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Propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester, PG) has an anti-proliferative effect in various cells. In this study, Calu-6 and A549 lung cancer cells were used to examine the anti-proliferative effect of PG in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PG (100-1,600 μM) dose-dependently inhibited the proliferation of Calu-6 and A549 cells at 24 h, and PG at 800-1,600 μM strongly induced cell death in both cell lines. PG (800-1,600 μM) increased cellular metabolism in Calu-6 but not A549 cells at 4 h. PG either increased or decreased ROS levels, including O2 ˙- and ˙OH, depending on the incubation doses and times of 1 or 24 h. Even these effects differed between Calu-6 and A549 cell types. PG reduced the activity of superoxide dismutase (SOD) in Calu-6 cells, and it augmented the activity of catalase in A549 cells. PG dose-dependently increased the number of GSH depleted cells in both Calu-6 and A549 cells at 24 h. In addition, PG decreased GSH levels in both lung cancer cells at 1 h. Furthermore, diethyldithiocarbamate (DDC; an inhibitor of SOD) and 3-amino-1,2,4-triazole (AT; an inhibitor of catalase) differently affected cellular metabolism, ROS and GSH levels in PG-treated and PG-untreated Calu-6 and A549 cells at 1 h. In conclusion, PG dose-dependently decreased the proliferation of Calu-6 and A549 lung cancer cells, which was related to changes in ROS levels and the depletion of GSH.

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, cell-permeable redox-cycling nitroxide water-soluble superoxide dismutase (SOD) mimetic agent. However, little is known about its cytotoxic effects on lung-related cells. Thus, the present study investigated the effects of Tempol on cell growth and death as well as changes in reactive oxygen species (ROS) and glutathione (GSH) levels in Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblast cells. Results showed that Tempol (0.5~4 mM) dose-dependently inhibited the growth of lung cancer and normal cells with an IC

Auranofin, as a thioredoxin reductase (TrxR) inhibitor, has promising anti-cancer activity in several cancer types. However, little is known about the inhibitory effect of auranofin on lung cancer cell growth. We, therefore, investigated the antigrowth effects of auranofin in various lung cancer cells with respect to cell death, reactive oxygen species (ROS), and glutathione (GSH) levels. Treatment with 0~5 µM auranofin decreased cell proliferation and induced cell death in Calu-6, A549, SK-LU-1, NCI-H460, and NCI-H1299 lung cancer cells at 24 h. In addition, 0~5 µM auranofin increased ROS levels, including O2•−, and depleted GSH levels in these cells. N-acetyl cysteine (NAC) prevented growth inhibition and mitochondrial membrane potential (MMP, ∆Ψm) loss in 3 and 5 µM auranofin-treated Calu-6 and A549 cells at 24 h, respectively, and decreased ROS levels and GSH depletion in these cells. In contrast, L-buthionine sulfoximine (BSO) enhanced cell death, MMP (∆Ψm) loss, ROS levels, and GSH depletion in auranofin-treated Calu-6 and A549 cells. Treatment with 3 and 5 µM auranofin induced caspase-3 activation and poly (ADP ribose) polymerase (PARP) cleavage in Calu-6 and A549 cells, respectively. Both were prevented by NAC, but enhanced by BSO. Moreover, TrxR activity was reduced in auranofin-treated Calu-6 and A549 cells. That activity was decreased by BSO, but increased by NAC. In conclusion, these findings demonstrate that auranofin-induced cell death is closely related to oxidative stress resulted from increased ROS levels and GSH depletion in lung cancer cells.

Propyl gallate [3,4,5-trihydroxybenzoic acid propyl ester; PG] exhibits an anti-growth effect in various cells. In this study, the anti-apoptotic effects of various caspase inhibitors were evaluated in PG-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Treatment with 800 μM PG inhibited the proliferation and induced the cell death of both Calu-6 and A549 cells at 24 h. Each inhibitor of pan-caspase, caspase-3, caspase-8, and caspase-9 reduced the number of dead and sub-G1 cells in both PG-treated cells at 24 h. PG increased ROS levels, including O2∙−, in both lung cancer cell lines at 24 h. Generally, caspase inhibitors appeared to decrease ROS levels in PG-treated lung cancer cells at 24 h and somewhat reduced O2∙− levels. PG augmented the number of GSH-depleted Calu-6 and A549 cells at 24 h. Caspase inhibitors did not affect the level of GSH depletion in PG-treated A549 cells but differently and partially altered the depletion level in PG-treated Calu-6 cells. In conclusion, PG exhibits an anti-proliferative effect in Calu-6 and A549 lung cancer cells and induced their cell death. PG-induced lung cancer death was accompanied by increases in ROS levels and GSH depletion. Therefore, the anti-apoptotic effects of caspase inhibitors were, at least in part, related to changes in ROS and GSH levels.

Propyl gallate (3,4,5‑trihydroxybenzoic acid propyl ester; PG) is a synthetic phenolic antioxidant which exerts many effects on tissue and cell functions. In the present study, Calu‑6 and A549 lung cancer cells were used to examine the molecular mechanism of the anti‑growth effects of PG in relation to apoptosis and cell cycle arrest. PG inhibited the growth of both lung cancer cell types in a dose‑dependent manner with an IC50 of 800 µM at 24 h based on MTT assays. DNA flow cytometry showed that PG induced G1 phase arrest of the cell cycle in Calu‑6 and A549 cells. In addition, PG induced apoptosis in both lung cancer cell types, as evidenced by sub‑G1 cell population and Annexin V‑stained cells. Western blot results demonstrated that PG decreased the Bcl‑2 level which was accompanied by an increase in the cleaved form of poly(ADP‑ribose) polymerase (PARP). PG also triggered loss of mitochondrial membrane potential (MMP; ∆Ψm) and decreased MMP (∆Ψm) levels in both lung cancer cell types, as assessed by FACS analysis. Furthermore, PG upregulated the activities of caspase‑3 and caspase‑8 in Calu‑6 cells. In conclusion, PG treatment inhibited the growth of lung cancer cells, especially Calu‑6 cells via caspase‑dependent apoptosis as well as G1 phase arrest of the cell cycle.

Background Cigarette smoking is a major health risk, particularly in male South Koreans. Smoking cessation can benefit health; however, the process of quitting smoking is difficult to some smokers and shows its relationship to their stress level. The hypothesis of this study is that who has failed attempts to stop smoking induce more stress than habitual smoking. Methods To test this, the analysis on the association between smoking cessation attempts and stress levels in smokers was performed. The Korean Community Health Survey (2011–2016) data with the total of 488,417 participants’ data were used for this study. Survey data were analyzed using the chi-square test and logistic regression. As the dependent variable, self-reported level of stress was selected. Results Of the subject population, 78.3% (63.3% males, 81.4% females) felt stressed. Among participants who successfully stopped smoking, 73.0% (72.6% males, 78.1% females) reported feeling stressed. In contrast, of those who failed to stop smoking, 83.3% (83.6% males, 86.3% females) reported high stress levels. Among those who did not attempt smoking cessation, 81.1% (81.2% males, 80.3% females) responded that they experienced stress. Those who failed to stop smoking had higher odds of stress than those who did not attempt smoking cessation [odds ratio (OR) 1.11, 95% confidence interval (CI) 1.09–1.14, p

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a potential redox agent in cells. The present study investigated changes in cellular reactive oxygen species (ROS) and glutathione (GSH) levels and in antioxidant enzymes, in Tempol-treated Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblasts. Results demonstrated that Tempol (0.5–4 mM) either increased or decreased general ROS levels in lung cancer and normal cells at 48 h and specifically increased O2•− levels in these cells. In addition, Tempol differentially altered the expression and activity of antioxidant enzymes such as superoxide dismutase, catalase, and thioredoxin reductase1 (TrxR1) in A549, Calu-6, and WI-38 VA-13 cells. In particular, Tempol treatment increased TrxR1 protein levels in these cells. Tempol at 1 mM inhibited the growth of lung cancer and normal cells by about 50% at 48 h but also significantly induced cell death, as evidenced by annexin V-positive cells. Furthermore, down-regulation of TrxR1 by siRNA had some effect on ROS levels as well as cell growth inhibition and death in Tempol-treated or -untreated lung cells. In addition, some doses of Tempol significantly increased the numbers of GSH-depleted cells in both cancer cells and normal cells at 48 h. In conclusion, Tempol differentially increased or decreased levels of ROS and various antioxidant enzymes in lung cancer and normal cells, and induced growth inhibition and death in all lung cells along with an increase in O2•− levels and GSH depletion.

Propyl gallate (PG) has an anti-growth effect in lung cancer cells. The present study investigated the effects of mitogen-activated protein kinase (MAPK; MEK, JNK, and p38) inhibitors on PG-treated Calu-6 and A549 lung cancer cells in relation to cell death as well as reactive oxygen species (ROS) and glutathione (GSH) levels. PG induced cell death in both Calu-6 and A549 lung cancer cells at 24 h, which was accompanied by loss of mitochondrial membrane potential (MMP; ΔΨm). All of the tested MAPK inhibitors increased cell death in both PG-treated lung cancer cell lines. In particular, MEK inhibitor strongly enhanced cell death and MMP (ΔΨm) loss in PG-treated Calu-6 cells and p38 inhibitor had the same effects in A549 cells as well. PG increased ROS levels and caused GSH depletion in both cell lines at 24 h. MAPK inhibitors increased O2•- levels and GSH depletion in PG-treated Calu-6 cells, and JNK and p38 inhibitors increased ROS levels and GSH depletion in PG-treated A549 cells. In conclusion, MAPK inhibitors increased cell death in PG-treated Calu-6 and A549 lung cancer cells. Enhanced cell death and GSH depletion in Calu-6 cells caused by the MEK inhibitor were related to increased O2•- levels, and the effects of the p38 inhibitor in A549 cells were correlated with increased general ROS levels.

Auranofin, an inhibitor of thioredoxin reductase (TrxR), inhibits the growth of a variety of cancer cells. In the present study, various lung cancer cells were used to investigate the molecular basis of anti‑cancer effects of auranofin, including cell death via apoptosis or necrosis and cell cycle arrest. Generally, auranofin inhibited the growth of the tested lung cancer cell lines in a dose‑dependent manner with an IC50 of 3‑4 µM at 24 h. This agent significantly decreased the activity of TrxR in Calu‑6 and A549 lung cancer cells. In addition, auranofin (3‑5 µM) triggered necrosis in lung cancer cells measured by the release of lactate dehydrogenase (LDH) into culture media. Auranofin increased the percentages of sub‑G1 cells in Calu‑6 and A549 cells. DNA flow cytometry showed that auranofin induced G2/M phase arrest of Calu‑6 cells. This agent also efficiently induced apoptosis, accompanied by loss of mitochondrial membrane potential (MMP; ∆Ψm), increases in cleavage forms of caspase‑3 and poly (ADP‑ribose) polymerase (PARP), and a high ratio of BAX to Bcl‑2 proteins. Furthermore, various caspase inhibitors reduced apoptosis and MMP (∆Ψm) loss in auranofin‑treated Calu‑6 cells. In particular, the pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone (Z‑VAD), decreased cleavage forms of caspase‑3, ‑8, and ‑9 in these cells. In conclusion, auranofin inhibited the proliferation of lung cancer cells, especially Calu‑6 cells, via cell cycle arrest and cell death due to necrosis or caspase‑dependent apoptosis.

Arsenic trioxide (ATO, As2O3) has anti-cancer effects in various solid tumors as well as hematological malignancy. Valproic acid (VPA), which is known to be a histone deacetylase inhibitor, has also anti-cancer properties in several cancer cells including lung cancer cells. Combined treatment of ATO and VPA (ATO/VPA) could synergistically enhance anti-cancer effects and reduce ATO toxicity ATO. In this study, the combined anti-cancer effects of ATO and VPA (ATO/VPA) was investigated in NCI-H460 and NCI-H1299 lung cancer cells in vitro and in vivo. A combination of 3 &mu, M ATO and 3 mM VPA (ATO/VPA) strongly inhibited the growths of both lung cancer cell types. DNA flow cytometry indicated that ATO/VPA significantly induced G2/M-phase arrest in both cell lines. In addition, ATO/VPA strongly increased the percentages of sub-G1 cells and annexin V-FITC positive cells in both cells. However, lactate dehydrogenase (LDH) release from cells was not increased in ATO/VPA-treated cells. In addition, ATO/VPA increased apoptosis in both cell types, accompanied by loss of mitochondrial membrane potential (MMP, ∆&Psi, m), activation of caspases, and cleavage of anti-poly ADP ribose polymerase-1. Moreover, a pan-caspase inhibitor, Z-VAD, significantly reduced apoptotic cell death induced by ATO/VPA. In the xenograft model, ATO/VPA synergistically inhibited growth of NCI-H460-derived xenograft tumors. In conclusion, the combination of ATO/VPA effectively inhibited the growth of lung cancer cells through G2/M-phase arrest and apoptotic cell death, and had a synergistic antitumor effect in vivo.

// Bo Ra You 1 , Bo Ram Han 1 , Woo Hyun Park 1 1 Department of Physiology, Medical School, Institute for Medical Sciences, Chonbuk National University, Jeonju, 561-180, Republic of Korea Correspondence to: Woo Hyun Park, email: parkwh71@chonbuk.ac.kr Keywords: lung cancer, histone deacetylase, suberoylanilide hydroxamic acid, tumor necrosis factor-α, apoptosis Received: December 08, 2015 Accepted: January 05, 2017 Published: January 13, 2017 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.

The thioredoxin (Trx) system controls cellular redox in vascular smooth muscle cells. The present study investigated the roles of Trx1 and Trx reductase1 (TrxR1) proteins in regulation of cell growth, death, reactive oxygen species (ROS) and glutathione (GSH) levels in hydrogen peroxide (H

The thioredoxin (Trx) system is an important enzymatic complex involved in cellular redox homeostasis. Arsenic trioxide (ATO; As2O3) is known to trigger cell death in vascular smooth muscle cells (VSMCs) via oxidative stress. In the present study, the effects of changes in thioredoxin 1 (Trx1) and Trx reductase1 (TrxR1) on cell growth, death, reactive oxygen species (ROS), and glutathione (GSH) levels were evaluated in ATO‑treated human pulmonary artery smooth muscle cells (HPASMCs). ATO inhibited growth and induced cell death in the HPASMCs at 24 h. Overexpression of Trx1 and TrxR1 using adenoviruses attenuated cell growth inhibition caused by ATO and partially prevented cell death. ATO increased ROS levels including the mitochondrial superoxide anion (O2•-) at 5 min. Administration of adTrx1 or adTrxR1 reduced the increased mitochondrial O2•- level in these cells. HPASMCs treated with Trx1 or TrxR1 siRNA showed increases in ROS levels with or without treatment of ATO at 5 min. Although ATO transiently increased GSH levels at 5 min, Trx1 and TrxR1 siRNAs reduced the increased GSH levels in these cells. In addition, PX‑12 (a Trx1 inhibitor) and auranofin (a TrxR1 inhibitor) diminished the cellular metabolism in HPASMCs at 4 h, accompanied by an increase in ROS level and a decrease in GSH level. In conclusion, upregulation of Trx1 and TrxR1 somewhat decreased cell growth inhibition and death in ATO‑treated HPASMCs, which was accompanied by reduced oxidative stress.

Reactive oxygen species (ROS) are associated with various pathophysiological processes of vascular smooth muscle cells (VSMCs). Pyrogallol (PG) induces the superoxide anion (O2•‑)‑mediated cell death of numerous cell types. The present study aimed to investigate the effects of exogenous hydrogen peroxide (H2O2) and PG treatment on the cell growth and death of human pulmonary artery smooth muscle cells (HPASMCs), with regards to intracellular ROS and glutathione (GSH) levels, as determined by MTT and cell number assays. H2O2 led to reduced growth of HPASMCs, with a half maximal inhibitory concentration of 250‑500 µM at 24 h, and induced apoptosis, as determined by Annexin V‑staining and benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone treatment. However, PG did not strongly induce growth inhibition and death of HPASMCs. In addition, H2O2 led to increased ROS levels, including mitochondrial O2•‑, and induced GSH depletion in HPASMCs. Treatment with N‑acetyl cysteine (NAC) attenuated apoptotic cell death and ROS levels in H2O2‑treated HPASMCs, and also prevented GSH depletion. Notably, PG treatment did not increase ROS levels, including mitochondrial O2•‑. Furthermore, NAC induced a significant increase in mitochondrial O2•‑ levels in PG‑treated HPASMCs, and cell death and GSH depletion were significantly increased. L‑buthionine sulfoximine intensified cell death and GSH depletion in PG‑treated HPASMCs. In conclusion, exogenous H2O2 induced growth inhibition and cell death of HPASMCs via GSH depletion.

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.

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.

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.

Hydrogen peroxide (H2O2) is frequently applied to cultured cells to induce oxidative stress. The present study investigated the molecular and cellular effects of exogenous H2O2 on Calu‑6 and A549 lung cancer cells. Based on MTT assays, H2O2 inhibited the growth of Calu‑6 and A549 cells with IC50 values of ~50 and 100 µM at 24 h, respectively. Cells treated with H2O2 demonstrated a considerable G1‑phase arrest of the cell cycle. H2O2 dose‑dependently augmented the numbers of dead (trypan blue‑positive) and Annexin V‑FITC‑stained cells in these cells, which was accompanied by the reduction of Bcl‑2 and pro‑caspase‑3 levels, as well as the upregulation of caspase‑3 and ‑8 activities. In addition, H2O2 triggered the failure of mitochondrial membrane potential (MMP; ΔΨm). However, relatively higher doses of H2O2 did not raise the percentages of sub‑G1 cells in these cell lines. All the tested caspase inhibitors (Z‑VAD for pan‑caspases, Z‑DEVD for caspase‑3, Z‑IETD for caspase‑8 and Z‑LEHD for caspase‑9) decreased the percentages of sub‑G1 and Annexin V‑FITC‑stained cells in the H2O2‑treated Calu‑6 and A549 cells. However, caspase inhibitors did not significantly prevent the loss of MMP (ΔΨm) in H2O2‑treated lung cancer cells. In conclusion, H2O2 inhibited the growth of Calu‑6 and A549 lung cancer cells through cell death and G1‑phase arrest. H2O2‑induced cell death resulted from necrosis, as well as caspase‑dependent apoptosis.

Reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), induce apoptosis in cancer cells by regulating mitogen-activated protein kinase (MAPK) signaling pathways. The present study investigated the effects of MAPK inhibitors on cell growth and death as well as changes in ROS and glutathione (GSH) levels in H2O2-treated Calu-6 and A549 lung cancer cells. H2O2 inhibited growth and induced death of Calu-6 and A549 lung cancer cells. All MAPK inhibitors appeared to enhance growth inhibition in H2O2-treated Calu-6 and A549 lung cancer cells and increased the percentage of Annexin V-FITC-positive cells in these cancer cells. Among the MAPK inhibitors, a JNK inhibitor significantly augmented the loss of mitochondrial membrane potential (MMP; ΔΨm) in H2O2-treated Calu-6 and A549 lung cancer cells. Intracellular ROS levels were significantly increased in the H2O2-treated cells at 1 and 24 h. Only the JNK inhibitor increased ROS levels in the H2O2-treated cells at 1 h and all MAPK inhibitors raised superoxide anion levels in these cells at 24 h. In addition, H2O2 induced GSH depletion in Calu-6 and A549 cells and the JNK inhibitor significantly enhanced GSH depletion in H2O2‑treated cells. Each of the MAPK inhibitors altered ROS and GSH levels differently in the Calu-6 and A549 control cells. In conclusion, H2O2 induced growth inhibition and death in lung cancer cells through oxidative stress and depletion of GSH. The enhanced effect of MAPK inhibitors, especially the JNK inhibitor, on cell death in H2O2-treated lung cancer cells was correlated with increased O2•- levels and GSH depletion.

Exogenous hydrogen peroxide (H

Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth 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 (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, 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 pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

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.

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.

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.

Enhanced oxidative stress contributes to endothelial dysfunction via the apoptotic induction of endothelial cells (ECs). However, the precise molecular mechanisms underlying its important effect remain unclear. Here, we evaluated the effects of exogenous hydrogen peroxide (H(2)O(2)) on cell growth and death in ECs such as calf pulmonary artery endothelial cells (CPAECs) and human umbilical vein endothelial cells (HUVECs) and investigated its mechanism of action in CPAECs. H(2)O(2) inhibited the growth of CPAECs and HUVECs at 24 h with IC50 of approximately 20 and 300 µM, respectively. H(2)O(2) induced cell death in both ECs, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). H(2)O(2)-induced CPAEC death occurred via apoptosis, demonstrated by Annexin V-staining cells and Z-VAD (a pan-caspase inhibitor) treatment. H(2)O(2) increased superoxide anion levels in HUVECs but not in CPAECs. Treatment with 30 µM H(2)O(2) significantly decreased the activities of superoxide dismutases and catalase in CPAECs. H(2)O(2) induced glutathione (GSH) depletion in both ECs. Z-VAD and N-acetyl cysteine (NAC; a well-known antioxidant) attenuated apoptotic cell death and GSH depletion in H(2)O(2)-treated CPAECs. In conclusion, H(2)O(2) induced growth inhibition and death in ECs via GSH depletion. HUVECs were relatively resistant to H(2)O(2) compared with CPAECs. H(2)O(2)-induced CPAEC apoptosis required the activation of various caspases.

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.

Gallic acid (GA; 3,4,5-triphydroxyl-benzoic acid) is widely dispersed in various plants, fruits and foods and it shows various biological properties including anticancer effects. This study investigated the effects of GA on HeLa cervical cancer cells in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). GA dose-dependently inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC) at 24 or 72 h. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA induced apoptosis in HeLa cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm). GA increased ROS levels including O2•- in HeLa cells at 24 h and it also induced GSH depletion. N-acetyl cysteine (NAC) increased the growth inhibition of GA-treated HeLa cells and enhanced the death of these cells. NAC differently influenced ROS levels in GA-treated HeLa cells and significantly increased GSH depletion in these cells. L-buthionine sulfoximine (BSO) increased MMP (∆ψm) loss, ROS levels and GSH depletion in GA-treated HeLa cells. In conclusion, GA significantly inhibited the growth of HeLa cells. GA-induced HeLa cell death was tightly related to GSH depletion rather than ROS level changes.

Pyrogallol (PG) inhibits the growth of various cells via stimulating O2•--mediated death. This study investigated the effects of PG on cell death in human pulmonary fibroblast (HPF) cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PG inhibited the growth of HPF cells with an IC50 of ~50-100 µM at 24 h. PG induced a G1 phase arrest of the cell cycle and also triggered cell death accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm), Bcl-2 decrease, p53 increase and the activation of caspase-3. PG increased O2•- level in HPF cells and depleted GSH content in these cells. Z-VAD (a pan-caspase inhibitor) did not significantly change cell growth inhibition, death and MMP (∆ψm) loss in PG-treated HPF cells. N-acetylcysteine (NAC) attenuated growth inhibition, death and MMP (∆ψm) loss in PG-treated HPF cells and it decreased O2•- level in these cells as well. However, L-buthionine sulfoximine (BSO) strongly increased ROS level in PG-treated HPF cells and it intensified growth inhibition, cell death, MMP (∆ψm) loss and GSH depletion in these cells. In conclusion, PG-induced HPF cell death was closely related to increases in ROS level and GSH depletion.

Zebularine (Zeb) as a DNA methyltrasferase (DNMT) inhibitor has various cellular effects such as cell growth inhibition and apoptosis. In the present study, we evaluated the effects of Zeb on the growth and death of HeLa cervical cancer cells. Zeb inhibited the growth of HeLa cells with an IC50 of approximately 130 μM at 72 h in a dose-dependent manner. DNA flow cytometric analysis indicated that Zeb induced an S phase arrest of the cell cycle, which was accompanied by the increased levels of cdk2 and cyclin A proteins. This agent also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (Ψm), PARP-1 cleavage and the activation of caspase-3, -8 and -9. All of the tested caspase inhibitors significantly rescued some cells from Zeb-induced HeLa cell death. In relation to reactive oxygen species (ROS) and glutathione (GSH) levels, O 2 •− level was significantly increased in 100 μM Zeb-treated HeLa cells and caspase inhibitors reduced O 2 •− level in these cells. Zeb induced GSH depletion in HeLa cells, which was attenuated by caspase inhibitors. In conclusion, this is the first report that Zeb inhibited the growth of HeLa cells via cell cycle arrest and apoptosis.

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.

Gallic acid (GA) has various biological effects including apoptosis. In this study, we investigated the effects of GA on human primary umbilical vein endothelial cells (HUVECs) in relation to cell growth, cell death, reactive oxygen species (ROS) and glutathione (GSH). Treatment with 200 or 400 µM GA inhibited the growth of HUVECs at 24 h and induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ∆Ψm). GA decreased or increased ROS levels including O2•-. It dose-dependently increased GSH depleted cell numbers. Pan-caspase inhibitor (Z-VAD) did not affect cell growth inhibition, cell death, ROS and GSH levels in GA-treated HUVECs. However, N-acetyl-cysteine (NAC; a well known antioxidant) and L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) enhanced cell growth inhibition, cell death and MMP (∆Ψm) loss in GA-treated HUVECs. NAC decreased general ROS levels in GA-treated HUVECs, but strongly increased O2•- levels in these cells. Both NAC and BSO intensified the GSH depletion of GA-treated HUVECs. In conclusion, GA treatment induced growth inhibition and death of HUVECs. The changes of ROS and GSH levels by NAC and BSO influenced cell growth and death in GA-treated HUVECs.

Parthenolide (PT), a sesquiterpene lactone derived from the plant feverfew, has pro-apoptotic activity in a number of cancer cell types. We assessed whether PT induces the apoptosis of hepatic stellate cells (HCSs) and examined its effects on hepatic fibrosis in an in vivo model. The effects of PT on rat HSCs were investigated in relation to cell growth inhibition, apoptosis, NF-κB binding activity, intracellular reactive oxygen species (ROS) generation, and glutathione (GSH) levels. In addition, the anti-fibrotic effects of PT were investigated in a thioacetamide-treated rat model. PT induced growth inhibition and apoptosis in HSCs, as evidenced by cell growth inhibition and apoptosis assays. PT increased the expression of Bax proteins during apoptosis, but decreased the expression of Bcl-2 and Bcl-X(L) proteins. PT also induced a reduction in mitochondrial membrane potential, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. PT inhibited TNF-α-stimulated NF-κB binding activity in HSCs. The pro-apoptotic activity of PT in HSCs was associated with increased intracellular oxidative stress as evidenced by increased intracellular ROS levels and depleted intracellular GSH levels. Furthermore, PT ameliorated hepatic fibrosis significantly in a thioacetamide- treated rat model. In conclusion, PT exhibited pro-apoptotic effects in rat HSCs and ameliorated hepatic fibrosis in a thioacetamide-induced rat model.

Gallic acid (GA) is generally distributed in a variety of plants and foods, and its various biological effects have been reported. Here, we investigated the effects of GA and/or caspase inhibitors on Calu-6 and A549 lung cancer cells in relation to cell death and reactive oxygen species (ROS). The growths of Calu-6 and A549 cells were diminished with an IC(50) of approximately 30 and 150 μM GA at 24 h, respectively. GA also inhibited the growth of primary human pulmonary fibroblast (HPF) cells with an IC(50) of about 300 μM. GA induced apoptosis and/or necrosis in lung cancer cells, which was accompanied by the loss of mitochondrial membrane potential (MMP, ΔΨ(m)). The percents of MMP (ΔΨ(m)) loss and death cells by GA were lower in A549 cells than in Calu-6 cells. Caspase inhibitors did not significantly rescued lung cancer cells from GA-induced cell death. GA increased ROS levels including O(2) (•-) and induced GSH depletion in both lung cancer cells. Z-VAD (pan-caspase inhibitor) did not decrease ROS levels and GSH depleted cell number in GA-treated lung cancer cells. In conclusion, GA inhibited the growth of lung cancer and normal cells. GA-induced lung cancer cell death was accompanied by ROS increase and GSH depletion.

Gallic acid (GA) is widely distributed in various plants and foods and has various biological properties including anticancer effects. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) [MAP 20 kinase or ERK kinase (MEK), c-Jun N-terminal kinase (JNK), or p38)] inhibitors or small interfering RNAs (siRNAs) on GA-induced HeLa cell death in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. GA dose dependently inhibited the growth of HeLa cells via apoptosis and/or necrosis at 24 h, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). Treatment with 70 μM GA increased the ROS level including O(2)(•-) and significantly induced GSH depletion in HeLa cells. GA decreased the activity of extracellular signal-regulated kinase (ERK) at 24 h, whereas it increased that of JNK at the same time. While the MEK inhibitor or ERK siRNA did not affect cell growth and death in 70 μM GA-treated HeLa cells at 24 h, JNK and p38 inhibitors enhanced cell growth inhibition and death in these cells. Additionally, p38 siRNA administration augmented growth inhibition, death, and MMP (ΔΨ(m)) loss in 70 μM GA-treated HeLa cells. In relation to ROS and GSH levels, JNK and p38 inhibitors increased ROS levels, and GSH-depleted cell numbers in GA-treated HeLa cells. Moreover, p38 siRNA increased O(2)(•-) levels and GSH depletion in GA-treated HeLa cells. Each MAPK inhibitor and siRNA differentially affected ROS and GSH levels in HeLa control cells. Conclusively, JNK and p38 inhibitors and p38 siRNA enhanced growth inhibition and cell death in GA-treated HeLa cells, which were to some extent related to GSH depletion and ROS levels, especially O(2)(•-).

Urotensin II (UII) is a vasoactive peptide with potent cardiovascular effects through a G protein-coupled receptor. Hypoxia stimulates the secretion of UII and atrial natriuretic peptide (ANP). However, the effect of UII on hypoxia-induced cardiac hypertrophy is still controversial. The present study was conducted to determine whether human UII (hUII)-mediated ANP secretion influences hypoxia-induced cardiac hypertrophy using in vitro and in vivo models. Hypoxia caused an increase in ANP secretion and a decrease in atrial contractility in isolated perfused beating rat atria. hUII (0.01 and 0.1 nM) attenuated hypoxia-induced ANP secretion without changing the atrial contractility, and the hUII effect was mediated by the UII receptor signaling involving phospholipase C, inositol 1,3,4 trisphosphate receptor, and protein kinase C. Rats treated with monocrotaline (MCT, 60 mg/kg) showed right ventricular hypertrophy with increases in pulmonary arterial pressure and its diameter and plasma levels of UII and ANP that were attenuated by the pretreatment with an UII receptor antagonist, urantide. An acute administration of hUII (5 μM injection plus 2.5 μM infusion for 15 min) decreased the plasma ANP level in MCT-treated rats but increased the plasma ANP level in MCT plus urantide-treated and sham-operated rats. These results suggest that hUII may deteriorate MCT-induced cardiac hypertrophy mainly through a vasoconstriction of the pulmonary artery and partly through the suppression of ANP secretion.

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we investigated the effects of MAPK (MEK, JNK and p38) inhibitors on PG-treated HeLa cells in relation to cell death, ROS and GSH levels. PG induced cell growth inhibition and apoptosis in HeLa cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). ROS levels were increased or decreased in PG-treated HeLa cells depending on the incubation times. PG also increased GSH depleted cell numbers in HeLa cells. All the MAPK inhibitors slightly enhanced cell growth inhibition, death and MMP (ΔΨm) loss, and increased ROS levels in PG-treated HeLa cells. However, MAPK inhibitors did not significantly affect GSH depletion in PG-treated cells. In conclusion, the enhanced effect of MAPK inhibitors on PG-induced HeLa cell death was accompanied by increasing ROS levels but the effect was not related to changes of GSH level.

Gallic acid (GA) has various biological properties including anti-cancer effect. However, little is known about the toxicological effect of GA in primary normal cells. Here, we evaluated the effects of GA on human pulmonary fibroblast (HPF) cells in relation to reactive oxygen species (ROS) and glutathione (GSH). GA inhibited the growth of HPF cells at 24 hours in a dose-dependent manner. GA also induced HPF cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). GA increased ROS levels including O2•- and GSH-depleted cell numbers in HPF cells at 24 hours. Treatment with 2 mM N-acetyl-cysteine (NAC) intensified growth inhibition and death in GA-treated HPF cells. NAC decreased ROS levels and increased GSH depletion in these cells. Treatment with 10 μM L-buthionine sulfoximine (BSO) also enhanced growth inhibition and death in GA-treated HPF cells. BSO increased ROS levels and GSH depletion in these cells. In conclusion, GA-induced HPF cell death was accompanied by ROS increase and GSH depletion. The changes of ROS and GSH levels by NAC and BSO appeared to affect cell growth and death in GA-treated HPF cells.

Gallic acid (GA) widely distributed in plants and foods has its various biological effects. Here, we investigated the anti-cancer effects of GA on Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). GA dose-dependently decreased the growth of Calu-6 and A549 cells with an IC(50) of approximately 10-50 microM and 100-200 microM GA at 24h, respectively. GA also induced cell death in lung cancer cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)). The percents of MMP (DeltaPsi(m)) loss and death cells were lower in A549 cells than Calu-6 cells. GA increased ROS levels including O(2)(-) in lung cancer cells at 24h and also GSH depleted cell numbers at this time. N-acetyl-cysteine (NAC; a well-known antioxidant) intensified growth inhibition and death in GA-treated lung cancer cells. NAC changed ROS levels and increased GSH depletion in these cells. Vitamin C significantly attenuated cell death, ROS levels and GSH depletion in GA-treated lung cancer cells. L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) slightly enhanced growth inhibition and death in GA-treated lung cancer cells and also mildly increased ROS levels and GSH depletion in these cells. In conclusion, GA inhibited the growth of lung cancer cells. GA-induced lung cancer cell death was related to GSH depletion as well as ROS level changes.

MG132 as a proteasome inhibitor has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth of endothelial cells (ECs), especially calf pulmonary artery endothelial cells (CPAECs), in relation to cell death, ROS, and glutathione (GSH) levels. MG132 dose dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVECs) at 24 hours. MG132 also induced apoptotic cell death in CPAEC, which were accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)). MG132 increased ROS levels, including O(2)(*-) in CPAEC, but not in HUVEC. MG132 also dose dependently increased GSH-depleted cells in both ECs. N-acetyl-cysteine (NAC; a well-known antioxidant) reduced ROS levels in MG132-treated CPAEC with the slight prevention of cell death and GSH depletion. Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) increased ROS levels and decreased GSH levels in MG132-treated CPAEC without the enhancement of cell death. In conclusion, MG132 inhibited the growth of ECs, especially CPAEC. The changes of ROS and GSH levels by MG132 partially affect CPAEC death.

MG132 (carbobenzoxy-Leu-Leu-leucinal) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, we investigated the effects of MEK (mitogen-activated protein [MAP] kinase or extracellular signal-regulated kinase [ERK] kinase) or p38 inhibitor on MG132-treated Calu-6 lung cancer cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. Treatment with 10 mumol/L MG132 inhibited the growth of Calu-6 cells at 24 hours. MG132 induced apoptosis in Calu-6 cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)). ROS were increased in MG132-treated Calu-6 cells. MG132 also induced GSH depletion in Calu-6 cells. Treatment with MEK inhibitor did not significantly affect cell growth, cell death, ROS, and GSH levels in MG132-treated Calu-6 cells. Furthermore, MG132 increased the phosphorylation of p38 in Calu-6 cells at 1 and 24 hours. Treatment with p38 inhibitor significantly prevented cell growth inhibition, MMP (DeltaPsi(m)) loss and apoptosis in MG132-treated Calu-6 cells. This inhibitor increased ROS level and decreased GSH depletion in these cells. In conclusion, p38 inhibitor partially prevented Calu-6 cell death by MG132, which might be affected by GSH level changes.

The inhibition of proteasome function has emerged as a useful strategy to maneuver apoptosis. In the present study, we evaluated the effects of MG132 as a proteasome inhibitor on the growth of Calu-6 lung cancer cells in relation to the cell cycle, cell death, reactive oxygen species (ROS) and glutathione (GSH) levels. MG132 dose-dependently inhibited the growth of Calu-6 cells at 24h. DNA flow cytometric analysis indicated that 1-30 microM MG132 induced an S phase arrest in Calu-6 cells. MG132 also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; Deltapsi(m)). The pan-caspase inhibitor (Z-VAD) significantly rescued Calu-6 cells from MG132-induced cell death. The intracellular ROS levels including O(2)(-) were increased in MG132-treated Calu-6 cells. MG132 also increased GSH-depleted cell numbers in Calu-6 cells. Z-VAD significantly decreased O(2)(-) levels and GSH-depleted cell numbers in MG132-treated Calu-6 cells. In conclusion, MG132 inhibited the growth of Calu-6 cells via apoptosis and GSH depletion.

Pyrogallol (PG) as a polyphenol induces apoptosis in cells. Here, we evaluated the effects of PG on the growth and death of endothelial cells (ECs). PG dose-dependently inhibited the growth of calf pulmonary artery endothelial cells (CPAEC) and human umbilical vein endothelial cells (HUVEC). PG also induced apoptosis in both cells accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)). CPAEC were more sensitive to PG than HUVEC concerning cell growth and death. Caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) did not affect the growth inhibition of CPAEC by PG. However, pan-caspase inhibitor (Z-VAD) significantly reduced apoptosis and the loss of DeltaPsi(m) in PG-treated CPAEC. PG reduced ROS level and increased GSH depleted cell numbers in CPAEC. While Z-VAD increased ROS levels in PG-treated CPAEC, it decreased GSH depleted cell numbers. In conclusion, PG inhibited the growth of ECs, especially CPAEC via caspase-dependent apoptosis and GSH depletion.

Antimycin A (AMA) inhibits succinate oxidase, NADH oxidase, and mitochondrial electron transport chain between cytochrome b and c. We recently demonstrated that AMA inhibited the growth of Calu-6 lung cancer cells through apoptosis. Here, we investigated the effects of AMA and/or MAPK inhibitors on Calu-6 lung cancer cells in relation to cell growth, cell death, reactive oxygen species (ROS), and GSH levels. Treatment with AMA inhibited the growth of Calu-6 cells at 72 h. AMA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; Delta Psi m). While ROS were decreased in AMA-treated Calu-6 cells, O2.- among ROS was increased. AMA also induced GSH depletion in Calu-6 cells. Treatment with MEK inhibitor intensified cell death, MMP (Delta Psi m) loss, and GSH depletion in AMA-treated Calu-6 cells. JNK inhibitor also increased cell death, MMP (Delta Psi m) loss, and ROS levels in these cells. Treatment with p38 inhibitor magnified cell growth inhibition by AMA and increased cell death, MMP (Delta Psi m) loss, ROS level, and GSH depletion in AMA-treated cells. Conclusively, all the MAPK inhibitors slightly intensified cell death in AMA-treated Calu-6 cells. The changes of ROS and GSH by AMA and/or MAPK inhibitors were in part involved in cell growth and death in Calu-6 cells.

The authors report a rare case of diffuse microscopic angiodysplasia and nodular lymphoid hyperplasia involving the distal ileum and total colon in a 13-month-old girl who had recurrent episodes of massive lower gastrointestinal bleeding of obscure origin. Colonoscopy showed multiple nodular hyperplasia and mucosal erosions, and all other diagnostic studies were negative. At laparotomy, intraoperative transluminal endoscopic transillumination was of benefit in identifying the bleeding foci but could not detect every angiodysplastic lesion.

Pyrogallol (PG) is a polyphenol compound and a known O 2 − generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (Δ Ψ m ), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (Δ Ψ m ). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We evaluated the effects of ATO on the viability, cell cycle and apoptosis of human pulmonary adenocarcinoma, Calu-6 and A549 cells. ATO reduced the viability of Calu-6 cells with an IC50 of approximately 3 or 4 microM. However, A549 cells were very resistant to ATO. Calu-6 cells treated with 1, 3 or 5 microM ATO showed a G2 phase arrest of the cell cycle at 72 h. The G2 phase arrest was accompanied with the down-regulation of cdc2 protein. Treatment with ATO-induced apoptosis in Calu-6 cells. The apoptotic process was accompanied by the down-regulation of Bcl-2 protein, the activation of caspase-3, and the loss of the mitochondrial membrane potential (Delta Psi m). All of the caspase inhibitors, especially pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from ATO-induced cell death. Caspase inhibitors also prevented the loss of mitochondrial membrane potential (Delta Psi m). The inhibitors significantly increased the number of G2 phase cells in 10 microM ATO-treated cells. In addition, the levels of O2- were significantly increased in 10 microM ATO-treated cells. However, the changes of ROS by 10 microM ATO are not correlated with apoptosis in Calu-6 cells. Treatment with 10 microM ATO depleted GSH content in Calu-6 cells and caspase inhibitors significantly prevented the GSH depletion in these cells. In conclusion, we have demonstrated that ATO inhibits the growth of Calu-6 cells by inducing a G2 arrest of the cell cycle and by triggering apoptosis accompanied with the depletion of GSH.

Pyrogallol (PG) is a polyphenol compound and has been known to be an O(2)(.-) generator. We evaluated the effects of PG on the growth of human pulmonary adenocarcinoma Calu-6 cells in relation to the cell cycle. DNA flow cytometric analysis indicated that PG induced a G2 phase arrest of the cell cycle in Calu-6 cells at 72 h. PG down-regulated the expression of CDKI (p27), CDK2, CDK4 and CDK6 as well as cyclin D1, and increased cyclin A and cyclin B1 proteins. In addition, O(2)(.-) levels were significantly increased in PG-treated cells. Treatment with catalase rescued Calu-6 cells from PG-induced apoptosis, and also prevented the growth inhibition as well as a G2 phase arrest by PG, which were accompanied with the down-regulation of O(2)(.-) levels. In conclusion, PG inhibited the growth of Calu-6 cells by inducing the cell cycle arrest, accompanied with an increase in O(2)(.-) levels.

2,4-dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in the mitochondria. Here, we investigated the effect of DNP on the growth of Calu-6 lung cancer cells in view of cell cycle, apoptosis, ROS production and GSH content. DNP dose-dependently decreased cell viability at 72 h (EC50 of about 200 microM) as measured by a MTT assay. The lower doses of DNP induced a G1 arrest of the cell cycle in Calu-6 cells. Analysis of the cell cycle regulatory proteins demonstrated that DNP decreased the steady-state levels of cyclin proteins and cyclin dependent kinase (CDK), but increased the protein levels of cyclin dependent kinase inhibitor (CDKI) p27. DNP also caused a marked increase in apoptosis, as evidenced by DNA fragmentation (sub-G1 DNA content), DAPI staining, the loss of mitochondrial membrane potential (DeltaPsim), externalization of phosphatidylserine (PS). In addition, DNP-treated cells significantly increased the intracellular H2O2 and O2.- levels. All of caspase inhibitors could markedly rescue Calu-6 cells from DNP-induced cell death and only pan-caspase inhibitor, Z-VAD-FMK, could slightly prevent the loss of mitochondrial membrane potential (DeltaPsim). However, none of the caspase inhibitors reduced the increased H2O2 levels, but the increased O2.- levels was slightly attenuated by pan-caspase inhibitor. In addition, the depletion of GSH content in DNP-treated cells was prevented by all of caspase inhibitors. In conclusion, DNP, which induced ROS and reduced GSH content, inhibited the growth of Calu-6 cells via cell cycle arrest at G1 phase and apoptosis.

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We investigated an involvement of ROS such as H(2)O(2) and O(2)(*-), and GSH in ATO-treated Calu-6 cell death. The levels of intracellular H(2)O(2) were decreased in ATO-treated Calu-6 cells at 72 h. However, the levels of O(2)(*-) were significantly increased. ATO reduced the intracellular GSH content. Many of the cells having depleted GSH contents were dead, as evidenced by the propidium iodine staining. The activity of CuZn-SOD was strongly down-regulated by ATO at 72 h while the activity of Mn-SOD was weakly up-regulated. The activity of catalase was decreased by ATO. ROS scavengers, Tiron and Trimetazidine did not reduce levels of apoptosis and intracellular O(2)(*-) in ATO-treated Calu-6 cells. Tempol showing a decrease in intracellular O(2)(*-) levels reduced the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment with NAC showing the recovery of GSH depletion and the decreased effect on O(2)(*-) levels in ATO-treated cells significantly inhibited apoptosis. In addition, BSO significantly increased the depletion of GSH content and apoptosis in ATO-treated cells. Treatment with SOD and catalase significantly reduced the levels of O(2)(*-) levels in ATO-treated cells, but did not inhibit apoptosis along with non-effect on the recovery of GSH depletion. Taken together, our results suggest that ATO induces apoptosis in Calu-6 cells via the depletion of the intracellular GSH contents rather than the changes of ROS levels.

We investigated the in vitro effects of pyrogallol on cell growth, cell cycle regulation, and apoptosis in HeLa cells. Pyrogallol inhibited the growth of HeLa cells with an IC(50) of approximately 45 microM. Pyrogallol induced arrest during all phases of the cell cycle and also very efficiently resulted in apoptosis in HeLa cells, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation, and PARP cleavage. Pan-caspase inhibitor (Z-VAD) could rescue some HeLa cells from pyrogallol-induced cell death, while caspase-8 and -9 inhibitors unexpectedly enhanced the apoptosis. When we examined the changes of the ROS, H(2)O(2) or O(2)(*-) in pyrogallol-treated cells, H(2)O(2) was slightly increased and O(2)(*-) significantly was increased. In addition, we detected a decreased GSH content in pyrogallol-treated cells. Only pan-caspase inhibitor showing recovery of GSH depletion and reduced intracellular O(2)(*-) level decreased PI staining in pyrogallol-treated HeLa cells, which indicates dead cells. In summary, we have demonstrated that pyrogallol as a generator of ROS, especially O(2) (*-), potently inhibited the growth of HeLa cells through arrests during all phases of the cell cycle and apoptosis.