several reports indicated elevation of intracellular calcium ([Ca²⁺]_i) levels during oncogene-, rotenone-induced as well as replicative senescence [59, 60], there is no clear evidence about the exact relationship between [Ca²⁺]_i and senescence. [Ca²⁺]_i is implicated in various biological functions such as protein secretion, exocytosis, gene transcription, and cell growth and proliferation [61]. [Ca²⁺]_i has been highlighted as an important factor affecting different pathological conditions such as autophagy and apoptosis [62], and its impact in senescence has recently been unveiled. The small mitochondrial protein Fus1 (also known as Tusc2, tumor suppressor candidate 2) is a potential Ca²⁺-binding protein involved in the regulation of mitochondrial Ca²⁺ transport, and it is confirmed that in the absence of Fus1, mitochondrial efficiency in handling Ca²⁺ levels can be completely impaired, thus resulting in mitochondrial Ca²⁺ overload [63]. Uzhachenko et al. [63] provided the evidence that loss of Fus1 triggered mitochondrial disequilibrium of calcium homeostasis, thus leading to premature senescence and aging-associated pathologies. They also showed that Fus1KO mice developed multiple early aging signs such as lordokyphosis, lack of vigor, inability to accumulate fat, reduced ability to tolerate stress, and premature death. Sublethal H₂O₂ treatment induced a rapid calcium release from intracellular stores and resulted in further senescence development in human endometrium-derived stem cells, which was accompanied by persistently elevated [Ca²⁺]_i levels. [Ca²⁺]_i chelation by a selective calcium chelator BAPTA-AM was found sufficient to block the expansion of the senescence phenotype, to decrease endogenous ROS levels, to avoid G1 cell cycle arrest, and finally to retain proliferation state [64]. We found that Cr(VI)-induced Ca²⁺ overload depended on the release of Ca²⁺ from the endoplasmic reticulum mediated by inositol 1,4,5-trisphosphate receptor (IP3R), and store-operated calcium channels (SOCC) can mediate the influx of Ca²⁺ from the extracellular space [65].

Activation of transforming growth factor-β (TGF-β) signaling

TGF-β signaling is known to play critical roles in embryogenesis and adult tissue homeostasis by regulating cell growth, proliferation, differentiation, and death [66]. TGF-β 1–3 are unique multifunctional growth factors that are present only in mammals and stored in the extracellular matrix [67]. TGF-β1 overexpression is responsible for the induction of several biomarkers of replicative senescence after subcytotoxic H₂O₂ stress [68]. Human mesenchymal stem cells (hMSCs) can develop senescence phenotype due to long-term culture, which was companied with the increased expression of TGF-β2. Fibroblast growth factor-2 (FGF-2) stimulates the growth of hMSCs in vitro by suppressing TGF-β2 expression [69]. It is also confirmed that TGF-β2 could trigger senescence-associated changes, including SA-β-Gal activity, lipid peroxidation, and the mRNA expressions of senescence biomarkers Apo J and smooth muscle 22 (SM22) in human trabecular meshwork (TM) cells in vitro. Chatterjee et al. [70] also demonstrated that senescence-associated G1 cell cycle arrest induced by rapamycin is due to upregulation of TGF-β signaling. TGF-β was found to be highly expressed in the elderly donor corneal epithelium. In human corneal epithelial cell (HCEC) models, TGF-β could induce cellular senescence, characterized by increased SA-β-gal activity and elevated p16 and p21 expressions, and pharmacological inhibition of TGF-β signaling alleviated TGF-β-induced senescence.

Blockage of aryl hydrocarbon receptor pathway

Aryl hydrocarbon receptor (AHR) is known to be a ligand-dependent transcription factor that plays a role in the differentiation of various developmental pathways and also initiates the xenobiotic clearance program upon exposure to environmental contaminant agonists [71]. Cigarette smoke condensate (CSC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are known AHRs. Both TCDD and CSC are capable of suppressing premature senescence by increasing levels of the AHR target gene, CYP1A1, and the application of the AHR antagonist, 30-methoxy-40-nitroflavone (MNF) blocked the effects of TCDD and CSC on CYP1A1 expression and induced premature senescence. It is thus concluded that AHR-mediated inhibition of senescence may contribute to the progression of CSC-induced tumorigenesis, and the agents that block the actions of AHR may represent promising cancer therapeutics [72].

Conclusion and prospect

In this review, we summarized the possible cellular and molecular mechanisms of xenobiotics-induced premature senescence, including induction of ROS, tumor suppressors, and DNA damage; disequilibrium of calcium homeostasis; activation of TGF-β; and blockage of AHR pathway (Table 1). However, we should take into account that these players may be interconnected because of the signaling network, which means the continuous effort should be made to better demonstrate the mechanism of premature senescence. A deeper understanding of the molecular mechanisms underlying xenobiotics-induced senescence may shed light on new therapeutic strategies for age-related pathologies and extend healthy lifespan. After being exposed to various xenobiotics, cells may become senescent because of DNA damage or the induction of ROS, but a small subset of cells can bypass the senescence process to become survivors [73, 74]. If the xenobiotic were antibiotics and anticancer drugs, the survivors will become drug-resistant cells. If the xenobiotic were toxins like Cr(VI), the survivors will either remain normal function or become pre-tumor cells. Since the drug dosage required for senescence induction are much lower than those necessitated for cell death induction, it is believed that forcing cancer cells to undergo premature senescence is a less aggressive approach to inhibit tumor progression. Thus, we think the ability of cancer cells to escape senescence and become drug resistant must be inhibited, and a deep understanding of the mechanisms that govern drug-induced senescence may lead to discovery of novel approaches to suppress drug resistance.

Table 1.

Cellular and molecular mechanisms induced by different xenobiotics

Xenobiotic	Target	Cell	Mechanism	Reference
BU	GSH	L-02	ROS	[27]
Cr(VI)			ROS	[31]
Salidroside			ROS	[33]
NO	NADPH		ROS	[35]
MTX	p53, p21	C85	Cell cycle	[42]
GOIF		HepG2	Cell cycle	[43]
Hydroxyurea	p53		Cell cycle	[45]
t-BHP		Human diploid fibroblast	Cell cycle	[46]
H2O2	DDR-ATM-p53	Human diploid fibroblast	DNA damage	[54]
Nevirapine			DNA damage	[55]
Resveratrol	H2AX, p53, WRN	Osteosarcoma cell	DNA damage	[56]
Axitinib	γ-H2AX, Chk1	A-498, Caki-2	DNA damage	[57]
H2O2			Calcium homeostasis	[64]
Cr(VI)	IP3R, SOCC	L-02	Calcium homeostasis	[65]
TCDD, CSC	CYP1A1		AHR	[72]

Senescence bypass appears to be an important step in the development of cancer [75]. Indeed, lots of findings raise the possibility that senescent cells create an environment that limits the proliferative capacity of an individual cell, thereby functioning as tumor-suppressive mechanism. However, we think the utility of senescence as an antineoplastic therapy should be approached with the utmost caution because it is still not clear to what extent senescent cells alter the surrounding microenvironment and what impact these alterations will have on spontaneous tumor rates. Both the protective and the possible deleterious effects of senescence on autochthonously arising tumors deserve further investigation.

Conflict of Interest Statement

The authors have no conflicts of interest to declare in relation to this article.