N-acetylcysteine (NAC) has been used as a direct scavenger of reactive oxygen species (hydrogen peroxide, in particular) and an antioxidant in cancer biology and immuno-oncology. NAC is the antioxidant drug most frequently employed in studies using tumor cells, immune cells, and preclinical mouse xenografts. Most studies use redox-active fluorescent probes such as dichlorodihydrofluorescein, hydroethidine, mitochondria-targeted hydroethidine, and proprietary kit-based probes (i.e., CellROX Green and CellROX Red) for intracellular detection of superoxide or hydrogen peroxide. Inhibition of fluorescence by NAC was used as a key experimental observation to support the formation of reactive oxygen species and redox mechanisms proposed for ferroptosis, tumor metastasis, and redox signaling in the tumor microenvironment. Reactive oxygen species such as superoxide and hydrogen peroxide stimulate or abrogate tumor cells and immune cells depending on multiple factors. Understanding the mechanism of antioxidants is crucial for interpretation of the results. Because neither NAC nor the fluorescent probes indicated above react directly with hydrogen peroxide, it is critically important to reinterpret the results to advance our understanding of the mechanism of action of NAC and shed additional mechanistic insight on redox-regulated signaling in tumor biology. To this end, this review is focused on how NAC could affect multiple pathways in cancer cells, including iron signaling, ferroptosis, and the glutathione-dependent antioxidant and redox signaling mechanism, and how NAC could inhibit oxidation of the fluorescent probes through multiple mechanisms.
Abstract Many human malignancies such as prostate cancer (CaP) and primary brain tumor (GBM) exhibit high oxidative stress. Developing a successful therapeutic drug to treat advanced metastatic CaP and GBM is a major unmet medical need. It has been demonstrated that in certain chemo- and/or radiation resistant cancer cells with high oxidative stress, cells depend on reactive oxygen species (ROS) for survival and proliferation. Therefore, reducing ROS can be a successful strategy for developing chemotherapeutic drugs for use against chemo- and/or radiation-resistant tumors. The mitochondria are a major source of superoxide and other ROS. We focused on targeting spin-trapping nitroxide analogs at the mitochondrial interstitial space with appropriate linker length in order to remove the superoxide in order to reduce the cellular ROS levels without interfering with the mitochondrial electron transport chain. Our lead compound in this new class of drugs is Mito-tempol-C10. The drug successfully blocked ROS production in human CaP and GBM cell lines as determined by DCF dye oxidation assay. It exhibited marked growth inhibitory effect against androgen-dependent as well as androgen-independent cultured human CaP cells (IC50 < 1 µM) and similar growth inhibitory activity against several human GBM cells lines as well as cultured primary brain tumor stem cells as determined by DNA fluorescence measurement assay. Preliminary pharmacokinetic (PK) study show that the drug has serum half-life of approximately 45 minutes, but is retained by the tumor and other animal tissue for more than 24 hours. The drug is tolerated by ICR white mice, nude mice and NOD-SCID mice at a dose of 15 mg/kg i.p. given once every week for four weeks without any overt systemic toxicity. Preliminary efficacy study at this treatment condition showed marked effect on the growth of intracranially implanted, radiation-resistant U-87 MG human GBM cells in NOD-SCID mice and a small, but significant effect on the growth of androgen-independent PC-3 human prostate cancer cell xenografts growing on the flanks of nude mice. These results confirm that mitochondria-targeted spin-traps that reduce cellular ROS can be therapeutically effective in treatment of advanced, hard-to-treat human tumors with high oxidative stress. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B203.
ESR and deuteration studies of x-irradiated trifluroacetamide crystals at 77 /sup 0/K show that the CF/sub 3/(A) radical initially formed interacts with a fluorine nucleus on a neighboring molecule, resulting in an additional fluorine doublet of A/sub z/=10 G, A/sub y/< or =A/sub x/< or =1 G. This previously unobserved spectrum decays with time at 77 /sup 0/K and is irreversibly replaced by the ESR spectrum of CF/sub 3/(B) that is stable to 200 /sup 0/K. Based on an INDO calculation, the formation of CF/sub 3/(A) occurs when the crystallographic intermolecular FxxxCF/sub 3/ distance in the parent compound decreases from 3.77 to 2.4 A upon radical formation at 77 /sup 0/K. Following the formation of CF/sub 3/(A), the FxxxCF/sub 3/ distance increases with time or temperature to approximately 3.0 A.
Previously we showed that hypoxia‐induced mitochondrial respiratory stress in RAW 264.7 macrophages and other cells caused activation of retrograde signaling (also known as mitochondrial respiratory stress signaling) and the appearance of tartrate‐resistant acid phosphatase (TRAP)‐positive cells. In the present study, we used N‐acetyl cysteine and ascorbate (general antioxidants) and MitoQ, a mitochondria‐specific antioxidant, to investigate the role of intracellular reactive oxygen species (ROS) in osteoclast differentiation. Our results show that hypoxia‐mediated mitochondrial dysfunction, as tested by disruption of mitochondrial transmembrane potential, was suppressed by MitoQ as well as by the other antioxidants. These agents also suppressed the activation of mitochondrial retrograde signaling. Interestingly, in terms of molar concentrations, MitoQ was more than 1000‐fold more effective than general antioxidants in suppressing the receptor activator of nuclear factor‐B ligand‐induced differentiation of RAW 264.7 cells into multinucleated and TRAP‐positive osteoclasts. We propose that mitochondrial function and intramitochondrial ROS play important roles in osteoclastogenesis.
