The anti-tumour compound, RH1, causes mitochondria-mediated apoptosis by activating c-Jun N-terminal kinase

Background and purpose: 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.

Experimental approach: 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.

Key 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 and implications: 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone activated JNK, resulting in mitochondria-mediated apoptotic cell death that was NQO1-dependent.