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1.
Biochim Biophys Acta Mol Basis Dis ; 1866(3): 165605, 2020 03 01.
Article in English | MEDLINE | ID: mdl-31740402

ABSTRACT

Activation of costimulatory receptor 4-1BB enhances T helper 1 (Th1) and CD8 T cell responses in protective immunity, and prevents or attenuates several autoimmune diseases by increasing Treg numbers and suppressing Th17 or Th2 effector response. We undertook this study to elucidate the impact of enforced 4-1BB activation on the development of Sjögren's syndrome (SS)-like sialadenitis in non-obese diabetic (NOD) model of this disease. An anti-4-1BB agnostic antibody was intraperitoneally injected to female NOD mice aged 7 weeks, prior to the disease onset that occurs around 10-11 weeks of age, 3 times weekly for 2 weeks, and the mice were analyzed for SS pathologies at age 11 weeks. The salivary flow rate was markedly higher in the anti-4-1BB-treated NOD mice compared to the IgG-treated controls. Anti-4-1BB treatment significantly reduced the leukocyte infiltration of the submandibular glands (SMGs) and the levels of serum antinuclear antibodies. Flow cytometric analysis showed that the percentages of CD4 T cells, Th17 cells and plasmacytoid dendritic cells among SMG leukocytes were markedly reduced by anti-4-1BB treatment, in conjunction with a reduction in SMG IL-23p19 mRNA levels and serum IL-17 concentrations. Although the proportion of Tregs and IL-10 mRNA levels in SMGs were not altered by 4-1BB activation, IL-10 mRNA levels in salivary gland-draining lymph nodes and serum IL-10 concentrations were both markedly increased. While anti-4-1BB treatment did not affect the amount of Th1 cells and IFNγ mRNA in the SMGs, it increased these measurables in salivary gland-draining lymph nodes. Hence, agonistic activation of 4-1BB impedes the development of SS-like sialadenitis and hyposalivation.


Subject(s)
Antibodies, Monoclonal/pharmacology , Sialadenitis/prevention & control , Sjogren's Syndrome/prevention & control , Th1 Cells/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/agonists , Xerostomia/prevention & control , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Disease Models, Animal , Female , Interleukin-17/metabolism , Mice , Mice, Inbred NOD , Sialadenitis/etiology , Sialadenitis/pathology , Sjogren's Syndrome/etiology , Sjogren's Syndrome/pathology , Th1 Cells/drug effects , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Xerostomia/etiology , Xerostomia/pathology
2.
Mol Carcinog ; 56(12): 2566-2577, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28667779

ABSTRACT

Histone deacetylase (HDAC) inhibitors, especially suberoylanilide hydroxamic acid (SAHA) induce apoptosis in various cancer cells. Here, we investigated the effect of SAHA on apoptosis in lung cancer cells and addressed the role of reactive oxygen species (ROS), glutathione (GSH), and thioredoxin1 (Trx1) levels in this process. We also identified the miRNAs that down-regulate Trx1 expression at RNA level and thereby influence apoptotic cell death of SAHA increased intracellular ROS levels and promoted apoptotic cell death in cancerous cells but not in non-cancerous normal lung cells. Likewise, SAHA induced GSH depletion specifically in cancerous cells. While N-acetyl cysteine (NAC) reduced ROS level and reversed the effect of SAHA on cell death, L-buthionine sulfoximine (BSO) further enhanced GSH depletion, and promoted cell death. SAHA decreased the mRNA and protein levels of Trx1 in lung cancer cells. Knockdown/suppression of Trx1 intensified apoptosis in SAHA-treated lung cancer cells whereas overexpression of Trx1 prevented the cell death in these cells. SAHA up-regulated the level of miR-129-5p, which binds to 3' untranslated region (3'UTR) of Trx1 and down-regulates Trx1 expression. Down-regulation of Trx1 led to activation of apoptosis-signal regulating kinase (ASK), which induced apoptotic cell death by triggering ASK-JNK or ASK-p38 kinase pathway. In conclusion, changes in ROS and GSH levels in SAHA-treated lung cancer cells partially co-related with cell death. SAHA induced apoptosis via the down-regulation of Trx1, which was regulated by miR-129-5p.


Subject(s)
Apoptosis/drug effects , Hydroxamic Acids/pharmacology , MicroRNAs/genetics , Thioredoxins/genetics , Up-Regulation/drug effects , 3' Untranslated Regions/genetics , A549 Cells , Acetylcysteine/pharmacology , Blotting, Western , Buthionine Sulfoximine/pharmacology , Cell Line , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Glutathione/metabolism , Histone Deacetylase Inhibitors/pharmacology , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , MAP Kinase Kinase Kinase 5/metabolism , MAP Kinase Signaling System/drug effects , Reactive Oxygen Species/metabolism , Thioredoxins/metabolism , Vorinostat
3.
Oncotarget ; 8(11): 17726-17737, 2017 Mar 14.
Article in English | MEDLINE | ID: mdl-28099148

ABSTRACT

Suberoylanilide hydroxamic acid (SAHA) as a histone deacetylase (HDAC) inhibitor has anti-cancer effect. Here, we evaluated the effect of SAHA on HDAC activity and cell growth in many normal lung and cancer cells. We observed that the HDAC activities of lung cancer cells were higher than that of normal lung cells. SAHA inhibited the growth of lung cancer cells regardless of the inhibitory effect on HDAC. This agent induced a G2/M phase arrest and apoptosis, which was accompanied by mitochondrial membrane potential (MMP: ΔΨm) loss in lung cancer cells. However, SAHA did not induce cell death in normal lung cells. All tested caspase inhibitors prevented apoptotic cell death in SAHA-treated A549 and Calu-6 lung cancer cells. Treatment with tumor necrosis factor-alpha (TNF-α) enhanced apoptosis in SAHA-treated lung cancer cells through caspase-8 and caspase-9 activations. Especially, SAHA increased the expression level of TNF-α receptor 1 (TNFR1), especially acetylation of the region of TNFR1 promoter -223/-29 in lung cancer cells. The down-regulation of TNFR1 suppressed apoptosis in TNF-α and SAHA-treated lung cancer cells. In conclusion, SAHA inhibited the growth of lung cancer cells via a G2/M phase arrest and caspase-dependent apoptosis. SAHA also enhanced apoptotic effect of TNF-α in human lung cancer cells through up-regulation of TNFR1. TNF-α may be a key to improve anti-cancer effect of HDAC inhibitors.


Subject(s)
Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Lung Neoplasms/pathology , Receptors, Tumor Necrosis Factor, Type I/biosynthesis , Tumor Necrosis Factor-alpha/metabolism , A549 Cells , Acetylation , Apoptosis/drug effects , Caspase 8/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Activation/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , Histone Deacetylases/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Up-Regulation , Vorinostat
4.
Int J Oncol ; 48(5): 2197-204, 2016 May.
Article in English | MEDLINE | ID: mdl-26936390

ABSTRACT

Mesothelioma is an aggressive tumor which is mainly derived from the pleura of lung. In the present study, we evaluated the anticancer effect of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor on human mesothelioma cells in relation to the levels of HDAC1, reactive oxygen species (ROS) and thioredoxin (Trx). While 1 µM SAHA inhibited cell growth in Phi and ROB cells at 24 h, it did not affect the growth in ADA and Mill cells. Notably, the level of HDAC1 was relatively overexpressed among Phi, REN and ROB cells. SAHA induced necrosis and apoptosis, which was accompanied by the cleavages of PARP and caspase-3 in Phi cells. This agent also increased the loss of mitochondrial membrane potential (MMP, ΔΨm) in Phi cells. All the tested caspase inhibitors attenuated apoptosis in SAHA-treated Phi cells whereas HDAC1 siRNA enhanced the apoptotic cell death. SAHA increased intracellular ROS levels including O2•- in Phi cells. N-acetyl cysteine (NAC) and vitamin C (Vit.C) significantly reduced the growth inhibition and death of Phi cells caused by SAHA. This drug decreased the mRNA and protein levels of Trx1 in Phi and ROB cells. Furthermore, Trx1 siRNA increased cell death and O2•- level in SAHA-treated Phi cells. In conclusion, SAHA selectively inhibited the growth of Phi and ROB mesothelioma cells, which showed the higher basal level of HDAC1. SAHA-induced Phi cell death was related to oxidative stress and Trx1 levels.


Subject(s)
Histone Deacetylase 1/metabolism , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Mesothelioma/metabolism , Thioredoxins/metabolism , Apoptosis , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Membrane Potential, Mitochondrial/drug effects , Mesothelioma/drug therapy , Mesothelioma/genetics , Reactive Oxygen Species/metabolism , Vorinostat
5.
Oncol Rep ; 35(1): 546-51, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26530353

ABSTRACT

Mesothelioma is an aggressive tumor associated with asbestos exposure. Auranofin as an inhibitor of thioredoxin reductase (TrxR) affects many biological processes such as inflammation and proliferation. In the present study, we investigated the cellular effects of auranofin on patient-derived mesothelioma cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Basal TrxR1 levels have no difference between mesothelial cells and certain mesothelioma cells. In particular, ADA, CON and Hmeso mesothelioma cells showed lower levels of TrxR1 expression. Auranofin inhibited the proliferation of mesothelioma cells in a dose-dependent manner. Among mesothelioma cells were ADA and CON cells sensitive to auranofin. This agent also induced caspase-independent apoptosis and necrosis in ADA cells. In addition, auranofin increased ROS levels including O2(•-) and induced GSH depletion in mesothelioma cells. While N-acetyl cysteine (NAC) prevented cell death and decreased ROS levels in auranofin-treated mesothelioma cells, L-buthionine sulfoximine (BSO) intensified apoptosis and GSH depletion in these cells. In conclusion, auranofin induced mesothelioma cell death through oxidative stress and the death was regulated by the status of GSH content.


Subject(s)
Auranofin/pharmacology , Glutathione/metabolism , Mesothelioma/metabolism , Oxidative Stress/drug effects , Apoptosis , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Thioredoxin Reductase 1/metabolism
6.
J Cell Biochem ; 117(5): 1250-61, 2016 May.
Article in English | MEDLINE | ID: mdl-26460805

ABSTRACT

Suberoyl bishydroxamic acid (SBHA), a histone deacetylase (HDAC) inhibitor, can show an anticancer effect. In this study, we investigated the effects of SBHA on the growth inhibition and death of Calu-6 and NCI-H1299 cells in relation to reactive oxygen species (ROS) and antioxidant levels. SBHA inhibited the growth of Calu-6 and NCI-H1299 lung cancer cells with an IC50 of 50 µM at 72 h. This agent induced apoptosis in Calu-6 cells and triggered to a G2/M phase arrest in NCI-H1299 cells. Although it also reduced the growth of normal human pulmonary fibroblast (HPF) cells, the susceptibility of Calu-6 cells to SBHA was higher than that of HPF cells. In addition, SBHA did not affect the growth of human small airway epithelial cells (HSAEC). Regarding ROS and antioxidant levels, SBHA increased ROS level and glutathione (GSH) depletion in Calu-6 and NCI-H1299 cells whereas it decreased ROS levels in HPF and HSAEC. SBHA also decreased thioredoxin1 (Trx1) level in Calu-6 cells. Although the down-regulation of Trx1 intensified apoptosis and ROS level in SBHA-treated Calu-6 cells, the overexpression of Trx1 attenuated apoptosis and ROS level in these cells. This down-regulation of Trx1 did not affect apoptosis-signaling regulating kinase1 (ASK1) activation. In conclusion, the down-regulation of Trx1 by SBHA was closely involved in cell death in Calu-6 cells.


Subject(s)
Apoptosis/drug effects , Down-Regulation/drug effects , Hydroxamic Acids/pharmacology , Thioredoxins/metabolism , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , G2 Phase Cell Cycle Checkpoints/drug effects , Glutathione/metabolism , Humans , Lung/cytology , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , RNA Interference , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thioredoxins/genetics
7.
Int J Oncol ; 48(2): 813-20, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26647857

ABSTRACT

Antimycin A (AMA) inhibits the growth of various cells via stimulating oxidative stress-mediated death. However, little is known about the anti-growth effect of AMA on normal primary lung cells. Here, we investigated the effects of AMA on cell growth inhibition and death in human pulmonary fibroblast (HPF) cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. AMA inhibited the growth of HPF cells with an IC50 of ~150 µM at 24 h. AMA induced a G1 phase arrest of the cell cycle and it also triggered apoptosis accompanied by the loss of mitochondrial membrane potential (MMP; ∆Ψm). AMA increased ROS levels including O2᛫- in HPF cells from the early time point of 25 min. It induced GSH depletion in HPF cells in a dose-dependent manner. Z-VAD (a pan-caspase inhibitor) did not significantly prevent cell death and MMP (∆Ψm) loss induced by AMA. N-acetylcysteine (NAC; an antioxidant) attenuated cell growth inhibition, death and MMP (∆Ψm) loss in AMA-treated HPF cells and NAC generally decreased the ROS level in these cells as well. Vitamin C enhanced cell growth inhibition, death, GSH depletion and O2᛫- levels in 100 µM AMA-treated HPF cells whereas this agent strongly attenuated these effects in 200 µM AMA-treated cells. In conclusion, AMA inhibited the growth of HPF cells via apoptosis as well as a G1 phase arrest of the cell cycle. AMA-induced HPF cell death was related to increased ROS levels and GSH depletion.


Subject(s)
Antimycin A/pharmacology , Cell Death/drug effects , Fibroblasts/drug effects , Glutathione/metabolism , Lung/drug effects , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Antioxidants/metabolism , Apoptosis/drug effects , Ascorbic Acid/metabolism , Caspase Inhibitors/pharmacology , Cell Line , Cell Proliferation/drug effects , Fibroblasts/metabolism , G1 Phase/drug effects , Growth Inhibitors/pharmacology , Humans , Lung/metabolism , Membrane Potential, Mitochondrial/drug effects , Oxidative Stress/drug effects
8.
Mol Med Rep ; 11(2): 1428-34, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25370167

ABSTRACT

Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti­cancer drug. In the present study, the anti­growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP­ribose) polymerase and loss of mitochondrial membrane potential. The pan­caspase inhibitor, benzyloxycarbonyl­Val­Ala­Asp­fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan­caspase inhibitor reduced the levels of O2•- and GSH depletion in Au­treated HeLa cells. The antioxidant, N­acetyl cysteine, not only attenuated apoptosis and necrosis in the Au­treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L­buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au­treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Auranofin/pharmacology , Glutathione/metabolism , Oxidative Stress/drug effects , Acetylcysteine/pharmacology , Antioxidants/pharmacology , Buthionine Sulfoximine/pharmacology , Caspase Inhibitors/pharmacology , Cell Proliferation/drug effects , Glutathione/analysis , Glutathione/deficiency , HeLa Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Necrosis , Oligopeptides/pharmacology , Singlet Oxygen/analysis , Singlet Oxygen/metabolism
9.
Tumour Biol ; 36(3): 2087-95, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25391429

ABSTRACT

PX-12 (1-methylpropyl 2-imidazolyl disulfide) as a thioredoxin (Trx) inhibitor has an anti-tumor effect. However, there is no report about the toxicological effect of PX-12 on lung cancer cells. Here, we investigated the anti-growth effects of PX-12 on Calu-6 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PX-12 induced the growth inhibition of Calu-6 cells with IC50 of nearly 3 µM at 72 h. In contrast, PX-12 did not affect the growth of human small airway epithelial cells (HSAECs). Cell cycle distribution analysis indicated that PX-12 significantly induced a G2/M phase arrest in Calu-6 cells. PX-12 also increased the number of annexin V-FITC-positive cells in Calu-6 cells. All the tested caspase inhibitors markedly prevented Calu-6 cell death induced by PX-12. With regard to ROS and GSH levels, PX-12 increased ROS levels containing O2(·-) in Calu-6 cells and induced the depletion of GSH. N-acetyl cysteine (NAC), which is a well-known antioxidant, significantly reduced O2(·-) level in PX-12-treated Calu-6 cells and prevented apoptosis and GSH depletion in these cells. In conclusion, it is the first report that PX-12 inhibited the growth of Calu-6 cells via a G2/M phase arrest as well as apoptosis, which effect was related to the intracellular increases in ROS levels.


Subject(s)
Apoptosis/drug effects , Disulfides/pharmacology , Imidazoles/pharmacology , Oxidative Stress/drug effects , Acetylcysteine/metabolism , Annexin A5/metabolism , Antineoplastic Agents/pharmacology , Antioxidants/metabolism , Cell Death/drug effects , Cell Line, Tumor , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , G2 Phase Cell Cycle Checkpoints/drug effects , Glutathione/metabolism , Glutathione/pharmacology , Humans , M Phase Cell Cycle Checkpoints/drug effects , Reactive Oxygen Species/metabolism
10.
Tumour Biol ; 36(5): 3429-39, 2015 May.
Article in English | MEDLINE | ID: mdl-25537089

ABSTRACT

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor can induce apoptosis through the formation of reactive oxygen species (ROS). However, there is no report about the regulation of ROS and antioxidant enzymes in SBHA-treated lung cancer cells. Here, we investigated the toxicological effects of SBHA on the regulations of ROS, glutathione (GSH), and antioxidant enzymes, especially thioredoxin (Trx) in A549 lung cancer cells. SBHA inhibited the growth of A549 cells in time- and dose-dependent manners, and it induced apoptosis which accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). SBHA significantly increased ROS levels including O2 (•-) level at 72 h whereas it decreased ROS levels at the early time points (30 min to 3 h). SBHA also induced GSH depletion at 24 and 72 h. N-acetyl cysteine (NAC; a well-known antioxidant) prevented apoptotic cell death and GSH depletion via decreasing ROS in SBHA-treated A549 cells. In addition, SBHA changed the levels of antioxidant-related proteins, especially Trx1. The expression and activity of Trx1 in A549 cells were reduced by SBHA. While the downregulation of Trx1 enhanced cell death, ROS level, and GSH depletion in SBHA-treated A549 cells, the overexpression of Trx1 decreased ROS level in these cells without the prevention of cell death and GSH depletion. In conclusion, SBHA-induced A549 cell death was influenced by changes in ROS and GSH levels. The basal status of Trx1 among other antioxidant proteins was closely correlated with the survival of A549 cells.


Subject(s)
Apoptosis/drug effects , Glutathione/physiology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Lung Neoplasms/drug therapy , Reactive Oxygen Species/metabolism , Thioredoxins/physiology , Acetylcysteine/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Glutathione/analysis , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Thioredoxins/analysis
11.
Int J Oncol ; 44(5): 1745-55, 2014 May.
Article in English | MEDLINE | ID: mdl-24626405

ABSTRACT

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor which has anticancer effects. We evaluated the growth inhibitory effects of SAHA on HeLa cervical cancer cells in relation to reactive oxygen species (ROS) levels. SAHA inhibited the growth of HeLa cells with an IC(50) of approximately 10 µM at 24 h, and induced apoptosis which was accompanied by the cleavage of PARP, caspase-3 activation and loss of mitochondrial membrane potential (MMP; ∆ψ(m)). All the tested caspase inhibitors prevented HeLa cell death induced by SAHA whereas TNF-α intensified apoptotic cell death in SAHA-treated HeLa cells. With respect to ROS and glutathione (GSH) levels, SAHA increased ROS levels, especially mitochondrial O(2)•- in HeLa cells and also induced GSH depletion. Caspase inhibitors reduced the levels of ROS and GSH depletion in SAHA-treated HeLa cells whereas TNF-α enhanced the levels in these cells. The well-known antioxidant N-acetyl cysteine (NAC) attenuated cell death and an increase in ROS levels was caused by SAHA. Moreover, SAHA decreased the levels of thioredoxin 1 (Trx1) in HeLa cells. While the downregulation of Trx1 enhanced cell death and ROS levels in SAHA-treated HeLa cells, the overexpression of Trx1 attenuated the levels in these cells. In conclusion, SAHA inhibited the growth of HeLa cell via caspase-dependent apoptosis, which was influenced by the mitochondrial O(2)•- and Trx1 levels.


Subject(s)
Adenocarcinoma/pathology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Hydroxamic Acids/pharmacology , Reactive Oxygen Species/metabolism , Thioredoxins/metabolism , Uterine Cervical Neoplasms/pathology , Adenocarcinoma/metabolism , Caspase Inhibitors/pharmacology , Caspases/metabolism , Cell Cycle/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Humans , In Vitro Techniques , Membrane Potential, Mitochondrial/drug effects , Uterine Cervical Neoplasms/metabolism , Vorinostat
12.
Vaccine ; 32(15): 1670-7, 2014 Mar 26.
Article in English | MEDLINE | ID: mdl-24513010

ABSTRACT

DNA vaccination with plasmid has conventionally involved vectors designed for transient expression of antigens in injected tissues. Next generation plasmids are being developed for site-directed integration of transgenes into safe sites in host genomes and may provide an innovative approach for stable and sustained expression of antigens for vaccination. The goal of this study was to evaluate in vivo antigen expression and the generation of cell mediated immunity in mice injected with a non-integrating plasmid compared to a plasmid with integrating potential. Hyperactive piggyBac transposase-based integrating vectors (pmhyGENIE-3) contained a transgene encoding either eGFP (pmhyGENIE-3-eGFP) or luciferase (pmhyGENIE-3-GL3), and were compared to transposase-deficient plasmids with the same transgene and DNA backbone. Both non-integrating and integrating plasmids were equivalent at day 1 for protein expression at the site of injection. While protein expression from the non-integrating plasmid was lost by day 14, the pmhyGENIE-3 was found to exhibit sustained protein expression up to 28 days post-injection. Vaccination with pmhyGENIE-3-eGFP resulted in a robust CD8(+) T cell response that was three-fold higher than that of non-integrating plasmid vaccinations. Additionally we observed in splenocyte restimulation experiments that only the vaccination with pmhyGENIE-3-eGFP was characterized by IFNγ producing CD8(+) T cells. Overall, these findings suggest that plasmids designed to direct integration of transgenes into the host genome are a promising approach for designing DNA vaccines. Robust cell mediated CD8(+) T cell responses generated using integrating plasmids may provide effective, sustained protection against intracellular pathogens or tumor antigens.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunity, Cellular , Vaccines, DNA/immunology , Animals , Female , Genes, Reporter , Genetic Vectors , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Plasmids , Transfection , Transgenes , Vaccines, DNA/genetics
13.
Mol Carcinog ; 53(11): 847-57, 2014 Nov.
Article in English | MEDLINE | ID: mdl-23661569

ABSTRACT

Zebularine (Zeb) is a DNA methyltransferase (DNMT) inhibitor to that has an anti-tumor effect. Here, we evaluated the anti-growth effect of Zeb on A549 lung cancer cells in relation to reactive oxygen species (ROS) levels. Zeb inhibited the growth of A549 cells with an IC50 of approximately 70 µM at 72 h. Cell cycle analysis indicated that Zeb induced an S phase arrest in A549 cells. Zeb also induced A549 cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm ), Bcl-2 decrease, Bax increase, p53 increase and activation of caspase-3 and -8. In contrast, Zeb mildly inhibited the growth of human pulmonary fibroblast (HPF) normal cells and lead to a G1 phase arrest. Zeb did not induce apoptosis in HPF cells. In relation to ROS level, Zeb increased ROS level in A549 cells and induced glutathione (GSH) depletion. The well-known antioxidant, N-acetyl cysteine (NAC) prevented the death of Zeb-treated A549 cells. Moreover, Zeb increased the level of thioredoxin reductase 1 (TrxR1) in A549 cells. While the overexpression of TrxR1 attenuated death and ROS level in Zeb-treated A549 cells, the downregulation of TrxR1 intensified death and ROS level in these cells. In conclusion, Zeb inhibited the growth of A549 lung cancer cells via cell cycle arrest and apoptosis. The inhibition was influenced by ROS and TrxR1 levels.


Subject(s)
Apoptosis/drug effects , Cytidine/analogs & derivatives , Lung Neoplasms/pathology , Thioredoxin Reductase 1/biosynthesis , Acetylcysteine/pharmacology , Antioxidants/pharmacology , Caspase 3/biosynthesis , Caspase 8/biosynthesis , Cell Line, Tumor , Cell Proliferation/drug effects , Cytidine/pharmacology , DNA Modification Methylases/antagonists & inhibitors , Down-Regulation , Fibroblasts/drug effects , Fibroblasts/metabolism , G1 Phase Cell Cycle Checkpoints/drug effects , Glutathione/analysis , Glutathione/metabolism , Humans , Lung Neoplasms/metabolism , Membrane Potential, Mitochondrial/drug effects , Proto-Oncogene Proteins c-bcl-2/biosynthesis , RNA Interference , RNA, Small Interfering , Reactive Oxygen Species/metabolism , S Phase Cell Cycle Checkpoints/drug effects , Thioredoxin Reductase 1/genetics , Tumor Suppressor Protein p53/biosynthesis
14.
Int J Oncol ; 44(1): 301-8, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24172913

ABSTRACT

PX-12 (1-methylpropyl 2-imidazolyl disulfide) is an inhibitor of thioredoxin (Trx-1), which has antitumor effects. However, little is known about the toxicological effect of PX-12 on cancer cells. We investigated the anti-growth effects of PX-12 on A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Based on MTT assays, PX-12 inhibited the growth of A549 cells with an IC50 of approximately 20 µM at 72 h. DNA flow cytometric analysis indicated that PX-12 significantly induced the G2/M phase arrest of the cell cycle in A549 cells. This agent also induced apoptotic cell death, as demonstrated by Annexin V-FITC staining cells and the loss of mitochondrial membrane potential MMP (∆ψm). In addition, the administration of Bax siRNA attenuated PX-12-induced A549 cell death. All the tested caspase inhibitors, especially Z-VAD significantly prevented apoptosis induced by PX-12. With respect to ROS and GSH levels, PX-12 increased ROS levels including O2(•)- in A549 cells and induced GSH depletion. N-acetyl cysteine (NAC) markedly reduced ROS levels in PX-12-treated A549 cells. NAC also prevented apoptotic cell death and GSH depletion induced by PX-12. This is the first report to show that PX-12 inhibits the growth of A549 cells via G2/M phase arrest, and Bax-mediated and ROS-dependent apoptosis.


Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Disulfides/administration & dosage , Imidazoles/administration & dosage , Lung Neoplasms/drug therapy , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , G2 Phase Cell Cycle Checkpoints/drug effects , Glutathione/metabolism , Humans , Lung Neoplasms/pathology , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolism
15.
Oncol Lett ; 6(6): 1804-1810, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24260080

ABSTRACT

PX-12, as an inhibitor of thioredoxin (Trx), has antitumor activity. However, little is known about the toxicological effect of PX-12 on cervical cancer cells. In the present study, the growth inhibitory effects of PX-12 on HeLa cervical cancer cells in association with reactive oxygen species (ROS) and glutathione (GSH) levels were investigated. Based on MTT assays, PX-12 inhibited the growth of HeLa cells with an IC50 value of ~7 µM at 72 h. DNA flow cytometry analysis indicated that 5 and 10 µM PX-12 significantly induced a G2/M phase arrest of the cell cycle. PX-12 also increased the number of dead cells and annexin V-fluorescein isothiocyanate-positive cells, which was accompanied by the loss of mitochondrial membrane potential. All the investigated caspase inhibitors significantly rescued certain cells from PX-12-induced HeLa cell death. With respect to ROS and GSH levels, PX-12 increased ROS levels (including O2•-) in HeLa cells and induced GSH depletion. N-acetyl cysteine markedly reduced the levels of O2•- in PX-12-treated HeLa cells, and prevented apoptotic cell death and GSH depletion in these cells. By contrast, L-buthionine sulfoximine intensified cell death and GSH depletion in PX-12-treated HeLa cells. To conclude, this is the first study to demonstrate that PX-12 inhibits the growth of HeLa cells via G2/M phase arrest, as well as inhibiting apoptosis; the effect was associated with intracellular increases in ROS levels and GSH depletion.

16.
Oncol Rep ; 30(6): 2999-3005, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24064712

ABSTRACT

Valproic acid (VPA) as a histone deacetylase (HDAC) inhibitor has an anticancer effect. In the present study, we evaluated the effects of VPA on the growth and death of HeLa cervical cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Dose- and time-dependent growth inhibition was observed in HeLa cells with an IC50 of approximately 10 mM at 24 h. DNA flow cytometric analysis indicated that 10 mM VPA induced a G2/M phase arrest of the cell cycle. This agent also induced apoptosis, which was accompanied by the cleavage of PARP, the activation of caspase-3, -8 and -9, and the loss of mitochondrial membrane potential (MMP; ∆Ψm). All the tested caspase inhibitors significantly prevented HeLa apoptotic cell death induced by VPA, whereas TNF-α intensified the apoptotic cell death. With respect to ROS and GSH levels, VPA increased ROS levels and induced GSH depletion. However, N-acetyl cysteine (NAC; an antioxidant) and L-buthionine sulfoximine (BSO; a GSH synthesis inhibitor) did not significantly affect cell death in VPA-treated HeLa cells. In conclusion, VPA inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis and the growth inhibition is independent of ROS and GSH level changes.


Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Uterine Cervical Neoplasms/drug therapy , Valproic Acid/administration & dosage , Caspase Inhibitors/administration & dosage , Caspases/metabolism , Female , Glutathione/metabolism , HeLa Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolism , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology
17.
Tumour Biol ; 34(2): 1145-53, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23338718

ABSTRACT

Zebularine (Zeb) is a DNA methyltransferase (DNMT) inhibitor that has various biological properties including anti-cancer effect. In the present study, we evaluated the effects of Zeb on the growth and death of Calu-6 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Zeb inhibited the growth of Calu-6 cells with an IC50 of approximately 150 µM at 72 h in a dose-dependent manner. Zeb induced an S phase arrest of the cell cycle and apoptosis in Calu-6 cells. Pan-caspase inhibitor (Z-VAD) and caspase-8 inhibitor (Z-IETD) significantly rescued some cells from Zeb-induced Calu-6 cell death. In relation to ROS and GSH levels, O2 (•-) level was significantly increased in Zeb-treated Calu-6 cells and caspase inhibitors reduced O2 (•-) level in these cells. Zeb induced GSH depletion in HeLa cells, which was attenuated by caspase inhibitors. L-buthionine sulfoximine (BSO), a GSH synthesis inhibitor, intensified the apoptotic cell death, ROS level, and GSH depletion in Zeb-treated Calu-6 cells. In addition, BSO increased Bax protein and decreased Bcl-2 protein in Zeb-treated Calu-6 cells. In conclusion, Zeb inhibited the growth of Calu-6 lung cancer cells via cell cycle arrest and caspase-dependent apoptosis and its cell death was influenced by ROS and GSH level changes.


Subject(s)
Apoptosis/drug effects , Cytidine/analogs & derivatives , Glutathione/metabolism , Lung Neoplasms/pathology , Reactive Oxygen Species/metabolism , Blotting, Western , Caspases/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cytidine/pharmacology , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Membrane Potential, Mitochondrial/drug effects , Tumor Cells, Cultured
18.
Mol Biol Rep ; 40(5): 3807-16, 2013 May.
Article in English | MEDLINE | ID: mdl-23269626

ABSTRACT

Suberoyl bishydroxamic acid (SBHA) is a HDAC inhibitor that can regulate many biological functions including apoptosis and proliferation in various cancer cells. Here, we evaluated the effect of SBHA on the growth of HeLa cervical cancer cells in relation to apoptosis, reactive oxygen species (ROS) and glutathione (GSH) levels. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC50 of approximately 15 µM at 72 h. SBHA also induced apoptosis in HeLa cells, as evidenced by sub-G1 cells, annexin V-FITC staining cells, activations of caspase 3 and 8, and the loss of mitochondrial membrane potential (ΔΨm). In addition, all of the tested caspase inhibitors rescued some cells from SBHA-induced HeLa cell death. SBHA increased ROS levels including O2(•-) and induced GSH depletion in HeLa cells. Generally, caspase inhibitors did not affect ROS levels in SBHA-treated HeLa cells, but they significantly prevented GSH depletion in these cells. Furthermore, while the well-known antioxidants, N-acetyl cysteine and vitamin C, did not affect cell death, ROS level or GSH depletion in SBHA-treated HeLa cells, L-buthionine sulfoximine, a GSH synthesis inhibitor, enhanced cell death and GSH depletion in these cells. In conclusion, SBHA inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis, and the inhibition is independent of ROS level changes, but dependent on GSH level changes.


Subject(s)
Apoptosis/drug effects , Apoptosis/physiology , Glutathione/metabolism , Hydroxamic Acids/pharmacology , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Ascorbic Acid/pharmacology , Buthionine Sulfoximine/pharmacology , Caspase Inhibitors/pharmacology , Cell Proliferation/drug effects , HeLa Cells , Histone Deacetylases/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects
19.
Int J Oncol ; 42(1): 359-66, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23165748

ABSTRACT

Trichostatin A (TSA) as a HDAC inhibitor can regulate many biological properties including apoptosis and cell proliferation in various cancer cells. Here, we evaluated the effect of TSA on the growth and death of HeLa cervical cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Dose- and time-dependent growth inhibition was observed in HeLa cells with an IC50 of approximately 20 nM at 72 h. This agent also induced apoptotic cell death, as evidenced by annexin V-FITC staining cells, caspase-3 activation and the loss of mitochondrial membrane potential (MMP; ∆ψm). In addition, the administration of Bcl-2 siRNA intensified TSA-induced HeLa cell death. All of the tested caspase inhibitors significantly rescued some cells from TSA-induced HeLa cell death. TSA increased O2•- level and induced GSH depletion in HeLa cells. Caspase inhibitors significantly attenuated O2•- level and GSH depletion in TSA-treated HeLa cells. In addition, N-acetyl cysteine (NAC; a well known antioxidant) significantly prevented cell death and GSH depletion in TSA-treated HeLa cells via decreasing O2•- level. In conclusion, TSA inhibited the growth of HeLa cells via Bcl-2-mediated apoptosis, which was closely related to O2•- and GSH content levels.


Subject(s)
Apoptosis/drug effects , Glutathione/metabolism , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Oxidative Stress/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Antioxidants/pharmacology , Blotting, Western , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Proliferation/drug effects , Flow Cytometry , HeLa Cells , Histone Deacetylases/chemistry , Histone Deacetylases/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Small Interfering/genetics
20.
Oncol Rep ; 28(2): 749-57, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22684917

ABSTRACT

Arsenic trioxide (ATO; As2O3) induces apoptotic cell death in various cancer cells including lung cancer via the induction of reactive oxygen species (ROS). However, little is known about the toxicological effects of ATO on normal primary lung cells. Here, we investigated the effects of N-acetyl cysteine (NAC) and vitamin C (well-known antioxidants) or L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) on ATO-treated human pulmonary fibroblast (HPF) cells in relation to cell death, ROS and glutathione (GSH). ATO induced growth inhibition and death in HPF cells, accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm). ATO increased ROS levels including O2•- and GSH depleted cell numbers. NAC attenuated the growth inhibition, death and MMP (∆ψm) loss in ATO-treated HPF cells and also decreased the ROS levels in these cells. However, vitamin C enhanced the growth inhibition, death, MMP (∆ψm) loss and GSH depletion by ATO and even strongly increased mitochondrial O2•- levels in ATO-treated HPF cells. BSO showed a strong increase in ROS levels in ATO-treated HPF cells and intensified the growth inhibition, cell death, MMP (∆ψm) loss and GSH depletion. Moreover, superoxide dismutase (SOD2) or thioredoxin (TXN) siRNAs attenuated HPF cell death by ATO, which was not correlated with ROS and GSH level changes. In conclusion, ATO induced the growth inhibition and death of HPF cells, accompanied by increasing ROS levels and GSH depletion. NAC attenuated HPF cell death by ATO whereas vitamin C and BSO enhanced the death.


Subject(s)
Antineoplastic Agents/pharmacology , Arsenicals/pharmacology , Fibroblasts/drug effects , Glutathione/metabolism , Lung/drug effects , Oxides/pharmacology , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Antioxidants/metabolism , Apoptosis/drug effects , Arsenic Trioxide , Ascorbic Acid/pharmacology , Buthionine Sulfoximine/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Fibroblasts/cytology , Glutathione/deficiency , Humans , Lung/cytology , Lung/metabolism , Transfection
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