Abstract
PX-12 (1-methylpropyl 2-imidazolyl disulfide) as a thioredoxin (Trx) inhibitor has an anti-tumor effect. However, there is no report about the toxicological effect of PX-12 on lung cancer cells. Here, we investigated the anti-growth effects of PX-12 on Calu-6 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. PX-12 induced the growth inhibition of Calu-6 cells with IC50 of nearly 3 μM at 72 h. In contrast, PX-12 did not affect the growth of human small airway epithelial cells (HSAECs). Cell cycle distribution analysis indicated that PX-12 significantly induced a G2/M phase arrest in Calu-6 cells. PX-12 also increased the number of annexin V-FITC-positive cells in Calu-6 cells. All the tested caspase inhibitors markedly prevented Calu-6 cell death induced by PX-12. With regard to ROS and GSH levels, PX-12 increased ROS levels containing O2 ·− in Calu-6 cells and induced the depletion of GSH. N-acetyl cysteine (NAC), which is a well-known antioxidant, significantly reduced O2 ·− level in PX-12-treated Calu-6 cells and prevented apoptosis and GSH depletion in these cells. In conclusion, it is the first report that PX-12 inhibited the growth of Calu-6 cells via a G2/M phase arrest as well as apoptosis, which effect was related to the intracellular increases in ROS levels.
Similar content being viewed by others
Abbreviations
- ROS:
-
Reactive oxygen species
- Trx:
-
Thioredoxin
- GSH:
-
Glutathione
- Z-VAD-FMK:
-
Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
- Z-DEVD-FMK:
-
Benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone
- Z-IETD-FMK:
-
Benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone
- Z-LEHD-FMK:
-
Benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone
- NAC:
-
N-Acetyl cysteine
- MMP (ΔΨm):
-
Mitochondrial membrane potential
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- FITC:
-
Fluorescein isothiocyanate
- PI:
-
Propidium iodide
- H2DCFDA:
-
2′,7′-dichlorodihydrofluorescein diacetate
- DHE:
-
Dihydroethidium
- CMFDA:
-
5-Chloromethylfluorescein diacetate
References
Shi Y, Tang B, Yu PW, Hao YX, Lei X, Luo HX, et al. Autophagy protects against oxaliplatin-induced cell death via ER stress and ROS in Caco-2 cells. PLoS One. 2012;7:e51076.
Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta. 2006;1757:509–17.
Gonzalez C, Sanz-Alfayate G, Agapito MT, Gomez-Nino A, Rocher A, Obeso A. Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia. Respir Phys Neurobiol. 2002;132:17–41.
Baran CP, Zeigler MM, Tridandapani S, Marsh CB. The role of ROS and RNS in regulating life and death of blood monocytes. Curr Pharm Des. 2004;10:855–66.
Lo YL, Wang W, Ho CT. 7,3′,4′-trihydroxyisoflavone modulates multidrug resistance transporters and induces apoptosis via production of reactive oxygen species. Toxicology. 2012;302:221–32.
Bell EL, Emerling BM, Ricoult SJ, Guarente L. SirT3 suppresses hypoxia inducible factor 1alpha and tumor growth by inhibiting mitochondrial ROS production. Oncogene. 2011;30:2986–96.
Li C, Thompson MA, Tamayo AT, Zuo Z, Lee J, Vega F, et al. Over-expression of thioredoxin-1 mediates growth, survival, and chemoresistance and is a druggable target in diffuse large b-cell lymphoma. Oncotarget. 2012;3:314–26.
Yang J, Li C, Ding L, Guo Q, You Q, ** S. Gambogic acid deactivates cytosolic and mitochondrial thioredoxins by covalent binding to the functional domain. J Nat Prod. 2012;75:1108–16.
Chae JS, Gil Hwang S, Lim DS, Choi EJ. Thioredoxin-1 functions as a molecular switch regulating the oxidative stress-induced activation of MST1. Free Radic Biol Med. 2012;53:2335–43.
Ungerstedt J, Du Y, Zhang H, Nair D, 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. 2012;53:2002–7.
Lim JY, Yoon SO, Hong SW, Kim JW, Choi SH, Cho JY. Thioredoxin and thioredoxin-interacting protein as prognostic markers for gastric cancer recurrence. World J Gastroenterol: WJG. 2012;18:5581–8.
Pramanik KC, Srivastava SK. Apoptosis signal-regulating kinase 1-thioredoxin complex dissociation by capsaicin causes pancreatic tumor growth suppression by inducing apoptosis. Antioxid Redox Signal. 2012;17:1417–32.
Dunn LL, Buckle AM, Cooke JP, Ng MK. The emerging role of the thioredoxin system in angiogenesis. Arterioscler Thromb Vasc Biol. 2010;30:2089–98.
Cha MK, Suh KH, Kim IH. Overexpression of peroxiredoxin i and thioredoxin1 in human breast carcinoma. J Exp Clin Cancer Res: CR. 2009;28:93.
Wondrak GT. Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal. 2009;11:3013–69.
Welsh SJ, Williams RR, Birmingham A, Newman DJ, Kirkpatrick DL, 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. 2003;2:235–43.
Mukherjee A, Martin SG. The thioredoxin system: a key target in tumour and endothelial cells. Br J Radiol. 2008;81(Spec No 1):S57–68.
Baker AF, Adab KN, Raghunand N, Chow HH, Stratton SP, Squire SW, Boice M, Pestano LA, Kirkpatrick DL, Dragovich T. A phase ib trial of 24-hour intravenous px-12, a thioredoxin-1 inhibitor, in patients with advanced gastrointestinal cancers. Investig New Drugs. 2012.
Ramanathan RK, Kirkpatrick DL, Belani CP, Friedland D, Green SB, Chow HH, 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: Off J Am Assoc Cancer Res. 2007;13:2109–14.
Petty RD, Nicolson MC, Kerr KM, Collie-Duguid E, Murray GI. Gene expression profiling in non-small cell lung cancer: from molecular mechanisms to clinical application. Clin Cancer Res: Off J Am Assoc Cancer Res. 2004;10:3237–48.
Fernandes AP, Capitanio A, Selenius M, Brodin O, Rundlof AK, Bjornstedt M. Expression profiles of thioredoxin family proteins in human lung cancer tissue: correlation with proliferation and differentiation. Histopathology. 2009;55:313–20.
Wangpaichitr M, Sullivan EJ, Theodoropoulos G, Wu C, You M, Feun LG, et al. The relationship of thioredoxin-1 and cisplatin resistance: its impact on ros and oxidative metabolism in lung cancer cells. Mol Cancer Ther. 2012;11:604–15.
Han YH, Kim SZ, Kim SH, Park WH. Pyrogallol inhibits the growth of lung cancer calu-6 cells via caspase-dependent apoptosis. Chem Biol Interact. 2009;177:107–14.
Han YH, 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. 2009;22:385–91.
Han YH, Moon HJ, You BR, Kim SZ, Kim SH, Park WH. Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells. Toxicology. 2009;265:101–7.
You BR, Park WH. Zebularine inhibits the growth of hela cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis. Mol Biol Rep. 2012;39:9723–31.
Harlow E, Lane D: Bradford assay. CSH Protoc. 2006;2006.
Han YH, Moon HJ, You BR, Park WH. The effect of mg132, a proteasome inhibitor on hela cells in relation to cell growth, reactive oxygen species and gsh. Oncol Rep. 2009;22:215–21.
Han YH, Kim SH, Kim SZ, 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. 2009;63:201–9.
Han YH, Park WH. Propyl gallate inhibits the growth of hela cells via regulating intracellular GSH level. Food Chem Toxicol: Int J Publ Br Ind Biol Res Assoc. 2009;47:2531–8.
You BR, Park WH. Gallic acid-induced lung cancer cell death is related to glutathione depletion as well as reactive oxygen species increase. Toxicol In Vitro: Int J Publ Assoc BIBRA. 2010;24:1356–62.
Griffiths EJ. Mitochondria—potential role in cell life and death. Cardiovasc Res. 2000;46:24–7.
Testa B, Kramer SD. The biochemistry of drug metabolism—an introduction: Part 4. Reactions of conjugation and their enzymes. Chem Biodivers. 2008;5:2171–336.
Vogt A, Tamura K, Watson S, 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. 2000;294:1070–5.
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, et al. Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997;275:1129–32.
Lee YJ, Kim JH, Chen J, 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. 2002;62:1409–17.
Estrela JM, Ortega A, Obrador E. Glutathione in cancer biology and therapy. Crit Rev Clin Lab Sci. 2006;43:143–81.
You BR, Park WH. Arsenic trioxide induces human pulmonary fibroblast cell death via increasing ros levels and GSH depletion. Oncol Rep. 2012;28:749–57.
Acknowledgments
This work 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). This paper was supported by research funds of Chonbuk National University in 2014.
Conflicts of interest
None
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
You, B.R., Shin, H.R., Han, B.R. et al. PX-12 induces apoptosis in Calu-6 cells in an oxidative stress-dependent manner. Tumor Biol. 36, 2087–2095 (2015). https://doi.org/10.1007/s13277-014-2816-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13277-014-2816-x