A catalytic redox-cycling nanoreactor enables robust oxidative stress amplification for synergistic tumor apoptosis and ferroptosis.

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Insights on alpha lipoic and dihydrolipoic acids as promising scavengers of oxidative stress and possible chelators in mercury toxicology

The pharmacology of the antioxidant lipoic acid

Open AccessCCS ChemistryRESEARCH ARTICLE7 Dec 2022Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy Xuan Xiao†, Qingyu Zong†, Jisi Li and Youyong Yuan Xuan Xiao† School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou 511442 National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006 †X. Xiao and Q. Zong contributed equally to this work.Google Scholar More articles by this author , Qingyu Zong† National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006 School of Medicine, South China University of Technology, Guangzhou 510006 †X. Xiao and Q. Zong contributed equally to this work.Google Scholar More articles by this author , Jisi Li National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006 School of Medicine, South China University of Technology, Guangzhou 510006 Google Scholar More articles by this author and Youyong Yuan *Corresponding author: E-mail Address: [email protected] School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou 511442 National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006 Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology, Guangzhou 510006 Guangdong Provincial Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.022.202101564 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Efficacy of prodrugs in cancer therapy requires selective and efficient drug activation in cancer cells. Here, we report a novel dual-prodrug delivery system with tumor-selective cascade-amplified prodrug activation for synergistic oxidation-chemotherapy. Cancer cells overexpressing cathepsin B-activatable near-infrared (NIR) hemicyanine prodrug (CyNH-Citval) were encapsulated by the reactive oxygen species (ROS)-responsive polyprodrug of doxorubicin (DOX) (PTKDOX) to obtain PTKDOX/Cy. Upon uptake of PTKDOX/Cy by cancer cells and subsequent prodrug CyNH-Citval activation, NIR fluorescence was turned on and toxicity toward mitochondria was restored, thereby elevating intracellular ROS levels, which subsequently activated the polyprodrug PTKDOX to initiate the cascade and amplify DOX. Overall, these results indicate that enzyme-mediated initiation of drug activation and amplification of cascade ROS ultimately causes selective and efficient prodrug activation in tumors with synergistic oxidation and chemotherapy. These findings provide new insights to inform precise cooperative cancer therapy. Download figure Download PowerPoint Introduction Although chemotherapy is a major clinical approach for tumor therapy, its application remains constrained by poor selectivity and serious side effects.1–3 To improve the selectivity and therapeutic efficacy of chemotherapy, various stimulus-responsive drug delivery systems (DDSs) have been developed in the past decade.4–6 For example, numerous research has focused on prodrugs that are specifically activated by tumor associated stimuli to release the potent naïve drug which allows them to improve selectivity of chemotherapy.6–8 Various stimuli, including pH,9,10 glutathione,11,12 reactive oxygen species (ROS),13–15 and enzymes,16,17 are present in tumor microenvironments. Thus, tumor-associated enzyme-activated prodrugs have received numerous attention due to the high selectivity of enzymes overexpressed in cancer cells.18–23 However, use of enzyme-activated prodrugs is limited by ineffective drug activation owing to the paucity of tumor-associated enzymes that represent an essential step for the functioning of the prodrug.24,25 For example, Chen and co-workers26 amplified prodrug activation by sequentially delivering combretastatin A4 to upregulate metalloproteinase 9 (MMP9) and MMP9-activated doxorubicin (DOX) prodrug and promoted activation of tumor-selective prodrug for cancer therapy. In addition, Yin and co-workers27 reported that a pro-protein therapy was activated by self-amplified hypoxia associate enzymes. Consequently, development of an enzyme-responsive prodrug, which can simultaneously retain selectivity of enzyme-responsiveness for tumor targeting and amplification of the enzyme response signal for enhanced therapeutic efficiency, remains a great challenge. Recently, numerous research groups have exploited the ability of cancer cells to overproduce ROS to develop ROS-responsive DDSs containing oxidation-labile groups, such as thioketal, boronic ester, and proline, for cancer treatment.28 However, intracellular concentration of ROS is still not high enough for efficient drug activation, which represents an intrinsic limitation for the ROS-responsive systems despite their great potential.29–31 For example, Mokhir and co-workers29 reported an ROS-dependent aminoferrocene-based prodrug that amplified intracellular ROS level for efficient cancer therapy. Therefore, development of new strategies for enzyme-activated ROS generation is imperative to improvement of tumor selectivity. The generated ROS can be further utilized for efficient prodrug activation. In this study, we developed a tumor-selective cascade-amplified dual-prodrug activation system (denoted PTKDOX/Cy) consisting of cancer cells overexpressing cathepsin B (CTB), CTB-activated hemicyanine (CyNH2) prodrug (CyNH-Citval), and ROS-responsive polyprodrug of DOX (PTKDOX) conjugated on the side chain of poly(thioketal) (PTK; Schemes 1a and 1b). Previous studies have shown that amino containing near-infrared (NIR) CyNH2 can selectively accumulate in mitochondria and efficiently lower their membrane potential, thereby increasing intracellular ROS levels to cause oxidation-induced cell death.32 Prior to activation, the prodrug CyNH-Citval shows a weak fluorescence due to intramolecular charge transfer (ICT) and low toxicity, which can reduce its toxicity to normal cells.33,34 Upon interacting with CTB overexpressed in cancer cells, CyNH2 is activated to restore toxicity, and NIR fluorescence is triggered for drug activation monitoring. Activated CyNH2 leads to mitochondrial dysfunction in cancer cells, thereby elevating levels of intracellular ROS. Consequently, high ROS levels mediate activation of the polyprodrug PTKDOX, thereby initiating a cascade and amplifying the DOX prodrug. More importantly, CyNH2 and DOX showed synergistic oxidation and chemotherapy. Moreover, CyNH-Citval exhibited low toxicity and insignificant elevation of ROS levels in normal cells due to the low CTB expression. This phenomenon disrupted initiation of cascade DOX activation and resulted in low cytotoxicity of PTKDOX/Cy to normal cells. Integrating dual prodrugs into a single PTKDOX/Cy with cascade and amplified drug activation may increase tumor selectivity and efficiency of drug activation for synergistic oxidation-chemotherapy of cancer. Scheme 1 | Profile of the tumor-selective cascade-amplified dual-prodrug activation system (PTKDOX/Cy) developed in this research. (a) Chemical structure. (b) Schematic representation of the tumor-selective cascade-amplified dual-prodrugs activation for synergistic oxidation-chemotherapy. Download figure Download PowerPoint Experimental Methods Details of the materials and instruments utilized are provided in the Supporting Information. Preparation of PTKDOX and PTKDOX/Cy CyNH-Citval was synthesized by conjugating CyNH2 with CTB-specific citrulline-valine (Cit-Val) peptide linker. PTK was obtained by fast polycondensation of 1,3-dimercapto-2-propanol and acetone, with a molar ratio of 1∶1.05, in the presence of concentrated hydrogen chloride (HCl). Then PTK pyridine (PTK-SS) was obtained by the disulfide-thiol exchange reaction of PTK with 2,2′-dithiodipyridine at a molar ratio of 1∶3. Finally, PEG-TK-DOX (TK = polythioketone) was first synthesized via conjugating DOX and PEG with a fixed ratio of 10∶1 to the hydroxyl groups of PTK-SS. Then PEG-PTK-DOX (30 mg) was dissolved in 1 mL of dimethyl sulfoxide (DMSO), and then gradually added into 9 mL of ultrapure water under stirring. After additional stirring for 2 h, the solution was transferred into a dialysis bag (MWCO 3500) to remove DMSO against ultrapure water for 24 h, and then the solution was filtered through a 0.45 μm filter to obtain PTKDOX. The preparation of PTKDOX/Cy was similar to that for PTKDOX, but the polymer PEG-PTK-DOX was replaced with PEG-PTK-DOX and CyNH-Citval (1.0 mg). Cell culture and tumor model Mouse breast cancer cell line 4T1 cells were cultured in Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum and 1% penicillin-streptomycin. Cell cultures were incubated in a 5% CO2 and 21% O2 incubator at 37 °C. Female BALB/c mice and BALB/c nude mice (20 ± 2 g, 6–8 weeks old) were purchased from Hunan SJA Laboratory Animal Co. Ltd (Hunan, China). 4T1 cells (1 × 106) were injected into the right mammary fat pads to establish an orthotopic 4T1 tumor model. After the tumor volumes reached 100 mm3, the mice were used for subsequent experiments. At the end of experiments, all mice were killed by CO2 inhalation. All animal experiments were approved by the Ethics Committee of the South China University of Technology (Guangzhou, China). All detailed experimental methods are available in the Supporting Information. Results and Discussion Preparation and characterization of PEG-TK-DOX and CyNH-Citval A summary of synthetic routes to the NIR CyNH2 is presented in Supporting Information Scheme S1. The structure and purity of CyNH2 and intermediates were confirmed by 1H NMR spectra ( Supporting Information Figures S1–S6). The synthetic method for preparation of the prodrug CyNH-Citval is displayed in Supporting Information Scheme S2. Briefly, CyNH2 was conjugated with CTB-specific Cit-Val peptide linker to obtain the prodrug CyNH-Citval with a yield of 11.6%. Thereafter, the prodrug and its intermediates were verified via 1H NMR spectra ( Supporting Information Figures S7 and S8). The synthetic route of polyprodrug PTKDOX is shown in Supporting Information Scheme S3. In brief, PTK was obtained by rapid polycondensation of 1,3-dimercapto-2-propanol and acetone, with a molar ratio of 1∶1.05, in the presence of concentrated HCl. PTK appeared as a colorless waxy solid, with a 47% yield and 14 repetitive units, after contrastive analysis of integration intensities of peaks 1 (methylene protons of PTK) and 2 (sulfhydryl protons of 1,3-dimercapto-2-propanol) from the 1H NMR spectra ( Supporting Information Figures S9 and S10). Subsequently, PTK-SS was obtained, as a light-yellow solid, by the disulfide-thiol exchange reaction of PTK with 2,2′-dithiodipyridine, at a molar ratio of 1∶3. Next, PTK-SS was activated with N,N′-carbonyldiimidazole (CDI) then conjugated with DOX and amino-terminated methoxy poly(ethylene glycol) (PEG) to obtain polyprodrug PTKDOX. 1H NMR spectra revealed that the grafting rate for DOX was about 50% ( Supporting Information Figure S13). Moreover, 1H NMR spectra, 13C NMR spectra, MS spectra, and gel permeation chromatography studies were used for characterization analysis of the new compounds, polymers, and their intermediates ( Supporting Information Figures S1–S32 and S34A). The fluorescence change of CyNH-Citval in response to papain Results of analysis of CyNH2 and CyNH-Citval absorption are shown in Supporting Information Figure S34b. Summarily, CyNH2 had a maximum absorption of 710 nm, whereas that of CyNH-Citval blue-shifted to 615 nm, which is attributed to ICT of CyNH-Citval.33,34 In addition, CyNH2 exhibited a strong fluorescence intensity, whereas that of CyNH-Citval was weak, further affirming the ICT of CyNH-Citval ( Supporting Information Figure S34c). Next, we chose papain as a substitute enzyme for analysis of CyNH-Citval’s enzyme-response behavior, due to its similar enzyme activity to CTB,35 and investigated fluorescence changes of CyNH-Citval after treatment with different concentrations of papain over time. CyNH-Citval’s fluorescence intensity increased with prolonged incubation times, reaching saturation after 6 h with a papain concentration of 10 mM ( Supporting Information Figure S34d). In addition, CyNH-Citval’s fluorescence intensity increased upon increased papain concentration, reaching saturation upon addition of 10 μM papain after 6 h (Figure 1a). Notably, this fluorescence intensity increased ∼15-fold and exhibited papain concentration dependence over a wide range (0–10 μM). These results indicated that papain could effectively cleave the amide bond of CyNH-Citval, and release CyNH2 with strong fluorescence activation. Therefore, a high concentration of CTB in cancer cells may cause a release of CyNH2 and turn-on NIR fluorescence. Figure 1 | (a) Fluorescence spectra of CyNH-Citval treated with different concentrations of papain. (b) UV–vis absorbance spectra of DOX, CyNH-Citval, and PTKDOX/Cy. (c) Changes in hydrodynamic diameter of PTKDOX/Cy after treatment with H2O2, ClO−, or ·OH. (d) 1H NMR spectrum for H2O2-responsive degradation of PTK-SS with generation of acetone after treatment with DMSO-d6 and H2O2 (10 mM) at 37 °C. (e) Fluorescence spectra for DOX, PTKDOX, and PTKDOX/Cy. (f) Cumulative release of DOX from PTKDOX/Cy in the presence of different concentrations of H2O2. Download figure Download PowerPoint In vitro ROS-responsive degradation and DOX release Next, we employed a nanoprecipitation method to prepare PTKDOX/Cy by self-assembly from PEG-PTK-DOX via encapsulation of CyNH-Citval. Results showed that PTKDOX/Cy had a hydrodynamic diameter of ∼107 nm in phosphate-buffered saline (PBS) (Figure 1c). In addition, PTKDOX/Cy exhibited an absorbance spectrum with similar absorbance to DOX and CyNH-Citval, with maximum absorption at 480 and 615 nm, respectively (Figure 1b and Supporting Information Figure S34b), indicating that DOX and CyNH-Citval were successfully loaded. DOX and CyNH-Citval had loading capacities of 33.67 ± 0.23 and 9.13 ± 0.25%, respectively. Meanwhile, PTKDOX/Cy’s hydrodynamic diameter changed from 107 nm in PBS, to 10 nm after treatment with H2O2, ClO− or ·OH (Figure 1c), indicating that it was degraded in response to ROS. Also, transmission electron microscopy (TEM) and scanning electron microscopy images recorded for PTKDOX/Cy are shown in Supporting Information Figures S33a and S33c, and the TEM image recorded for PTKDOX/Cy after treatment with 10 mM H2O2 and 10 μM papain is shown in Supporting Information Figure S33b. Furthermore, 1H NMR spectra revealed H2O2-triggered degradation of PTK-SS (Figure 1d) as well as the disassociation mechanism of PTKDOX ( Supporting Information Figure S35). Degradation of PTK-SS (15 mg mL−1) was detected using a commixture of DMSO-d6 and H2O2 (10 mM), while the thioketal of PTK-SS eventually turned into acetone (Figure 1d). The fluorescence of DOX in PTKDOX/Cy was inhibited by the Förster resonance energy transfer of DOX to CyNH2 and aggregation-caused quenching of DOX (Figure 1e). Next, we studied drug release of the PTKDOX/Cy and found almost no or moderate release of free DOX in the presence of PBS and 1 mM H2O2, respectively (Figure 1f). Conversely, large amounts of DOX were released in the presence of 10 mM H2O2, indicating that more drug could be released in cells with high H2O2 concentrations. The critical micelle concentration of PTKDOX PTKDOX can itself induce the formation of polymeric nanoparticles. To evaluate the critical micelle concentration (CMC), we measured the count rates of nanoparticles at different concentrations according to the previous literature.36 As shown in Supporting Information Figure S36a, the CMCs of PTKDOX nano-assembly is 0.0728 mg/mL. The dynamic light scattering data revealed that the size of obtained PTKDOX nanoparticles was about 73.2 nm ( Supporting Information Figure S36b). In vitro CyNH2 release and the cellular uptake mechanism for the PTKDOX/Cy Analysis of the release behavior of CyNH2 from PTKDOX/Cy in the presence of both ROS (10 mM H2O2) and enzyme (10 μM papain) ( Supporting Information Figure S37a) shows the cumulative release of CyNH2 was <5% in phosphate buffer at 37 °C after 48 h, which means negligible leakage before the prodrug reaches the tumor site. When H2O2 or papain was added, the release of CyNH2 was <10%. In contrast, the cumulative release amount of CyNH2 was 72.4% after 48 h in the presence of H2O2 and papain. These results indicate that CyNH2 only can be released when H2O2 and papain were simultaneously present. The endocytic pathway of PTKDOX/Cy by cancer cells was investigated in the presence of several endocytosis inhibitors including chlorpromazine (inhibitor of clathrin-mediated endocytosis), methyl-β-cyclodextrin (inhibitor of caveolae-mediated endocytosis), and amiloride (inhibitor of giant pinocytosis). As shown in Supporting Information Figure S37b, the cells treated with chlorpromazine at 4 °C showed lower nanoparticle internalization, suggesting that PTKDOX/Cy is susceptible to a clathrin-mediated endocytotic pathway in 4T1 cells in an energy-dependent manner. Intracellular fluorescence recovery of PTKDOX/Cy Next, we performed confocal image analysis on mouse breast cancer cell line 4T1 and the mouse embryonic fibroblast (MEF) cell line MEF to evaluate PTKDOX/Cy’s applicability in cancer therapy and imaging. Results revealed strong red fluorescence signals in both CyNH-Citval and PTKDOX/Cy-treated 4T1 cells, with ∼10- and 6-fold increases in the mean fluorescence intensity (MFI), respectively, relative to MEF cells (Figure 2a and Supporting Information Figure S38). In contrast, pretreatment of 4T1 cells with CA-074 methyl ester (CA-074-Me), a CTB inhibitor, resulted in diminished fluorescence. Overall, these results indicated selective activation of CyNH-Citval and PTKDOX/Cy fluorescence in 4T1 tumor cells, making it promising for tumor-specific intelligent images. Figure 2 | (a) Confocal microscopy images showing fluorescence of CyNH2 (red) in 4T1 and MEF cells after incubation with CyNH-Citval or PTKDOX/Cy for 6 h. Inhi. represents the CTB inhibitor CA-074-Me, which was preincubated with the cells for 2 h. (b) Confocal microscopy images showing intracellular ROS levels stained with DCFH-DA (green) in 4T1 and MEF cells after different treatments. VC represents ROS scavenger vitamin C. (c) Cytotoxicity of 4T1 and MEF cells incubated with CyNH2 or CyNH-Citval. (d) Cytotoxicity of 4T1 cells treated with PTKDOX, PTKDOX/Cy, and PTKDOX/Cy+VC. Statistical significance: *P < 0.05, **P < 0.01. Download figure Download PowerPoint Colocalization of CyNH2 with mitochondria and mitochondrial membrane potential study Furthermore, we found good colocalization between CyNH2 and MitoTracker Green-labeled mitochondria, as evidenced by a colocalization coefficient of 0.83 ( Supporting Information Figure S39). Next, we explored mitochondrial membrane potentials (MMPs) using in 4T1 cells the probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1). Results showed that JC-1 could assemble into J-aggregates, with red fluorescence in high MMP mitochondrial matrix, but dispersed into the cytoplasm in a monomeric form with green fluorescence in low MMP mitochondrial matrix. Notably, cells treated with CyNH-Citval and CyNH2 exhibited marked green fluorescence ( Supporting Information Figure S40), indicative of a gradual decrease in MMP, although cells treated with CyNH-Citval+Inhi CTB inhibitor exhibited a weak green fluorescence. Intracellular ROS level The decrease in MMP, by may be attributed to ROS explored and ROS in 4T1 cells using the ROS which is by ROS and subsequently into with green fluorescence. Results revealed strong green fluorescence in cells treated with CyNH2 and PTKDOX/Cy, with an and increase in the respectively, relative to PBS or DOX (Figure and Supporting Information Figure in the which had been with ROS scavenger vitamin and CTB inhibitor CA-074-Me, exhibited a weak fluorescence these results confirmed that CyNH2 can intracellular ROS which can be further utilized to DOX. Intracellular DOX release To ROS levels by released CyNH2 could amplify DOX activation, we employed confocal scanning to intracellular DOX release from PTKDOX and PTKDOX/Cy in 4T1 cells. Results showed that 4T1 cells exhibited red fluorescence after incubation with PTKDOX and PTKDOX/Cy for 6 h (Figure A further h incubation resulted in a weak red fluorescence signal in the with indicating that only a amount of DOX was released in cells treated with PTKDOX. Conversely, a strong red fluorescence signal was in the of cells treated with PTKDOX/Cy, indicative of enhanced activation of DOX with PTKDOX/Cy. Next, we employed to evaluate concentrations of DOX in 4T1 cells, after incubation with PTKDOX and PTKDOX/Cy over time. Results showed that cells treated with PTKDOX/Cy had a increase in the amount of released DOX relative to treated with PTKDOX at h ( Supporting Information Figure These results indicated that the released CyNH2 from PTKDOX/Cy with ROS levels amplified the DOX activation in the tumor cells. In vitro cytotoxicity and cellular Next, we employed the to the cytotoxicity of CyNH-Citval and CyNH2 to more of cell cancer cell mouse cancer cell line and mouse cell line cells and cells had lower MEF and cells (Figure and Supporting Information Figure indicating that CTB-activated prodrug CyNH-Citval tumor-specific Moreover, DOX and CyNH2 had a synergistic on 4T1 cells, as evidenced by a of ( Supporting Information Figure Furthermore, we investigated PTKDOX, PTKDOX/Cy, and cytotoxicity on 4T1 cells, at different DOX and found that PTKDOX whereas PTKDOX/Cy had toxicity PTKDOX and the concentration of in 4T1 cells (Figure In contrast, PTKDOX/Cy had low activity in the presence of indicating that increased activation of DOX was on this marked cytotoxicity of PTKDOX/Cy to 4T1 cells, we explored cellular using the Results showed that PTKDOX/Cy treatment cellular PTKDOX, whereas cell increased addition of VC or the inhibitor ( Supporting Information Figure which results from the In study of PTKDOX/Cy Results from in NIR fluorescence images of PTKDOX/Cy in 4T1 nude mice revealed strong fluorescence in tumors of mice injected with PTKDOX/Cy to treated with and the fluorescence was in the tumors to 48 h (Figure and Supporting Information Figure the tumors and major 48 h after of PTKDOX/Cy and to obtain images. Results revealed fluorescence intensity in tumors of mice treated with PTKDOX/Cy relative to the while a weak intensity was in the treated with ( Supporting Information Figure These results indicated that PTKDOX/Cy was selectively in and precise in images. To the efficacy of PTKDOX/Cy in we fluorescence intensity of CyNH2 and ROS levels in tumor after treatment with PTKDOX/Cy and Results showed strong fluorescence intensities in and CyNH2 from PTKDOX/Cy-treated but weak in ( Supporting Information Figure the release of CyNH2 and elevation of ROS levels in tumor which was with results from the in vitro cell these results provide further that CyNH2 from PTKDOX/Cy is activated with NIR fluorescence for drug activation and the activated CyNH2 causes mitochondria dysfunction in cancer cells and increases levels of intracellular ROS in tumor Figure | (a) In NIR fluorescence images of nude mice 4T1 after with PTKDOX/Cy and fluorescence images. (b) Changes in tumor volumes in 4T1 mice under different treatments. (c) tumor of mice under different treatments. (d) of tumors in mice after and at the end of the therapy in response to different treatments. = 100 Statistical significance: *P < 0.05, **P < < Download figure Download PowerPoint In of PTKDOX/Cy Next, we efficacy of PTKDOX/Cy in using mice 4T1 The mice were the tumors to mm3, into groups, and treated with PBS, free DOX, PTKDOX, and PTKDOX/Cy mg respectively, via Thereafter, we measured and recorded the tumor volumes and 2 Results showed that mice treated with DOX and PTKDOX exhibited tumor to relative to in the PBS (Figure However, tumors in mice treated with PTKDOX/Cy and were Notably, PTKDOX/Cy treatment exhibited the therapeutic efficiency, as evidenced by a tumor rate of with the tumor relative to the results were in tumor and images (Figure and Supporting Information Figure Notably, of mice treated with PTKDOX/Cy were almost indicating great of the prodrug PTKDOX/Cy to mice ( Supporting Information Figure Moreover, results from and of major revealed a negligible after PTKDOX/Cy relative to PBS, which PTKDOX/Cy’s ( Supporting Information Figure Furthermore, analysis of tumors from mice in the PTKDOX/Cy revealed that a great of tumor cells was and the (Figure results were obtained after end as evidenced by the green fluorescence. successfully a dual-prodrug delivery a tumor-selective cascade amplified prodrug activation, for synergistic oxidation-chemotherapy. prodrug, not only therapy with high tumor selectively but ROS for efficient activation of prodrug DOX. CyNH-Citval could be activated by CTB overexpressed in 4T1 tumor cells, relative to normal MEF cells, whereas activated CyNH2 a increase in intracellular ROS which further enhanced DOX activation by to PTKDOX. Moreover, PTKDOX/Cy resulted in the in therapeutic efficacy against 4T1 as evidenced by a tumor which was in to PTKDOX these findings indicate that dual-prodrugs with enzyme-responsive drug release is a promising for selectivity and therapeutic efficacy of cancer therapy. The was through of all All have to the of the Supporting Information Supporting Information is available and the of the and TEM images as well as the of in vitro DOX the fluorescence change of CyNH-Citval in response to confocal image and colocalization of CyNH2 with mitochondria, MMP study, intracellular ROS level intracellular DOX in vitro cytotoxicity and cellular in study, in of PTKDOX/Cy, and additional Figures of The no This was by the National of China and the and Technology of Guangzhou Guangdong Provincial the for the of Key in Guangzhou Key Laboratory of and as an for Cancer Google Scholar and Google Scholar on A in the against Cancer with a Google Scholar for Google Scholar in Google Scholar Xiao Yuan with a for Synergistic Google Scholar for and Cancer Google Scholar Chen for the of and of Google Scholar Li A for to in Cancer Google Scholar C. of to Google Scholar for and Cancer Google Scholar Xiao Zong Yuan with and Activation for Google Scholar for Google Scholar a Google Scholar Chen Chen Materials for and Google Scholar Chen of for Google Scholar and of into Google Scholar in Cancer Google Scholar and Google Scholar for and Google Scholar for Google Scholar of Cancer and Google Scholar Park Park of for Cancer Google Scholar Park that in to Google Scholar for Google Scholar Li Chen A4 of Google Scholar Li Yin by a Google Scholar Chen Google Scholar Mokhir of as Google Scholar Li of a H2O2 for Google Scholar Chen by Google Scholar Cancer a with Google Scholar for of Google Scholar Yuan for between the and Google Scholar Li Q. and for and Google Scholar C. 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Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained some recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O2-), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap, were eliminated by DHLA but not by TA. The sulfhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O2-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O2-, suggesting the reduction of O2- by DHLA. Competition of O2- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O2- and DHLA, which was found to be a 3.3 x 10(5) M-1 s-1. On the other hand, the DMPO signal of hydroxyl radicals (HO.) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO.. There was no electron transfer from Fe2+ to TA or from DHLA to Fe3+ detected by measuring the Fe(2+)-phenanthroline complex. DHLA did not potentiate the DMPO signal of HO. indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular, DHLA is very effective as shown by its dual capability by eliminating both O2- and HO..

Cancer stem cells (CSCs) in a tumor bulk exhibit self-renewal capacity and contribute to tumor aggressiveness that leads to metastasis, cancer therapeutic resistance and relapse. The normal or tumor cells and CSCs are sensitive to the level of reactive oxygen species (ROS) in them. ROS are highly reactive molecules that are generated as byproducts of the endogenous reduction-oxidation (redox) metabolic reactions. In cells maintaining their redox homeostasis, the normal levels of ROS are involved in various cellular functions, including signaling. However, disruption of the redox homeostasis because of incompetent scavenging mechanism elevates the level of ROS, leading to oxidative stress and irreversible damage to the genomic DNA and/or other biomolecules. Subsequently, this results in altered gene expression, genomic instability, mutations, cellular transformation of normal cells to tumor cells or CSCs, survival of CSCs, and tumorigenesis. These aforementioned abnormalities are associated with increased rate of proliferation of tumor cells, hypoxia, angiogenesis, and epithelial to mesenchymal transition (EMT), tumor aggressiveness, and metastasis in several cancers. CSCs and tumor cells with the elevated levels of ROS, hijack various signaling molecules, thus alter multiple signaling cascades or molecular pathways including the stem cell regulatory pathways, which favor the invasiveness of these cells and their tumor microenvironment (TME). Importantly, the elevated levels of ROS can exhibit antitumorigenic activity to suppress tumorigenesis or protumorigenic activity to promote tumorigenesis, metastasis, and cancer treatment resistance and relapse. Moreover, the interactions between the elevated levels of ROS and CSCs create new networks with other signaling cascades, which ultimately leads to poor prognosis, therapeutic resistance and recurrence of cancer. In this chapter, we focus on the molecular interactions between ROS and CSCs and their effects on the tumor aggressiveness and TME. Furthermore, we discuss about the impact of elevated levels of ROS on the cellular transformation of normal or tumor cells to CSCs, cell signaling in CSCs and tumor cells, and their implications in cancer and its therapy.KeywordsCancer stem cells (CSCs)Reactive oxygen species (ROS)Redox-dependent signalingCancer progressionEpithelial to mesenchymal transition (EMT)InvasivenessMesenchymal to epithelial transition (MET)MetastasisTherapeutic resistanceRelapseDrug development

Intensive blood glucose control can prevent the initiation and progression of diabetic complications. However, the impacts of intensive therapy against diabetic complications might be limited, because of difficulty in maintaining blood glucose concentrations close to the normal range or other unknown reasons. Thus, another approach based on the elucidation of mechanisms of diabetic complications might be required to prevent the progression of the complications. Production of reactive oxygen species (ROS) and lipid peroxidation are increased in diabetic patients, especially in those with poor glycemic control. Oxidative stress can be crucial for the development of diabetic vascular complications. Thus, there is interest in determining whether antioxidant therapy can complement intensive blood glucose control. In fact, a large number of studies evaluating the efficacy of antioxidants have been carried out. However, the efficacy of these antioxidant-based therapies is still uncertain in relation to preventing diabetic complications in clinical practice. Certainly, a number of experimental studies suggest that some natural antioxidants, such as α-tocopherol (vitamin E), ascorbate (vitamin C), coenzyme Q (CoQ), taurine, glutathione or α lipoic acid, showed beneficial effects on diabetic complications. However, the results from large, long-term clinical trials using α-tocopherol were disappointing. Among the various antioxidant-based therapies, only α-lipoic acid might be somewhat useful in preventing diabetic complications. Meta-analysis has provided evidence that intravenous treatment with 600 mg/day α-lipoic acid over 3 weeks significantly improves both positive neuropathic symptoms and neuropathic deficits in diabetic patients with symptomatic polyneuropathy. α-Lipoic acid is approved in Germany as an agent for the treatment of diabetic neuropathy. The effectiveness of natural antioxidants in preventing diabetic complications is still uncertain. Therefore, new strategies for controlling oxidative stress, such as development of new mechanism-based antioxidants, will be required to prevent diabetic complications. In addition, to develop these agents, we should investigate the mechanisms that underlie the association between diabetes and oxidative stress. There are several potential mechanisms by which hyperglycemia can lead to oxidative stress1: We previously reported that hyperglycemia could increase the production of ROS from the mitochondrial electron transport chain (mitochondrial ROS)2. In addition, the normalization of the mitochondrial ROS production prevented the glucose-induced activation of PKC and polyol pathway, and the formation of advanced glycation end-products (AGEs), all of which are known to be involved in the development of diabetic complications (Figure 1). Because the production of mitochondrial ROS is thought to be one of the key events in the pathogenesis of diabetic complications and other mitochondria-related diseases, mitochondria-targeted antioxidants, such as idebenone and mitoquinone, have been developed. Idebenone is a synthetic short chain analog of CoQ10, initially patented to provide a suitable medical composition for treating and improving the after-effects of cerebral infarction. It was reported that idebenone acts as an antioxidant and protects the mitochondrial membrane against lipid peroxidation. Interestingly, it was reported that in clinical trials, idebenone is useful in controlling cardiac hypertrophy in Friedreich's ataxia (FRDA), recovery of visual acuity in Leber's hereditary optic neuropathy (LHON), and improvement of mitochondrial oxidative metabolism in the brain of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Furthermore, a phase 3 study of idebenone in Duchenne muscular dystrophy (DMD) is ongoing now. Subcellular localization of idebenone is thought to be in mitochondria. However, because it distributes through the extracellular and intracellular compartments, its effectiveness might be uncertain. Thus, mitoquinone, which is a mitochondria-targeted antioxidant analog of idebenone, has been newly developed. Because mitoquinone possesses a terminal triphenylphosphonium group instead of a hydroxyl group, it is accumulated several hundred-fold within mitochondria, enhancing the protection of mitochondria from oxidative damage. A phase 2 study of mitoquinone in chronic hepatitis C virus (HCV) infection was carried out, and mitoquinone could decrease the liver damage associated with chronic HCV infection3. Disappointingly, to our knowledge, there is no clinical evidence of idebenone and mitoquinone in preventing diabetic complications. However, FRDA, LHON and MELAS are clinical syndromes of mitochondrial disorders, in which respiratory chain functions are defective. In addition, molecular pathology of DMD is reported to be associated with increased ROS production and mitochondrial dysfunction. Furthermore, in HCV infection, there is considerable evidence for increased mitochondrial ROS and damage leading to cell death and tissue fibrosis. Therefore, similar to those mitochondria-related diseases, both idebenone and mitoquinone might show benefits in preventing diabetic complications (Figure 1). By the way, in classical natural antioxidants, why could α-lipoic acid show some effectiveness in preventing diabetic complications when α-tocopherol could not? One possible explanation for the difference might be as a result of the difference in antioxidant capacity between α-lipoic acid and α-tocopherol. α-Lipoic acid is believed to be a powerful antioxidant compared with α-tocopherol. Another explanation might be that α-lipoic acid has additional effects in preventing diabetic complications. We previously reported that metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) activate adenosine monophosphate-activated protein kinase (AMPK), normalize hyperglycemia-induced mitochondrial ROS production and promote mitochondrial biogenesis in cultured human umbilical vein endothelial cells. Because an overexpression of dominant negative AMPKα1 (T172A) attenuated metformin-induced and AICAR-induced inhibition of mitochondrial ROS and production of mitochondrial biogenesis, the effects of metformin and AICAR were dependent on the activation of AMPK4. AMPK might be one of the molecular targets for attenuating hyperglycemia-induced overproduction of mitochondrial ROS. Interestingly, it was recently reported that α-lipoic acid could activate AMPK in multiple peripheral tissues, including skeletal muscle, liver and adipocytes5. In addition, it was reported that α-lipoic acid improves mitochondrial dysfunction and oxidative damage in aging. Taking all this together, administrated α-lipoic acid might reduce hyperglycemia-induced mitochondrial ROS through AMPK activation in peripheral tissues, and prevent diabetic complications. Because oxidative stress is generally defined as an imbalance that favors the production of ROS over the antioxidant defense system, there is another strategy to reinforce the antioxidant defense system. Nuclear factor E2-related factor 2 (Nrf2) is one of the most important cellular defense mechanisms to cope with oxidative stress. Thus, Nrf2-targeted agents, such as bardoxolone methyl and sulforaphane, have been developed to prevent or slow down the progression of oxidative stress-related diseases. Nrf2 is a transactivator of genes containing an antioxidant response element (ARE) in their promoter. Such genes code for a number of antioxidative enzymes including NADPH: quinone oxidoreductase, glutathione S-transferases, aldo-keto reductases and heme oxygenase-1. Under normal physiological conditions, Nrf2 is anchored in the cytoplasm by binding to Kelchlike ECH-associated protein 1 (Keap1), which promotes the ubiquitination and subsequent proteolytic degradation of Nrf2; whereas both bardoxolone methyl and sulforaphane interact with cysteine residues on Keap1, allowing Nrf2 translocation to the nucleus and subsequent upregulation of a number of genes of antioxidative enzymes. Recently, the 52-Week Bardoxolene Methyl Treatment: Renal Function in CKD/Type 2 Diabetes (BEAM) study was carried out in patients with moderate to severe chronic kidney disease (CKD) and type 2 diabetes as a double-blind, randomized, placebo-controlled phase 2 clinical trial6. After 24 weeks of the treatment, the patients treated with bardoxolone methyl showed a significant increase in the mean estimated glomerular filtration rate compared with those treated with placebo. Although it is unknown whether bardoxolone methyl could reduce hyperglycemia-induced mitochondrial ROS, it was reported that heme oxygenase-1 regulates cardiac mitochondrial biogenesis through Nrf2-mediated transcriptional control of nuclear respiratory factor-1 (NRF-1). In addition, activation of Nrf2 by sulforaphane increased ARE-linked gene expression of transketolase and glutathione reductase, and ameliorated hyperglycemia-induced production of ROS, activation of hexosamine and PKC pathways, and prevented increased cellular accumulation and excretion of the glycating agent methylglyoxal. Furthermore, Nrf2 was reported to regulate promoter activity of the aldose reductase gene, which is the key enzyme of the polyol pathway. Because activation of Nrf2 might prevent hyperglycemia-induced ROS production and metabolic dysfunctions, such as activation of hexosamine, PKC and polyol pathways and accumulation of intracellular AGEs, bardoxolone methyl or sulforaphane might have promise for the treatment of diabetic complications (Figure 1). Considered together, although oxidative stress has been implicated in the pathology of diabetic complications, the efficacy of classical natural antioxidants in preventing diabetic complications is still uncertain. However, mechanism-based antioxidants, such as idebenone, mitoquinone, bardoxolone methyl and sulforaphane, have been developed. These mechanism-based strategies might suggest the potential for better treatment approaches to reduce the burden of oxidative stress and to prevent diabetic complications in clinical practice. In particular, because mitochondrial ROS production in response to hyperglycemia might be the central in the pathogenesis of diabetic complications, reduction of mitochondrial ROS might be the important therapeutic strategy to prevent diabetic complications.

Lipoic acid: a unique antioxidant in the detoxification of activated oxygen species

Cancer stem cells (CSCs) in a tumor bulk exhibit self-renewal capacity and contribute to tumor aggressiveness that leads to metastasis, cancer therapeutic resistance and relapse. The normal or tumor cells and CSCs are sensitive to the level of reactive oxygen species (ROS) in them. ROS are highly reactive molecules that are generated as byproducts of the endogenous reduction-oxidation (redox) metabolic reactions. In cells maintaining their redox homeostasis, the normal levels of ROS are involved in various cellular functions, including signaling. However, disruption of the redox homeostasis because of incompetent scavenging mechanism elevates the level of ROS, leading to oxidative stress and irreversible damage to the genomic DNA and/or other biomolecules. Subsequently, this results in altered gene expression, genomic instability, mutations, cellular transformation of normal cells to tumor cells or CSCs, survival of CSCs, and tumorigenesis. These aforementioned abnormalities are associated with increased rate of proliferation of tumor cells, hypoxia, angiogenesis, and epithelial to mesenchymal transition (EMT), tumor aggressiveness, and metastasis in several cancers. CSCs and tumor cells with the elevated levels of ROS, hijack various signaling molecules, thus alter multiple signaling cascades or molecular pathways including the stem cell regulatory pathways, which favor the invasiveness of these cells and their tumor microenvironment (TME). Importantly, the elevated levels of ROS can exhibit antitumorigenic activity to suppress tumorigenesis or protumorigenic activity to promote tumorigenesis, metastasis, and cancer treatment resistance and relapse. Moreover, the interactions between the elevated levels of ROS and CSCs create new networks with other signaling cascades, which ultimately leads to poor prognosis, therapeutic resistance and recurrence of cancer. In this chapter, we focus on the molecular interactions between ROS and CSCs and their effects on the tumor aggressiveness and TME. Furthermore, we discuss about the impact of elevated levels of ROS on the cellular transformation of normal or tumor cells to CSCs, cell signaling in CSCs and tumor cells, and their implications in cancer and its therapy.

Dextran-engineered Cu-MOF nanozyme with multi-enzyme mimetic cascade for cuproptosis-enhanced synergistic therapy in triple-negative breast cancer.

Cancer is a complex disease and is currently the leading cause of mortality and morbidity across the globe. Dysregulated bioenergetics is one of the hallmarks of cancer cells and is characterized by increased activity of several enzymes of metabolic pathways. Consequently, cancer cells produce higher levels of reactive oxygen species (ROS) which contribute to their enhanced proliferation and survival over normal cells. Elevated levels of ROS cause oxidative stress, redox imbalance, DNA damage, activation of oncogenes, chronic inflammation and eventually cancer. Additionally, ROS mediated oxidative stress activates several oncogenic signaling cascades including PI3K/Akt pathway, NF-κB pathway, cyclooxygenase pathway, JAK/STAT pathway, angiogenesis and metastasis. To maintain redox balance and neutralize the detrimental effects of ROS, normal cells exhibit an antioxidant defence system, comprising of both enzymatic and non-enzymatic division. Activation of Nrf2 signaling pathway is the key regulatory pathway that helps in restoring the cellular redox homeostasis. Extensive research in the past decades has witnessed the potential health benefits of dietary antioxidants alone or in combination in the prevention of several chronic diseases, including cancer. A number of antioxidants from dietary backgrounds such as epigallocatechin gallate, resveratrol, curcumin, phloretin, berberine and lycopene have shown appreciable potential as a chemopreventive agent without causing significant toxicity. This chapter presents an extensive analysis of existing knowledge on the protective effects of various dietary antioxidants against cancer with a focus on oxidative stress, redox homeostasis and dysregulated cellular signaling leading to cancer cell proliferation, survival and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related mortalities in the Western world and continues to be a major unresolvable health problem at the start of the 21st century. Resistance to the currently available treatment options has led to development of new approaches, such as personalized medicine and immunotherapy. However, new therapeutic strategies based on the unique molecular biology and physiology of pancreatic cancer hold the greatest promise. Glutathione S-transferase pi 1 (GSTP1) is a key detoxification enzyme which metabolizes xenobiotic compounds and byproducts of cellular metabolism. GSTP1 is overexpressed in many tumors, particularly ovarian, non-small cell lung, breast, colon, and pancreas. Moreover, GSTP1 is overexpressed in drug-resistant cancer cell lines. The reasons for increased expression ratios compared to normal tissues or wild-type cell lines are not well understood. To investigate the role of GSTP1 in PDAC pathogenicity, we generated two knockdown lines of GSTP1 in metabolically diverse PDAC cells. We showed that GSTP1 knockdown impairs the growth and proliferation of PDAC cells. Additionally, GSTP1 knockdown cells exhibit elevated reactive oxygen species (ROS) levels and a prolonged G0/G1 phase of the cell cycle. Intrigued by these results, we next examined whether pharmacological inhibition with a selective GSTP1 inhibitor, Ezatiostat (TLK199), also impaired PDAC pathogenicity. Ezatiostat is a small molecule drug and is novel glutathione analog, which selectively binds and inhibits GSTP1. Ezatiostat treatment in PDAC cells recapitulated the proliferation impairments observed with genetic inactivation of GSTP1 and elevated ROS levels. Orthotopic implantation of GSTP1 knockdown cells in athymic nude mice resulted in reduced tumor weight and volume compared to the control. The growth trajectory of the tumors was monitored via Vevo-3100 ultrasound imaging system. Our preliminary data indicate enhanced sensitivity of glycolytic cancer cells towards an ROS-inducing agent and a GSTP1 inhibitor, piperlongumine (PL). Interestingly, we have found PL is less cytotoxic to cells with reduced GSTP1 levels, indicating that PL primarily works by inhibiting GSTP1 activity. Together, these data suggest that GSTP1 knockdown and inhibition impairs the growth and survival of phenotypically diverse PDAC cells in vitro and in vivo. Moreover, GSTP1 knockdown results in elevated ROS levels and an extended G0/G1 phase of the cell cycle. With these data, we propose that GSTP1 is a novel therapeutic target for PDAC. Citation Format: Rahul Raj Singh, Katie M. Reindl. GSTP1 knockdown and inhibition impairs pancreatic ductal adenocarcinoma (PDAC) growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 764.

Phagocytic cells contain NADPH oxidase that they use for host defense by catalyzing the production of superoxide. Bacterial lipopolysaccharide (LPS) has been found to stimulate NADPH oxidase in mobile and sessile macrophages and microglia. It also evokes fever in homeothermic animals and men, a reaction mediated by central nervous system (CNS) activities. The purpose of the present study was to determine whether reactive oxygen species are involved in LPS-induced fever. In rabbits we found that plasma hydroperoxide levels increased and catalase activity decreased 15 min after LPS injection and that fever started with a similar latency, while plasma levels of tumor necrosis factor-alpha (TNFalpha) increased 30 min after the injection. Treating rabbits with methylene blue or aspirin did not affect TNFalpha secretion but prevented the LPS-induced rise of hydroperoxides and the inactivation of catalase, abolishing fever. Incubation of human blood with nitroblue tetrazolium and LPS increased the number of formazan-positive neutrophils from 10 +/- 5 to 52 +/- 9%. Adding LPS to blood preincubated with either methylene blue, alpha-lipoic acid, or aspirin respectively decreased the number of formazan-positive neutrophils to 0.9 +/- 0.8, 0.8 +/- 0.9, or 2.0 +/- 0.9%, disclosing the antioxidant capacity of these drugs. Systemic application of 80 mg/kg alpha-lipoic acid elicited heat-loss reactions within 15 min and decreased core temperature by 2.2 +/- 0.3 degrees C within 2 h. Alpha-lipoic acid applied 45 min after LPS induced antipyresis within 15 min, and this antipyresis was associated with a decrease of elevated hydroperoxide levels and restoration of catalase activity. Our results show that fever is prevented when the production of reactive oxygen species is blocked and that an elevated body temperature returns to normal when oxygen radical production decreases. Estimation of plasma dihydrolipoic acid (DHLA) levels following injection of 80 mg/kg alpha-lipoic acid in afebrile and febrile rabbits revealed that this acid is converted into DHLA, which in afebrile rabbits increased the plasma DHLA concentration from 2.22 +/- 0.26 microg/ml to peak values of 8.60 +/- 2.28 microg/ml DHLA within 30 min and which in febrile rabbits increased it from 0.84 +/- 0.22 microg/ml to peak values of 3.90 +/- 0.94 microg/ml within 15 min. Methylene blue, aspirin, and alpha-lipoic acid, which all cross the blood-brain barrier, seem to act not only on peripheral tissues but also on the CNS. Brain structures that have been shown to sense oxidative stress are vicinal thiol groups attached to the NMDA subtype of glutamate receptor. Their reduction by thiol-reducing drugs like dithiothreitol or DHLA has been found to increase glutamate-mediated neuronal excitability, while the opposite effect has been observed after their oxidation. Because we found that systemic application of alpha-lipoic acid in the afebrile state elicits hypothermia and in the febrile state is antipyretic, we think this type of NMDA receptor is involved in thermoregulation and that oxidation of its thiol groups induces fever. It appears that temperature homeostasis can be maintained only if the redox homeostasis of the brain is guaranteed.

The induction of senescence, an irreversible growth arrest, in cancer cells is regarded as a mean to halt tumor progression. The phytoalexin resveratrol (RV) is known to possess a variety of cancer-preventive, -therapeutic, and -chemosensitizing properties. We report here that chronic treatment with RV in a subapoptotic concentration induces senescence-like growth arrest in tumor cells. In contrast to the widely accepted antioxidant property of RV, we demonstrate that one causative stimulus for senescence induction by chronic RV is an increased level of reactive oxygen species (ROS). The ROS formed upon RV exposure include hydrogen peroxide and superoxide and originate largely from mitochondria. Consistently, co-incubation with the antioxidant N-acetyl cysteine interfered with RV-mediated reactivation of the senescence program. Molecular mediators on the way from increased ROS levels to the observed growth arrest include p38 MAPK, p53, and p21. Moreover, we provide evidence that RV-initiated replication stress, apparent by activation of the ataxia telangiectasia-mutated kinase pathway, is associated with increased ROS levels and senescence induction. This is the first report linking cell cycle effects with a pro-oxidant and pro-senescent effect of RV in cancer cells.