2-Aminophenoxazine-3-one-induced apoptosis via generation of reactive oxygen species followed by c-jun N-terminal kinase activation in the human glioblastoma cell line LN229

Che,Xiao-Fang; Moriya,Shota; Zheng,Chun-Lei; Abe,Akihisa; Tomoda,Akio; Miyazawa,Keisuke

doi:10.3892/ijo.2013.2088

2-Aminophenoxazine-3-one-induced apoptosis via generation of reactive oxygen species followed by c-jun N-terminal kinase activation in the human glioblastoma cell line LN229

Authors:
- Xiao-Fang Che
- Shota Moriya
- Chun-Lei Zheng
- Akihisa Abe
- Akio Tomoda
- Keisuke Miyazawa
View Affiliations

Affiliations: Department of Biochemistry, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402, Japan, Department of Medical Oncology, Cancer Hospital, Fudan University, Shanghai 200032, P.R. China
Published online on: September 4, 2013 https://doi.org/10.3892/ijo.2013.2088
Pages: 1456-1466

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

2-Aminophenoxazine-3-one (Phx-3) induces apoptosis in several types of cancer cell lines. However, the mechanism of apoptosis induction by Phx-3 has not been fully elucidated. In this study, we investigated the anticancer effects of Phx-3 in the glioblastoma cell line LN229 and analyzed its molecular mechanism. The results indicated that 6- and 20-h treatment with Phx-3 significantly induced apoptosis in LN229 cells, with downregulation of survivin and XIAP. Both ERK and JNK, which are the members of the MAPK family, were activated after treatment with Phx-3. Inhibition of ERK using the specific inhibitor U0126 blocked the Phx-3-induced apoptosis only in part. However, inhibition of JNK using the specific inhibitor SP600125 completely prevented Phx-3-induced apoptosis and restored the phosphorylation states of ERK to the control levels. Enhanced generation of reactive oxygen species (ROS) was detected after 3-h treatment with Phx-3. In addition, the ROS scavenger melatonin almost completely blocked Phx-3-induced JNK activation and apoptosis. This suggests that JNK activation was mediated by Phx-3-induced ROS generation. Although SP600125 and melatonin completely blocked the reduction of mitochondrial membrane potential after a 3-h treatment with Phx-3, extension of Phx-3 exposure time to 20 h resulted in no cancelation of mitochondrial depolarization by these reagents. These reagents also had little effect on the decreased expression of survivin and XIAP during a 3-20-h exposure to Phx-3. These results indicate that the production of ROS following JNK activation is the main axis of Phx-3-induced apoptosis in LN229 cells for short-term exposure to Phx-3, whereas alternative mechanism(s) appear to be involved in apoptosis induction during long-term exposure to Phx-3.

View Figures

View References

1.	Bidros DS and Vogelbaum MA: Novel drug delivery strategies in neuro-oncology. Neurotherapeutics. 6:539–546. 2009. View Article : Google Scholar : PubMed/NCBI
2.	Tomoda A, Arai S, Ishida R, Shimamoto T and Ohyashiki K: An improved method for the rapid preparation of 2-amino-4,4·-di-hydro-4,7-dimethyl-3Hphenoxazine-3-one, a novel antitumor agent. Bioorg Med Chem Lett. 11:1057–1058. 2001.PubMed/NCBI
3.	Shimizu S, Suzuki M, Tomoda A, Arai S, Taguchi H, Hanawa T and Kamiya S: Phenoxazine compounds produced by the reactions with bovine hemoglobin show antimicrobial activity against non-tuberculosis mycobacteria. Tohoku J Exp Med. 203:47–52. 2004. View Article : Google Scholar
4.	Shimamoto T, Tomoda A, Ishida R and Ohyashiki K: Antitumor effects of a novel phenoxazine derivative on human leukemia cell lines in vitro and in vivo. Clin Cancer Res. 7:704–708. 2001.PubMed/NCBI
5.	Shirato K, Imaizumi K, Miyazawa K, Takasaki A, Mizuguchi J, Che XF, Akiyama S and Tomoda A: Apoptosis induction preceded by mitochondrial depolarization in multiple myeloma cell line U266 by 2-aminophenoxazine-3-one. Biol Pharm Bull. 31:62–67. 2008. View Article : Google Scholar : PubMed/NCBI
6.	Shirato K, Imaizumi K, Abe A and Tomoda A: Phenoxazine derivatives induce caspase-independent cell death in human glioblastoma cell lines, A-172 and U-251MG. Oncol Rep. 17:201–208. 2007.PubMed/NCBI
7.	Miyano-Kurosaki N, Kurosaki K, Hayaashi M, Takaku H, Hayafune M, Shrato K, Kasuga T, Endo T and Tomoda A: 2-Aminophenoxazine-3-one suppresses the growth of mouse malignant melanoma B16 cells transplanted into C57BL/6Cr Slc mice. Biol Pharm Bull. 29:2197–2201. 2006. View Article : Google Scholar : PubMed/NCBI
8.	Che XF, Zheng CL, Akiyama S and Tomoda A: 2-Amino phenoxazine-3-one and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one cause cellular apoptosis by reducing higher intracellular pH in cancer cells. Proc Jpn Acad Ser B Phys Biol Sci. 87:199–213. 2011.
9.	Takasaki A, Hanyu H, Iwamoto T, Shirato K, Izumi R, Toyota H, Izuguchi J, Miyazawa K and Tomoda A: Mitochondrial depolarization and apoptosis associated with sustained activation of c-jun-N-terminal kinasein the human multiple myeloma cell line U266 induced by 2-aminophenoxazine-3-one. Mol Med Rep. 2:199–203. 2009.PubMed/NCBI
10.	Zheng CL, Che XF, Akiyama S, Miyazawa K and Tomoda A: 2-Aminophenoxazine-3-one induces cellular apoptosis by causing rapid intracellular acidification and generating reactive oxygen species in human lung adenocarcinoma cells. Int J Oncol. 36:641–650. 2010.
11.	Azuine MA, Tokuda H, Takayasu J, Enjyo F, Mukainaka T, Konoshima T, Nishino H and Kapadia GJ: Cancer chemopreventive effect of phenothiazines and related tri-heterocyclic analogues in the 12-O-tetradecanoylphorbol-13-acetate promoted Epstein-Barr virus early antigen activation and the mouse skin two-stage carcinogenesis models. Pharmacol Res. 49:161–169. 2004. View Article : Google Scholar
12.	Nakada M, Kita D, Watanabe T, Hayashi Y, Teng L, Pyko IV and Hamada J: Aberrant signaling pathways in glioma. Cancers. 3:3242–3278. 2011. View Article : Google Scholar : PubMed/NCBI
13.	Soni D, King JA, Kaye AH and Hovens CM: Genetics of glioblastoma multiforme: mitogenic signaling and cell cycle pathways converge. J Clin Neurosci. 12:1–5. 2005. View Article : Google Scholar : PubMed/NCBI
14.	Los M, Maddika S, Erb B and Schulze-Osthoff K: Switching Akt: from survival signaling to deadly response. Bioessays. 31:492–495. 2009. View Article : Google Scholar : PubMed/NCBI
15.	Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K and Cobb MH: Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 22:153–183. 2001.PubMed/NCBI
16.	Davis RJ: Signal transduction by the JNK group of MAP kinases. Cell. 103:239–252. 2000. View Article : Google Scholar : PubMed/NCBI
17.	Nakachi T, Tabuchi T, Takasaki A, Arai S, Miyazawa K and Tomoda A: Anticancer activity of phenoxazines produced by bovine erythrocytes on colon cancer cells. Oncol Rep. 23:1517–1522. 2010.PubMed/NCBI
18.	Kawaguchi T, Miyazawa K, Moriya S, Ohtomo T, Che XF, Naito M, Itoh M and Tomoda A: Combined treatment with bortezomib plus bafilomycin A1 enhances the cytocidal effect and induces endoplasmic reticulum stress in U266 myeloma cells: crosstalk among proteasome, autophagy-lysosome and ER stress. Int J Oncol. 38:643–654. 2011.
19.	Nohl H, Gille L and Staniek K: Intracellular generation of reactive oxygen species by mitochondria. Biochem Pharmacol. 69:719–723. 2005. View Article : Google Scholar : PubMed/NCBI
20.	Gogvadze V, Orrenius S and Zhivotovsky B: Mitochondria as targets for cancer chemotherapy. Semin Cancer Biol. 19:57–66. 2009. View Article : Google Scholar : PubMed/NCBI
21.	Maundrell K, Antonsson B, Magnenat E, Camps M, Muda M, Chabert C, Gillieron C, Boschert U, Vial-Knecht E, Martinou JC and Arkinstall S: Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1. J Biol Chem. 272:25238–25242. 1997. View Article : Google Scholar
22.	Basu A and Haldar S: Identification of a novel Bcl-xL phospho rylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis. FEBS Lett. 538:41–47. 2003. View Article : Google Scholar : PubMed/NCBI
23.	Fulda S, Gorman AM, Hori O and Samali A: Cellular stress responses: cell survival and cell death. Int J Cell Biol. 2010:2140742010. View Article : Google Scholar : PubMed/NCBI
24.	Trachootham D, Lu W, Ogasawara MA, Valle NR and Huang P: Redox regulation of cell survival. Antioxid Redox Signal. 10:1343–1374. 2008. View Article : Google Scholar : PubMed/NCBI
25.	Feligioni M, Brambilla E, Camassa A, Sclip A, Arnaboldi A, Morelli F, Antoniou X and Borsello T: Crosstalk between JNK and SUMO signaling pathways: deSUMOylation is protective against H₂O₂-induced cell injury. PLoS One. 6:e281852011. View Article : Google Scholar : PubMed/NCBI
26.	Conde de la Rosa L, Schoemaker MH, Vrenken TE, Buist-Homan M, Havinga R, Jansen PL and Moshage H: Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases. J Hepatol. 44:918–929. 2006.PubMed/NCBI
27.	Matos TJ, Duarte CB, Goncalo M and Lopes MC: Role of oxidative stress in ERK and p38 MAPK activation induced by the chemical sensitizer DNFB in a fetal skin dendritic cell line. Immunol Cell Biol. 83:607–614. 2005. View Article : Google Scholar : PubMed/NCBI
28.	Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, Takeda K, Minowa O, Miyazono K, Noda T and Ichijo H: ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep. 2:222–228. 2001. View Article : Google Scholar : PubMed/NCBI
29.	Song JJ, Rhee JG, Suntharalingam M, Walsh SA, Spitz DR and Lee YJ: Role of glutaredoxin in metabolic oxidative stress: glutaredoxin as a sensor of oxidative stress mediated by H₂O₂. J Biol Chem. 277:46566–46575. 2002. View Article : Google Scholar : PubMed/NCBI
30.	Yoshizumi M, Abe J, Haendeler J, Huang Q and Berk BC: Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem. 275:11706–11712. 2000. View Article : Google Scholar : PubMed/NCBI
31.	Wang T, Arifoglu P, Ronai Z and Tew KD: Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus. J Biol Chem. 276:20999–21003. 2001. View Article : Google Scholar : PubMed/NCBI
32.	Sinha D, Bannergee S, Schwartz JH, Lieberthal W and Levine JS: Inhibition of ligand-independent ERK1/2 activity in kidney proximal tubular cells deprived of soluble survival factors upregulates Akt and prevents apoptosis. J Biol Chem. 279:10962–10972. 2004. View Article : Google Scholar