The anti-tumour compound, RH1, causes mitochondria-mediated apoptosis by activating c-Jun N-terminal kinase.
OBJECTIVE
2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although the cytotoxic efficacy of RH1 against tumours has been studied extensively, the molecular mechanisms underlying this anti-cancer activity have not yet been fully elucidated.


METHODS
2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone-induced apoptosis and related signalling pathways in NQO1-negative and NQO1-overexpressing cells were evaluated. The role of p53 in RH1-induced cell death was investigated using parental and p53-deficient RKO human colorectal cancer cells by assaying clonogenic cell survival. Specific inhibitors and siRNAs targeting factors involved in RH1-induced apoptosis were used to clarify the roles played by such factors in RH1-activated apoptotic signalling pathways.


RESULTS
2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone induced apoptosis and clonogenic death, dependent on NQO1 and p53. Treatment of NQO1-overexpressing cells with RH1 caused rapid disruption of mitochondrial membrane potential, nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNA targeting AIF and Endo G effectively attenuated RH1-induced apoptotic cell death. Moreover, RH1 induced cleavage of Bax, which targets mitochondria. RH1 significantly activated the c-Jun N-terminal kinase (JNK) pathway, and inhibition of this pathway suppressed RH1-induced mitochondria-mediated apoptosis. RH1-induced generation and mitochondrial translocation of cleaved Bax were blocked by the JNK inhibitor, SP600125. Inhibition of JNK with SP600125 attenuated the mitochondrial translocation of JNK.


CONCLUSIONS
2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone activated JNK, resulting in mitochondria-mediated apoptotic cell death that was NQO1-dependent.
Department of Microbiology, Center for Advanced Medical Education by BK21 Project, College of Medicine, Inha University, Incheon, Korea.
Moon-Taek
MT
Park
Min-Jeong
MJ
Song
Eun-Taex
ET
Oh
Hyemi
H
Lee
Bo-Hwa
BH
Choi
Seong-Yun
SY
Jeong
Eun-Kyung
EK
Choi
Heon Joo
HJ
Park
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D001388:Aziridines / Q000494:pharmacology*; D016227:Benzoquinones / Q000494:pharmacology*; D020169:Caspases / Q000502:physiology; D045744:Cell Line, Tumor; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000502:physiology*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000502:physiology; D021381:Protein Transport; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C117776:2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone; D000970:Antineoplastic Agents; D051033:Apoptosis Inducing Factor; D001388:Aziridines; C494826:BAX protein, human; D016227:Benzoquinones; D051028:bcl-2-Associated X Protein; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D048031:JNK Mitogen-Activated Protein Kinases; D004706:Endodeoxyribonucleases; C052391:endonuclease G; D020169:Caspases
Cancer Lett. 2002 Aug 8;182(1):83-92
12175527
J Biol Chem. 2003 Dec 12;278(50):50624-34
14522966
J Biol Chem. 2000 Jan 7;275(1):322-7
10617621
J Cell Physiol. 2006 Nov;209(2):457-67
16894574
Nature. 1998 Jan 1;391(6662):96-9
9422513
J Cell Biochem. 2010 Jun 1;110(3):725-31
20512932
Blood. 2003 Oct 1;102(7):2605-14
12816867
J Biol Chem. 2005 Apr 15;280(15):14716-22
15689616
J Pharmacol Exp Ther. 2005 May;313(2):771-9
15665137
J Mol Biol. 2010 Mar 5;396(4):858-69
20070945
Blood. 2005 Feb 15;105(4):1724-33
15486061
J Cell Physiol. 2008 Nov;217(2):423-32
18543249
J Biol Chem. 2003 May 16;278(20):17593-6
12665525
J Biol Chem. 2003 Aug 22;278(34):32448-56
12794087
Hum Mutat. 2002 Jun;19(6):607-14
12007217
Cell. 2000 Dec 8;103(6):839-42
11136969
Biochem J. 2003 Jun 1;372(Pt 2):359-69
12614194
Cancer Chemother Pharmacol. 2007 Nov;60(6):851-61
17333193
Int J Radiat Oncol Biol Phys. 2004 Feb 1;58(2):376-85
14751506
Cell. 1997 Nov 14;91(4):479-89
9390557
Biochim Biophys Acta. 1965 Oct 11;108(2):275-84
5865510
J Biol Chem. 2007 Sep 28;282(39):28960-28970
17686764
Mol Biol Cell. 2000 Nov;11(11):3751-63
11071904
J Biol Chem. 2002 Mar 22;277(12):10244-50
11786558
DNA Repair (Amst). 2009 Jun 4;8(6):704-19
19272840
Mol Cell. 2003 Mar;11(3):577-90
12667443
Oncogene. 2004 Apr 12;23(16):2838-49
15077147
Anticancer Drugs. 2004 Jan;15(1):71-7
15090746
J Biol Chem. 2000 Dec 15;275(50):39435-43
10993883
Transplantation. 2008 May 27;85(10):1500-4
18497693
J Interferon Cytokine Res. 2000 Dec;20(12):1121-9
11152579
Nature. 2000 Oct 12;407(6805):770-6
11048727
J Biol Chem. 2001 Aug 24;276(34):32353-61
11418614
Mol Cell Biol. 2004 Mar;24(6):2499-512
14993287
Oncogene. 2001 Jan 11;20(2):147-55
11313944
Proc Natl Acad Sci U S A. 1980 Sep;77(9):5216-20
6933553
Nature. 2001 Mar 29;410(6828):549-54
11279485
J Cell Sci. 2003 Oct 15;116(Pt 20):4077-85
12972501
J Cell Biol. 2001 May 28;153(5):999-1010
11381085
Autophagy. 2009 Jan;5(1):103-5
19115484
Cell. 1993 Sep 24;74(6):957-67
8402885
J Cell Biochem. 2006 Mar 1;97(4):724-34
16229013
Proc Natl Acad Sci U S A. 2001 Jan 30;98(3):1188-93
11158615
Biochem Biophys Res Commun. 2003 May 23;305(1):176-85
12732214
Cancer Res. 2002 Jul 1;62(13):3615-9
12097262
Science. 1995 Nov 24;270(5240):1326-31
7481820
J Biol Chem. 2000 Sep 29;275(39):29980-5
10913135
Cancer Lett. 2003 Jan 28;189(2):221-30
12490315
FEBS Lett. 2000 Jul 7;476(3):118-23
10913597
J Biol Chem. 2001 Jun 29;276(26):23341-8
11319218
Anticancer Res. 2003 Sep-Oct;23(5A):3979-83
14666706
J Cell Physiol. 2008 Oct;217(1):23-33
18412143
Clin Cancer Res. 1998 Dec;4(12):3083-8
9865924
Nature. 2000 Oct 12;407(6805):810-6
11048733
Cancer Treat Rev. 2004 Aug;30(5):437-49
15245776
Apoptosis. 2008 Sep;13(9):1111-20
18651223
Clin Cancer Res. 2005 Apr 1;11(7):2695-701
15814651
Br J Cancer. 2009 Jul 7;101(1):55-63
19491903
Mutat Res. 2004 Nov 2;555(1-2):149-71
15476858