Sage Journals: Discover world-class research

Abstract

Acute inhalation exposure to high levels of manganese (Mn) is associated with pulmonary edema and impaired function. The immune-mediated lung epithelium injury of Mn in vivo and in vitro experiments has been well characterized, whereas its apoptotic effect is not well defined. Our results show that human bronchial epithelial (16HBE) cells undergo caspase-9-mediated cell death in response to Mn. Loss of mitochondrial membrane potential (ΔΨm), the formation of reactive oxygen species and release of cytochrome c were regulated during this process. In addition, decreasing c-Myc level and increasing of phosphorylated p53 (Ser 15) and WAF1/p21 were also taken part in Mn-mediated lung toxicity. Proteasome inhibitor MG132 could increase c-Myc protein in abundance. Taking together, our results demonstrate that caspase-9-dependent intrinsic pathway, the downregulation of c-Myc and the upregulation of p53 and phosphorylated p53 might be responsible for Mn-mediated apoptosis in 16HBE cells. Moreover, c-Myc decrease might be due to increased degradation through the ubiquitin–proteasome pathway.

Keywords

Manganese apoptosis mitochondria caspase-9

Introduction

As a constituent of many enzymes, manganese (Mn) plays an important role in a number of physiological processes by serving as a constituent of some enzymes and an activator of others involved in the regulation of amino acid, protein, lipid and carbohydrate metabolisms.¹ Mn is also a heavily used metal in industry. Occupational or environmental exposure to excessive Mn is toxic. In addition to causing neurotoxicity, when inhaled in excess, Mn has also been implicated in inducing lung disorders, such as emphysema, lung stress and apoptosis.^2

–5 Normal human lung epithelial cells (small airway epithelial cell and BEAS-2B) were used to determine the cytotoxicity of Mn, and the results showed that the toxicity of Mn in lung epithelial cells can result in recruitment of cells in the immune system.⁶ Recent studies have suggested that Mn mediates potential pulmonary toxicity by upregulation of vascular endothelia growth factor, a hypertension factor in adult mouse.⁷ In 2008, Zhao et al. used human lung carcinoma A549 cells as a model to study Mn-induced lung toxicity and the results indicated that manganese chloride (MnCl₂) induced A549 cells G0/G1 and S phase arrest.⁸ In spite of a considerable wealth of researches, the molecular mechanisms underlying this Mn lung toxicity are poorly understood.

Apoptotic pathways are activated in the lungs of patients with acute injury, in part by activation of the membrane Fas death receptor by soluble Fas ligand, which results in the activation of caspase-8.⁹ The second pathway (the mitochondrial intrinsic pathway) is activated in response to chemical and physical stress, which triggers the cytoplasmic release of proapoptotic mitochondrial proteins leading to activation of caspase-9.¹⁰ Mn accumulates specifically within mitochondria and adversely affects mitochondrial function both in vivo and in vitro, followed by the dynamics of mitochondrial morphology and mitochondrial respiratory function.^11,12 In 2005, Suzuki and Tsukamoto indicated that the activation of JNK and upregulation of c-Jun, p53 and p21^WAF1/CIPI were involved in the apoptosis of hepatocytes induced by Mn.¹³ Another study showed that Mn activated the mitochondrial apoptotic pathway in rat astrocytes by modulating the expression of proteins of Bcl-2 family.¹⁴ One recent study found that the extrinsic and intrinsic apoptotic pathways were involved in Mn toxicity in rat astrocytoma C6 cells.² Otherwise, research by Oubrahim et al. demonstrated that mitochondria played no roles in Mn(α)-induced apoptosis in Hela cells.¹⁵ We considered that the altered responses noted above could be a result of different cell types.

Epithelial injury is a critical event in the development of acute lung injury, but the mechanisms that cause death of the alveolar epithelium are not completely understood. Mn(α), a transition metal, causes pulmonary injury upon environmental or occupational inhalation in excess.¹⁶ Therefore, MnCl2, one of the most diffused and extensively studied Mn compounds, was chosen in this study, in an attempt to identify the potential toxicological mechanisms of Mn-mediated pulmonary toxicity.

Materials and methods

Cell culture and materials

Human bronchial epithelial (16HBE) cells were purchased from Tumor Marker Research Center (Beijing, China). The cell line is a simian virus 40-immortalized human bronchial epithelial cell line and retains the function of normal epithelium. Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum from Gibco (Grand Island, New York, USA) in a humidified atmosphere of 5% CO₂ at 37°C. Primary antibodies of c-Myc, cytochrome c, caspase-3, caspase-9, p53, phospho-p53 (Ser 15), p21 and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). Mouse polyclonal anticaspase-8 was purchased from Cell Signaling Technology Inc. (Danver, Massachusetts, USA). Mouse monoclonal cytochrome c oxidase 4 (Cox IV) antibody was purchased from Abcam Inc. (Cambridge, UK). Mitochondria/Cytosol Fractionation Kit was purchased from Pierce (Florida, USA). Reactive Oxygen Species Assay Kit and JC-1 Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Other chemicals used in this study were purchased from Sigma (St Louis, Missouri, USA), unless specified.

Cytotoxicity assay

Cytotoxicity of MnCl₂ was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded in 96-well plates (1 × 10⁴ cells), 36 h later, various doses of MnCl₂ (0, 0.1875, 0.375, 0.75, 1.50 or 3.0 mM) were applied for 12 or 24 h, and cytotoxicity was assessed using MTT assay. The plates were read at 570 nm using a microplate reader. The relative cell viability (%) related to control was calculated by [OD]_test/[OD]_control × 100%.

Apoptosis assay

Apoptosis was assessed by Annexin V-FITC Apoptosis Detection Kit (KeyGene institute of Biotechnology, Shanghai, China) according to the manufacture’s protocol. After exposure, the cells were harvested and washed twice with phosphate-buffered saline and gently resuspended in 500 µl binding buffer with 5 µl annexin V-FITC and 5 µl propidium iodide (PI). After incubation for 15 min in the dark, cells were analyzed using flow cytometry. Annexin V and PI double-negative cells were defined as live cells, annexin V-positive and PI-negative cells as early apoptotic cells and annexin V and PI double-positive cells as late apoptotic cells.

Measurement of reactive oxygen species generation

Reactive oxygen species (ROS) were detected with Reactive Oxygen Species Assay Kit (KeyGene institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. After treatment with MnCl₂ (0 or 0.375 mM) for 6, 12 or 24 h, cells were washed twice and loaded with 10 μM 2′,7′-dichlorofluorescein diacetate for 20 min at 37°C in the dark. The formation of the fluorescent-oxidized derivative of dichlorofluorescein was monitored by flow cytometry (excitation at 488 nm, emission at 525 nm).[Please check the expansion for the term ‘’DCF’.] Finally, ROS generation was quantified by the median fluorescence intensity of 10,000 cells. The endogenous ROS hydrogen peroxide was used as a positive control (200 μM).

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨ _m) was investigated using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). JC-1 color alters from red to green with the decrease of ΔΨ_m . Cells incubated with MnCl₂ (0 or 0.375 mM) for 12 or 24 h in 6-well plates were harvested and resuspended in 0.5 ml fresh medium. After addition of 0.5 ml JC-1 working solution, the cells were incubated at 37°C for 20 min. Then the cells were washed with JC-1 staining buffer twice. ΔΨ_m was measured using a laser scanning confocal microscope (TCS SP5 α; Leica, Mannheim, Germany) with excitation at 488 nm and emission at 515–545 nm (green fluorescence) and 570–610 nm (red fluorescence).

Western blot

Western blot was performed using a standard protocol. The samples were separated on sodium dodecyl sulfate–polyacrylamide gels and then transferred to polyvinylidene fluoride membrane. Membranes were incubated with primary antibodies overnight and subsequently with peroxidase-labeled secondary antibody. Immunoreactive bands were visualized using enhanced chemiluminescence reagents. The images were analyzed by Quantity One software (Bio-Rad, Hercules, California, USA).

Statistical analysis

Statistical analysis was performed using SPSS 14.0 (SPSS, Inc., Chicago, Illinois, USA). Data are expressed as the mean ± SEM of the indicated number (n) of experiments. Results were analyzed with one-way analysis of variance.

Results

Inhibition of MnCl₂ on 16HBE cell proliferation

As shown in Figure 1, slight inhibition effects were observed in proliferation among cells exposed to 0.375 mM MnCl₂ for more than 12 h. Twenty-four hours after treatment, a significant reduction was found in 16HBE cells, with a decrease of 88.5%, 78.8% and 68.4% at concentrations of 0.375, 0.75 and 1.50 mM MnCl₂, respectively. These data indicated that MnCl₂ inhibited 16HBE cells proliferation in a dose- and time-dependent behavior. In 16HBE cells at 0.375 mM concentration of MnCl₂, the inhibition growth effects are moderate, so we selected 0.375 mM MnCl₂ in the subsequent experiments.

Figure 1.

Effects of MnCl₂ on cytotoxicity in 16HBE cells. Cells were treated with 0, 0.1875, 0.375, 1.50 and 3.0 mM MnCl₂ for 12 or 24 h. Cytotoxicity was measured by the MTT assay. Results are expressed as the SD of four independent experiments. *p < 0.05, **p < 0.01 versus control group. MnCl₂: manganese chloride; 16HBE: human bronchial epithelial; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

MnCl₂ induces apoptosis in 16HBE cells

As indicated in Figure 2(a), the annexin/PI point diagram displayed a significant time-dependent increase in apoptotic cells. The percentage of apoptotic cells increased approximately 7.2%, 10.1% and 24.8% in 16HBE cells treated with 0.375 mM MnCl₂ for 12, 24 and 48 h, respectively, compared with the control (Figure 2(b)). Apart from annexin/PI, we also detected the cleavage of caspase-3 by Western blotting analysis using an antibody specific for the detection of 20 kDa cleaved caspase-3 protein. Mn exposure induced cleavage of caspase-3 after 12 and 24 h (Figure 2(c)). Upon Mn treatment, full-length caspase-3, which exists in the isoform of 32 kDa, was cleaved into p20 intermediate fragments. Mn induced an increase in p20 kDa levels 128 ± 12% and 181 ± 39% for 12 and 24 h, respectively (Figure 2(d)). These results indicate that MnCl₂ induces apoptosis in a time-dependent manner in 16HBE cells.

Figure 2.

Effects of MnCl₂ on apoptosis and caspase-3 activation in 16HBE cells. (a) Cells were treated with MnCl₂ for 12, 24 or 48 h, apoptosis was detected by annexin V/PI assay using flow cytometry. Q1 represents necrotic cells; Q2 represents live cells; Q3 represents early apoptotic cells; Q4 represents late apoptotic cells. (b) The percentage of total apoptotic cells (Q3 + Q4) was calculated and shown in histogram. (c) After MnCl₂ treatment for 12 or 24 h, the cleavage of caspase-3 was analyzed. (d) Densitometry of protein level. β-Actin was used as the equal loading control. The results presented are typical of four separate experiments. Each value is the mean ± SD (n = 4). *p < 0.05, **p < 0.01 versus control group. MnCl₂: manganese chloride; PI: propidium iodide; 16HBE: human bronchial epithelial.

Mitochondrial dysfunction triggered by MnCl₂

It has been hypothesized that Mn toxicity is associated with mitochondrial dysfunction, because Mn accumulates specifically within mitochondria and adversely affects mitochondrial function both in vivo and in vitro. Hence, we want to further clarify MnCl₂-mediated mitochondria injury in 16HBE cells. As shown in Figure 3(a), after treatment using 0.375 mM MnCl₂ a time dependent ROS generation was detected. The rate of ROS generation was enhanced by Mn, reaching a 30 ± 9% (p < 0.01) increase for 0.375 mM at 12 h. At 24 h, the rate of ROS generation was further enhanced 24 ± 3%. These results provide evidence about the occurrence of oxidative stress in our experimental model. The decline in ΔΨ_m is the marker for viable apoptotic cells. After exposure to Mn for 12 h or 24 h, we found that ΔΨ_m of 16HBE cells was significantly decreased in a time-dependent manner (Figure 3(b)). It has been suggested that a critical event leading to cell apoptosis is the release of cytochrome c from mitochondria into cytosol. In order to gain more insight into the triggering of apoptotic pathway in our model, we studied cytochrome c subcellular location in cells exposed to Mn. The supernatant was centrifuged for 1 h at 100,000 × g, and both the pellet (mitochondria-enriched membrane fraction) and supernatant (cytosolic fraction) were collected separately and used for Western blotting. Our data indicate that after Mn exposure for 12 h, the expression of cytochrome c in cytosol began to increase (Figure 3(c)). Consequently, at 24 h, 22.2% increase in the cytosolic accumulation of cytochrome c was observed. COX IV was used as the mitochondrial loading control. The absence of COX IV in the cytosol ensures the success of separation between cytosol and pellet.

Figure 3.

Effects of MnCl₂ on ROS, mitochondrial membrane potential (ΔΨ_m ) and release of cytochrome c in 16HBE cells. (a) Cells were treated with MnCl₂ for 6, 12 or 24 h and ROS were detected using flow cytometry with quantification. (b) After MnCl₂ treatment for 12 or 24 h, ΔΨ _m was obtained using laser scanning confocal microscope and (c) the level of cytochrome c in pellet and cytosol was analyzed using Western blotting. COX IV was the loading control in pellet. Results are expressed as the ratio between each protein and COX IV (pellet) or β-actin (cytosol) relative quantities, respectively. Values are expressed as mean ± SD (n = 4). **p < 0.01 versus control group. ROS: reactive oxygen species; MnCl₂: manganese chloride; COX IV: cytochrome c oxidase 4; 16HBE: human bronchial epithelial.

Taken together, these data suggest that the formation of ROS, the reduction in ΔΨ_m and the release of cytochrome c to the cytosol are involved in MnCl₂-mediated apoptosis in 16HBE cells.

The activation of caspase-9, c-Myc decrease, p53, p-p53 and p21 increase are involved in MnCl₂-induced apoptosis in 16HBE cells

Mitochondrial apoptosis induction by different stimuli is mediated by intrinsic caspase-9 pathway and/or death receptor-mediated extrinsic caspase-8 pathway. Next, we designed experiments to gain insights into the relative contribution of intrinsic and extrinsic caspase pathways in MnCl₂-induced apoptosis. Positive control group referred to Jurkat cells is treated with 25 µM etoposide for 5 h. Using Jurkat lysates, full caspase-9, which existed as an isoform of 46 kDa, was cleaved into p35 kDa intermediate fragment. As can be seen in Figure 4(a), at both 12 and 24 h, MnCl₂ exposure resulted in the cleavage of caspase-9 in 16HBE cells. However, no cleavage of caspase-8 (43 kDa) was determined in MnCl₂-mediated apoptosis, at 12 or 24 h. These results suggest that MnCl₂-induced apoptosis in 16HBE cells was probably mediated via caspase-9-dependent intrinsic pathway.

Figure 4.

Effect of MnCl₂ on expression of apoptosis-related protein in 16HBE cells. (a) Cells were treated with 0.375 mM MnCl₂ for 12 or 24 h, c-Myc, p53, p-p53 (Ser 15), p21 and the cleavage of caspases-9 and caspase-8 were analyzed. Positive control group referred to Jurkat cells that was treated with 25 µM etoposide for 5 h. (b) Densitometric analysis of (a). (c) Effect of MG132 with or without 0.375 mM MnCl₂ on c-Myc level at 12 h. (d) Densitometric analysis of (c). (e) Effect of MG132 with or without 0.375 mM MnCl₂ on apoptosis. Results are expressed as the ratio between each protein and β-actin relative quantities. Values are expressed as the mean ± SD (n = 4). **p < 0.01 versus control group. MnCl₂: manganese chloride; 16HBE: human bronchial epithelial.

To further identify proteins that participate in MnCl₂-induced apoptosis in 16HBE cells, the levels of c-Myc, p53, p-p53 (Ser 15) and p21 were determined and the results are shown in Figure 4(a). The cellular levels of c-Myc decreased significantly, whereas the levels of p53 and p21 increased in Mn-treated cells at 12 or 24 h. When compared with the control, the cells exposed to MnCl₂ increased by 37% and 51% of p53 and p21, respectively, at 12 h. At 24 h, in the presence of MnCl₂, the levels of p53 and p21 further increased by 46% and 137%, respectively (p < 0.01; Figure 4(b)). In 16HBE cells treated with 0.375 mM MnCl₂ for 12 and 24 h, the expression of p53 phosphorylated at Ser 15 was increased in a dose-dependent manner (Figure 4(a)).

To further confirm that c-Myc stability is regulated as a function of the mitochondrial apoptotic pathway induced by MnCl₂, we studied the effect of MG132, a specific 26S proteasome inhibitor, on the level of c-Myc and apoptosis induced by Mn. We treated 16HBE cells with 1 µM MG132 plus 0.375 mM MnCl₂ for 12 h, and the result indicated that c-Myc obviously increased in lysate from cells treated with MG132 plus MnCl₂ in comparison with MnCl₂-treated cells (p < 0.01, Figure 4(d)). The results of flow cytometry showed that the percentage of apoptotic cells increased approximately 7.2% of 16HBE cells treated with 0.375 mM MnCl₂ for 12 h, compared with the control (p < 0.01); apoptosis in the cells treated with Mn plus MG132 were obviously decreased, compared with the cells treated with Mn alone (Figure 4(e)).

Discussion

Despite being an essential element, Mn causes respiratory and neurological diseases when present in the body in large amounts. Inhalation of particulate Mn leads to an inflammatory response in the lungs of animals. Several acute and intermediate duration studies in animals report various signs of lung inflammation following periods ranging from 1 day to 10 months at Mn concentrations ranging from 0.7 to 69 mg/m³.^17,18 Recent studies further indicated that Mn induced pulmonary inflammation in vitro using lung carcinoma cells lines and normal human lung epithelial cells.^6,19,20 Apoptosis is an important contributor to the pathophysiology of pulmonary inflammation. Therefore, understanding the mechanism of Mn-induced apoptosis would pave the way to a further understanding of Mn-induced lung disease. Up to now, the results about the effect of Mn on apoptosis obtained in different cells are inconsistent. This is the first evidence that Mn induces apoptosis in lung epithelial cells in vitro. To understand the mechanisms further by which Mn induces apoptotic events, the intrinsic and extrinsic apoptotic pathways are also examined.

According to the scatter plot, the nonviable apoptotic cells increased in a time-dependent manner. On the other hand, we also found that even at 48 h, there was no significant difference in necrosis between the Mn-treated cells and the control. This suggests MnCl₂-induced 16HBE cell death depends on the apoptotic pathway. Previous studies have shown that Mn would elicit apoptosis in A549 cells, rat pheochromocytoma (PC12) cells, neural stem cells and rat astrocytes,^2,5,21 which further supports our observation and suggests that Mn-induced apoptosis exists not only in neural and cancer cells but also in normal lung cells.

Cells undergo apoptosis through two major pathways, namely the extrinsic pathway or the intrinsic pathway. In both apoptotic pathways, caspase-3 is the core protein, therefore we detected the cleavage of caspase-3 using Western blotting analysis. The level of cleaved caspase-3 increased significantly in a time-dependent manner. To further understand the apoptotic pathway mediated by Mn in 16HBE cells, we detected the intrinsic and the extrinsic pathway-related proteins. It is possible that caspase-9-dependent pathway plays the primary role in the execution of Mn-induced 16HBE cell apoptosis, since caspase-8 cleavage was not detected. In this study, although we did not evaluate any other factors responsible for mitochondrial apoptosis, we demonstrated caspase-9 activation and the significant apoptosis prevention induced by its specific inhibitor (data not shown). These results suggest that Mn-induced apoptosis in 16HBE cells occurs via caspase-9-dependent intrinsic pathway. Mitochondria are the central regulators for intrinsic apoptotic pathways by integrating diverse pro- and antiapoptotic signals. Apoptotic signals initiate the opening of the outer mitochondrial membrane and the release of caspase-activating molecules such as cytochrome c into the cytosol.^22–25 Following Mn treatment, a significant time-dependent increase in the formation of ROS, loss of ΔΨ_m and release of cytochrome c to the cytosol were observed. All these results supported our assumption that the mitochondrial pathway takes part in MnCl₂-induced apoptosis in 16HBE cells.

The mitochondrial apoptotic pathway participates in MYC-mediated apoptosis. A great deal of evidence has demonstrated that c-Myc can alter the balance of pro- and antiapoptotic members of the Bcl-2 family, in parallel with or independent of p53, influencing the intrinsic apoptotic pathway.^26–30 Overexpression of c-Myc sensitizes cells to apoptosis by a variety of stimuli.³¹ ^,32 In some cancer cell lines, downregulation of c-Myc is critical for compound-induced apoptosis.³³ ^,34 Our results suggest that c-Myc decrease took part in MnCl₂-induced mitochondrial apoptosis in 16HBE cells. The degradation of c-Myc is through the ubiquitin–proteasome pathway.³⁵ ^,36 We investigated the effect of specific proteasome inhibitor MG132 on c-Myc in Mn-induced apoptosis. Western blotting analysis showed that abundance of c-Myc in the cells treated with MnCl₂ plus MG132 were obviously increased, compared with the cells treated with MnCl₂ alone. The results indicate that in MnCl₂-induced apoptosis, the decrease in c-Myc level is caused by the increase of its degradation through the ubiquitin–proteasome pathway. The results of flow cytometry showed that the percentage of apoptotic cells induced by Mn was decreased by MG132, which further indicated that the restoration of c-Myc protein by MG132 could prevent Mn-induced apoptosis.

The p53 tumor suppressor is a major mediator of cell cycle arrest and/or apoptosis in the response of mammalian cells to stress stimuli. p53 activation is accompanied by downregulation of c-Myc expression.³⁰ Clearly, the level of p53 and c-Myc should determine whether p21 is induced or suppressed and whether cells undergo apoptosis or growth arrest. In the current study, the increased expression of WAF1/p21 was observed in Mn-treated cells at both 12 and 24 h. Although p21 is a p53 transcription target implicated in both major functions of tumor suppressor cell cycle arrest and apoptosis, the mechanism by which p21 can regulate the apoptotic pathway is not well understood. Prabhu et al. and Lincet et al. demonstrated the antiapoptotic effect of p21, whereas Shibata et al. showed the proapoptotic effect.^37
–39 Thus, the influence and activity of p21 on the apoptosis signal cascade may depend on the cell type or cell conditions. However, in lung injury, p21 acts as an antiapoptotic factor protecting lung from oxidative stress.⁴⁰ In our study, although p21 expression was also induced in 16HBE cells treated with Mn, it failed to prevent the cells from undergoing apoptosis. This observation also suggests that the mechanism mediating the apoptotic effect of 0.375 mM MnCl₂ on 16HBE cells was in this instance not regulated by p21 but by another, unidentified, factor(s) that was defective in the cells.

This article provides evidence that MnCl₂ induced apoptosis in 16HBE cells associated with the release of cytochrome c from the intermembrane space of mitochondria into the cytosol, the activation of caspase-9, c-Myc decrease and the upregulation of p53 and p-p53 expression. The c-Myc decrease might be due to increased degradation through the ubiquitin–proteasome pathway. This will be helpful for designing novel molecular intervention targets in Mn-induced lung disease.

Footnotes

Funding

This study was supported by grants from the Nature Science Foundation of China (no. 81001252), Ministry of Education of China (no. 20100072120053) and International Science &Technology Cooperation Program of China (no. 2011DFB30010). Fundamental Research Funds for the Central Universities (Study on the key technology of disaster warning and relief for rural remote areas).

Conflict of interest

The authors declared no conflicts of interest.

References

Holley

Bakthavatchalu

Velez-Roman

St Clair

. Manganese superoxide dismutase: guardian of the powerhouse. Int J Mol Sci 2011; 12: 7114–7162.

Alaimo

Gorojod

Kotler

. The extrinsic and intrinsic apoptotic pathways are involved in manganese toxicity in rat astrocytoma C6 cells. Neurochem Int 2011; 59: 297–308.

Antonini

Sriram

Benkovic

Roberts

Stone

Chen

Mild steel welding fume causes manganese accumulation and subtle neuroinflammatory changes but not overt neuronal damage in discrete brain regions of rats after short-term inhalation exposure. Neurotoxicology 2009; 30: 915–925.

Corradi

Mutti

. Metal ions affecting the pulmonary and cardiovascular systems. Met Ions Life Sci 2011; 8: 81–105.

Tamm

Sabri

Ceccatelli

. Mitochondrial-mediated apoptosis in neural stem cells exposed to manganese. Toxicol Sci 2008; 101: 310–320.

Pascal

Tessier

. Cytotoxicity of chromium and manganese to lung epithelial cells in vitro. Toxicol Lett 2004; 2: 143–151.

Bredow

Falgout

March

Yingling

Malkoski

Aden

Subchronic inhalation of soluble manganese induces expression of hypoxia-associated angiogenic genes in adult mouse lungs. Toxicol Appl Pharmacol 2007; 221: 148–157.

Zhao

Zhong

Ying

Yuan

Zhou

. Manganese chloride-induced G0/G1 and S phase arrest in A549 cells. Toxicology 2008; 250: 39–46.

Martin

Haqimoto

Nakamura

Matute-Bello

. Apoptosis and epithelia injury in the lungs. Proc Am Thorac Soc 2005; 2: 214–220.

10.

de Souza

Lindsay

. Apoptosis as a therapeutic target for the treatment of lung disease. Curr Opin Pharmacol 2005; 5: 232–237.

11.

Gunter

Gavin

Gunter

. The case for manganese interaction with mitochondria. Neurotoxicology 2009; 30: 727–729.

12.

Kalia

Zheng

Jiang

. Importance of mitochondria in manganese-induced cellular toxicity. Neurotoxicology 2009; 30: 727.

13.

Suzuki

Tsukamoto

. Manganese-induced apoptosis in hepatocytes after partial hepatectomy. Eur J Pharmacol 2005; 525: 48–55.

14.

Gonzalez

Juknat

Venosa

Verrengia

Kotler

. Manganese activates the mitochondrial apoptotic pathway in rat astrocytes by modulating the expression of proteins of the Bcl-2 family. Neurochem Int 2008; 53: 408–415.

15.

Oubrahim

Stadtman

Chock

. Mitochondria play no roles in Mn(II)-induced apoptosis in HeLa cells. Proc Natl Acad Sci USA 2001; 98: 9505–9510.

16.

Han

Lee

Choi

Lee

Hong

Choi

Manganese (II) induces chemical hypoxia by inhibiting HIF-prolyl hydroxylase: implication in manganese-induced pulmonary inflammation. Toxicol Appl Pharmacol 2009; 235: 261–267.

17.

Camner

Curstedt

Jarstrand

Johannsson

Robertson

Wiernik

. Rabbit lung after inhalation of manganese chloride: a comparison with the effects of chlorides of nickel, cadmium, cobalt, and copper. Environ Res 1985; 38: 301–309.

18.

Ulrich

Rinehart

Brandt

Evaluation of the chronic inhalation toxicity of a manganese oxide aerosol. III. Pulmonary function, electromyograms, limb tremor, and tissue manganese data. Am Ind Hyg Assoc J 1979; 40: 349–353.

19.

Han

Lee

Choi

Lee

Hong

Choi

Manganese (II) induces chemical hypoxia by inhibiting HIF-prolyl hydroxylase: implication in manganese-induced pulmonary inflammation. Toxicol Appl Pharmacol 2009; 235: 261–267.

20.

Rice

Clarke

Godleski

AI-Mutairi

Jiang

Hauser

Differential ability of transition metals to induce pulmonary inflammation. Toxicol Appl Pharmacol 2001; 177: 46–53.

21.

Catassi

Cesario

Arzani

Menichini

Alama

Bruzzo

Characterization of apoptosis induced by marine natural products in non small cell lung cancer A549 cells. Cell Mol Life Sci 2006; 63: 2377–2386.

22.

Nasirudeen

Wang

Liu

. Induction of p53-dependent and mitochondria-mediated cell death pathway by dengue virus infection of human and animal cells. Microbes Infect 2008; 10: 1124–1132.

23.

Green

Hao

Mak

. Cytochrome c: functions beyond respiration. Nat Rev Mol Cell Biol 2008; 9: 532–542.

24.

Zhao

Inagaki

Wang

Cinobufacini, an aqueous extract from Bufo bufo gargarizans Cantor, induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells. J Ethnopharmacol 2010; 128: 654–661.

25.

Suen

Norris

Youle

. Mitochondrial dynamics and apoptosis. Genes Dev 2008; 22: 1577–1590.

26.

Albihn

Johnsen

Henriksson

. MYC in oncogenesis and as a target for cancer therapies. Adv Cancer Res 2010; 107: 162–224.

27.

Dang

O'donnell

Juopperi

. The great MYC escape in tumorigenesis. Cancer Cell 2005; 8: 177–178.

28.

Maclean

Dorsey

Cleveland

Kastan

. Targeting lysosomal degradation induces p53-dependent cell death and prevents cancer in mouse models of lymphomagenesis. J Clin Invest 2008; 118: 79–88.

29.

Sachdeva

Zhu

Walia

Kumar

P53 represses c-Myc through induction of the tumor suppressor miR-145. Proc Natl Acad Sci USA 2009; 106: 3207–3212.

30.

Sakamuro

Eviner

Elliott

Showe

White

Prendergast

GC.

c-Myc induces apoptosis in epithelial cells by both p53-dependent and p53-independent mechanisms. Oncogene 1995; 11: 2411–2418.

31.

Hoffman

Liebermann

. Apoptotic signaling by c-MYC. Oncogene 2008; 27: 6462–6472.

32.

Klefstrom

Verschuren

Evan

c-Myc augments the apoptotic activity of cytosolic death receptor signaling proteins by engaging the mitochondrial apoptotic pathway. J Biol Chem 2002; 277: 43224–43232.

33.

Chow

Liu

Lin

Lee

Chengg

Lin

Downregulation of c-Myc determines sensitivity to 2-methoxyestradiol-induced apoptosis in human acute myeloid leukemia. Exp Hematol 2008; 36: 140–148.

34.

Russo

Arzani

Trombino

Falugi

c-Myc down-regulation induces apoptosis in human cancer cell lines exposed to RPR-115135 (C₃₁H₂₉NO₄), a non-peptidomimetic farnesyltransferase inhibitor. J Pharmacol Exp Ther 2003; 304: 37–47.

35.

Gregory

Hann

SR.

c-Myc proteolysis by the ubiquitin–proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells. Mol Cell Biol 2000; 20: 2423–2435.

36.

Kluz

Sun

Costa

. Mechanisms of c-Myc degradation by nickel compounds and hypoxia. PLoS One 2009; 4: e8531.

37.

Prabhu

Somasundaram

Tian

Enders

Satyamoorthy

Herlyn

The administration schedule of cyclin-dependent kinase inhibitor gene therapy and etoposide chemotherapy is a major determinant of cytotoxicity. Int J Oncol 1999; 1: 209–216.

38.

Shibata

Yoshidome

Shibata

Jorcyk

Green

. Suppression of mammary carcinoma growth in vitro and in vivo by inducible expression of the CDK inhibitor p21. Cancer Gene Ther 2001; 8: 23–35.

39.

Lincet

Poulain

Remy

Deslandes

Duigou

Gauduchon

The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells. Cancer Lett 2000; 161: 17–26.

40.

Helt

Rancourt

Staversky

O'Reilly

. P53-dependent induction of p21(Cip1/WAF1/Sdi1) protects against oxygen-induced toxicity. Toxicol Sci 2001; 63: 214–222.

Manganese activates caspase-9-dependent apoptosis in human bronchial epithelial cells

Abstract

Keywords

Introduction

Materials and methods

Cell culture and materials

Cytotoxicity assay

Apoptosis assay

Measurement of reactive oxygen species generation

Measurement of mitochondrial membrane potential

Western blot

Statistical analysis

Results

Inhibition of MnCl2 on 16HBE cell proliferation

MnCl2 induces apoptosis in 16HBE cells

Mitochondrial dysfunction triggered by MnCl2

The activation of caspase-9, c-Myc decrease, p53, p-p53 and p21 increase are involved in MnCl2-induced apoptosis in 16HBE cells

Discussion

Footnotes

Funding

Conflict of interest

References

Inhibition of MnCl₂ on 16HBE cell proliferation

MnCl₂ induces apoptosis in 16HBE cells

Mitochondrial dysfunction triggered by MnCl₂

The activation of caspase-9, c-Myc decrease, p53, p-p53 and p21 increase are involved in MnCl₂-induced apoptosis in 16HBE cells