A single-center, retrospective study-spring-evaluating the efficacy and safety of recombinant human vascular endothelial inhibitor combined with anti-PD-1 in elderly patients aged 80 and above with NSCLC.

Aim: To investigate the efficacy and safety of combining Recombinant Human Endostatin Injection (marketed as Endo) with anti-PD-1 in elderly patients aged 80 and above with non-small cell lung cancer (NSCLC). Methods: Retrospective analysis of 181 patients with NSCLC aged 80 and above treated in the Department of Respiratory and Critical Care Medicine at Chaohu Hospital, affiliated with Anhui Medical University, from June 2019 to January 2024. Patients who received at least one cycle of combined Endo with anti-PD-1 were included based on inclusion criteria. Clinical and pathological data were collected, including complete blood count, liver and kidney function, electrocardiogram, coagulation function, thyroid function, cardiac enzymes, and whole-body imaging. Adverse events were recorded with a final follow-up on January 25, 2024. The primary endpoints were progression-free survival (PFS) and overall survival (OS), with safety as a secondary endpoint. Results: This study involved 14 elderly patients with NSCLC aged over 80. Median progression-free survival (mPFS) was 102 days, and median overall survival (mOS) was 311 days. Subgroup analyses based on treatment cycles showed a non-significant 441-day mPFS increase in the long-term group (≥6 cycles, 5 patients) compared to the short-term group (<6 cycles, 9 patients). However, the mOS in the long-term group significantly exceeded the short-term group by 141 days, with statistical significance (P=0.048). Further categorization revealed a 204-day shorter mPFS in the monotherapy maintenance group (Endo or Immunol) compared to the combination maintenance group (Endo combined with Immunol, 441 days). The mOS of the monotherapy maintenance group was longer (686 days) than the combination maintenance group (311 days), but no statistical significance (P= 0.710, 0.920). Throughout the treatment, 77 adverse events were recorded, mainly grade 1-2, with no new treatment-related reactions occurred. Overall, the safety of Endo combined with anti-PD-1 was considered good and manageable. Conclusion: The combination of Endo and anti-PD-1 could be an effective treatment choice for patients with NSCLC aged 80 and above.

[Se-ZnCu-65P enhances mouse peritoneal macrophage phagocytosis and inhibits secretion of NO and H2O2 in vitro].

OBJECTIVE: To investigate the effect of selenopeptide on phagocytosis, NO and H2O2 secretion of mouse peritoneal macrophages. METHODS: Mouse peritoneal macrophages induced by lipopolysaccharide (LPS) were cultured for 24 hours by various concentrations of Se-ZnCu-65P, which is a selenopeptide with double antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Then, the relative cell activity was determined by methyl thiazolyl tetrazolium (MTT) assays, the phagocytic ability of macrophages was evaluated by neutral red uptake assay, nitric oxide (NO) content was examined by nitrate reductase method, and hydrogen peroxide (H2O2) content was detected by molybdate colorimetric method. RESULTS: When Se-ZnCu-65P acted on macrophages alone, the relative cell viability was enhanced, the phagocytic ability was not affected, H2O2 content was reduced, and NO level was almost unchanged. However, the phagocytic ability of macrophages induced by LPS was raised, meanwhile, secretion of NO and H2O2 was promoted. When Se-ZnCu-65P acted on macrophages induced by LPS, the phagocytic ability were further enhanced, and secretion of NO and H2O2 was inhibited significantly. CONCLUSION: Se-ZnCu-65P can effectively improve the relative cell activity of mouse peritoneal macrophages induced by LPS, enhance the phagocytic ability of macrophages, and reduce the secretion levels of NO and H2O2.

The protective effects of 6-CySeCD with GPx activity against UVB-induced injury in HaCaT cells.

BACKGROUND: The generation of harmful reactive oxygen species (ROS) induced by UVB irradiation could induce cell apoptosis and change the cell cycle. 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-ß-cyclodextrin (6-CySeCD) is a novel glutathione peroxidase (GPx; EC 1.11.1.9) mimic. The aim of this study was to investigate the anti-oxidative effects of 6-CySeCD in cultured immortalised human keratinocyte cells (HaCaT). METHODS: HaCaT cells were treated with 30 mJ/cm(2) UVB to establish a damage model. The cultured HaCaT cells were randomly assigned to the control, UVB and treatment groups. The treatment group was incubated with 20 µmol/L of GPx mimics before UVB irradiation. Cell viability was detected by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the level of lipid peroxidation was determined by the formation of malondialdehyde (MDA), DNA fragmentation was observed using agarose gel electrophoresis and the levels of intracellular ROS and cell cycle progression were measured by flow cytometry. RESULTS: The levels of cytotoxicity, intracellular ROS, lipid peroxidation and oxidative DNA damage significantly increased after UVB irradiation in the HaCaT cells. UVB irradiation caused pre-G1 -phase arrest in HaCaT cells and significantly reduced the number of HaCaT cells in the S phase. The GPx mimics 6-CySeCD and 2-phenyl-l,2-benzisoselenazol-3(2H)-one (ebselen) significantly blocked UVB-induced apoptosis and changed the cell cycle of the HaCaT cells. The blocked effect of pretreatment 6-CySeCD in UVB-irradiated HaCaT cells was better than that of pretreatment with ebselen. CONCLUSION: 6-CySeCD can relieve the damage induced by UVB irradiation in HaCaT cells.

A novel 65-mer peptide imitates the synergism of superoxide dismutase and glutathione peroxidase.

Reactive oxygen species (ROS) are involved in cell growth, differentiation, and death. Excessive amounts of ROS (e.g., O(2)(-), H(2)O(2), and HO) play a role in aging as well as in many human diseases. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) are critical antioxidant enzymes in living organisms. SOD catalyzes the dismutation of O(2)(-) to H(2)O(2), and GPx catalyzes the reduction of H(2)O(2) and other harmful peroxides by glutathione (GSH). They not only function in catalytic processes but also protect each other, resulting in more efficient removal of ROS, protection of cells against injury, and maintenance of the normal metabolism of ROS. To imitate the synergism of SOD and GPx, a 65-mer peptide (65P), containing sequences that form the domains of the active center of SOD and the catalytic triad of GPx upon the incorporation of some metals, was designed on the basis of native enzyme structural models; 65P was expressed in the cysteine auxotrophic expression system to obtain Se-65P. Se-65P was converted into Se-CuZn-65P by incorporating Cu(2+) and Zn(2+). Se-CuZn-65P exhibited high SOD and GPx activities because it has a delicate dual-activity center. The synergism of the enzyme mimic was evaluated by using an in vitro model and a xanthine/xanthine oxidase/Fe(2+)-induced mitochondrial damage model system. We anticipate that the peptide enzyme mimic with synergism is promising for the treatment of human diseases and has potential applications in medicine as a potent antioxidant.

Antioxidant enzyme mimics with synergism.

The antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase contribute dominatingly to enhance cellular antioxidant defense against oxidative stress. They act cooperatively to scavenge reactive oxygen species, and not one of them can singlehandedly clear all forms of reactive oxygen species. On the basis of the structural understanding for these natural enzymes, many mimics with multifunctional activities had been obtained by chemical synthesis, biosynthesis, and protein fusion techniques. Some of them display remarkable antioxidant cooperative effect in living model which possess potential application in medicine as potent antioxidants. This review summarizes aspects of some multifunctional mimics which have been reported so far.

UV-B induced thymocytes apoptosis blocked by dicyclodextrinyl ditelluride-A GPX mimic.

Apoptosis is known to occur after ultraviolet-B (UV-B) radiation. It was found that UV-B could induce cell apoptosis and change cell cycle progression. After exposure to 100J/m(2) of UV-B, pre-G1 phase thymocytes were increased significantly and S phase thymocytes were decreased significantly. UV-B could also induce lipid peroxidation of thymocytes to have their MDA amount increased. These phenomena could be explained by production of reactive oxygen species (ROS), which were induced by UV-B radiation. In this study, we examined the protective effect of dicyclodextrinyl ditelluride (2-TeCD), the glutathione peroxidase (GPX, EC 1.11.1.9) mimics, on thymocytes apoptosis induced by UV-B radiation. The experimental results showed that 2-TeCD protects thymocytes from apoptosis. Moreover, 2-TeCD inhibits lipid peroxidation of thymocytes and displayed great antioxidant ability. Furthermore, 2-TeCD blocks the accumulation of wild-type-p53 (wt-p53) tumor-suppressor gene product caused by UV-B radiation.

Human catalytic antibody Se-scFv-B3 with high glutathione peroxidase activity.

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol.

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity.

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.

A novel selenium-containing glutathione transferase zeta1-1, the activity of which surpasses the level of some native glutathione peroxidases.

Glutathione peroxidase (GPX) is a critical antioxidant selenoenzyme in organisms that protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). Thus, some GPX mimics have been generated because of their potential therapeutic value. The generation of a semisynthetic selenoenzyme with peroxidase activity, which matches the catalytic efficiencies of naturally evolved GPX, has been a great challenge. Previously, we semisynthesized a GPX mimetic with high catalytic efficiency using a rat theta class glutathione transferase (rGST T2-2) as a scaffold, in which the highly specific GSH-binding site is adjacent to an active site serine residue that can be chemically modified to selenocysteine (Sec). In this study, we have taken advantage of a new scaffold, hGSTZ1-1, in which there are two serine residues in the active site, to achieve both high thiol selectivity and highly catalytic efficiency. The GPX activity of Se-hGSTZ1-1 is about 1.5 times that of rabbit liver GPX, indicating that the selenium content at the active site plays an important role in enhancement of catalytic performance. Kinetic studies revealed that the catalytic mechanism of Se-hGSTZ1-1 belong in a ping-pong mechanism similar to that of the natural GPX.

Screening of single-chain variable fragments against TSP50 from a phage display antibody library and their expression as soluble proteins.

A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

The molecular mechanism of protecting cells against oxidative stress by 2-selenium-bridged beta-cyclodextrin with glutathione peroxidase activity.

Ultraviolet B (UVB) induces apoptosis and lipid peroxidation of NIH3T3 cells by producing reactive oxygen species (ROS). Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in organism and it can scavenge ROS. 2-selenium-bridged beta-cyclodextrin (2-SeCD) is a GPX mimic generated in our lab. Its GPX activity is 7.4 U/mumol, which is 7.5 times as much as that of ebselen. In this paper, we have established a cell damage system using UVB radiation. Using this system, we have determined antioxidant effect of 2-SeCD by comparison of malondialdehyde (MDA) and H(2)O(2) contents in NIH3T3 cells before and after UVB radiation. Experimental results indicate that 2-SeCD can inhibit lipid peroxidation and protect the cells from the damage generated by UVB radiation. To evaluate the molecular mechanism of this protection, we determined the effect of 2-SeCD on the expression of p53 and Bcl-2 in NIH3T3 cells. The results showed that 2-SeCD inhibits the increase of p53 expression level and the decrease of expression of Bcl-2 induced by UVB radiation. Thus, we have concluded that protection of NIH3T3 cells against oxidative stress by 2-SeCD was carried out by regulation of the expression of Bcl-2 and p53.

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli.

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.

Selenium-containing 15-mer peptides with high glutathione peroxidase-like activity.

Glutathione peroxidase (GPX) is one of the most crucial antioxidant enzymes in a variety of organisms. Here we described a new strategy for generating a novel GPX mimic by combination of a phage-displayed random 15-mer peptide library followed by computer-aided rational design and chemical mutation. The novel GPX mimic is a homodimer consisting of a 15-mer selenopeptide with an appropriate catalytic center, a specific binding site for substrates, and high catalytic efficiency. Its steady state kinetics was also studied, and the values of k(cat)/K(m)(GSH) and k(cat)/ K(mH(2)O(2)) were found to be similar to that of native GPX and the highest among the existing GPX mimics. Moreover, the novel GPX mimic was confirmed to have a strong antioxidant ability to inhibit lipid peroxidation by measuring the content of malondialdehyde, cell viability, and lactate dehydrogenase activity. Importantly, the novel GPX mimic can penetrate into the cell membrane because of its small molecular size. These characteristics endue the novel mimic with potential perspective for pharmaceutical applications.

Protection of epidermal cells against UVB injury by the antioxidant selenium-containing single-chain Fv catalytic antibody.

The antioxidant effect of selenium-containing single-chain Fv catalytic antibody (Se-scFv2F3), a new mimic of glutathione peroxidase, was confirmed using a model system in which cultured rat skin epidermal cells were injured by ultraviolet B (UVB). The cell damage was characterized in terms of lipid peroxidation of the cells, cell viability, and cell membrane integrity. The injury effects of UVB and protection effects of Se-scFv2F3 on the cells were studied using the model system. UVB can damage the cells severely. Upon precultivation of the cells with 0.4U/ml Se-scFv2F3, however, the damage was significantly reduced as shown by the increase in cell viability, the decrease in the malondialdehyde and hydrogen peroxide levels, and the normalization of lactate dehydrogenase activity. In addition, a novel finding that Se-scFv2F3 can stimulate cultured epidermal cells to proliferate under certain conditions was observed.

UV-B induced keratinocyte apoptosis is blocked by 2-selenium-bridged beta-cyclodextrin, a GPX mimic.

Cell proliferation and cell death of keratinocytes are tightly regulated to ensure epidermal homeostasis. UV-B induces keratinocyte apoptosis. UV-B also induces lipid peroxidation of keratinocytes to increase their amount of malondialdehyde (MDA). These phenomena can be explained by the production of reactive oxygen species (ROS) induced by UV-B radiation. We synthesized 2-selenium-bridged beta-cyclodextrin (2-SeCD) to imitate glutathione peroxidase (GPX), an important antioxidant and established a damage system, in which keratinocytes can be damaged by Ultraviolet B (UV-B) radiation. Using this damage system we studied 2-SeCD protection of keratinocytes against injury induced by UV-B. Experimental results showed that 2-SeCD could protect keratinocytes from apoptosis. Moreover, 2-SeCD inhibits lipid peroxidation of keratinocytes and scavenges ROS. 2-SeCD inhibits the UV-B induced apoptotic signal transduction. This antiapoptotic mechanism may be partly related to the elimination of hydrogen peroxide.

The progress in mechanism of selenoprotein biosynthesis.

As the twenty-first amino acid, selenocysteine can be co-translationally incorporated into the polypeptide chain at UGA codon in the coding region of selenoprotein mRNA. The incorporation of selenocysteine needs a cis-acting element SECIS and four gene products: SelA, SelB, SelC and SelD. The position of SECIS in the mRNA of prokaryote and its structural features are greatly different from that of eukaryote. The researchers have made exploration in selenoprotein engineering by virtue of the mechanism of selenocysteine incorporation in Escherichia coli.

[Studies on the optimal expression condition, purification and its characterization of ScFv-2F3].

The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.