Effects of cytokine-induced macrophages on the response of tumor cells to banoxantrone (AQ4N)

Abstract

Tumor-associated macrophages (TAMs) are found in many solid tumors and have often been shown to accumulate in the hypoxic regions surrounding areas of necrosis. TAMs are the major site of expression of nitric oxide synthase (NOS), a heme-containing homodimeric enzyme consisting of oxygenase and reductase domains. The latter has a high degree of sequence homology to cytochrome P450 reductase and a functional consequence of this is the ability of NOS, under hypoxic conditions, to activate the bioreductive drugs tirapazamine and RSU1069. Banoxantrone (AQ4N) is a bioreductive prodrug activated in hypoxia by an oxygen-dependent two-electron reductive process to yield the topoisomerase II inhibitor AQ4. A feature of this process is that the final product could potentially show bystander cell killing. Thus, in this study, we investigated the ability of inducible NOS (iNOS)-expressing TAMs to activate AQ4N and elicit toxicity in cocultured human tumor cells. Murine macrophages were induced to overexpress iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumor cells, and the toxicity of AQ4N was determined under aerobic or hypoxic conditions. The aerobic toxicity of AQ4N toward tumor cells was not affected through coculturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated with an increase in AQ4N metabolism and a substantial increase in tumor cell toxicity, which was dependent on the proportion of macrophages in the culture. This study is the first demonstration of TAM-mediated prodrug activation to result in bystander killing of human tumor cells.

In vivo activation of the hypoxia-targeted cytotoxin AQ4N in human tumor xenografts

“…A number of studies have shown that cytochrome P450 enzymes (CYP) and other haem-containing reductases, suchas nitric oxide synthase, contribute to the bioreduction of AQ4N under anoxic conditions (16)(17)(18). The ability of S9 or microsome fractions to activate AQ4N to AQ4 ex vivo has been shown in human liver microsomes (16), activated macrophages (18), and murine tumors (19).…”

“…The ability of S9 or microsome fractions to activate AQ4N to AQ4 ex vivo has been shown in human liver microsomes (16), activated macrophages (18), and murine tumors (19). Recent work has shown that AQ4N can be activated in vivo in tumor xenograft samples (3,20), and in clinical samples from a phase I study (12).…”

Impact of Tumor Blood Flow Modulation on Tumor Sensitivity to the Bioreductive Drug Banoxantrone

“…BxPC-3 cells are intrinsically sensitive to the active metabolite AQ4 in vitro, and they respond favorably to AQ4N when grown as solid tumors in vivo (Lalani et al, 2007), indicating that AQ4N activation is not deficient in BxPC-3 tumors in vivo, in contrast to our findings in culture. Conceivably, enzymes active in AQ4N bioactivation, such as cytochromes P450 2S1 and 2W1 (Nishida et al, 2010), might be more highly expressed in the context of the tumor environment in vivo; alternatively, AQ4N activation might occur in tumorassociated host cells such as macrophages, which have the capacity to activate AQ4N by iNOS (Mehibel et al, 2009). Conversely, whereas cultured 9L cells responded well to AQ4N, the 9L tumor xenografts were largely unresponsive (Figs.…”

“…Certain cytochrome P450 enzymes (McErlane et al, 2005;Nishida et al, 2010;Xiao et al, 2011) and iNOS (Fitzpatrick et al, 2008;Nishida and Ortiz de Montellano, 2008;Mehibel et al, 2009) can activate AQ4N via a two-step reduction of the di-N-oxide to the di-amino compound AQ4. Other pathways of reductive metabolism demonstrated for related quinones include one-electron reduction to a semiquinone radical and two-electron reduction to a hydroquinone, both of which can be cytotoxic (Nguyen and Gutierrez, 1990;Ross et al, 1996).…”

“…AQ4 intercalates into DNA and inhibits topoisomerase II; moreover, it can passively diffuse into neighboring cells and exert bystander cytotoxicity (Patterson, 2002). AQ4N has a bis-N-oxide quinone structure with the potential to be reduced under hypoxic conditions by several cellular enzymes, including cytochrome P450 (McCarthy et al, 2003;Nishida et al, 2010) and inducible nitric oxide synthase (iNOS) (Nishida and Ortiz de Montellano, 2008;Mehibel et al, 2009). AQ4N is considered an ideal bioreductive drug in that it penetrates deep into tumor tissue (Tredan et al, 2009).…”