RÉSUMÉ
Acetaminophen (AAP) is metabolized by a variety of pathways such as sulfation, glucuronidation, and fatty acid amide hydrolase-mediated conversion to the active analgesic metabolite AM404. CYP2E1-mediated metabolism to the hepatotoxic reactive metabolite NAPQI (N-acetyl-p-benzoquinone imine) is a minor metabolic pathway that has not been linked to AAP therapeutic benefits yet clearly leads to AAP liver toxicity. N-acetylcysteine (NAC) (an antioxidant) and fomepizole (a CYP2E1 inhibitor) are clinically used for the treatment of AAP toxicity. Mice treated with AAP in combination with fomepizole (plus or minus NAC) were assessed for liver toxicity by histology and serum chemistry. The anticancer activity of AAP with NAC and fomepizole rescue was assessed in vitro and in vivo. Fomepizole with or without NAC completely prevented AAP-induced liver toxicity. In vivo, high-dose AAP with NAC/fomepizole rescue had profound antitumor activity against commonly used 4T1 breast tumor and lewis lung carcinoma lung tumor models, and no liver toxicity was detected. The antitumor efficacy was reduced in immune-compromised NOD-scid IL2Rgammanull mice, suggesting an immune-mediated mechanism of action. In conclusion, using fomepizole-based rescue, we were able to treat mice with 100-fold higher than standard dosing of AAP (650 mg/kg) without any detected liver toxicity and substantial antitumor activity. SIGNIFICANCE STATEMENT: High-dose acetaminophen can be given concurrently with CYP2E1 inhibition to allow for safe dose escalation to levels needed for anticancer activity without detected evidence of toxicity.
Sujet(s)
Acétaminophène , Cytochrome P-450 CYP2E1 , Souris , Animaux , Acétaminophène/toxicité , Cytochrome P-450 CYP2E1/métabolisme , Fomépizole , Souris de lignée NOD , Foie/métabolisme , Acétylcystéine/pharmacologieRÉSUMÉ
High-dose acetaminophen (AAP) with N-acetylcysteine (NAC) rescue is among the few treatments that has shown activity in phase I trials without achieving dose-limiting toxicity that has not progressed to evaluation in later line studies. While the anti-tumor effects of AAP/NAC appear not to be mediated by glutathione depletion and free radical injury, the mechanism of anti-tumor effects of AAP/NAC has not been definitively characterized. In vitro, the effects of AAP/NAC were evaluated on bone marrow derived macrophages. Effects of AAP on IL-4/STAT6 (M2) or IFN/LPS/STAT1 (M1) signaling and downstream gene and protein expression were studied. NAC reversed the AAP toxicity in the normal liver but did not reverse AAP cytotoxicity against tumor cells in vitro. AAP/NAC selectively inhibited IL-4-induced STAT6 phosphorylation but not IFN/LPS-induced STAT1 phosphorylation. Downstream, AAP/NAC inhibited IL-4 induction of M2-associated genes and proteins but did not inhibit the IFN/LPS induction of M1-associated genes and proteins. In vivo, AAP/NAC inhibited tumor growth in EF43.fgf4 and 4T1 triple-negative breast tumors. Flow cytometry of tumor-associated macrophages revealed that AAP/NAC selectively inhibited M2 polarization. The anti-tumor activity of high-dose AAP/NAC is lost in macrophage-depleted mouse syngeneic tumor models, suggesting a macrophage-dependent mechanism of action. In conclusion, our study is the first to show that high-dose AAP/NAC has profound effects on the tumor immune microenvironment that facilitates immune-mediated inhibition of tumor growth.
RÉSUMÉ
High-dose acetaminophen (AAP) with delayed rescue using n-acetylcysteine (NAC), the FDA-approved antidote to AAP overdose, has demonstrated promising antitumor efficacy in early phase clinical trials. However, the mechanism of action (MOA) of AAP's anticancer effects remains elusive. Using clinically relevant AAP concentrations, we evaluated cancer stem cell (CSC) phenotype in vitro and in vivo in lung cancer and melanoma cells with diverse driver mutations. Associated mechanisms were also studied. Our results demonstrated that AAP inhibited 3D spheroid formation, self-renewal, and expression of CSC markers when human cancer cells were grown in serum-free CSC media. Similarly, anti-CSC activity was demonstrated in vivo in xenograft models - tumor formation following in vitro treatment and ex-vivo spheroid formation following in vivo treatment. Intriguingly, NAC, used to mitigate AAP's liver toxicity, did not rescue cells from AAP's anti-CSC effects, and AAP failed to reduce glutathione levels in tumor xenograft in contrast to mice liver tissue suggesting nonglutathione-related MOA. In fact, AAP mediates its anti-CSC effect via inhibition of STAT3. AAP directly binds to STAT3 with an affinity in the low micromolar range and a high degree of specificity for STAT3 relative to STAT1. These findings have high immediate translational significance concerning advancing AAP with NAC rescue to selectively rescue hepatotoxicity while inhibiting CSCs. The novel mechanism of selective STAT3 inhibition has implications for developing rational anticancer combinations and better patient selection (predictive biomarkers) for clinical studies and developing novel selective STAT3 inhibitors using AAP's molecular scaffold.