Treatment with p38 inhibitor intensifies the death of MG132-treated As4.1 juxtaglomerular cells via the enhancement of GSH depletion

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MG132 and/or MAPK inhibitors on As4.1 juxtaglomerular cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m) and activation of caspase-3 and -8. MG132 increased ROS levels, and GSH depleted cell numbers. The MEK inhibitor slightly reduced cell growth and caspase-3 activity in MG132-treated As4.1 cells and mildly increased MMP (ΔΨ_m) loss and O₂^?- level. However, it did not increase apoptosis and GSH depletion. The JNK inhibitor did not strongly influence cell growth, cell death, and GSH depletion by MG132, but increased caspase-3 activity, MMP (ΔΨ_m) loss, and O₂^?- level. Treatment with the p38 inhibitor magnified cell-growth inhibition and apoptosis by MG132. This agent also strongly increased caspase-8 activity, MMP (ΔΨ_m) loss, O₂^?- level, and GSH depletion. Conclusively, the p38 inhibitor strongly intensified cell death in MG132-treated As4.1 cells. The changes of GSH content by MG132 and/or MAPK inhibitors were closely related to the death of As4.1 cells.     

MG132, a proteasome inhibitor-induced calf pulmonary arterial endothelial cell growth and death,are changed by MAPK inhibitors

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK inhibitors on MG132-treated calf pulmonary artery endothelial cells (CPAECs) in relation to cell death, ROS, and glutathione (GSH). MG132 inhibited the growth of CPAEC and also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). MG132 increased ROS levels and GSH-depleted cell numbers in CPAEC. Treatment with MAPK (MEK, JNK, and p38) inhibitors showed a slight enhancement of cell-growth inhibition by MG132. All the MAPK inhibitors decreased cell death by MG132. Especially, the JNK inhibitor showed a strong effect. They all did not affect ROS levels and GSH depletion in MG132-treated CPAEC, but increased ROS and GSH levels in MG132-untreated CPAEC. In conclusion, MG132 induced apoptosis in CPAEC, which was accompanied by ROS increase and GSH depletion. The changes of MG132-induced CPAEC growth inhibition and death by MAPK inhibitors were not tightly correlated to ROS and GSH levels.     

Mitogen-activated protein kinase inhibitors differently affect the growth inhibition and death of a proteasome inhibitor, MG132-treated human pulmonary fibroblast cells

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor can induce growth inhibition and death in lung cancer or normal cells. However, little is known about relationship between proteasome inhibition and mitogen-activated protein kinase (MAPK) signaling in normal lung cells. Thus, in the present study, we investigated the effects of MAPK inhibitors on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition, cell death, reactive oxygen species (ROS) and glutathione (GSH). Treatment with 15 μM MG132 increased ROS levels including mitochondrial O(2?)(-) and GSH depleted cell numbers in HPF cells at 24 hours. MAP kinase or ERK kinase (MEK) inhibitor did not significantly affect cell growth inhibition, cell death, the loss of mitochondrial membrane potential (MMP; ΔΨ(m)), ROS level and GSH depletion in MG132-treated HPF cells. c-Jun N-terminal kinase (JNK) inhibitor attenuated the growth inhibition and death by MG132. This inhibitor also significantly decreased O(2?)(-) level in MG132-treated HPF cells. Although p38 inhibitor slightly enhanced HPF cell growth inhibition by MG132, this inhibitor and siRNA prevented HPF cell death induced by MG132. p38 inhibitor also attenuated d O(2?)(-) level and GSH depletion. Moreover, extracellular signal regulated kinase (ERK), JNK or p38 siRNA did not strongly affect ROS levels in MG132-treated HPF cells. ERK and JNK siRNAs decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. In conclusion, MAPK inhibitors differently affected the growth inhibition and death of MG132-treated HPF cells. Especially, p38 inhibitor attenuated HPF cell death by MG132, which was in part related to changes in ROS and GSH levels.     

Pyrogallol-induced As4.1 juxtaglomerular cell death is attenuated by MAPK inhibitors via preventing GSH depletion

Pyrogallol (PG) induces apoptosis in several types of cells mediated by superoxide anion (O₂^•−). Here, we investigated the effects of PG and/or MAPK (MEK, JNK, and p38) inhibitors on the changes in cell growth, cell death, reactive oxygen species (ROS), and GSH levels in As4.1 juxtaglomerular (JG) cells. PG inhibited the growth of As4.1 cells. It also induced apoptosis and the loss of mitochondrial membrane potential (MMP; ΔΨ_m) and increased the level of p53 protein. Intracellular O₂^•− level was increased in PG-treated As4.1 cells. PG also increased the number of GSH deleted cells in As4.1 cells. All the MAPK inhibitors significantly attenuated the growth inhibition and death mediated by PG. They decreased the levels of p53 protein and MMP (ΔΨ_m) loss in PG-treated As4.1 cells. They also reduced O₂^•− level and GSH-depleted cell number in these cells. In conclusion, MAPK inhibitors attenuated As4.1 cell growth inhibition and death mediated by PG treatment. The changes in O₂^•− and GSH levels by PG and/or MAPK inhibitors appeared to affect the growth and death of As4.1 cells.     

MG132, a proteasome inhibitor-induced calf pulmonary arterial endothelial cell growth and death, are changed by MAPK inhibitors

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK inhibitors on MG132-treated calf pulmonary artery endothelial cells (CPAECs) in relation to cell death, ROS, and glutathione (GSH). MG132 inhibited the growth of CPAEC and also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). MG132 increased ROS levels and GSH-depleted cell numbers in CPAEC. Treatment with MAPK (MEK, JNK, and p38) inhibitors showed a slight enhancement of cell-growth inhibition by MG132. All the MAPK inhibitors decreased cell death by MG132. Especially, the JNK inhibitor showed a strong effect. They all did not affect ROS levels and GSH depletion in MG132-treated CPAEC, but increased ROS and GSH levels in MG132-untreated CPAEC. In conclusion, MG132 induced apoptosis in CPAEC, which was accompanied by ROS increase and GSH depletion. The changes of MG132-induced CPAEC growth inhibition and death by MAPK inhibitors were not tightly correlated to ROS and GSH levels.     

The effects of antimycin A on endothelial cells in cell death,reactive oxygen species and GSH levels

Antimycin A (AMA) inhibits mitochondrial electron transport chain between cytochrome b and c. Here, we evaluated the effects of AMA on the growth and death of endothelial cells (ECs) in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. AMA inhibited the growth of calf pulmonary artery endothelial cells (CPAEC) and human umbilical vein endothelial cells (HUVEC). AMA also induced apoptosis in both ECs which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). HUVEC were more sensitive to AMA than CPAEC. AMA increased ROS level in CPAEC but decreased the levels in HUVEC. Z-VAD (pan-caspase inhibitor) mildly prevented apoptosis in AMA-treated ECs without the significant changes of ROS. N-acetyl-cysteine (NAC; a well-known antioxidant) decreased ROS levels in AMA-treated ECs. NAC reduced CPAEC death by AMA but enhanced HUVEC death by it. Furthermore, AMA increased GSH depleted cell numbers in ECs. Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis), showing a pro-apoptotic effect on AMA-treated HUVEC, significantly increased GSH depleted cell number but it did not affect cell death and GSH depletion in AMA-treated CPAEC. In conclusion, AMA inhibited the growth of ECs via caspase-dependent apoptosis. ROS level change by AMA was partially related to CPAEC death, but did not affect HUVEC death. The change of GSH contents by AMA influenced the death of ECs.     

Differential response of MG132 cytotoxicity against small cell lung cancer cells to changes in cellular GSH contents

The effect of the depletion or oxidation of cellular GSH on cytotoxicity of MG132 was assessed. Viability loss and decrease in GSH contents in small cell lung cancer (SCLC) cells treated with MG132 was attenuated by caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk). Thiol compounds (N-acetylcysteine and N-(2-mercaptopropionyl)glycine) and free radical scavengers reduced MG132-induced cell death. Antioxidants, including N-acetylcysteine, inhibited the MG132-induced nuclear damage, loss in mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c and caspase-3 activation. Depletion of GSH due to buthionine sulfoxime did not affect the cell viability loss, ROS formation and GSH depletion due to MG132 in SCLC cells. A thiol oxidant monochloramine, p-chloromercuribenzoate and N-ethylmaleiamide also did not affect cytotoxicity of MG132. The results suggest that the toxicity of MG132 on SCLC cells is mediated by activation of caspase-8, -9 and -3. Removal of free radicals and recovery of GSH contents may attenuate MG132-induced apoptotic cell death. Nevertheless, depletion or oxidation of cellular GSH may not affect toxicity of MG132.     

Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis

Gallic acid (GA) is widely distributed in various plants and foods, and its various biological effects have been reported. Here, we evaluated the effects of GA on HeLa cells in relation to cell growth inhibition and death. HeLa cell growth was diminished with an IC₅₀ of approximately 80 μM GA at 24 h whereas an IC₅₀ of GA in human umbilical vein endothelial cells (HUVEC) was approximately 400 μM. GA-induced apoptosis and/or necrosis in HeLa cells and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). The percents of MMP (ΔΨ_m) loss cells and death cells were lower in HUVEC than HeLa cells. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from GA-induced cell death. GA increased reactive oxygen species (ROS) level and GSH (glutathione) depleted cell number in HeLa cells. Caspase inhibitors reduced GSH depleted cell number but not ROS level in GA-treated HeLa cells. In conclusion, GA inhibited the growth of HeLa cells and HUVEC via apoptosis and/or necrosis. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA-induced HeLa cell death was accompanied by ROS increase and GSH depletion.     

Pyrogallol-induced calf pulmonary arterial endothelial cell death via caspase-dependent 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 (ΔΨ_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 ΔΨ_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.     

Enhancement of gallic acid-induced human pulmonary fibroblast cell death by N-acetyl cysteine and L-buthionine sulfoximine

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; ΔΨ(m)). GA increased ROS levels including O(2)(?-) 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.     

The anti-apoptotic effects of caspase inhibitors on propyl gallate-treated HeLa cells in relation to reactive oxygen species and glutathione levels

Propyl gallate (PG) as a synthetic antioxidant is widely used in processed food, cosmetics and medicinal preparations. Despite the assumed low toxicity of PG, it exerts a variety of effects on tissue and cell functions. In the present study, we evaluated the anti-apoptotic effects of caspase inhibitors on PG-treated human cervix adenocarcinoma HeLa cells in relation to the changes of reactive oxygen species (ROS) and glutathione (GSH) levels. PG induced apoptosis in a dose-dependent manner, as evidenced by sub-G1 cells and annexin V staining cells. Treatment with pan-caspase inhibitor, caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor significantly prevented apoptosis in PG-treated HeLa cells at 24 h. The intracellular ROS levels including O₂^•− were increased or decreased in PG-treated HeLa cells depending on the incubation times (1 or 24 h). PG depleted intracellular GSH content in HeLa cells at 24 h. Treatment with caspase inhibitor reduced ROS levels and significantly prevented GSH depletion in PG-treated HeLa cells at 24 h. In conclusion, PG induced apoptosis in HeLa cells. The anti-apoptotic effect of caspase inhibitor on PG-induced HeLa cell death was closely related to the reduction of ROS levels, especially mitochondrial O₂^•−, as well as to the inhibition of GSH depletion.     

Enhanced cell death effects of MAP kinase inhibitors in propyl gallate-treated lung cancer cells are related to increased ROS 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; ΔΨ_m). 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 (ΔΨ_m) 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.     

Gallic acid-induced human pulmonary fibroblast cell death is accompanied by increases in ROS level and GSH depletion

Gallic acid (GA), as a polyhydroxylphenolic compound, has various biological properties, including an anticancer effect. However, little is known about the toxicological effect of GA in primary normal cells. In the present study, we investigated the molecular mechanisms of GA on human pulmonary fibroblast (HPF) cell death in relation to apoptosis. HPF cell growth was dose dependently diminished with an IC(50) of approximately 400 μM of GA at 24 hours. GA-induced HPF cell death was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). All the tested caspase inhibitors (e.g., pan-caspase, caspase-3, -8, or -9 inhibitor) did not rescue HPF cells from GA-induced cell death. GA increased reactive oxygen species (ROS) levels and glutathione (GSH)-depleted cell numbers. Caspase inhibitors partially altered ROS levels, but did not reduce GSH-depleted cell number, in GA-treated HPF cells. In conclusion, we demonstrated that GA induced the growth inhibition and death of HPF cells, which was accompanied by ROS increase and GSH depletion.     

Pyrogallol, ROS generator inhibits As4.1 juxtaglomerular cells via cell cycle arrest of G2 phase and apoptosis

Pyrogallol as a catechin compound has been employed as an O(2)(*-) generator and often used to investigate the role of ROS in the biological system. Here, we investigated the in vitro effect of pyrogallol on cell growth, cell cycle and apoptosis in As4.1 juxtaglomerular cells. Dose-dependent inhibition of cell growth was observed with IC(50) of about 60 microM for 48 h using MTT assay. Pyrogallol (100 microM) did not alter intracellular H(2)O(2) level and catalase activity, but increased the intracellular O(2)(-) level and decreased SOD activity in As4.1 cells. DNA flow cytometric analysis indicated that 50 and 100 microM pyrogallol significantly increased G2 phase cells as compared with those of pyrogallol-untreated cells. Also, pyrogallol induced apoptosis as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptosis process was accompanied with the loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation and PARP cleavage. Pan caspase inhibitor (Z-VAD) could significantly rescue As4.1 cells from pyrogallol-induced cell death. But, the inhibitors of caspase-3, caspase-8, and caspase-9 did not prevent apoptotic events in pyrogallol-treated As4.1 cells. Taken together, we have demonstrated that an ROS inducer, pyrogallol inhibits the growth of As4.1 JG cells via cell cycle arrest and apoptosis, and suggest that the compound exhibits an anti-proliferative efficacy on these cells.     

Propyl gallate inhibits the growth of calf pulmonary arterial endothelial cells via glutathione depletion

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we evaluated the effects of PG on the growth and death of endothelial cells (ECs), especially calf pulmonary artery endothelial cells (CPAEC) in relation to reactive oxygen species (ROS) and glutathione (GSH). PG dose-dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVEC) at 24 h. PG induced cell death in CPAEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). PG generally increased ROS level in CPAEC but not in HUVEC. PG also dose-dependently increased GSH depleted cells in both ECs. The treatment with antioxidant of N-acetyl-cysteine (NAC) or ascorbate acid (AA) prevented CPAEC growth inhibition and death by PG, which was accompanied by the attenuation of GSH depletion but not by the reduction of ROS level. In conclusion, PG induced growth inhibition and death of ECs, especially CPAEC via GSH depletion.     

Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou-Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O₂⁻) levels. Our results showed that combined arsenite + MG132 produced low levels of O₂⁻ at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O₂⁻ levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O₂⁻ levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O₂⁻ at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O₂⁻ production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O₂⁻ levels at early time points after exposure.     

Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC₅₀ of approximately 6.64 ± 1.84 μM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 μM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.     

T-2 toxin cytotoxicity mediated by directly perturbing mitochondria in human gastric epithelium GES-1 cells

T-2 toxin is one of the most toxic trichothecenes and harmful to human health and animal husbandry. The mechanism underlying its growth suppression remains unclear, especially for mitochondrial damage in human gastric epithelial cells. In the present study, we investigated cell death caused by T-2 toxin in a human gastric epithelial cell line (GES-1) and the possible mechanism of T-2-induced cytotoxicity. T-2 strongly reduced the viability of GES-1 cells in a time- and dose-dependent manner within a small range of concentrations. However, when the concentrations of T-2 were >40 nM, there was no concentration dependence, only time dependence. Moreover, T-2 induced apoptosis, with the activation of caspase-3 in GES-1 and mitochondrial membrane potential (MMP) decrease and cytochrome c release. T-2 also resulted in the accumulation of reactive oxygen species (ROS) and DNA damage with a positive signal of p-H2A.X in GES-1 cells. While T-2 caused a MMP decrease, DNA damage and cell death were not blocked by pretreatment with 3 mM glutathione (GSH), a typical scavenger of ROS. The induction of mitochondrial permeability transition pore (mPTP) regulators voltage-dependent anion channel (VDAC1) and cyclophilin D (CypD) were also observed in T-2-treated cells. Interestingly, cyclosporine A (CsA), a CypD inhibitor, significantly reversed the drop in MMP and the DNA damage, as well as ROS accumulation caused by T-2. Additionally, GES-1 cell death could also be protected to some extent by 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), an inhibitor of VDAC1, especially the combination of CsA and DIDS, and 3 mM GSH could further enhance the effect of CsA + DIDS on cell viability. In conclusion, our present findings indicate that the T-2 induced MMP decrease, DNA damage and cell death, as well as ROS accumulation in GES-1 cells, starts with T-2 directly perturbing the mitochondria triggering ROS generation by acting on CypD and VDAC1. This study presents a new viewpoint for evaluating the toxicity of T-2 toxin.     

Arsenic trioxide induces growth inhibition and death in human pulmonary artery smooth muscle cells accompanied by mitochondrial increase and GSH depletion

Arsenic trioxide (ATO; As₂O₃) 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 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 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 level and GSH depletion.