Synthesis, Structural Characterization, and Apoptosis Cell Death Facilitated Palladium(II) Schiff Base Complexes Derived From Salicylaldehyde and Aminoguanidine

The condensation reaction of 1,2-bis(2′-aminophenoxy)benzene with 2-pyridinecarbaldehyde in a mole ratio of 1:2 gives a new Schiff base ligand ( L ). Four Schiff base complexes, CoL(NO 3 ) 2 ( 1 ), NiLCl 2 ( 2 ), ZnL(NO 3 ) 2 ( 3 ) and Pd 2 LCl 4 ( 4 ) have been prepared by direct reaction of the ligand ( L ) and appropriate metal salts. The Schiff base ligand ( L ) has been characterized by IR, 1 H NMR and 13 C NMR spectroscopy and elemental analysis. Also, all complexes have been characterized by IR and XRD spectroscopy techniques and elemental analysis. The synthesized complexes have very poor solubility in all polar and non-polar solvents such as: H 2 O, MeOH, EtOH, CH 3 CN, DMSO, DMF, CHCl 3 , CH 2 Cl 2 , THF, etc; therefore, they have been used as heterogeneous catalysts. Catalytic performance of the complexes was studied in oxidation of thioanisole using hydrogen peroxide (H 2 O 2 ) as the oxidant. Various factors including the reaction temperature, amount of oxidant and catalyst amount were optimized. The palladium Schiff base complex, Pd 2 LCl 4 ( 4 ), shows better catalytic activity than other complexes. Therefore, the Pd(II) Schiff base complex has been used as a catalyst for oxidation of different sulfides to their corresponding sulfones in acetonitrile with hydrogen peroxide as the oxidant. The palladium Schiff base complex, Pd 2 LCl 4 ( 4 ), has shown a very good recyclability, up to five times, without any appreciable decreases in catalytic activity and selectivity.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs invivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

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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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.

Dinuclear Pd(II) complexes containing bis-O,N-bidentate Schiff base ligands: Synthesis, characterization, DFT study and application as Suzuki–Miyaura coupling catalysts

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.

Palladium is arguably the most versatile and most widely applied catalytic metal in the field of fine chemicals due to its high selectivity and activity. Palladium catalyst offers an abundance of possibilities of carbon-carbon bond formation in organic synthesis. In this research, three different Schiff base ligands have been prepared by condensation reaction between appropriate aldehyde or ketone with amine namely 2,2-dimethyl-1,3-propanediamine in the molar ratio of 2:1. The corresponding palladium (II) Schiff base complexes were prepared through the reaction between the Schiff base ligand with palladium (II) acetate in a molar ratio 1:1. FTIR, 1H-NMR and 13C-NMR spectroscopic data revealed that the ligands are N,N,O,O-tetradentate coordinated to the Pd atom through both the azomethine N atoms and phenolic O atoms. From X-ray Crystallographic analysis, it showed that the complex exists as square planar geometry. The synthesized palladium (II) Schiff base complexes were then subjected in catalytic Heck and Suzuki reaction of iodobenzene.

Benzyl isothiocyanate (BITC), a dietary cancer chemopreventive agent, causes apoptosis in MDA-MB-231 and MCF-7 human breast cancer cells, but the mechanism of cell death is not fully understood. We now demonstrate that the BITC-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) due to inhibition of complex III of the mitochondrial respiratory chain. The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase and pharmacological inhibition of the mitochondrial respiratory chain. The mitochondrial DNA-deficient Rho-0 variant of MDA-MB-231 cells was nearly completely resistant to BITC-mediated ROS generation and apoptosis. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of Bax. Biochemical assays revealed inhibition of complex III activity in BITC-treated MDA-MB-231 cells as early as at 1 h of treatment. The BITC treatment caused activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which function upstream of Bax activation in apoptotic response to various stimuli. Pharmacological inhibition of both JNK and p38 MAPK conferred partial yet significant protection against BITC-induced apoptosis. Activation of JNK and p38 MAPK resulting from BITC exposure was abolished by overexpression of catalase. The BITC-mediated conformational change of Bax was markedly suppressed by ectopic expression of catalytically inactive mutant of JNK kinase 2 (JNKK2(AA)). Interestingly, a normal human mammary epithelial cell line was resistant to BITC-mediated ROS generation, JNK/p38 MAPK activation, and apoptosis. In conclusion, the present study indicates that the BITC-induced apoptosis in human breast cancer cells is initiated by mitochondria-derived ROS.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

BackgroundCurrently available treatments for colorectal cancer (CRC) associate with numerous side-effects that reduce patients’ quality of life. The effective nutraceuticals with high anti-proliferative efficacy and low side-effects are desirable. Our previous study has reported that free fatty acids extract (FFAE) of krill oil induced apoptosis of CRC cells, possibly associated with changes in mitochondrial membrane potential (MMP). The aims of this study were to compare the anti-proliferative efficacy of FFAE from krill oil on CRC cells with commonly used chemotherapeutic drug, Oxaliplatin, and to investigate the molecular mechanisms underlying the anti-proliferative effects of krill oil with a focus on intrinsic mitochondrial death pathway.MethodsThree human CRC cell lines, including DLD-1, HT-29 and LIM-2405, and one mouse CRC cell line, CT-26, were treated with FFAE of KO and the bioactive components of krill oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for 24 h and 48 h. Similarly, these cell lines were treated with Oxaliplatin, a commonly used drug for CRC treatment, for 24 h. The effects of FFAE of KO, EPA, DHA and Oxaliplatin on cell proliferation, mitochondrial membrane potential and reactive oxygen species (ROS) were determined via WST-1, JC-10, and ROS assays respectively. The expression of caspase-3, caspase-9 and DNA damage following treatments of FFAE of KO was investigated via western blotting and immunohistochemistry.ResultsThe FFAE of KO, EPA and DHA significantly inhibited cell proliferation and increased formation of ROS in all four cell lines (P < 0.01). A small dose of FFAE from KO ranged from 0.06 μL/100 μL to 0.12 μL/100 μL containing low concentrations of EPA (0.13–0.52 μM) and DHA (0.06–0.26 μM) achieved similar anti-proliferative effect as Oxaliplatin (P > 0.05). Treatments with the FFAE of KO, EPA and DHA (2:1 ratio) resulted in a significant increase in the mitochondrial membrane potential (P < 0.001). Furthermore, the expression of active forms of caspase-3 and caspase-9 was significantly increased following the treatment of FFAE of KO.ConclusionsThe present study has demonstrated that the anti-proliferative effects of krill oil on CRC cells are comparable with that of Oxaliplatin, and its anti-proliferative property is associated with the activation of caspase 3/9 in the CRC cells.

ObjectiveThe first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin.Approach and ResultsTo address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-α, VCAM, IL-6, and IL-1β) or cellular composition (smooth muscle cell, macrophage, and T-cell content).ConclusionsAortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells

Radiotherapy is the primary treatment for nasopharyngeal carcinoma while radioresistance can hinder efficient treatment. To explore the role of annexin A1 and its potential mechanisms in radioresistance of nasopharyngeal carcinoma, human nasopharyngeal carcinoma cell line CNE2-sh annexin A1 (knockdown of annexin A1) and the control cell line CNE2-pLKO.1 were constituted and CNE2-sh annexin A1 xenograft mouse model was generated. The effect of annexin A1 knockdown on the growth of xenograft tumor after irradiation and radiation-induced DNA damage and repair was analyzed. The results of immunohistochemistry assays and Western blotting showed that the level of annexin A1 was significantly downregulated in the radioresistant nasopharyngeal carcinoma tissues or cell line compared to the radiosensitive nasopharyngeal carcinoma tissues or cell line. Knockdown of annexin A1 significantly promoted CNE2-sh annexin A1 xenograft tumor growth compared to the control groups after irradiation. Moreover, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays revealed that knockdown of annexin A1 significantly inhibited apoptosis in vivo compared to the control groups. We assessed the intracellular reactive oxygen species levels and the extent of radiation-induced DNA damage and repair using reactive oxygen species assay, comet assays, and immunohistochemistry assay. The results showed that knockdown of annexin A1 remarkedly reduced the intracellular reactive oxygen species levels, level of DNA double-strand breaks, and the phosphorylation level of H2AX and increased the accumulation of DNA-dependent protein kinase in nasopharyngeal carcinoma cells after irradiation. The findings suggest that knockdown of annexin A1 inhibits DNA damage via decreasing the generation of intracellular reactive oxygen species and the formation of γ-H2AX and promotes DNA repair via increasing DNA-dependent protein kinase activity and therefore improves the radioresistance in nasopharyngeal carcinoma cells. Together, our findings suggest that knockdown of annexin A1 promotes radioresistance in nasopharyngeal carcinoma and provides insights into therapeutic targets for nasopharyngeal carcinoma radiotherapy.

Acetaminophen (APAP) is a common over-the-counter medication used to treat pain and fever. It can be taken orally, topically, or intravenously and is considered a safe and effective drug when taken at therapeutic doses. However, recent research suggests that taking APAP regularly can result in increased blood pressure and cardiovascular risk. Previous publications found that Non-Steroidal-Anti-Inflammatory-Drugs (NSAIDs) increased Reactive Oxygen Species (ROS) in cardiomyocytes, resulting in cardiac dysfunction. Although APAP is not a NSAID, APAP likely has similar effects. We hypothesized that regular APAP usage resulted in increased ROS levels, leading to potential dysfunction in hearts and other organs. We used the embryonic rat heart cell line H9C2 to test our hypothesis. We conducted a ROS Assay using H9C2 cells treated with dichlorodihydrofluorescein diacetate (DFCDA). The cells were then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM hydrogen peroxide for 1.5 hours before measuring the ROS levels of the cells. We also measured the cell viability of H9C2 cells. Cells were treated with 10μM Alamar Blue, then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM or 200μM hydrogen peroxide for 48 hours before detecting the percentage of viable cells. The mitochondrial membrane potential (MMP) of cells treated with APAP was also measured. Cells were treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 250μM p-triflouromethoxyphenylhydrazone (FCCP) for 24 hours before being incubated with JC-10 for one hour. The cells were measured using a fluorescent reader at excitation/emission 490/525 nm and 540/590 nm. For the ROS assay, the cells treated with 100μM APAP, 200μM APAP, and 100μM Hydrogen Peroxide significantly increased total ROS compared to the control. The cell viability assay resulted in a significant decrease in percent viability in cells treated with 200μM APAP, 100μM hydrogen peroxide, and 200μM hydrogen peroxide compared to the control. The MMP Assay resulted in a significant decrease in MMP at all concentrations of APAP and 250μM FCCP compared to the control. These results suggest that treatment of H9C2 cardiac cells with physiological concentrations of APAP causes an increase in intracellular ROS levels, decreased cell viability, and decreased MMP. Overall, APAP causes mitochondrial dysfunction, which is likely to cause cardiomyocyte dysfunction. This research is funded by the NIEHS/Superfund Research Program (P42 ES004699). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The aim of this study was to optimize the formulation of a C60-modified self-microemulsifying drug delivery system loaded with triptolide (C60-SMEDDS/TP) and evaluate the cytoprotective effect of the C60-SMEDDS/TP on normal human cells. The C60-SMEDDS/TP exhibited rapid emulsification, an optimal particle size distribution of 50 ± 0.19 nm (PDI 0.211 ± 0.049), and a near-neutral zeta potential of -1.60 mV. The release kinetics of TP from the C60-SMEDDS/TP exhibited a sustained release profile and followed pseudo-first-order release kinetics. Cellular proliferation and apoptosis analysis indicated that the C60-SMEDDS/TP (with a mass ratio of TP: DSPE-PEG-C60 = 1:10) exhibited lower toxicity towards L02 and GES-1 cells. This was demonstrated by a higher IC50 (40.88 nM on L02 cells and 17.22 nM on GES-1 cells) compared to free TP (21.3 nM and 11.1 nM), and a lower apoptosis rate (20.8% on L02 cells and 26.3% on GES-1 cells, respectively) compared to free TP (50.5% and 47.0%) at a concentration of 50 nM. In comparison to the free TP group, L02 cells and GES-1 cells exposed to the C60-SMEDDS/TP exhibited a significant decrease in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM). On the other hand, the C60-SMEDDS/TP demonstrated a similar inhibitory effect on BEL-7402 cells (IC50 = 28.9 nM) and HepG2 cells (IC50 = 107.6 nM), comparable to that of the free TP (27.2 nM and 90.4 nM). The C60-SMEDDS/TP group also exhibited a similar intracellular level of ROS and mitochondrial membrane potential compared to the SMEDDS/TP and free TP groups. Fullerenol-Grafted Distearoyl Phosphatidylethanolamine-Polyethylene Glycol (DSPE-PEG-C60) was synthesized and applied in the self-microemulsifying drug delivery system. The C60-SMEDDS/TP was formulated using Cremophor EL, medium-chain triglycerides (MCT), PEG-400, and DSPE-PEG-C60, and loaded with triptolide (TP). The toxicity and bioactivity of the C60-SMEDDS/TP were assessed using normal human liver cell lines (L02 cells), normal human gastric mucosal epithelial cell lines (GES-1 cells), and liver cancer cell lines (BEL-7402 cells and HepG2 cells). The production of reactive oxygen species (ROS) after the C60-SMEDDS/TP treatment was assessed using 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The alterations in mitochondrial membrane potential (ΔψM) were assessed by measuring JC-1 fluorescence. The cytoprotection provided by the C60-SMEDDS/TP favored normal cells (L02 and GES-1) over tumor cells (BEL-7402 and HepG2 cells) in vitro. This suggests a promising approach for the safe and effective treatment of TP.