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N-acetyl cysteine inhibits human signet ring cell gastric cancer cell line (SJ-89) cell growth by inducing apoptosis and DNA synthesis arrest
Abstract
In this study, we investigated the inhibitory effects of N-acetyl cysteine (NAC) on the growth of the human signet ring cell from the gastric-cancer cell line SJ-89 , via the induction of apoptosis and the arrest of DNA synthesis.
SJ-89 cells were regularly incubated in the presence of NAC at 5, 10 and 20 mmol/l, and with IMDM as untreated control. Trypan blue-dye exclusion analysis and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay were applied to detect cell proliferation. Apoptotic morphology was observed by electron microscopy. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay were performed to detect NAC-triggered apoptosis.
NAC could inhibit proliferation of human gastric cancer SJ-89 cells in a dose-dependent and time-dependent manner. The growth curve showed suppression by 15.8, 37.6 and 66.3% following 72 h of NAC treatment at 5, 10 and 20 mmol/l, respectively, similar to the findings of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. DNA synthesis was evidently reduced by 25, 39 and 91% after 24 h NAC treated at 20 mmol/l and 5 days at 10 and 20 mmol/l, respectively. Cell growth was inhibited by 100% with the treatment of 20 mmol/l NAC on day 6. NAC-treated SJ-89 cells were characterized by typical apoptotic alterations, including morphological changes by electron microscopy, typical apoptotic sub-G1 peaking observed by flow cytometry and increase of apoptotic cells with the elevation of the concentration of NAC in a clearly dose-dependent manner by TUNEL assay. Electrophoresis analysis showed typical 'DNA ladder'.
The data above implicated that NAC inhibits human gastric-cancer SJ-89 cell growth by inducing apoptosis and DNA synthesis arrest. Although the exact mechanisms involved in NAC-induced apoptosis have not been known up to now, the ability to induce apoptosis in a tumor-cell population within 48 h is worth noting. It is also noteworthy that NAC can selectively inhibit the growth of tumor cells. Further studies are needed to elucidate the mechanisms.
... ROS inhibitors NAC and caVeic acid phenethyl ester sensitize astrocytoma cells to Fas-induced apoptosis in a redox-dependent manner, suggesting a potential use in the treatment of malignant brain tumors (Choi et al. 2007). NAC also inhibits human gastric-cancer SJ-89 cell growth by inducing apoptosis and DNA synthesis arrest (Li et al. 2007). On the other hand, the DNA damage response (DDR) has been recognized as an important oncogene-provoked anticancer barrier in early human tumorigenesis leading to apoptosis or cellular senescence. ...
... It has been also demonstrated that the extent of oxidative stress and DNA damage was greater in MDA- MB-231 cells than in MCF-7 cells, with a strong correlation to estrogen receptor status. In conclusions, Wndings add further support to the theme that ROS formation is a signiWcant determinant factor in mediating the induction of oxidative DNA damage in human breast cancer cells exposed to carcinogenesis (Lin et al. 2007). On the other hand, the role of reactive nitrogen species (RNS) in colon carcinogenesis is multifactorial and aVects diverse processes such as proliferation , apoptosis, diVerentiation, tumorigenesis, and metastases. ...
... Metal binding and oxidation–reduction may also aVect its activity in vivo. It is a tumor suppressor often inactivated in cancer, which controls cell proliferation and survival through several coordinated pathways and it is induced in response to many forms of cellular stress, genotoxic or not (Rahman- Roblick et al. 2007). There is clear evidence on the role of p53 as a transactivator or transrepressor of genes involved in the production and control of ROS. ...
Oxidative stress can be defined as the imbalance between cellular oxidant species production and antioxidant capability. Reactive oxygen species (ROS) are involved in a variety of different cellular processes ranging from apoptosis and necrosis to cell proliferation and carcinogenesis. In fact, molecular events, such as induction of cell proliferation, decreased apoptosis, and oxidative DNA damage have been proposed to be critically involved in carcinogenesis. Carcinogenicity and aging are characterized by a set of complex endpoints, which appear as a series of molecular reactions. ROS can modify many intracellular signaling pathways including protein phosphatases, protein kinases, and transcription factors, suggesting that the majority of the effects of ROS are through their actions on signaling pathways rather than via non-specific damage of macromolecules; however, exact mechanisms by which redox status induces cells to proliferate or to die, and how oxidative stress can lead to processes evoking tumor formation are still under investigation.
... The antineoplastic action of the nanocomposite arises through infinite possible routes, including antioxidant, antimitotic and/or antiangiogenic activity, as well as raising pH of body. NAC aids cellular health by deterring the replication of abnormal cellular growth [28]. ...
In present study the chemoprevention activity of N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate was studied in female albino rats. The experimental animals were divided in to four groups each containing fifteen animals. Group I served as normal control. the other three groups, viz. II, III and IV were, Group II at the end of the second week (On day 16) rats received single oral dose of DMBA diluted in Olive oil (1 ml) orally by stomach tube (100 mg/kg body weight), Group III received N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate at dose 50 mg/kg body weight for three times per week at the end of the second week (On day 16) the rats received single oral dose from 7-12 Dimethyl benzanthracene (DMBA) 100 mg/ kg body weight and then treated with N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate as group II, Group IV administrated with N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate at dose 50 mg/kg body weight three times per week for 18 weeks, After eighteen weeks, all the animals were sacrificed and the effects of the N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate and DMBA were monitored by assaying the levels of lipid peroxidation (MDA), Superoxide dismutase (SOD) and Catalase (CAT) in liver and mammary gland homogenates. In addition plasma Aspartate aminotransferase (AST), plasma Alanine amino transferase (ALT), Urea, Creatinine and histopathological of mammary gland were also studied.
... Kuyumcu et al. demonstrated that NAC supplementation has beneficial impact on OS parameters of patients with gastrointestinal system tumor that underwent to oncological surgery. However, in an experimental model by Li et al., it was shown that NAC supplementation may cause preventive effect [99]. ...
During cellular processes, free radicals are produced and removed which have potential to cause cellular damage via different pathways. Oxidative stress (OS) is a result of imbalance between free radical production and elimination, which the accumulation of free radicals may cause group disorders including atherosclerotic vascular disorders and cancer.Carcinogenesis is a multistep process including metaplasia to overt cancer that has significantly related to internal and external oxidative agents. Gastrointestinal tract is the susceptible area to dietary oxidants and microbial agents. Lack of available data exists with regard to exact relation of OS and common gastrointestinal system cancers. In this chapter, complex interaction of OS and five most common gastrointestinal system cancers were summarized.
... The antineoplastic action of the nanocomposite arises through infinite possible routes, including antioxidant, antimitotic and/or antiangiogenic activity, as well as raising pH of body. NAC aids cellular health by deterring the replication of abnormal cellular growth [28]. ...
I IS SS SN N: : 2 23 32 20 0-6 68 81 10 0 ABSTRACT In present study the chemoprevention activity of N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate was studied in female albino rats. The experimental animals were divided in to four groups each containing fifteen animals. Group I served as normal control. the other three groups, viz. II, III and IV were, Group II at the end of the second week (On day 16) rats received single oral dose of DMBA diluted in Olive oil (1 ml) orally by stomach tube (100 mg/kg body weight), Group III received N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate at dose 50 mg/kg body weight for three times per week at the end of the second week (On day 16) the rats received single oral dose from 7-12 Dimethyl benzanthracene (DMBA) 100 mg/ kg body weight and then treated with N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate as group II, Group IV administrated with N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate at dose 50 mg/kg body weight three times per week for 18 weeks, After eighteen weeks, all the animals were sacrificed and the effects of the N-acetyl cysteine/ zinc oxide nanocomposite combined with High ascorbate and DMBA were monitored by assaying the levels of lipid peroxidation (MDA), Superoxide dismutase (SOD) and Catalase (CAT) in liver and mammary gland homogenates. In addition plasma Aspartate aminotransferase (AST), plasma Alanine amino transferase (ALT), Urea, Creatinine and histopathological of mammary gland were also studied.
... The balance between proliferation and cell death is a decisive factor in the progression or inhibition of carcinogenesis, and a variety of mechanisms can be activated or inactivated to induce apoptosis [33]. Antioxidant molecules that have a thiol group, such as NAC, have the ability to promote several of these mechanisms in different types of human tumours [13,45]. One of these mechanisms refers to upregulation of pro-apoptotic genes together with the downregulation of inhibitors of apoptosis genes, often accompanied by increased permeability of the mitochondrial membrane and release of cytochrome c, activating the caspase cascade . ...
Background
Liver cancer is one of the most common malignancies in the world and at the moment, there is no drug intervention effective for the treatment of liver tumours. Investigate the effect of N-acetylcysteine (NAC), which has been studied for its antitumoural properties, on the toxicity of hepatocarcinoma (HCC) cells in vitro when used with the drug interferon alpha-2A (IFN), which is used clinically to treat HCC.
Results
NAC, IFN and NAC plus IFN reduced cell viability, as determined by MTT assay. More importantly, NAC potentiates the cytotoxic effect of IFN, with the best response achieved with 10 mM of NAC and 2.5 x 104 of IFN. These results were confirmed by Annexin/PI staining through flow cytometry and morphologic analyses. Co-treatment reduced the expression of the nuclear transcription factor kappa-B (NF-kB). In a similar way to NAC, RNAi against p65 potentiated the toxic effect of IFN, suggesting that, indeed, NAC may be enhancing the effect of IFN through inhibition of NF-kB.
Conclusions
Our results support the notion that NAC may be an important drug for the treatment of liver tumours as primary or adjuvant therapy. IFN has a limited clinical response, and therefore, the anti-proliferative properties of NAC in the liver should be explored further as an alternative for non-responders to IFN treatment.
... In a rat mammary cancer model, a low dose of N-acetylcysteine modestly decreased, and a high dose significantly increased, tumour occurrence [28]. The single study that we have identified which related to gastric cancer reported that N-acetylcysteine inhibited human gastric cancer SJ-89 cell growth by inducing apoptosis and DNA synthesis arrest [29]. A randomized trial of 2592 former or current smokers with head and neck or lung cancers found no effect on survival or the incidence of second primary tumours following 2 years of N-acetylcysteine supplementation [30]. ...
Cancers of the upper gastrointestinal tract remain a significant cause of morbidity and mortality. Cysteine, known to be involved in a myriad of immuno-modulatory, anti-oxidant, and anti-carcinogenic pathways, has not been investigated in the aetiology of oesophageal or gastric cancers. To examine the relationship between serum cysteine concentration and risk of these cancers we conducted a nested case-cohort study within the General Population Nutrition Intervention Trial in Linxian, China.
498 oesophageal squamous cell carcinomas (OSCCs) and 255 gastric cardia adenocarcinomas (GCAs) were matched by age and sex to 947 individuals from the wider cohort. We calculated HRs and 95% CIs using the case-cohort estimator for the Cox proportional hazards models, stratified on age and sex, with adjustment for potential confounders.
Higher concentrations of serum cysteine were significantly associated with a lower risk of both OSCC and GCA. For those in the highest quartile of serum cysteine, compared to those in the lowest, the multivariate HRs were 0.70 for OSCC (95% CI 0.51 to 0.98) and 0.59 for GCA (95% CI 0.38 to 0.91). These associations were dose dependent (p for trend=0.006 and 0.008, respectively). These inverse associations were not significantly modified by other risk factors, with the exception of age, where a stronger association was noted among persons in the older age strata.
Higher serum concentrations of cysteine were associated with a significantly reduced risk of OSCC and GCA. Cysteine should be further investigated for its potential as a chemopreventive agent for upper gastrointestinal cancers.
Purpose
Gastric cancer (GC) is a disease with high prevalence and mortality, but we lack convenient and accurate methods to screen for this disease. Thus, we aimed to search for some salivary biomarkers and explore changes in metabolites in patients’ saliva after radical gastrectomy.
Patients and Methods
A total of 152 subjects were divided into three groups (healthy group, GC group, and one-week postoperative group). After simple processing, saliva samples were analyzed by liquid chromatography–mass spectrometry. First, we used total ion chromatography and principal component analysis to determine the metabolite profiles. Next, t-test, partial least squares discriminant analysis, support vector machine, and receiver operating characteristics curve analysis were performed to identify biomarkers. Then, Fisher discriminant analysis and hierarchical clustering analysis were performed to determine the discriminating ability of biomarkers. Finally, we established a generalized linear model to predict GC based on biomarkers, and used bootstrapping for internal validation.
Results
Between the healthy and GC groups, we identified four biomarkers: lactic acid, kynurenic acid, 3-hydroxystachydrine, and S-(1,2,2-trichlorovinyl)-L-cysteine. We used stepwise regression to select five metabolites and develop a model with areas under the curve equal to 0.973 in the training dataset and 0.924 in the validation dataset. Between the GC and one-week postoperative groups, we found two differential metabolites: 19-hydroxyprostaglandin E2 and DG (14:0/0:0/18:2n6).
Conclusion
Differential metabolites were observed among the three groups. GC could be initially diagnosed on the basis of detection of these biomarkers. Moreover, changes in salivary metabolites in postoperative patients could provide important insights for basic studies.
This article covers the anticancer activities and mechanisms of action of Cu(II) complexes of flavonoid‐derived quercetin and 1,10‐phenanthroline ligands. The antiproliferative activity of the complex and its ligands was evaluated by MTT, ATP, and SRB viability assays in human lung cancer cells (A549, H1299). Findings for apoptosis were determined by fluorescent staining, flow cytometry analysis, and the M30 antigen method. In addition, the mechanism of action of the complex was investigated by Annexin V staining, caspase 3/7 activity, ROS formation, and cell cycle analysis. The involvement of caspases, thus, apoptosis was confirmed by rescuing cell death by using a pan‐caspase inhibitor (Z‐VAD‐FMK). Again, increased ROS levels in the cell showed that death may occur by apoptosis. For this reason, the accuracy of ROS‐induced apoptosis in cells has been proven as a result of the application of N‐acetylcysteine (NAC), which is a ROS inhibitor. The efficacy of the complex was compared with Cisplatin and ligands. The results showed that the Cu(II) flavonoid complex is cytotoxic on lung cancer cells and may have the potential to act as an effective metal‐based anticancer drug with a lower IC50 over Cisplatin.
N-acetylcysteine (NAC) is a recognized antioxidant in culture studies and treatments for oxidative stress-related diseases, but in some cases, NAC is a pro-oxidant. To study the effect of NAC on cell proliferation in the presence or absence of ROS stress, we used the stable ROS generator gallic acid (GA) to treat CL1-0 lung cancer cell models with different antioxidant activities. Different antioxidant activities were achieved through the ectopic expression of different PERP-428 single nucleotide polymorphisms. GA increased ROS levels in CL1-0/PERP-428C cells and caused cell death but had no effect on CL1-0/PERP-428G cells within 24 h. We found that 0.1 mM NAC eliminated GA-induced growth inhibition, but 0.5 mM NAC enhanced GA-induced CL1-0/PERP-428C cell death. However, in the absence of GA, NAC exceeding 2 mM inhibited the growth of CL1-0/PERP-428G cells more significantly than that of CL1-0/PERP-428C cells. Without GA, NAC has an antioxidant effect. Under GA-induced ROS stress, NAC may have pro-oxidant effects. Each cell type has a unique range of ROS levels for survival. The levels of ROS in the cell determines the sensitivity of the cell to an antioxidant or pro-oxidant. Cells with different antioxidant capacities were used to show that the intracellular ROS level affects NAC function and provides valuable information for the adjuvant clinical application of NAC.
Gastric cancer remains a major unmet clinical problem with over 1 million new cases worldwide. It is the fourth most commonly occurring cancer in men and the seventh most commonly occurring cancer in women. A major fraction of gastric cancer has been linked to variety of pathogenic infections including but not limited to Helicobacter pylori (H. pylori) or Epstein Barr virus (EBV). Strategies are being pursued to prevent gastric cancer development such as H. pylori eradication, which has helped to prevent significant proportion of gastric cancer. Today, treatments have helped to manage this disease and the 5-year survival for stage IA and IB tumors treated with surgery are between 60 and 80%. However, patients with stage III tumors undergoing surgery have a dismal 5-year survival rate between 18 and 50% depending on the dataset. These figures indicate the need for more effective molecularly driven treatment strategies. This review discusses the molecular profile of gastric tumors, the success, and challenges with available therapeutic targets along with newer biomarkers and emerging targets.
N-acetyl-l-cysteine, commonly known as N-acetylcysteine or NAC, is the acetylated derivative of the amino acid l-cysteine and a precursor to the powerful antioxidant glutathione. NAC has been in clinical practice for several decades. It was first introduced in the 1960s as a mucolytic to reduce the viscosity of abnormal respiratory tract secretions in various tracheobronchial and bronchopulmonary diseases. NAC is best known as an antidote to paracetamol poisoning and sometimes used as a dietary supplement. However, there has been a growing interest in the therapeutic potential of NAC in a range of diseases, in particular as an antioxidant where oxidative stress is a driver. By virtue of divergent biological functions, NAC has been shown to affect neoplastic growth in preventative, pre-neoplastic and treatment stages. In the present chapter, history, pharmacological features, potential and actual applications, and safety profile of NAC are reviewed.
In the present project, we focused on three common or unique types of secondary peritoneal surface malignancies. Peritoneal dissemination is the most frequent pattern of metastasis and recurrence of gastric cancer (GC). In the context of colorectal cancer (CRC), too, peritoneal carcinomatosis is a common mode of the disease progression and a frequent finding in the recurrent disease. Pseudomyxoma peritonei (PMP) is also believed to originate from an appendiceal mucinous tumor with a characteristic peritoneal dissemination pattern associated with copious secretion and multifocal accumulation of mucin. With respect to the natural history, peritoneal carcinomatosis from CRC and, in particular, GC follows a rapidly fatal course. By comparison, PMP is an indolent, less aggressive tumor with slow progression to death. However, it is a debilitating, recurring condition that severely impacts quality of life and is associated with morbid, even fatal, complications. The state-of-the-art, multidisciplinary approach to these conditions (extensive peritonectomy plus perioperative intraperitoneal chemotherapy) has been promising in select patients. However, current chemotherapy protocols usually fail to maintain the surgical complete response. In this chapter, results of our research project evaluating the efficacy of an experimental formulation composed of bromelain and NAC (two naturally occurred agents with good safety profiles) on both in vitro and in vivo models are presented and discussed. Taking into account the critical role of membrane-associated and secreted mucins in these pathological conditions, the effect of treatment on the expression and/or secretion of mucins, including the specific mucins MUC1, MUC2, and MUC5AC, was also explored. Based on our results, this formulation shows preclinical promise for intraperitoneal treatment of gastrointestinal PC and PMP, at least as an adjuvant, affecting tumor cells and their associated mucins both.
Background:
The nonessential amino acid cysteine is known to be involved in many antioxidant and anticarcinogenic pathways. Cysteinylglycine is a pro-oxidant metabolite of glutathione and a precursor of cysteine.
Objective:
To examine the relation between serum cysteine and cysteinylglycine and risk of gastric adenocarcinomas, esophageal squamous cell carcinomas, and head and neck squamous cell carcinomas, we conducted a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention study of male Finnish smokers aged 50-69 y at baseline.
Design:
In total, 170 gastric adenocarcinomas, 68 esophageal squamous cell carcinomas, and 270 head and neck squamous cell carcinomas (identified from the Finnish Cancer Registry) were matched one-to-one with cancer-free control subjects on age and the date of serum collection. We calculated ORs and 95% CIs with the use of a multivariate-adjusted conditional logistic regression.
Results:
Cysteine had a U-shaped association with gastric adenocarcinomas; a model that included a linear and a squared term had a significant global P-test (P = 0.036). Serum cysteinylglycine was inversely associated with adenocarcinomas of the gastric cardia (OR for above the median compared with below the median: 0.07; 95% CI: 0.01, 0.70; n = 38 cases) but not for other sites. Both cysteine and cysteinylglycine were not associated with esophageal squamous cell carcinoma or head and neck squamous cell carcinoma.
Conclusions:
We observed associations between serum cysteine and cysteinylglycine with upper gastrointestinal cancer risk. Future studies are needed to replicate these findings. This trial was registered at clininicaltrials.gov as NCT00342992.
Preliminary studies have suggested that the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) may be effective in inhibiting the growth of pancreatic cancer cells. In-depth cellular and molecular analyses were carried out to determine NAC's mode of action in inhibiting the growth of a well-characterized pancreatic cancer cell line (AsPC-1). Standardized assays were used to monitor cellular growth, apoptosis, levels of ROS, cellular senescence, migration, and invasiveness. Cell stiffness was measured using atomic force microscopy. Gene expression was monitored by quantitative PCR. NAC significantly inhibits the growth and metastatic potential of AsPC-1 cells by inducing cell-cycle arrest in G1 and subsequent cellular senescence and decreased invasiveness. These anticancer properties are associated with an unexpected increase in the intracellular concentrations of ROS. NAC does not decrease the susceptibility of AsPC-1 cells to the anticancer drugs gemcitabine, mitomycin C, and doxorubicin. NAC-induced changes in gene expression are consistent with the onset of mesenchymal-to-epithelial transition. In conclusion, our findings indicate that NAC induces an integrated series of responses in AsPC-1 cells that make it a highly promising candidate for development as a pancreatic cancer therapeutic.
Neointima formation, mainly characterized by smooth muscle cell proliferation, is an important cause of venous bypass graft failure. The therapeutic potential of the antioxidant N-acetylcysteine (NAC) to attenuate smooth muscle cell proliferation and neointima formation was examined in vivo. The effects of NAC on hyperoxia-induced venous smooth muscle cell (VSMC) cytokine production and proliferation were addressed in vitro.
Rats underwent autologous epigastric vein-to-femoral artery interposition grafting. Fourteen rats received oral NAC, and a similar control group received saline. Histomorphometric analysis was performed after 7 days or 3 weeks. Cytokine analysis and cell proliferation assay were performed in cultured human VSMCs after hyperoxic or normoxic exposure and NAC administration.
NAC-treated rats displayed a threefold reduction in neointimal area, a sixfold reduction in stenosis rate, and a twofold reduction in VSMC proliferation after vein graft surgery. Incubation of VSMCs in 70 per cent oxygen stimulated the release of mitogenic inflammatory cytokines interleukin (IL) 6 and IL-8. Cytokine-rich medium from these VSMCs induced proliferation of normoxic VSMCs. NAC inhibited hyperoxia-induced cytokine release and VSMC proliferation.
NAC attenuated neointima formation and vein graft stenosis by reducing VSMC proliferation in vivo, and prevented hyperoxia-induced cytokine production and VSMC proliferation in vitro.
In this study, we investigated the role of reduced glutathione (GSH) and nuclear factor-kappaB (NFkappaB) in hypoxia-induced apoptosis. Hypoxia caused p53-dependent apoptosis in murine embryonic fibroblasts transfected with Ras and E1A. N-Acetyl-l-cysteine (NAC) but not other antioxidants, such as the vitamin E analog trolox and epigallocatechin-3-gallate, enhanced hypoxia-induced caspase-3 activation and apoptosis. NAC also enhanced hypoxia-induced apoptosis in two human cancer cell lines, MIA PaCa-2 pancreatic cancer cells and A549 lung carcinoma cells. In murine embryonic fibroblasts, all three antioxidants blocked hypoxia-induced reactive oxygen species formation. NAC did not enhance hypoxia-induced cytochrome c release but did enhance poly-(ADP ribose) polymerase cleavage, indicating that NAC acted at a post-mitochondrial level. NAC-mediated enhancement of apoptosis was mimicked by incubating cells with GSH monoester, which increased intracellular GSH similarly to NAC. Hypoxia promoted degradation of an inhibitor of kappaB(IkappaBalpha), NFkappaB-p65 translocation into the nucleus, NFkappaB binding to DNA, and subsequent transactivation of NFkappaB, which increased X chromosome-linked inhibitor of apoptosis protein levels. NAC failed to block degradation by IkappaBalpha and sequestration of the p65 subunit of NFkappaB to the nucleus. However, NAC did abrogate hypoxia-induced NFkappaB binding to DNA, NFkappaB-dependent gene expression, and induction of X chromosome-linked inhibitor of apoptosis protein. In conclusion, NAC enhanced hypoxic apoptosis by a mechanism apparently involving GSH-dependent suppression of NFkappaB transactivation.
The imbalance between proliferation and programmed cell death (apoptosis) is one of the critical cellular events that lead to oncogenesis. While there is no doubt that uncontrolled cell proliferation is essential for the development of cancer, deregulation of apoptosis may play an equally important role in this process. Inhibition of apoptosis prevents the death of tumor cells with DNA damage either associated with carcinogenic initiation or cancer therapy. The transcription factor NF-κB is a key regulator in oncogenesis. By promoting proliferation and inhibiting apoptosis, NF-κB tips the balance between proliferation and apoptosis toward malignant growth in tumor cells.
It has been demonstrated that nitric oxide (NO) can promote apoptosis in human cancer cells. To test the protective effects of antioxidants (N-acetyl-L-cysteine (LNAC) and free-radical spin traps (5,5-dimethyl-1-pyrroline N-oxide and 2,2,6,6,-tetramethyl-1-piperidinyloxy) against NO-induced apoptosis, a human colon cancer cell line (COLO 205) was treated with NO, and its survival rate was evaluated both with and without antioxidant therapy. LNAC arrested the development of progression of apoptosis in COLO 205 cells in a dose-dependent manner, promoted long-term survival, and prevented the internucleosomal DNA cleavage induced by NO. The intracellular level of glutathione (GSH) was found to be elevated in cells after exposure to LNAC. The bax protein levels were elevated by NO treatment, and this effect was blocked by LNAC. On the other hand, the bcl-2 oncoprotein level in the LNAC-pretreated cells was significantly elevated in a time-dependent manner compared to cells that received NO pretreatment. In summary, our results suggest that the protective effect of LNAC may be linked to its inducement of increases in cellular GSH and bcl-2 protein levels and to its suppression of cellular bax protein in treated cells.
We have reported previously that N-acetyl-L-cysteine facilitated interleukin-1beta-induced nitric oxide synthase (iNOS) expression in rat vascular smooth muscle cells. The present study compares the effect of N-acetyl-L-cysteine with other antioxidants and tested the possibility that N-acetyl-L-cysteine potentiates iNOS induction by a mechanism involving activation of p44/42 mitogen-activated protein kinases (MAPKs). The effect of N-acetyl-L-cysteine on potentiating interleukin-1beta-induced nitrite production and iNOS expression was mimicked either by the enantiomers, L-cysteine and D-cysteine, or by a non-thiol-containing antioxidant, L-ascorbic acid. Interleukin-1beta activated p44/42 MAPK, and this activation was enhanced in the presence of N-acetyl-L-cysteine. Inhibition of p44/42 MAPK phosphorylation by the selective inhibitor PD98059 clearly inhibited iNOS expression induced by interleukin-1beta either in the absence or in the presence of N-acetyl-L-cysteine. These observations, combined with previous results, indicate that p44/42 MAPK activation is required for interleukin-1beta induction of iNOS and that N-acetyl-L-cysteine may act as a reducing agent and facilitate interleukin-1beta-induced iNOS expression through a reduction/oxidation-related mechanism involving potentiation of cytokine activation of the p44/42 MAPK signaling pathway.
We investigated whether the dissipation of mitochondrial transmembrane potential (Delta(Psi)(m)) was involved in apoptosis of cultured human aortic endothelial cells (HAECs) exposed to hyperglycemic conditions (30 mmol/L glucose). In parallel experiments, N-acetyl-L-cysteine (NAC) was added to the culture medium to verify whether this antioxidant may prevent apoptosis in these cells. The binding of annexin V and DNA fragmentation were measured, in addition to the production of reactive oxygen species (ROS), the number of cells with depolarized mitochondria, and the intracellular glutathione (GSH) content. As compared to the control (5 mmol/L glucose), high-glucose treatment increases both ROS generation and the number of cells binding annexin V. Moreover, a simultaneous decrease of intracellular GSH content was observed, which was accompanied by an increased number of cells showing both depolarized mitochondria and fragmented DNA. Incubation of HAECs with high glucose in the presence of 10 mmol/L NAC prevented the drop of intracellular GSH content, and decreased both ROS generation and the number of cells committed to apoptosis. These results suggest that high glucose triggers the same cascade of molecular events as do other apoptosis inducers in other cells. Among these events, the disruption of mitochondrial membrane barrier function might be decisive because it causes the release of soluble proteins from intermembrane space, which then induce nuclear apoptotic changes.
Oxidative stress generated by an imbalance between reactive oxygen species (ROS) and antioxidants contributes to the pathogenesis of arthritis, cancer, cardiovascular, liver and respiratory diseases. Proinflammatory cytokines and growth factors stimulate ROS production as signaling mediators. Antioxidants such as N-acetylcysteine (NAC) have been used as tools for investigating the role of ROS in numerous biological and pathological processes. NAC inhibits activation of c-Jun N-terminal kinase, p38 MAP kinase and redox-sensitive activating protein-1 and nuclear factor kappa B transcription factor activities regulating expression of numerous genes. NAC can also prevent apoptosis and promote cell survival by activating extracellular signal-regulated kinase pathway, a concept useful for treating certain degenerative diseases. NAC directly modifies the activity of several proteins by its reducing activity. Despite its nonspecificity, ability to modify DNA and multiple molecular modes of action, NAC has therapeutic value for reducing endothelial dysfunction, inflammation, fibrosis, invasion, cartilage erosion, acetaminophen detoxification and transplant prolongation.
The imbalance between proliferation and programmed cell death (apoptosis) is one of the critical cellular events that lead to oncogenesis. While there is no doubt that uncontrolled cell proliferation is essential for the development of cancer, deregulation of apoptosis may play an equally important role in this process. Inhibition of apoptosis prevents the death of tumor cells with DNA damage either associated with carcinogenic initiation or cancer therapy. The transcription factor NF-kappaB is a key regulator in oncogenesis. By promoting proliferation and inhibiting apoptosis, NF-kappaB tips the balance between proliferation and apoptosis toward malignant growth in tumor cells.
To investigate the effect of antioxidants N-acetyl-L-cysteine and catalase on H(2)O(2)-induced apoptosis of lens epithelial cells and the activity of caspase-3.
Rat lenses were incubated in MEM with 2 mmol/L H(2)O(2) (H(2)O(2) groups) or without H(2)O(2) (control groups), antioxidants 100 micromol/L N-acetyl-L-cysteine and 900 U/ml catalase were used to block the oxidative injury of lens epithelial cells respectively. Lens opacification and apoptosis of lens epithelial cells were detected using transmission electron microscope and AnnexinV-PI staining after 24 hours incubation. The activity of caspase-3 was analyzed by Western blot at the same time.
The observations revealed that 2 mmol/L H(2)O(2) could apparently induce lens opacification and lens epithelial cell apoptosis in vitro, the apoptosis rate increased to (31.20 +/- 3.31)% after 24 hours incubation, which is also the time caspase-3 was activated. Treatment with N-acetyl-L-cysteine and catalase could inhibit lens opacification, apoptosis and caspase-3 activation induced by H(2)O(2). The apoptosis rate decreased to (20.90 +/- 3.16)% and (15.02 +/- 2.41)% respectively (P < 0.01).
These data indicate that antioxidants N-acetyl-L-cysteine and catalase, possibly through regulation of the activity of caspase-3, can prevent lens opacification and apoptosis of lens epithelial cells.
Intense exercise leads to post-exercise lymphocytopenia and immunosuppression, possibly by triggering lymphocyte apoptosis. To test the role of oxidative stress on exercise-induced lymphocyte apoptosis, we administered the antioxidant N-acetyl--cysteine (NAC) and measured apoptosis in intestinal lymphocytes (IL) from exhaustively exercised animals. Eighty-seven female C57BL/6 mice were randomly assigned to receive NAC (1 g/kg) or saline 30 min prior to treadmill exercise for 90 min at 2degrees slope (30 min at 22 m min(-1), 30 min at 25 m min(-1), and 30 min at 28 m min(-1)) and sacrificed immediately (Imm) or 24 hours (24 h) after cessation of exercise. Control mice (nonexercised) were exposed to treadmill noise and vibration without running. Exercise increased IL phosphatidylserine externalization (p<0.001), mitochondrial membrane depolarization (p<0.05), and decreased intracellular glutathione concentrations (p<0.05) immediately following exercise in saline relative to nonexercised mice. At 24 h post-exercise, saline injected mice had fewer total (p<0.001) and CD3+ (p<0.005) IL compared to nonexercised animals. NAC injection in mice maintained intracellular glutathione levels, prevented phosphatidylserine externalization, mitochondrial membrane depolarization, and loss of IL immediately and 24 h after exercise. These data suggest that lymphocyte apoptosis precedes post-exercise lymphocytopenia and may be due to oxidative stress.
Chronic renal failure (CRF) is usually accompanied by abnormalities of both humoral and cellular immune response. The aim of the study was to investigate the influence of N-acetyl-cysteine (NAC) on intracellular oxidative stress and apoptosis rate of T lymphocytes in children with CRF. Twenty-two children (aged 4-16, mean 7.4) with CRF treated with dialysis were enrolled in the study. Intracellular reactive oxygen species (ROS) production was quantified by mean rhodamine 123 (RHO) fluorescence intensity with flow cytometry. Annexin V FITC was used for identifying apoptotic cells. Mean fluorescence intensity (MFI), which reflected intracellular oxidative stress in T lymphocytes, was increased in patients with CRF compared with the controls (CD3+: 31.58+/-11.58 vs 22.55+/-4.97, p = 0.043; CD3+CD4+: 32.50+/-8.59 vs 27.75+/-12.76, NS; CD3+CD8+: 32.10+/-11.85 vs 20.77+/- 4.89, p =0.012). Apoptotic T lymphocytes occurred more frequently in patients with CRF treated with hemodialysis (HD) (11.36+/-6.96%) than in the controls (6.14%+/-3.36%; p = 0.025). After 24 h incubation with NAC MFI and apoptosis rate decreased significantly in all subpopulations of lymphocytes. NAC, as a strong antioxidant, has a favorable effect on intracellular oxidative stress and apoptosis rate of T lymphocytes in patients with CRF. A decreased apoptosis rate may have positive effect on functional abnormalities of T cells already found in patients with CRF.
Redox imbalance due to oxidative stress or excessive antioxidant levels can alter apoptotic responses. Recently, antioxidants like N-acetylcysteine (NAC) were reported to inhibit H(2)O(2)-mediated necrotic cell death, although they were inactive against apoptosis induced by other agents like etoposide. NAC was also found to kill preferentially tumor cells compared to normal fibroblasts at 20-50mM, but these concentrations are lethal to normal splenocytes. We now demonstrate that 10mM NAC, a non-toxic concentration, can enhance the UV radiation-mediated apoptosis of human C8161 melanoma cells. Compared to treatment with UV radiation alone, combination treatment with NAC doubled the ratio of activated caspase-3 to pro-caspase-3 and produced greater fragmentation of the retinoblastoma protein and the E2F-4 transcription factor without affecting the E2F-1 protein. These effects of joint NAC-UV radiation treatment were counteracted by the overexpression of the bcl-2 gene. To our knowledge, this report is the first to: (i) demonstrate a synergy between DNA-damaging agents, like UV radiation, and antioxidants, like NAC, and (ii) show that a Bcl-2-inhibitable E2F-4 fragmentation occurs concurrently with caspase-3 activation and apoptosis.
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