PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis

  • Authors:
    • Bo Ra You
    • Hye Rim Shin
    • Woo Hyun Park
  • View Affiliations

  • Published online on: October 29, 2013     https://doi.org/10.3892/ijo.2013.2152
  • Pages: 301-308
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Abstract

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

Introduction

Thioredoxin (Trx) is a small redox protein (12 kDa) with two redox-active cysteine residues (C32 and C35) in the active site (1). There are two main isoforms of Trx in mammalian cells; the cytoplasmic form, Trx-1 and the mitochondrial form, Trx-2 (2). These Trxs are reduced by thioredoxin reductase and NADPH following the reduction of oxidative target proteins (3,4). Trx affects cell growth and proliferation via regulating the redox status in cells (5). Trx-1 is also implicated in cell survival, tumor development and angiogenesis (6,7). Numerous studies demonstrated that overexpression of Trx is observed in many cancers such as gastric and breast cancers (5,8). Therefore, Trx-1 inhibitors have been considered as novel anticancer drugs. In particular, PX-12 (1-methylpropyl 2-imidazolyl disulfide) is an irreversible Trx-1 inhibitor, which has an anti-tumor effect (9). PX-12 decreased the activity of Trx-1 by thioalkylating the critical cysteine residue (Cys73) in this protein or by increasing the dimerization of its oxidative form. In addition, PX-12 downregulates the expression of vascular endothelial growth factor via decreasing hypoxia-inducible factor-1α, consequently inhibiting metastasis of cancer cells (10,11). Recently, PX-12 has been clinically tested in colorectal and pancreatic cancers (12,13).

Lung cancer is a major cause of cancer death in the developed countries. Various novel therapeutic strategies are still currently under consideration since the clinical use of cytotoxic drugs is limited due to intrinsic or acquired resistant and toxicity (14). An increase in Trx-1 level is detected in lung cancer patients compared to the control group (15). In addition, it is reported that the high level of Trx-1 contributes to chemo-resistance in lung cancer cells (16). However, little is known about the cellular effect of PX-12 in lung cancer. Therefore, in the present study we investigated the effects of PX-12 on cell growth and death in human lung cancer A549 cells with respect to reactive oxygen species (ROS) and glutathione (GSH) levels.

Materials and methods

Cell culture

Human lung adenocarcinoma A549 cells from the American Type Culture Collection (ATCC, Manassas, VA, USA) were maintained in a humidified incubator containing 5% CO2 at 37°C. A549 cells were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin–streptomycin (Gibco BRL, Grand Island, NY, USA). Cells were routinely grown in 100-mm plastic tissue culture dishes (Nunc. Roskilde, Denmark) and harvested with a solution of trypsin-EDTA (Gibco BRL) while in a logarithmic phase of growth.

Reagents

PX-12 was purchased from Tocris Bioscience (Bristol, UK) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at 100 mM as a stock solution. The pan-caspase inhibitor (Z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), caspase-3 inhibitor (Z-DEVD-FMK; benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), caspase-8 inhibitor (Z-IETD-FMK; benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone) and caspase-9 inhibitor (Z-LEHDFMK; benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylke-tone) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA) and were dissolved in DMSO at 10 mM to serve as stock solutions. NAC was dissolved in the buffer [20 mM HEPES (pH 7.0)]. Based on the previous studies (17,18), cells were pretreated with 15 μM caspase inhibitors or 2 mM NAC for 1 h prior to treatment with PX-12. DMSO (0.03%) was used as a control vehicle and it did not affect cell growth or death.

Growth inhibition assay

The effect of PX-12 on cell growth was determined by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) absorbance in living cells as described previously (19). In brief, 1×104 cells per well were seeded in 96-well microtiter plates (Nunc). Following exposure to the designated doses of PX-12 for the indicated times, MTT solution [20 μl: 2 mg/ml in phosphate-buffered saline (PBS)] was added to each well of the 96-well plates. The plates were additionally incubated for 3 h at 37°C. Medium was withdrawn from the plates by pipetting and 200 μl DMSO was added to each well to solubilize the formazan crystals. The optical density was measured at 570 nm using a microplate reader (Synergy 2, BioTek Instruments Inc., Winooski, VT, USA).

Western blot analysis

The expression of proteins was evaluated using western blot analysis, as previously described (20). In brief, 1×106 cells in a 60-mm culture dish (Nunc) were incubated with the designated doses of PX-12 for 72 h. The cells were then washed in PBS and suspended in 5 Vol of lysis buffer (20 mM HEPES. pH 7.9, 20% glycerol, 200 mM KCl, 0.5 mM EDTA, 0.5% NP40, 0.5 mM DTT, 1% protease inhibitor cocktail). Supernatant protein concentrations were determined using the Bradford method. Supernatant samples containing 30 μg total protein were resolved by 15% SDS-PAGE gels depending on the size of target proteins, transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting and then probed with anti-PARP, anti-c-PARP, anti-Bcl-2, anti-Bax (Cell Signaling Technology Inc., Danvers, MA, USA), anti-Trx-1 and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an ECL kit (Amersham, Arlington Heights, IL, USA).

Measurement of Trx-1 activity

The Trx-1 activity was assessed using the ProteoStat Thioredoxin-1 assay kit according to the manufacturer’s instructions (EnZo Life Science, Plymouth Meeting, PA, USA). In brief, 1×106 cells in 60-mm culture dish (Nunc) were incubated with the indicated doses of PX-12 for 72 h. The cells were then washed in PBS and suspended in 5 Vol of lysis buffer (R&D Systems, Inc.). Protein concentrations were determined using the Bradford method. Supernatant samples containing 20 μg of total protein were used for determination of Trx-1 activity, and added to each well in 96-well microtiter plates (Nunc) with the insulin and DTT at 25°C for 30 min. The fluorescence intensity of each well was determined using a fluorescence reader (Synergy 2).

Cell cycle and sub-G1 cell analysis

Cell cycle and sub-G1 cell analysis were determined by propidium iodide (PI, Ex/Em=488/617 nm; Sigma-Aldrich) staining as described previously (20). In brief, 1×106 cells in 60-mm culture dish (Nunc) were incubated with the designated doses of PX-12 for 72 h. Total cells including floating cells were then washed with PBS and fixed in 70% (v/v) ethanol. Cells were washed again with PBS, then incubated with PI (10 μg/ml) with simultaneous RNase treatment at 37°C for 30 min. Cellular DNA content was measured using a FACStar flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and analyzed by using lysis II and cellfit software (Becton-Dickinson).

Annexin V-FITC/PI staining for cell death detection

Apoptotic cell death was determined by staining cells with Annexin V-fluorescein isothiocyanate (FITC, Invitrogen Life Technologies, Camarillo, CA, USA; Ex/Em=488/519 nm) as described previously (21). In brief, 1×106 cells in 60-mm culture dish (Nunc) were incubated with the designated doses of PX-12 for 72 h with or without 15 μM each caspase inhibitor or 2 mM NAC. Cells were washed twice with cold PBS and then resuspended in 500 μl binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1×106 cells/ml. Annexin V-FITC (5 μl) and PI (1 μg/ml) were then added and the cells were analyzed with a FACStar flow cytometer.

Measurement of the mitochondrial membrane potential (MMP; ΔΨm)

The MMP (ΔΨm) levels were measured by a rhodamine 123 fluorescent dye (Sigma-Aldrich; Ex/Em=485/535 nm) as described previously (21,22). In brief, 1×106 cells in 60-mm culture dish (Nunc) were incubated with the designated doses of PX-12 for 72 h with or without 15 μM each caspase inhibitor or 2 mM NAC. Cells were washed twice with PBS and incubated with rhodamine 123 (0.1 μg/ml) at 37°C for 30 min. Rhodamine 123 staining intensity was determined by a FACStar flow cytometer. The cells that were rhodamine 123-negative were indicated to have lost MMP (ΔΨm).

Transfection of cells with Bcl-2 and Bax siRNAs

Gene silencing of Bax and Bcl-2 was performed using a siRNA knockdown system. A non-specific control siRNA duplex [5′-CCUACGCC ACCAAUUUCGU(dTdT)-3′], Bcl-2 siRNA duplex [5′-CAGA AGUCUGGGAAUCGAU(dTdT)-3′] and Bax siRNA duplex [5′-GCUGGACAUUGGACUUCCU(dTdT)-3′] were purchased from the Bioneer Corp. (Daejeon, Korea). In brief, 2.5×105 cells in 6-well plates (Nunc) were incubated in RPMI-1640 supplemented with 10% FBS. Following 12 h, cells (∼30–40% confluence) in each well were transfected with the control, Bax or Bcl-2 siRNA [80 pmol in Opti-MEM (Gibco BRL)] using Lipofectamine 2000, according to the manufacturer’s instructions (Invitrogen, Branford, CT, USA). One day later, cells were treated with or without 20 μM PX-12 for additional 48 h. The transfected cells were collected and used for western blot analysis, sub-G1 cells, Annexin V-FITC staining and MMP (ΔΨm) level measurements.

Detection of intracellular ROS levels

Intracellular ROS were detected by means of an oxidation-sensitive fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (Ex/Em=495/529 nm; Invitrogen Life Technologies) and dihydroethidium (DHE, Ex/Em=518/605 nm; Invitrogen Life Technologies) as previously described (21,23). DHE is highly selective for O2•- among ROS. In brief, 1×106 cells in 60-mm culture dish (Nunc) were incubated with the designated doses of PX-12 for indicated times with or without 15 μM each caspase inhibitor or 2 mM NAC. Cells were then washed in PBS and incubated with 20 μM H2DCFDA or DHE at 37°C for 30 min. H2DCFDA or DHE fluorescence was assessed using a FACStar flow cytometer. ROS and O2•- levels were expressed as mean fluorescence intensity, which was calculated by CellQuest software.

Detection of intracellular GSH

Cellular GSH levels were analyzed using a 5-chloromethylfluorescein diacetate dye (CMFDA, Ex/Em=522/595 nm; Invitrogen Life Technologies) as previously described (23,24). In brief, 1×106 cells in 60-mm culture dish (Nunc) were incubated with the designated doses of PX-12 for 72 h with or without 15 μM each caspase inhibitor or 2 mM NAC. Cells were then washed with PBS and incubated with 5 μM CMFDA at 37°C for 30 min. CMF fluorescence intensity was determined using a FACStar flow cytometer. Negative CMF staining (GSH-depletion) of cells is expressed as the percentage of (-) CMF cells.

Statistical analysis

Results represent the mean of at least three independent experiments (mean ± SD). Data were analyzed using Instat software (GraphPad Prism4, San Diego, CA, USA). The Student’s t-test or one-way analysis of variance with post hoc analysis using Tukey’s multiple comparison test was used for parametric data. The statistical significance was defined as p<0.05.

Results

Effects of PX-12 on the growth of A549 cells

We first examined the effect of PX-12 on the growth of A549 cells using MTT assays. After exposure to PX-12 for 24, 48 and 72 h, the growth of A549 cells dose-dependently decreased with an IC50 of ∼20 μM at 48 and 72 h (Fig. 1A). In addition, it was observed that PX-12 as a Trx-1 inhibitor decreased the activity of Trx-1 in A549 cell lysate (Fig. 1B). Moreover, PX-12 decreased the level of Trx-1 expression in A549 cells at 72 h (Fig. 1B).

Effects of PX-12 on cell cycle distributions, cell death and MMP (ΔΨm) in A549 cells

Because the growth inhibition of A549 cells caused by PX-12 can be explained by an arrest during the cell cycle progression, cell cycle distributions were examined at 72 h. As shown in Fig. 2A, DNA flow cytometric analysis indicated that 5-20 μM PX-12 significantly induced a G2/M phase arrest of the cell cycle in A549 cells. In addition, PX-12 increased the percentages of sub-G1 cells in A549 cells in a dose-dependent manner at 72 h (Fig. 2B). This agent also increased the numbers of Annexin V-FITC-positive cells in A549 cells (Fig. 2C). The intact of poly(ADP-ribose) polymerase (PARP) and Bcl-2 levels were decreased by PX-12 (Fig. 2C). However, PX-12 increased Bax level in A549 cells at 72 h (Fig. 2C). Cell death is closely related to the collapse of the MMP (ΔΨm) (25). As expected, the loss of MMP (ΔΨm) was detected in PX-12 treated A549 cells (Fig. 2D).

Effect of Bcl-2 and Bax siRNAs on cell death and MMP (ΔΨm) in PX-12-treated A549 cells

To investigate the effects of Bcl-2 and Bax on A549 cell death, A549 cells were transfected with either non-target control siRNA, Bcl-2 or Bax siRNA. As shown in Fig. 3A, the expression of Bcl-2 and Bax was clearly downregulated by the administration with each siRNA as compared with cells transfected with control siRNA. While Bax siRNA significantly prevented A549 cell death induced by PX-12, Bcl-2 siRNA slightly increased cell death in PX-12-treated A549 cells (Fig. 3B and C). In addition, Bax siRNA significantly attenuated the loss of MMP (ΔΨm) in PX-12-treated A549 cells (Fig. 3D). However, Bcl-2 siRNA did not significantly affect the loss of MMP (ΔΨm) caused by PX-12 (Fig. 3D).

Effects of PX-12 on the intracellular ROS and GSH levels in A549 cells

The changes in intracellular ROS and GSH levels were investigated in A549 cells treated with PX-12 at 72 h. As shown in Fig. 4A, PX-12 increased the intracellular ROS (DCF) levels in A549 cells at 72 h. Moreover, red fluorescence derived from DHE reflecting the intracellular O2•- levels significantly increased in PX-12-treated A549 cells at 72 h (Fig. 4B). When intracellular GSH levels were measured in PX-12-treated A549 cells using a CMFDA dye, PX-12 significantly increased GSH-depleted cell number in A549 cells (Fig. 4C).

Effects of caspase inhibitors on cell death, MMP (ΔΨm), ROS and GSH levels in PX-12-treated A549 cells

Which caspase was involved in A549 cell death induced by PX-12, was determined. For this experiment, we chose 20 μM PX-12 as a suitable dose to differentiate the levels of cell death in the presence or absence of each caspase inhibitor. Based on a previous study (17), A549 cells were pretreated with 15 μM of each caspase inhibitor for 1 h prior to treatment with PX-12. This dose did not significantly affect cell death in the control A549 cells (Fig. 5A). Treatment with all the tested caspase inhibitors (Z-VAD for pan-caspases, Z-DEVD for caspase-3, Z-IETD for caspase-8 and Z-LEHD for caspase-9), especially Z-VAD showed significant rescue of A549 cells from PX-12-induced apoptosis at 72 h, as measured by the population of Annexin V-FITC-positive cells (Fig. 5A). Furthermore, all the caspase inhibitors significantly prevented the loss of MMP (ΔΨm) caused by PX-12 (Fig. 5B).

Whether the intracellular ROS and GSH levels in PX-12-treated A549 cells were changed by treatment with each caspase inhibitor was investigated. As shown in Fig. 5C, all the caspase inhibitors marginally reduced ROS levels in PX-12-treated A549 cells. Moreover, these caspase inhibitors, especially Z-DEVD and Z-LEHD prevented GSH depletion in these cells (Fig. 5D).

Effects of NAC on cell death and MMP (ΔΨm), ROS and GSH levels in PX-12-treated A549 cells

Next, the effects of NAC on cell death and MMP (ΔΨm) in 20 μM PX-12-treated A549 cells were assessed at 72 h. As shown in Fig. 6A and B, NAC significantly decreased the percentages of sub-G1 cells and Annexin V-FITC-positive cells in PX-12-treated A549 cells. With respect to MMP (ΔΨm), NAC significantly attenuated the loss of MMP (ΔΨm) caused by PX-12 (Fig. 6C).

Furthermore, it was determined whether the intracellular ROS and GSH levels in PX-12-treated A549 cells were changed by treatment with NAC. NAC markedly decreased ROS levels including O2•- in PX-12-treated A549 cells at 72 h (Fig. 6D and E). In relation to GSH levels, NAC markedly prevented GSH depletion caused by PX-12 in A549 cells (Fig. 6F).

Discussion

We investigated the effects of PX-12 in A549 lung cancer cells on cell growth and death in relation to ROS and GSH level. It was observed that PX-12 decreased the activity of Trx-1 in A549 cell lysate, indicating that PX-12 directly inhibited the activity of Trx-1 in this lysate. In addition, the level of Trx-1 expression was downregulated in PX12-treated A549 cells. Since the tight binding of PX-12 to Trx-1 leads to compositional changes in Trx-1 protein, it is possible that Trx-1 can be degraded via a ubiquitination system. However, the activity of Trx-1 was not reduced in PX-12-treated A549 cells at 72 h. These results suggest that the activity of Trx-1 does not closely correspond with its level and the different effects of PX-12 on the activity of Trx-1 in cell lysate and cells are probably derived from the different functional bioavailability of this drug.

PX-12 inhibited the growth of A549 cells time- and dose-dependently. PX-12 significantly induced a G2/M phase arrest of the cell cycle in A549 cells. Similarly, it has been found that PX-12 induces a G2/M phase arrest in cancer cells such as breast cancer, B-cell lymphoma (1,26) and Calu-6 lung cancer cells (unpublished data). Therefore, the G2/M phase arrest in PX-12-treated cancer cells was an underlying mechanism to suppress cell growth and prolifeation. PX-12 also induced apoptosis in A549 cells and this drug strongly triggered the loss of MMP (ΔΨm). These results supported that apoptosis is closely related to the collapse of MMP (ΔΨm) (27).

A high ratio of Bax to Bcl-2 has been known to be a main trigger in the collapse of MMP (ΔΨm) and apoptosis in cells (28). Likewise, the levels of Bcl-2 and Bax were downregulated and upregulated in PX-12-treated A549 cells, respectively. Moreover, the administration of Bax siRNA prevented A549 cell death caused by PX-12 whereas that of Bcl-2 siRNA did not strongly affect apoptosis and MMP (ΔΨm) in PX-12 treated A549 cells. Therefore, PX-12 seemed to induce apoptosis in A549 cells depending on the upregulation of Bax protein. When determined which caspases were involved in apoptosis in PX-12-treated A549 cells, all the tested caspase inhibitors, especially Z-VAD prevented PX-12-induced A549 cell death. These data demonstrated that mitochondrial intrinsic pathway as well as death receptor extrinsic pathway together are necessary for the complete induction of apoptosis in PX-12-treated A549 cells. In addition, all the caspase inhibitors markedly attenuated the loss of MMP (ΔΨm) caused by PX-12. These results suggest that the loss of MMP (ΔΨm) induced by PX-12 is a crucial step to fully induce apoptosis in A549 cells.

PX-12 as an inhibitor of Trx-1 can affect the status of redox in cells. It is reported that PX-12 induces oxidative stress (29). Likewise, the intracellular ROS levels including O2•- significantly increased in PX-12-treated A549 cells at 72 h. All caspase inhibitors showing anti-apoptotic effects attenuated ROS levels in PX-12-treated A549 cells. Furthermore, NAC markedly prevented apoptotic cell death and the loss of MMP (ΔΨm) in PX-12-treated A549 cells, accompanied by decreasing ROS levels including O2•- in these cells. Taken together, these results suggest that PX-12-induced cell death is mediated by oxidative stress. GSH is an important intracellular antioxidant that protects cells from damage caused by free radicals, peroxides and toxins. It is able to clear away O2•- and provide electrons for glutathione peroxidase to reduce H2O2 to H2O. Apoptotic effects are inversely comparative to GSH content (30,31). Similarly, PX-12 increased the percentages of GSH-depleted cells in A549 cells at 72 h. NAC and caspase inhibitors markedly prevented the depletion of GSH in PX-12-treated A549 cells. However, BSO, which is an inhibitor of GSH synthesis, did not affect apoptotic cell death in A549 cells (data not shown). Therefore, the loss of GSH content seemed to be necessary but not sufficient to induce apoptosis in PX-12-treated A549 cells.

In conclusion, depicted in Fig. 7, it is the first report that PX-12 inhibited the growth of A549 lung cancer cells via G2/M phase arrest, and Bax-mediated and ROS-dependent apoptosis.

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2008-0062279) and supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013006279).

References

1. 

Li C, Thompson MA, Tamayo AT, et al: Over-expression of Thioredoxin-1 mediates growth, survival, and chemoresistance and is a druggable target in diffuse large B-cell lymphoma. Oncotarget. 3:314–326. 2012.PubMed/NCBI

2. 

Yang J, Li C, Ding L, Guo Q, You Q and Jin S: Gambogic acid deactivates cytosolic and mitochondrial thioredoxins by covalent binding to the functional domain. J Nat Prod. 75:1108–1116. 2012. View Article : Google Scholar : PubMed/NCBI

3. 

Chae JS, Gil Hwang S, Lim DS and Choi EJ: Thioredoxin-1 functions as a molecular switch regulating the oxidative stress-induced activation of MST1. Free Radic Biol Med. 53:2335–2343. 2012. View Article : Google Scholar : PubMed/NCBI

4. 

Ungerstedt J, Du Y, Zhang H, Nair D and Holmgren A: In vivo redox state of human thioredoxin and redox shift by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Free Radic Biol Med. 53:2002–2007. 2012. View Article : Google Scholar : PubMed/NCBI

5. 

Lim JY, Yoon SO, Hong SW, Kim JW, Choi SH and Cho JY: Thioredoxin and thioredoxin-interacting protein as prognostic markers for gastric cancer recurrence. World J Gastroenterol. 18:5581–5588. 2012. View Article : Google Scholar : PubMed/NCBI

6. 

Pramanik KC and Srivastava SK: Apoptosis signal-regulating kinase 1-thioredoxin complex dissociation by capsaicin causes pancreatic tumor growth suppression by inducing apoptosis. Antioxid Redox Signal. 17:1417–1432. 2012. View Article : Google Scholar

7. 

Dunn LL, Buckle AM, Cooke JP and Ng MK: The emerging role of the thioredoxin system in angiogenesis. Arterioscler Thromb Vasc Biol. 30:2089–2098. 2010. View Article : Google Scholar : PubMed/NCBI

8. 

Cha MK, Suh KH and Kim IH: Overexpression of peroxiredoxin I and thioredoxin 1 in human breast carcinoma. J Exp Clin Cancer Res. 28:932009. View Article : Google Scholar : PubMed/NCBI

9. 

Wondrak GT: Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal. 11:3013–3069. 2009. View Article : Google Scholar : PubMed/NCBI

10. 

Welsh SJ, Williams RR, Birmingham A, Newman DJ, Kirkpatrick DL and Powis G: The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit hypoxia-induced factor 1alpha and vascular endothelial growth factor formation. Mol Cancer Ther. 2:235–243. 2003.

11. 

Mukherjee A and Martin SG: The thioredoxin system: a key target in tumour and endothelial cells. Br J Radiol. 81(Spec1): S57–S68. 2008. View Article : Google Scholar : PubMed/NCBI

12. 

Baker AF, Adab KN, Raghunand N, et al: A phase IB trial of 24-hour intravenous PX-12, a thioredoxin-1 inhibitor, in patients with advanced gastrointestinal cancers. Invest New Drugs. 31:631–641. 2013. View Article : Google Scholar : PubMed/NCBI

13. 

Ramanathan RK, Kirkpatrick DL, Belani CP, et al: A Phase I pharmacokinetic and pharmacodynamic study of PX-12, a novel inhibitor of thioredoxin-1, in patients with advanced solid tumors. Clin Cancer Res. 13:2109–2114. 2007. View Article : Google Scholar : PubMed/NCBI

14. 

Petty RD, Nicolson MC, Kerr KM, Collie-Duguid E and Murray GI: Gene expression profiling in non-small cell lung cancer: from molecular mechanisms to clinical application. Clin Cancer Res. 10:3237–3248. 2004. View Article : Google Scholar : PubMed/NCBI

15. 

Fernandes AP, Capitanio A, Selenius M, Brodin O, Rundlof AK and Bjornstedt M: Expression profiles of thioredoxin family proteins in human lung cancer tissue: correlation with proliferation and differentiation. Histopathology. 55:313–320. 2009. View Article : Google Scholar : PubMed/NCBI

16. 

Wangpaichitr M, Sullivan EJ, Theodoropoulos G, et al: The relationship of thioredoxin-1 and cisplatin resistance: its impact on ROS and oxidative metabolism in lung cancer cells. Mol Cancer Ther. 11:604–615. 2012. View Article : Google Scholar : PubMed/NCBI

17. 

Han YH, Kim SZ, Kim SH and Park WH: Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis. Chem Biol Interact. 177:107–114. 2009. View Article : Google Scholar : PubMed/NCBI

18. 

Han YH and Park WH: The effects of N-acetyl cysteine, buthionine sulfoximine, diethyldithiocarbamate or 3-amino-1,2,4-triazole on antimycin A-treated Calu-6 lung cells in relation to cell growth, reactive oxygen species and glutathione. Oncol Rep. 22:385–391. 2009.

19. 

Han YH, Moon HJ, You BR, Kim SZ, Kim SH and Park WH: Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells. Toxicology. 265:101–107. 2009. View Article : Google Scholar : PubMed/NCBI

20. 

You BR and Park WH: Zebularine inhibits the growth of HeLa cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis. Mol Biol Rep. 39:9723–9731. 2012. View Article : Google Scholar : PubMed/NCBI

21. 

Han YH, Moon HJ, You BR and Park WH: The effect of MG132, a proteasome inhibitor on HeLa cells in relation to cell growth, reactive oxygen species and GSH. Oncol Rep. 22:215–221. 2009.PubMed/NCBI

22. 

Han YH, Kim SH, Kim SZ and Park WH: Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) as an O2(*-) generator induces apoptosis via the depletion of intracellular GSH contents in Calu-6 cells. Lung Cancer. 63:201–209. 2009. View Article : Google Scholar : PubMed/NCBI

23. 

Han YH and Park WH: Propyl gallate inhibits the growth of HeLa cells via regulating intracellular GSH level. Food Chem Toxicol. 47:2531–2538. 2009. View Article : Google Scholar : PubMed/NCBI

24. 

You BR and Park WH: Gallic acid-induced lung cancer cell death is related to glutathione depletion as well as reactive oxygen species increase. Toxicol In Vitro. 24:1356–1362. 2010. View Article : Google Scholar : PubMed/NCBI

25. 

Griffiths EJ: Mitochondria - potential role in cell life and death. Cardiovasc Res. 46:24–27. 2000. View Article : Google Scholar : PubMed/NCBI

26. 

Vogt A, Tamura K, Watson S and Lazo JS: Antitumor imidazolyl disulfide IV-2 causes irreversible G(2)/M cell cycle arrest without hyperphosphorylation of cyclin-dependent kinase Cdk1. J Pharmacol Exp Ther. 294:1070–1075. 2000.PubMed/NCBI

27. 

Yang J, Liu X, Bhalla K, et al: Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 275:1129–1132. 1997. View Article : Google Scholar : PubMed/NCBI

28. 

Martinou JC and Youle RJ: Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell. 21:92–101. 2011. View Article : Google Scholar : PubMed/NCBI

29. 

Lee YJ, Kim JH, Chen J and Song JJ: Enhancement of metabolic oxidative stress-induced cytotoxicity by the thioredoxin inhibitor 1-methylpropyl 2-imidazolyl disulfide is mediated through the ASK1-SEK1-JNK1 pathway. Mol Pharmacol. 62:1409–1417. 2002. View Article : Google Scholar

30. 

Estrela JM, Ortega A and Obrador E: Glutathione in cancer biology and therapy. Crit Rev Clin Lab Sci. 43:143–181. 2006. View Article : Google Scholar

31. 

You BR and Park WH: Arsenic trioxide induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion. Oncol Rep. 28:749–757. 2012.PubMed/NCBI

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2014-January
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You BR, Shin HR and Park WH: PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis. Int J Oncol 44: 301-308, 2014
APA
You, B.R., Shin, H.R., & Park, W.H. (2014). PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis. International Journal of Oncology, 44, 301-308. https://doi.org/10.3892/ijo.2013.2152
MLA
You, B. R., Shin, H. R., Park, W. H."PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis". International Journal of Oncology 44.1 (2014): 301-308.
Chicago
You, B. R., Shin, H. R., Park, W. H."PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis". International Journal of Oncology 44, no. 1 (2014): 301-308. https://doi.org/10.3892/ijo.2013.2152