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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 IC50 of approximately 1~2 mM at 48 h. Tempol induced apoptosis in lung cells with loss of mitochondrial membrane potential (MMP; ∆Ψm) and activation of caspase-3. There was no significant difference in susceptibility to Tempol between lung cancer and normal cells. Z-VAD, a pan-caspase inhibitor, significantly decreased the number of annexin V-positive cells in Tempol-treated Calu-6, A549, and WI-38 VA-13 cells. A 2 mM concentration of Tempol increased ROS levels, including O2•− in A549 and WI-38 VA-13 cells after 48 h, and specifically increased O2•− levels in Calu-6 cells. In addition, Tempol increased the number of GSH-depleted cells in Calu-6, A549, and WI-38 VA-13 cells at 48 h. Z-VAD partially downregulated O2•− levels and GSH depletion in Tempol-treated these cells. In conclusion, treatment with Tempol inhibited the growth of both lung cancer and normal cells via apoptosis and/or necrosis, which was correlated with increased O2•− levels and GSH depletion.

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.

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.

Arsenic trioxide (ATO; As2 O3 ) induces cell death in various cells via oxidative stress. Expose to chronic arsenic is involved in the development of vascular diseases. However, little is known about the cytotoxic effects of ATO on human normal vascular smooth muscle cells (VSMCs). Thus, in this study, we investigated the effects of ATO on cell growth and death in human pulmonary artery smooth muscle (HPASM) cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. ATO treatment decreased the growth of HPASM cells with an IC50 of ∼30-50 μM at 24 h, and ATO induced HPASM cell death via apoptosis or necrosis dependent on the doses of it at this time. Treatment with 50 μM ATO did not increase ROS levels at the early time points, but it significantly increased mitochondrial O2•- levels at 24 h. ATO also induced GSH depletion in HPASM cells. N-acetyl cysteine (NAC; a well-known antioxidant) did not significantly affect apoptotic cell death, ROS levels, or GSH depletion in ATO-treated HPASM cells. However, l-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) intensified mitochondrial O2•- levels in ATO-treated HPASM cells, and significantly increased cell death and GSH depletion in these cells as well. In summary, we provided the first evidence that ATO inhibited the growth of HPASM cells, and induced apoptotic and/or necrotic cell death in these cells, accompanied by increases in mitochondrial O2•- level and GSH depletion.

Exogenous hydrogen peroxide (H2O2) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H2O2-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50–500 μM H2O2 inhibited the growth of Calu-6 and A549 cells at 24 h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H2O2-treated lung cancer cells. H2O2 increased intracellular ROS levels, including that of O 2 ·− , at 1 and 24 h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H2O2 triggered GSH deletion in Calu-6 and A549 cells at 24 h. It reduced GSH levels in Calu-6 cells at 1 h but increased them at 24 h. Caspase inhibitors decreased O 2 ·− levels in H2O2-treated Calu-6 cells at 1 h and these inhibitors decreased ROS levels, including that of O 2 ·− , in H2O2-treated A549 cells at 24 h. Caspase inhibitors partially attenuated GSH depletion in H2O2-treated A549 cells and increased GSH levels in these cells at 24 h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24 h. In conclusion, H2O2 induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H2O2-induced oxidative stress and GSH depletion.

Gallic acid (GA) exhibits a number of cellular effects, including apoptosis, which is associated with oxidative stress. The present study investigated the effects of GA on calf pulmonary arterial endothelial cell (CPAEC) growth and death, along with the levels of reactive oxygen species (ROS) and glutathione (GSH). GA treatment inhibited the growth of CPAECs at 24 h, and the half‑maximal inhibitory concentration (IC50) value of GA was ~30 µM. GA treatment also induced cell death, which was accompanied by a loss of mitochondrial membrane potential (ΔѰm). GA treatment in CPAECs resulted in decreased ROS levels, including O2•‑, whereas the number of GSH‑depleted cells increased. Neither a pan‑caspase inhibitor (benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone) nor buthionine sulfoximine treatment affected GA‑induced cell growth inhibition, cell death, ROS and GSH levels in CPAECs, whereas co‑treatment with N‑acetyl‑cysteine (NAC) resulted in enhanced cell growth inhibition, cell death and ΔѰm loss in these cells. Although NAC treatment did not significantly influence ROS levels in GA‑treated CPAECs, it significantly enhanced GSH depletion in these cells. In conclusion, GA inhibited the growth of CPAECs via cell death, which was associated with GSH depletion rather than alterations to ROS levels.

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.

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.

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.

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.

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.

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.

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.

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.

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. © 2013 Wiley Periodicals, Inc.

H2O2-induced cytotoxicity in normal human pulmonary fibroblasts (HPFs) is of interest in toxicological research since HPFs are involved in lung inflammation, fibrosis and cancer. The present study investigated the cytotoxic effects of H2O2 on normal HPFs in relation to reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) using the well-known antioxidants N-acetyl cysteine (NAC) and propyl gallate (PG), as well as MAPK inhibitors. Treatment with 50 μM H2O2 inhibited the growth of the HPFs by ∼45% in 24 h. H2O2 induced cell death via apoptosis and triggered the loss of mitochondrial membrane potential (MMP; Δψm) in the HPFs. H2O2 also increased the ROS levels, including O2•−, in the HPFs and induced glutathione (GSH) depletion. NAC and PG attenuated the death of the HPFs and the loss of MMP (Δψm) through the use of H2O2. NAC decreased the ROS levels in the H2O2-treated HPFs and PG markedly prevented an increase in O2•− levels in these cells. However, PG alone induced cell death in the HPF control cells and increased the ROS levels in these cells. None of the MAPK (MEK, JNK and p38) inhibitors affected cell growth inhibition or cell death by H2O2. In addition, these inhibitors did not significantly affect the ROS levels and GSH depletion in the H2O2-treated HPFs. In conclusion, H2O2 induced growth inhibition and cell death in the HPFs via GSH depletion. NAC and PG attenuated H2O2-induced HPF cell death but each regulated the ROS levels in a different manner. Treatment with MAPK inhibitors did not affect cell death or the ROS levels in the H2O2-treated HPFs.

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.

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)(•-).

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.

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we investigated the molecular mechanisms of MG132 in As4.1 juxtaglomerular cell death in relation to apoptosis and levels of ROS and glutathione (GSH). MG132 inhibited the growth of As4.1 cells with an IC(50) of approximately 0.3-0.4 microM at 48 h and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; Psi(m)), Bcl-2 decrease, activations of caspase-3 and caspase-8, and PARP cleavage. MG132 increased intracellular ROS levels and GSH-depleted cell numbers. However, caspase inhibitors, especially Z-VAD (pan-caspase inhibitor) intensified cell growth inhibition, cell death, MMP (Psi(m)) loss, and Bcl-2 decrease in MG132-treated As4.1 cells. Z-VAD also slightly intensified increases in ROS levels and GSH depletion in MG132-treated As4.1 cells. In conclusion, MG132 reduced the growth of As4.1 cells via caspase-independent apoptosis. The changes in ROS and GSH levels by MG132 and caspase inhibitors partially influenced the growth inhibition and death of As4.1 cells.

The proteasome inhibitor MG132 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 and death of As4.1 juxtaglomerular cells in relation to ROS and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells with an IC(50) of approximately 0.3-0.4microM at 48h and induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)), Bcl-2 decrease, activation of caspase-3 and -8, and PARP cleavage. MG132 increased intracellular ROS levels including O(2)(-) and GSH depleted cell numbers. N-acetyl cysteine (NAC, a well-known antioxidant) significantly decreased ROS level and GSH depleted cell numbers in MG132-treated As4.1 cells, along with the prevention of cell growth inhibition, cell death and MMP (DeltaPsi(m)) loss. NAC also decreased the caspase-3 activity of MG132. l-Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) did not affect cell growth, death, ROS and GSH levels in MG132-treated As4.1 cells. Conclusively, MG132 reduced the growth of As4.1 cells via apoptosis. The changes of ROS and GSH by MG132 were involved in As4.1 cell growth and death.

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.

2,4-Dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in mitochondria. Here, we investigated the in vitro effect of DNP on apoptosis and the involvement of reactive oxygen species (ROS) in As4.1 juxtaglomerular cell death. Dose- and time-dependent induction of apoptosis was evidenced by flow cytometric detection of sub-G1 DNA content and annexin V binding assay. The intracellular H(2)O(2) and O(2)(-) levels were markedly increased in DNP-treated cells. However, the reduction of intracellular H(2)O(2) level by Tiron and catalase did not prevent apoptosis induced by DNP. Moreover, DNP rapidly reduced intracellular GSH content in As4.1 cells. Taken together, apoptosis in DNP-treated As4.1 cells is correlated with the rapid change of intracellular GSH levels rather than ROS levels.

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.

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.

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.

We investigated an involvement of ROS, such as H 2 O 2 and O 2 − and GSH in the As4.1 cell death by antimycin A and examined whether ROS scavengers rescue antimycin A-induced As4.1 cell death and its mechanism. Levels of intracellular H 2 O 2 and O 2 − were markedly increased in antimycin A-treated cells. Antimycin A reduced the intracellular GSH content. A ROS scavenger, Tiron down-regulated the production of intracellular H 2 O 2 . However, the reduction of intracellular H 2 O 2 level did not change the apoptosis parameters, such as sub-G1 DNA content and annexin V binding. Interestingly, treatment of Tiron could partially prevent the loss of mitochondrial transmembrane potential (Δ Ψ m ). Treatment of SOD and catalase also reduced the intracellular H 2 O 2 and loss of mitochondrial transmembrane potential (Δ Ψ m ) without reducing O 2 − level and apoptosis in antimycin A-treated As4.1 cells. All the ROS scavengers, SOD and catalase did not inhibit GSH depletion induced by antimycin A, resulting in failure of preventing the apoptosis. In addition, all the reagents including antimycin A did not induce any specific phase arrest of cell cycle in As4.1 cells. In summary, these results demonstrate that antimycin A generates potently ROS, H 2 O 2 and O 2 − and induces the depletion of GSH content in As4.1 JG cells, and that Tiron, SOD and catalase inhibited partially the loss of mitochondrial transmembrane potential (Δ Ψ m ) via the reduction of intracellular H 2 O 2 level.