Abstract Real‐time monitoring of hydroxyl radical (⋅OH) generation is crucial for both the efficacy and safety of chemodynamic therapy (CDT). Although ⋅OH probe‐integrated CDT agents can track ⋅OH production by themselves, they often require complicated synthetic procedures and suffer from self‐consumption of ⋅OH. Here, we report the facile fabrication of a self‐monitored chemodynamic agent (denoted as Fc‐CD‐AuNCs) by incorporating ferrocene (Fc) into β‐cyclodextrin (CD)‐functionalized gold nanoclusters (AuNCs) via host–guest molecular recognition. The water‐soluble CD served not only as a capping agent to protect AuNCs but also as a macrocyclic host to encapsulate and solubilize hydrophobic Fc guest with high Fenton reactivity for in vivo CDT applications. Importantly, the encapsulated Fc inside CD possessed strong electron‐donating ability to effectively quench the second near‐infrared (NIR‐II) fluorescence of AuNCs through photoinduced electron transfer. After internalization of Fc‐CD‐AuNCs by cancer cells, Fenton reaction between redox‐active Fc quencher and endogenous hydrogen peroxide (H 2 O 2 ) caused Fc oxidation and subsequent NIR‐II fluorescence recovery, which was accompanied by the formation of cytotoxic ⋅OH and therefore allowed Fc‐CD‐AuNCs to in situ self‐report ⋅OH generation without undesired ⋅OH consumption. Such a NIR‐II fluorescence‐monitored CDT enabled the use of renal‐clearable Fc‐CD‐AuNCs for efficient tumor growth inhibition with minimal side effects in vivo.
The glutathione peroxidase Gpx3 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. Both gel-filtration and dynamic light-scattering (DLS) results indicate that Gpx3 is a monomer in solution at a concentration of about 2 mg ml(-1), whereas glutathione peroxidases are normally tetrameric or dimeric. X-ray diffraction data from a single crystal of Gpx3 have been collected to 2.6 A resolution. The crystals are triclinic and belong to space group P1, with unit-cell parameters a = 38.187, b = 43.372, c = 56.870 A, alpha = 71.405, beta = 73.376, gamma = 89.633 degrees. There are two Gpx3 monomers in a crystallographic asymmetric unit. Preliminary analyses show that the yeast Gpx3 is quite different from those of mammals.
Rationally constructing single-atom enzymes (SAEs) with superior activity, robust stability, and good biocompatibility is crucial for tumor therapy but still remains a substantial challenge. In this work, we adopt biocompatible carbon dots as the carrier material to load Ru single atoms, achieving Ru SAEs with superior multiple enzyme-like activity and stability. Ru SAEs behave as oxidase, peroxidase, and glutathione oxidase mimics to synchronously catalyze the generation of reactive oxygen species (ROS) and the depletion of glutathione, thus amplifying the ROS damage and finally causing the death of cancer cells. Notably, Ru SAEs exhibit excellent peroxidase-like activity with a specific activity of 7.5 U/mg, which surpasses most of the reported SAEs and is 20 times higher than that of Ru/C. Theoretical results reveal that the electrons of the Ru 4d orbital in Ru SAEs are transferred to O atoms in H2O2 and then efficiently activate H2O2 to produce •OH. Our work may provide some inspiration for the design of SAEs for cancer therapy.
The accumulation of reactive oxygen species (ROS) in aerobic organisms can cause oxidative stress which results in significant damages to cell constituents. These damages are believed to be related to a couple of degenerative diseases in human beings.1, 2 To eliminate the damages caused by ROS, cells have developed several defense mechanisms.3 As one of the key members during oxidative stress response, the yeast Saccharomyces cerevisiae Hyr1/YIR037W (formerly termed Gpx3) was reported to be a glutathione-dependent phospholipid peroxidase (PhGpx) that specifically detoxifies phospholipid peroxide.4 Moreover, Hyr1 has been identified to sense and transfer the oxidative signal to the transcription factor Yap1.5 Upon accumulation of H2O2, the Hyr1-Cys36 residue is oxidized to a cysteine sulfenic acid (Cys-SOH)6 and then forms a mixed disulfide bond with Yap1-Cys598. This disulfide bond is further resolved into a Yap1 Cys310-Cys598 intra-molecular disulfide bond, leaving a reduced Hyr1-Cys36 ready for the next cycle of signal sensing. When the oxidative stress is released, thioredoxin Trx2 can turn off this signal transduction pathway by reducing both Hyr1 and Yap1. In vivo experiments also suggested that Hyr1 is more likely an oxidative signal sensor and transducer, instead of a hydroperoxides scavenger.5 Compared to mammalian GPxs, Hyr1 has two distinct features. First, Hyr1 lacks an oligomerization loop corresponding to the tetramer interface of mammalian GPxs (except that human GPx4 is a monomeric enzyme lacking the tetrameric interface).7 Second, Hyr1 has a nonaligned second cysteine (Cys82) analogous to the resolving cysteine in the 2-Cys peroxiredoxins.8 Homology modeling suggests that these two features are important determinants to the thioredoxin specificity, which makes Hyr1 a thioredoxin-dependent peroxidase rather than a glutathione-dependent one as it was previously annotated.9 Here we reported the crystal structure of Hyr1 from Saccharomyces cerevisiae and activity assay of its active Cys82. Comprehensive structural analyses revealed that the Cys36 thiol adopts an orientation that favors the formation of Cys36-Cys82 intramolecular disulfide bond. The weak electron density of the Cys82-segment (residues 69–86) suggests a high degree of motion of this region, which might act as a mobile lid to control the recognition of substrates or protein partners. Furthermore, the enzymatic assay of wild-type Hyr1 and Cys82Ser mutant confirmed the importance of Cys82, consistent with previous reports.5 The open reading frame of Hyr1/YIR037W gene from Saccharomyces cerevisiae was cloned into a pET28a-derived vector. This construct adds a hexahistidine (6×His) tag to the N-terminus of the recombinant protein, which was overexpressed in E. coli Rosetta (DE3) strain using 2×YT culture medium (5 g of NaCl, 16 g of bactotrypton, and 10 g of yeast extract per liter). The cells were grown at 37°C up to an A600 nm of 0.6. Expression of recombinant Hyr1 was induced at exponential phase with 0.2 mM isopropyl-β-d-thiogalactoside (IPTG) and cell growth continued for another 20 h at 16°C before harvesting. Cells were collected by centrifugation and resuspended in lysis buffer (50 mM HEPES at pH 7.0, 250 mM NaCl). After 3 min of sonication and centrifugation, the supernatant containing the soluble target protein was collected and loaded to a Ni-NTA column (GE Healthcare) equilibrated with binding buffer (50 mM HEPES pH7.0, 250 mM NaCl). The target protein was eluted with a linear gradient of imidazole from 50 to 500 mM, and further purified by gel filtration in a Superdex 75 column (Amersham Biosciences) equilibrated with 50 mM HEPES pH 7.0 and 250 mM NaCl. The purified protein showed a single band in SDS-PAGE and the integrity was checked by mass spectrometry. The protein sample was concentrated to 1 mg/mL and the solvent-exposed lysines were methylated following the protocol published previously.10 The precipitated protein was removed by centrifugation before purification of the soluble fraction with a desalt column pre-equilibrated in 20 mM Tris-HCl pH 7.0, 50 mM NaCl, and 14 mM β-mercaptoethanol. Fractions containing the target protein were pooled and concentrated to 10 mg/mL. The crystals were grown at 289 K using the hanging drop vapor-diffusion techniques, with the initial condition by mixing 1 μL of the protein sample with equal volume of mother liquor (0.2M ammonium acetate, 0.1M sodium acetate, pH 4.6, 30% polyethylene glycol 4000). Typically, crystals appeared in 10 days. The crystal was transferred to the cryoprotectant of the reservoir solution supplemented with 15% glycerol and flash frozen with liquid nitrogen. The X-ray diffraction data were collected at 100 K in a liquid nitrogen stream using a Rigaku MM007 X-ray generator (λ = 1.5418) with a MarRearch 345 image-plate detector at School of Life Sciences, University of Science and Technology of China (USTC, Hefei, China). Data were processed with the Program MOSFLM11 and scaled with SCALA.12 The crystal structure of Hyr1 was solved by the molecular replacement method with the program MOLREP13 using the reduced form of poplar PtGpx5 (PDB code 2P5Q) as the search model. The dataset was severely anisotropic, with diffraction limits of 2.0 Å along the a* and b* directions, but only 2.80 Å along the c* direction. For this reason, the data was truncated along c* at 2.80 Å and scaled with the diffraction anisotropy server (see the Supporting Information table).14 Crystallographic refinement was performed using the program REFMAC5.15 After several rounds of manual rebuilding using the graphics program COOT,16 TLS parameters were analyzed with the TLSMD server and introduced in the refinement.17, 18 The water molecules were then placed into the electron density map, resulting in the final model. The structure was finally refined to 2.02 Å with the Rfactor 22.4% and Rfree 25.7%. The stereochemical quality of the final model was verified using the program PROCHECK.19 The structure factor and coordinate were deposited in the Protein Data Bank under the accession code of 3CMI. The final statistics and refinement parameters were listed in Table I. All figures of protein structure were prepared with PyMol.20 Peroxidase activity was monitored by the spectrometric determination of NADPH consumption at 340 nm following the previous method.5 The reaction was carried out in a buffer containing 100 mM Tris-HCl pH 8.0, 0.3 mM NADPH, and 2.68 μM yeast thioredoxin Trx2 and 0.18 μM yeast thioredoxin reductase Trr1. Purified Hyr1 was added to a 200 μL reaction mix to a final concentration of 1.35 μM, and the reaction was started 30 s later by adding H2O2 to 100 μM. The N-terminal 6×His tag and residues 69–86 including the resolving Cys82 can not be located because of weak electron density, suggesting a high degree of structural flexibility of these regions. The overall structure of Hyr1 adopts the typical thioredoxin-like fold,21 consisting of four β-strands clustered as the central β-sheet which is surrounded by three α-helices [Fig. 1(A)]. The crystal structure indicates that Hyr1 is a monomeric protein, which is consistent with our previous gel filtration result.22 Compared to mammalian tetrameric Gpx structures,23 Hyr1 lacks an oligomeration loop that is located at the C-terminus and an extended N-terminal 310 helix. Besides, the characteristic N-terminal β-hairpin in mammalian Gpxs or poplar Gpx5 is not preserved either.23-25 Hydrophobic residues near Cys36 such as Val32, Tyr42, Pro63, and Phe127 are partially exposed to solvent and form a hollow hydrophobic pocket, which suggests a potential role in the substrate binding. It is worth noticing that these residues are well conserved in the Gpx family, but they are shielded from solvent by helix α2 in mammalian Gpx structures and rPtGpx5 (see below for a detailed discussion). A: Cartoon representation of the overall fold of Hyr1, colored and labeled according to the secondary structures. Cys36 thiol was highlighted as sticks and the interrupted points were colored with purple and connected with a gray dashed line. B, C: Superposition of Hyr1 (red) upon rPtGpx5 (cyan) and oPtGpx5 (gray), respectively. The helix α2 of rPtGpx5 and peroxidatic and resolving cysteine of all structures were labeled. D: Hyr1 peroxidase activity assays. Assays were performed separately for three times in the presence of Trx2, Trr1, NADPH, and H2O2, with Hyr1 (filled squares), with Hyr1-Cys82Ser mutant (open triangles), without Hyr1 as a null control (filled circles). Data are expressed as the decrement of OD340 nm against time in minute. The error bars represent the standard deviations. The side chain of Cys36 is pointing toward the exterior of the protein and is solvent-exposed. Homology modeling and biochemical study of Hyr1 has suggested that Gln70 and Trp125 form hydrogen bonds with the Cys36 thiol in the reduced state and play an essential role in modulating the reactivity towards peroxide.6 However, in our structure the thiol of Cys36 is not hydrogen-bonded to any neighboring residues. The side chain of Trp125 is more than 10 Å away from the thiol of Cys36 and Gln70 is not positioned in the structure due to weak electron density. Compared to the mammalian homologue Gpx4 and reduced form of PtGpx5 (rPtGpx5) with sequence identity ranging from 44% to 50%,24, 25 the structure of Hyr1 undergoes large local conformation changes, especially at the loop between helix α1 and strand β1. Taking rPtGpx5 as the reference structure, the beginning of Hyr1 helix α1 undergoes a partial unwinding and rearrangement so that the main chain of Cys36 shift ∼6.0 Å toward the aligned helix α2 of rPtGpx5, with the thiol of Cys36 flipping over by ∼180° to the opposite and becoming more solvent-exposed [Fig. 1(B)]. The missing Cys82-segment corresponds to the rPtGpx5 helix α2, which is just aligned in the position to shield the hydrophobic pocket of Hyr1 from solvent. Superposition of Hyr1 onto the oxidized form of PtGpx5 (oPtGpx5) structure reveals that the Hyr1 Cys36 has a main chain shift of ∼3.7 Å from the oPtGpx5 Cys42, but the thiols of the two equivalent cysteines adopt the same orientation [Fig. 1(C)]. Moreover, oPtGpx5 helix α2 is completely unwound into less-ordered loop with a relatively higher B-factor,24 whereas in our structure this part shows weak electron density because of the high degree of motion. However, no obvious crystallographic evidence supports the presence of Cys36-Cys82 disulfide bond or Cys-SOH intermediate of Cys36 in Hyr1. Considering the lack of any reducing agent throughout purification and crystallization, the present crystal structure of Hyr1 may represent an intermediate state between the reduced and oxidized states, with the active site residues taking the conformations more favorable of forming Cys36-Cys82 disulfide bond. Taken together, the Cys82-segment of Hyr1 may act as a flexible lid covering the substrate binding site. Upon oxidation, it might be moved to facilitate the formation of intra-molecular disulfide bond, and/or the hydrophobic interaction with its substrate or protein partners, such as hydrophobic part of phospholipid peroxide or Yap1.4, 5 The equivalent parts of mammalian tetrameric Gpxs are not reported to show flexibility partially because they are involved in protein dimerization and thus stabilized.9 However, it remains unknown how the substrates or protein partners specifically interact with Hyr1 and what is the actual driven force of this flexible lid. To confirm whether the recombinant Gpx3 has a functional Cys82, we investigated the catalytic properties of Hyr1 and Cys82Ser mutant. In the presence of thioredoxin system (Trx2-Trr1-NADPH), wild-type Hyr1 could consume H2O2 rapidly, while the Cys82Ser mutant totally abolished the activity, indicating the important role of Cys82 in Trx2 specificity [Fig. 1(D)]. In summary, these results and previous reports5 further evidenced our structural analyses of the mobile active site. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Abstract Ferritin is an iron‐storage protein nanocage that is assembled from 24 subunits. The hollow cavity of ferritin enables its encapsulation of various therapeutic agents; therefore, ferritin has been intensively investigated for drug delivery. The use of antibody‐ferritin conjugates provides an effective approach for targeted drug delivery. However, the complicated preparation and limited protein stability hamper wide applications of this system. Herein, we designed a novel nanobody‐ferritin platform (Nb‐Ftn) for targeted drug delivery. The site‐specific conjugation between nanobody and ferritin is achieved by transglutaminase‐catalyzed protein ligation. This ligation strategy allows the Nb conjugation after drug loading in ferritin, which avoids deactivation of the nanobody under the harsh pH environment required for drug encapsulation. To verify the tumor targeting of this Nb‐Ftn platform, a photodynamic reagent, manganese phthalocyanine (MnPc), was loaded into the ferritin cavity, and an anti‐EGFR nanobody was conjugated to the surface of the ferritin. The ferritin nanocage can encapsulate about 82 MnPc molecules. This MnPc@Nb‐Ftn conjugate can be efficiently internalized by EGFR positive A431 cancer cells, but not by EGFR negative MCF‐7 cells. Upon 730 nm laser irradiation, MnPc@Nb‐Ftn selectively killed EGFR positive A431 cells by generating reactive oxygen species (ROS), whereas no obvious damage was observed on MCF‐7 cells. Given that ferritin can be used for encapsulation of various therapeutic agents, this work provides a strategy for facile construction of nanobody‐ferritin for targeted drug delivery.
Cellular immunotherapy has become a potential therapeutic method for different diseases.Herein, we reported clinical trial results of Cytokine-induced killer (CIK) cells used for patients with hepatitis B, cirrhosis and liver cancers from 2000 to 2015.Results showed CIK cell therapeutic effects were closely positively associated with CIK cell numbers, treated times and HBV genotypes.Different stages of HBV patients treated with > 10 10 CIK cells per time for more than ten times exhibited remarkable decrease of HBV DNA numbers (P < 0.01), ALT and AST gradually recovered to normal scope, cytokine factors such as IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-10, IL-22 and IL-27 exhibited obvious increase, lifespan of patients with cirrhosis and hepatocellular carcinoma were extended, and that all the patients felt better in sleep, diet and pain during the period of CIK therapy.In conclusion, CIK cell therapy is a good alternative therapeutic method and can be effectively used for treatment of different stages of HBV patients.
The combination of photodynamic therapy (PDT) and enzyme therapy is a highly desirable approach in malignant tumor therapies as it takes advantage of the spatial-controlled PDT and the effective enzyme-catalyzed bioreactions. However, it is a challenge to co-encapsulate hydrophilic enzymes and hydrophobic photosensitizers, and these two agents often interfere with each other. In this work, a protocell-like nanoreactor (GOx-MSN@MnPc-LP) has been designed for synergistic starvation therapy and PDT. In this nanoreactor, the hydrophilic glucose oxidase (GOx) is loaded in the pore of mesoporous silica nanoparticles (MSNs), while the hydrophobic manganese phthaleincyanide (MnPc) is loaded in the membrane layer of liposome. This spatial separation of two payloads protects GOx and MnPc from the cellular environment and avoids interference with each other. GOx catalyzes the oxidation of glucose, which generates hydrogen peroxide and gluconic acid, leading to the starvation therapy via glucose consumption in cancer cells, as well as the disruption of cellular redox balance. MnPc produces cytotoxic singlet oxygen under 730 nm laser irradiation, achieving PDT. The antitumor effects of the nanoreactor have been verified on tumor cells and tumor-bearing mice models. GOx-MSN@MnPc-LP efficiently inhibits tumor growth in vivo with a single treatment, indicating the robust synergy of starvation therapy and PDT treatment. This work also offers a versatile strategy for delivering hydrophilic enzymes and hydrophobic photosensitizers using a protocell-like nanoreactor for effective cancer treatment.
The aim of the present study was to identify potential human epidermal growth factor receptor 2 (HER2) amplification, according to American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) 2013 HER2 testing guidelines, in patients previously determined not to possess HER2 amplification, in accordance with previous 2007 guidelines. Potential discrepancies may arise from chromosome enumeration probe 17 (CEP17) amplification, deletion, polysomyor monosomy. HER2, CEP17, tumor protein p53 (TP53) and retinoic acid receptor α (RARA) genes from 67 patient specimens with suspected amplification, polysomy or monosomy of CEP17 were analyzed using fluorescence in situ hybridization. HER2 status was interpreted using 2007 and 2013 ASCO HER2 test guidelines as well as the reference genes TP53 and RARA. According to ASCO/CAP2007 HER2 guidelines, 20 patients exhibited HER2 amplification (29.85%), 41 were without HER2 amplification (including 25 with polysomy, 15 with monosomy and 1 with suspected monosomy plus co-amplification of HER2 and CEP17) and the remaining 6 patients were equivocal. Using ASCO/CAP 2013 HER2 guidelines, 49 patients exhibited HER2 gene amplification (73.1%). The 29-patient increase included 6 originally at equivocal levels but now demonstrating amplification, 22 originally with polysomy but now revealing co-amplification, and 1 with suspected monosomy plus co-amplification of HER2 and CEP17. According to TP53 and RARA, HER2 was amplified in 43 patients (64.1%). Using the revised guidelines, HER2, originally identified as amplified in 6 patients, was not amplified following the introduction of TP53 and RARA control genes. Among these 6, 4 possessed normal TP53 and RARA. The incidence of co-amplification of HER2 and CEP17 was 1.4% (21/1,518). RARA and TP53 are suitable control genes to evaluate HER2 status.
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