RH1 Induces Cellular Damage in an NAD(P)H:Quinone Oxidoreductase 1-Dependent Manner: Relationship between DNA Cross-linking, Cell Cycle Perturbations, and Apoptosis

Abstract

Structure-based development of NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones resulted in development of RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], a methyl-substituted diaziridinyl quinone. We conducted experiments to evaluate the mechanism of RH1-induced cytotoxicity and the inter-relationship between DNA cross-linking, cell cycle changes, and apoptosis using an isogenic cell line pair developed from the human breast cancer cell line MDA-MB-468 differing only in expression of wtNQO1 (NQ16 cells). Statistically significant DNA cross-linking was detected using a modified comet assay in cells with wtNQO1 within 1 h of dosing, whereas in parental cells, only marginal DNA cross-linking was observed and required a concentration up to 50 times higher. Cross-linking in NQ16 cells could be abrogated with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanismbased inhibitor of NQO1. RH1 prolonged S phase and caused a G 2 /M block. Cell cycle changes were observed up to 10-fold lower in RH1 concentrations in NQ16 cells relative to parental cells. Apoptosis was similarly observed morphologically in both cell lines after RH1 treatment but was induced preferentially in NQ16 cells at lower concentrations and earlier time points. Marked cleavage of caspase-3 was observed in NQ16 cells relative to parental cells using lower concentrations of RH1. Temporally, low doses of RH1-induced rapid DNA cross-linking in NQ16 cells followed by induction of apoptosis at times when a G 2 /M block was not observed. This suggests that cell cycle arrest is not required for RH1-induced apoptosis and that DNA damage may directly initiate apoptotic events. In summary, RH1-induced preferential DNA cross-linking, cell cycle changes, and apoptosis in an NQO1-dependent manner.The bioactivation of antitumor quinones by the two-electron reductase NQO1 has attracted considerable interest after it was found that NQO1 was expressed at high levels throughout many human solid tumors (Cresteil and Jaiswal, 1991;Malkinson et al., 1992;Mikami et al., 1998). The structure-based development of NQO1-directed antitumor quinones led to the identification and testing of a number of compounds, including the aziridinylbenzoquinone RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], which is currently in clinical trials in the UK. Upon twoelectron reduction of the quinone by NQO1, the two aziridinyl functional groups undergo facile protonation, leading to aziridine ring opening. The resulting bifunctional alkylating species can covalently bind to DNA (forming inter-and intrastrand cross-links) and other cellular nucleophiles (Hargreaves et al., 1999). It is presumed that the cytotoxicity of RH1 is related to its ability to cross-link DNA (Ward et al., 2000) after activation by NQO1 because it does not appreciably redox cycle , nor does one-electron reduction appear to contribute significantly to the cytotoxicity of RH1. Kim et al. (2004b) did not find a difference in RH1 toxicit...

Induction of Caspase-9, Biochemical Assessment and Morphological Changes Caused by Apoptosis in Cancer Cells Treated with Goniothalamin Extracted from Goniothalamus macrophyllus

“…Furthermore, we indicate that GTN induced DNA damage in more than 90% cells after 2h in low dose IC 10 and 100% in high dose (IC 25 ) however from MTT only about 25% of these cells died after 72 h. This is further supported by the proliferation assay which shows that the cells were still able to divide and proliferate in the same concentration of goniothalamin. This result was similar to a study where the anti-tumor quinone RH1 caused DNA damage in more than 80% of NQ16 breast cancer cells but only about 50% of the cells succumbed to cell death 48h later (Dehn et al, 2005) and similar to another study of the effect on GTN on VSMCs (Chan et al, 2006).…”

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

“…***Significant difference between si-cont RNA-and si-JNK2 RNA-transfected cells after RH1 treatment; P < 0.05. ᭣ cell death (Dehn et al, 2005). Although we show here that RH1 induced caspase activation and PARP cleavage, the apoptotic cell death caused by RH1 appeared to be caspaseindependent, because caspase inhibitors failed to attenuate the process.…”

“…Therefore, it was likely that not only NQO1 status, but also p53 expression, affects the sensitivity of cells towards RH1. Furthermore, as MDA-MB-468 cells overexpressing NQO1 could be killed by nanomolar doses of RH1 (Dehn et al, 2005), unlike what was seen in NQO1…”

“…In detail, this report suggested that the interaction between JNK and gH2AX during mitosis positively facilitate cell death signals. Furthermore, because RH1 has been already reported to induce DNA cross-linking, G2/M arrest and subsequent cell death in an NQO1-dependent manner (Dehn et al, 2005), RH1 is likely to cause mitotic catastrophe through the activation of JNK. Further investigation is in progress in our laboratory to shed light on the role of JNK in RH1-induced mitotic catastrophe.…”

Preclinical efficacy of the bioreductive alkylating agent RH1 against paediatric tumours

“…RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone) was identified through its very high affinity for DTD (Beall et al, 1995;Winski et al, 1998). Reduction of RH1 facilitates the formation of GCC sequence-specific DNA interstrand cross-linked adducts (Berardini et al, 1993), and the induction of apoptosis within 6 h of exposure (Dehn et al, 2005). Diphtheria toxin-diaphorase activity and expression correlate with RH1-induced cytotoxicity in colon cancer and non-small-cell lung cancer cell lines in vitro (Winski et al, 1998;Sharp et al, 2000).…”