Autophagy induction by SIRT6 is involved in oxidative stress-induced neuronal damage

SIRT6 is a NAD+-dependent histone deacetylase and has been implicated in the regulation of genomic stability, DNA repair, metabolic homeostasis and several diseases. The effect of SIRT6 in cerebral ischemia and oxygen/glucose deprivation (OGD) has been reported, however the role of SIRT6 in oxidative stress damage remains unclear. Here we used SH-SY5Y neuronal cells and found that overexpression of SIRT6 led to decreased cell viability and increased necrotic cell death and reactive oxygen species (ROS) production under oxidative stress. Mechanistic study revealed that SIRT6 induced autophagy via attenuation of AKT signaling and treatment with autophagy inhibitor 3-MA or knockdown of autophagy-related protein Atg5 rescued H2O2-induced neuronal injury. Conversely, SIRT6 inhibition suppressed autophagy and reduced oxidative stressinduced neuronal damage. These results suggest that SIRT6 might be a potential therapeutic target for neuroprotection.     

Inhibition of SIRT6 in prostate cancer reduces cell viability and increases sensitivity to chemotherapeutics

SI RT6 is an important histone modifying protein that regulates DNA repair, telomere maintenance, energy metabolism, and target gene expression. Recently SIRT6 has been identifi ed as a tumor suppressor and is downregulated in certain cancer types, but not in other cancers. From deposited gene profi ling studies we found that SIRT6 was overexpressed in prostate tumors, compared with normal or paratumor prostate tissues. Tissue microarray studies confi rmed the higher levels of SIRT6 in both prostate tumor tissues and prostate cancer cells than in their normal counterparts. Knockdown of SIRT6 in human prostate cancer cells led to sub-G1 phase arrest of cell cycle, increased apoptosis, elevated DNA damage level and decrease in BCL2 gene expression. Moreover, SIRT6-deficiency reduced cell viability and enhanced chemotherapeutics sensitivity. Taken together, this study provides the fi rst evidence of SIRT6 overexpression in human prostate cancer, and SIRT6 regulation could be exploited for prostate cancer therapy.     

SIRT1/PGC-1α signaling protects hepatocytes against mitochondrial oxidative stress induced by bile acids

Abstract

Oxidative stress and mitochondrial dysfunction are hypothesized to contribute to the pathogenesis of chronic cholestatic liver diseases. Silent information regulator 1 (SIRT1) attenuates oxidative stress and improves mitochondrial biogenesis in numerous mitochondrial-related diseases; however, a functional role for SIRT1 in chronic liver cholestasis, characterized by increased levels of toxic bile acids, remains unknown. We show decrease in SIRT1 levels and its activity and impairment of mitochondrial biogenesis in the liver of patients with extrahepatic cholestasis. Moreover, we found that glycochenodeoxycholic acid (GCDCA) stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content in L02 cells. Consistent with this finding, GCDCA was found to decrease SIRT1 protein expression and activity, thus promoting the deacetylation of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), a key enzyme involved in mitochondrial biogenesis and function. Conversely, GCDCA-induced mitochondrial injury was efficiently attenuated by SIRT1 overexpression. In summary, these findings indicate that the loss of SIRT1 may play a crucial role in the pathogenesis of liver damage observed in patients with extrahepatic cholestasis. The findings also indicate that genetic supplementation of SIRT1 can ameliorate GCDCA-induced hepatotoxicity through the activation of PGC-1α-dependent mitochondrial biogenesis.     

Measurement of the cellular deacetylase activity of SIRT1 on p53 via LanthaScreen® technology

Upon genomic insult, the tumor suppressor p53 is phosphorylated and acetylated at specific serine and lysine residues, increasing its stability and transactivation function. Deacetylases, including the type III histone deacetylase SIRT1, remove acetyl groups from p53 and counterbalance acetyltransferase activity during a DNA damage response. This report describes a series of high-throughput LanthaScreen? time-resolved F?rster resonance energy transfer (TR-FRET) immunoassays for detection of intracellular p53 phosphorylation of Ser15 and acetylation of Lys382 upon treatment with DNA damage agents, such as etoposide. These assays were used to measure the deacetylase activity of SIRT1 and/or Type I/II Histone deacetylases (HDACs). First, BacMam-mediated overexpression of SIRT1 resulted in dose-dependent deacetylation of GFP-p53 following etoposide treatment of U-2 OS cells, confirming that GFP-p53 serves as a SIRT1 substrate in this assay format. Further, overexpression of the acetyltransferase p300 via BacMam increased the acetylation of GFP-p53 at Lys382. Next, siRNA-mediated knockdown of SIRT1 resulted in increased GFP-p53 acetylation, indicating that endogenous SIRT1 activity can also be measured in U-2 OS cells. Consistent with these results, GFP-p53 acetylation was also increased upon treatment of cells with a small-molecule inhibitor of SIRT1, EX-527. The effect of this compound was dramatically increased when used in combination with chemotherapeutic drug and/or the HDAC inhibitor Trichostatin A, confirming a proposed synergistic mechanism of p53 deacetylation by SIRT1 and Type I/II HDACs. Taken together, the cellular assays described here can be used as high-throughput alternatives to traditional immunoassays such as western blotting for identifying pharmacological modulators of specific p53-modifying enzymes.     

SIRT7: a sentinel of genome stability

SIRT7 is a class III histone deacetylase that belongs to the sirtuin family. The past two decades have seen numerous breakthroughs in terms of understanding SIRT7 biological function. We now know that this enzyme is involved in diverse cellular processes, ranging from gene regulation to genome stability, ageing and tumorigenesis. Genomic instability is one hallmark of cancer and ageing; it occurs as a result of excessive DNA damage. To counteract such instability, cells have evolved a sophisticated regulated DNA damage response mechanism that restores normal gene function. SIRT7 seems to have a critical role in this response, and it is recruited to sites of DNA damage where it recruits downstream repair factors and directs chromatin regulation. In this review, we provide an overview of the role of SIRT7 in DNA repair and maintaining genome stability. We pay particular attention to the implications of SIRT7 function in cancer and ageing.     

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.     

Regulation of WRN protein cellular localization and enzymatic activities by SIRT1-mediated deacetylation

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.     

SIRT1 contributes to telomere maintenance and augments global homologous recombination

Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1(super)) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies.     

SIRT2 transgenic over-expression does not impact lifespan in mice

The NAD⁺-dependent deacylase family of sirtuin enzymes have been implicated in biological ageing, late-life health and overall lifespan, though of these members, a role for sirtuin-2 (SIRT2) is less clear. Transgenic overexpression of SIRT2 in the BubR1 hypomorph model of progeria can rescue many aspects of health and increase overall lifespan, due to a specific interaction between SIRT2 and BubR1 that improves the stability of this protein. It is less clear whether SIRT2 is relevant to biological ageing outside of a model where BubR1 is under-expressed. Here, we sought to test whether SIRT2 over-expression would impact the overall health and lifespan of mice on a nonprogeroid, wild-type background. While we previously found that SIRT2 transgenic overexpression prolonged female fertility, here, we did not observe any additional impact on health or lifespan, which was measured in both male and female mice on standard chow diets, and in males challenged with a high-fat diet. At the biochemical level, NMR studies revealed an increase in total levels of a number of metabolites in the brain of SIRT2-Tg animals, pointing to a potential impact in cell composition; however, this did not translate into functional differences. Overall, we conclude that strategies to enhance SIRT2 protein levels may not lead to increased longevity.     

SIRT3 deregulation is linked to mitochondrial dysfunction in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Despite decades of study, effective treatments for AD are lacking. Mitochondrial dysfunction has been closely linked to the pathogenesis of AD, but the relationship between mitochondrial pathology and neuronal damage is poorly understood. Sirtuins (SIRT, silent mating type information regulation 2 homolog in yeast) are NAD‐dependent histone deacetylases involved in aging and longevity. The objective of this study was to investigate the relationship between SIRT3 and mitochondrial function and neuronal activity in AD. SIRT3 mRNA and protein levels were significantly decreased in AD cerebral cortex, and Ac‐p53 K320 was significantly increased in AD mitochondria. SIRT3 prevented p53‐induced mitochondrial dysfunction and neuronal damage in a deacetylase activity‐dependent manner. Notably, mitochondrially targeted p53 (mito‐p53) directly reduced mitochondria DNA‐encoded ND2 and ND4 gene expression resulting in increased reactive oxygen species (ROS) and reduced mitochondrial oxygen consumption. ND2 and ND4 gene expressions were significantly decreased in patients with AD. p53‐ChIP analysis verified the presence of p53‐binding elements in the human mitochondrial genome and increased p53 occupancy of mitochondrial DNA in AD. SIRT3 overexpression restored the expression of ND2 and ND4 and improved mitochondrial oxygen consumption by repressing mito‐p53 activity. Our results indicate that SIRT3 dysfunction leads to p53‐mediated mitochondrial and neuronal damage in AD. Therapeutic modulation of SIRT3 activity may ameliorate mitochondrial pathology and neurodegeneration in AD.     

SIRT6 overexpression induces massive apoptosis in cancer cells but not in normal cells

Emerging evidence suggests that Sirtuin 6 (SIRT6) functions as a longevity assurance gene by promoting genomic stability, regulating metabolic processes and attenuating inflammation. Here, we examine the effect of SIRT6 activation on cancer cells. We show that SIRT6 overexpression induces massive apoptosis in a variety of cancer cell lines but not in normal, non-transformed cells. This cell death requires the mono-ADP-ribosyltransferase but not the deacetylase activity of SIRT6 and is mediated by the activation of both the p53 and p73 apoptotic signaling cascades in cancer cells by SIRT6. These results suggest that SIRT6 is an attractive target for pharmacological activation in cancer treatment.Key words: cancer, SIRT6, p53, p73, DNA damage     

A defect in the activity of Delta6 and Delta5 desaturases may be a factor predisposing to the development of insulin resistance syndrome

GLUT-4 (glucose transporter) receptor, tumor necrosis factor-alpha (TNF-alpha), interleukins-6 (IL-6), daf-genes and PPARs (peroxisomal proliferation activator receptors) play a role in the development of insulin resistance syndrome and associated conditions. But, the exact interaction between these molecules/factors and the mechanism(s) by which they produce insulin resistance syndrome is not clear. I propose that a defect in the activity of the enzymes Delta6 and Delta5 desaturases that are essential for the formation of long chain metabolites of essential fatty acids, linoleic acid and alpha-linolenic acid, is a factor in the development of insulin resistance syndrome. Long chain polyunsaturated fatty acids (LCPUFAs) increase cell membrane fluidity and enhance the number of insulin receptors and the affinity of insulin to its receptors; suppress TNF-alpha, IL-6, macrophage migration inhibitory factor (MIF) and leptin synthesis; increase the number of GLUT-4 receptors, serve as endogenous ligands of PPARs, modify lipolysis, and regulate the balance between pro- and anti-oxidants, and thus, play a critical role in the pathogenesis of insulin resistance. In the nematode, Caenorhabditis elegans, the protein encoded by daf-2 is 35% identical to the human insulin receptor; daf-7 codes a transforming growth factor-beta (TGF-beta) type signal and daf-16 enhances superoxide dismutase (SOD) expression. Melatonin has anti-oxidant actions similar to daf-16, TGF-beta and SOD. Calorie restriction enhances the activity of Delta6 and Delta5 desaturases, melatonin production, decreases daf-2 signaling, free radical generation, and augments anti-oxidant defenses that may explain the beneficial effect of diet control in the management of obesity, insulin resistance, and type II diabetes mellitus. These evidences suggest that the activities of Delta6 and Delta5 enzymes play a critical role in the expression and regulation of GLUT-4, TNF-alpha, IL-6, MIF, daf-genes, melatonin, and leptin by modulating the synthesis and tissue concentrations of LCPUFAs. Caloric restriction delays ageing by activating Sir 2 deacetylase in yeast, and expression of Sir 2 (SIRT1) in human cells. Both insulin and insulin-like growth factor-1 (IGF-1) attenuated this response. SIRT1 sequesters the proapoptotic factor Bax, prevents stress-induced apoptosis of cells, and thus, prolongs survival. In addition, SIRT1 repressed PPAR-gamma, and overexpression of SIRT1 attenuated adipogenesis, and upregulation of SIRT in differentiated fat cells triggered lipolysis and loss of fat, events that are known to attenuate insulin resistance and prolong life span. It remains to be seen whether LCPUFAs have a regulatory role in SIRT1 expression and control Sir 2 deacetylase activity. Thus, calorie restriction or reduced food intake has a role not only in the pathobiology of insulin resistance, but also in other associated conditions such as obesity, type II diabetes mellitus, ageing, and longevity.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.     

SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunit and cytotoxicity in renal proximal tubule cells

As the increased acetylation of p65 is linked to nuclear factor-κB (NF-κB) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-κB p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-κB during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-κB p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-κB through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.     

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.     

SIRT1 markedly extends replicative lifespan if the NAD salvage pathway is enhanced

Sir2 mediates lifespan extension in lower eukaryotes but whether its mammalian homolog, sirtuin 1, silent mating type information regulation 2 homolog (SIRT1), is a longevity protein is controversial. We stably introduced the SIRT1 gene into human vascular smooth muscle cells (SMCs) and observed minimal extension of replicative lifespan. However, SIRT1 activity was found to be exquisitely dependent on nicotinamide phosphoribosyltransferase (Nampt) activity. Moreover, overexpression of Nampt converted SIRT1-overexpressing SMCs to senescence-resistant cells together with heightened SIRT1 activity, suppressed p21, and strikingly lengthened replicative lifespan. Thus, SIRT1 can markedly postpone SMC senescence, but this requires overcoming an otherwise vulnerable nicotinamide adenine dinucleotide salvage reaction in aging SMCs.     

Aldehyde dehydrogenase 3A1 protects airway epithelial cells from cigarette smoke-induced DNA damage and cytotoxicity

Aldehyde dehydrogenase 3A1 (ALDH3A1), an ALDH superfamily member, catalyzes the oxidation of reactive aldehydes, highly toxic components of cigarette smoke (CS). Even so, the role of ALDH3A1 in CS-induced cytotoxicity and DNA damage has not been examined. Among all of the ALDH superfamily members, ALDH3A1 mRNA levels showed the greatest induction in response to CS extract (CSE) exposure of primary human bronchial epithelial cells (HBECs). ALDH3A1 protein accumulation was accompanied by increased ALDH enzymatic activity in CSE-exposed immortalized HBECs. The effects of overexpression or suppression of ALDH3A1 on CSE-induced cytotoxicity and DNA damage (γH2AX) were evaluated in cultured immortalized HBECs. Enforced expression of ALDH3A1 attenuated cytotoxicity and downregulated γH2AX. SiRNA-mediated suppression of ALDH3A1 blocked ALDH enzymatic activity and augmented cytotoxicity in CSE-exposed cells. Our results suggest that the availability of ALDH3A1 is important for cell survival against CSE in HBECs.