Lactic Acid Bacteria-Derived Postbiotics as Adjunctive Agents in Breast Cancer Treatment to Boost the Antineoplastic Effect of a Conventional Therapeutic Comprising Tamoxifen and a New Drug Candidate: An Aziridine-Hydrazide Hydrazone Derivative.

Breast cancer is associated with high mortality and morbidity rates. As about 20-30% of patients exhibiting ER-positive phenotype are resistant to hormonal treatment with the standard drug tamoxifen, finding new therapies is a necessity. Postbiotics, metabolites, and macromolecules isolated from probiotic bacteria cultures have been proven to have sufficient bioactivity to exert prohealth and anticancer effects, making them viable adjunctive agents for the treatment of various neoplasms, including breast cancer. In the current study, postbiotics derived from L. plantarum and L. rhamnosus cultures were assessed on an in vitro breast cancer model as potential adjunctive agents to therapy utilizing tamoxifen and a candidate aziridine-hydrazide hydrazone derivative drug. Cell viability and cell death processes, including apoptosis, were analyzed for neoplastic MCF-7 cells treated with postbiotics and synthetic compounds. Cell cycle progression and proliferation were analyzed by PI-based flow cytometry and Ki-67 immunostaining. Postbiotics decreased viability and triggered apoptosis in MCF-7, modestly affecting the cell cycle and showing a lack of negative impact on normal cell viability. Moreover, they enhanced the cytotoxic effect of tamoxifen and the new candidate drug toward MCF-7, accelerating apoptosis and the inhibition of proliferation. This illustrates postbiotics' potential as natural adjunctive agents supporting anticancer therapy based on synthetic drugs.

Deciphering the Role of miR-30a-5p and DLGAP1 Gene in Non-small Cell Lung Cancer.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide. Understanding the mechanisms of lung cancer development is vital for targeted therapy advancements. This article explores the little-known role of the guanylate kinase-associated protein (GKAP), encoded by the Disks large-associated protein 1 (DLGAP1) gene, in NSCLC along with assessing microRNA-30a-5p's influence on DLGAP1 gene expression in the A549 cell line. MATERIALS AND METHODS: Experiments were conducted on A549 cells transfected with synthetic oligonucleotides. The luciferase assay was employed to confirm the binding site of miR-30a-5p to the 3'UTR of DLGAP1 mRNA. The role of miRNA-30a-5p mimic in regulating potential target gene expression at the protein and mRNA levels was studied by performing RT-qPCR and western blot analyses. The effects of DLGAP1 knockdown and miRNA-30a-5p mimic on cell viability and the cell cycle were evaluated using the MTT test and flow cytometry with annexin/iodide cell staining. RESULTS: The luciferase assay indicated that miR-30a-5p has the ability to bind to the 3'UTR of DLGAP1 mRNA. RT-qPCR revealed that the overexpression of miR-30a-5p down-regulates DLGAP1 mRNA. Western blot analysis indicated that miR-30a-5p slightly reduces the level of the GKAP protein. Knockdown of DLGAP1 with synthetic oligonucleotides, as well as transfection with a miR-30a-5p mimic, significantly attenuates cell proliferation and increases the number of cells in the early and late stages of apoptosis. CONCLUSION: Our findings reveal the antiproliferative effect of miR-30a-5p and DLGAP1 gene knockdown on A549 cancer cells, implying that these elements could be considered as therapeutic targets for personalized medicine in NSCLC patients.

Autophagy Inhibition with Chloroquine Increased Pro-Apoptotic Potential of New Aziridine-Hydrazide Hydrazone Derivatives against Glioblastoma Cells.

Tumor therapy escape due to undesired side effects induced by treatment, such as prosurvival autophagy or cellular senescence, is one of the key mechanisms of resistance that eventually leads to tumor dormancy and recurrence. Glioblastoma is the most frequent and practically incurable neoplasm of the central nervous system; thus, new treatment modalities have been investigated to find a solution more effective than the currently applied standards based on temozolomide. The present study examined the newly synthesized compounds of aziridine-hydrazide hydrazone derivatives to determine their antineoplastic potential against glioblastoma cells in vitro. Although the output of our investigation clearly demonstrates their proapoptotic activity, the cytotoxic effect appeared to be blocked by treatment-induced autophagy, the phenomenon also detected in the case of temozolomide action. The addition of an autophagy inhibitor, chloroquine, resulted in a significant increase in apoptosis triggered by the tested compounds, as well as temozolomide. The new aziridine-hydrazide hydrazone derivatives, which present cytotoxic potential against glioblastoma cells comparable to or even higher than that of temozolomide, show promising results and, thus, should be further investigated as antineoplastic agents. Moreover, our findings suggest that the combination of an apoptosis inducer with an autophagy inhibitor could optimize chemotherapeutic efficiency, and the addition of an autophagy inhibitor should be considered as an optional adjunctive therapy minimizing the risk of tumor escape from treatment.

Antioxidant enzymes immobilized on gold and silver nanoparticles enhance DNA repairing systems of rat skin after exposure to ultraviolet radiation.

The aim of the study was to investigate in vivo whether the application of immobilized superoxide dismutase (SOD) and catalase (CAT) could enhance DNA repairing systems and reduce level of CPD (cyclobutane pyrimidine dimers) and 6-4PP ((6-4) pyrimidine-pyrimidone photoproducts), and whether the immobilization on gold (AuNPs) and silver (AgNPs) nanoparticles affects the outcome. The study presents secondary analysis of our previous research. Three-day application of SOD and CAT in all forms of solution decreased the levels of CPD and 6-4PP boosted by UV irradiation. The mRNA expression level of the nucleotide excision repair (NER) system genes (XPA, XPC, ERCC1, ERCC2, ERCC3, LIG1) increased after application of immobilized and free enzymes. Increased by UV irradiation, p53 mRNA expression level normalized with the enzyme application. In conclusion, application of free and immobilized antioxidant enzymes accelerates removal of harmful effects of UV radiation in the rat skin by increasing expression level of NER genes.

The effect of immobilized antioxidant enzymes on the oxidative stress in UV-irradiated rat skin.

Aim: Superoxide dismutase (SOD) and catalase (CAT) immobilized on gold nanoparticles (AuNP) and silver nanoparticles (AgNP) nanoparticles were used to reduce UV radiation-induced oxidative stress in rat skin. Materials & methods: The antioxidant influence of the enzymes was investigated on level of malondialdehyde, 8-hydroksy-2'deoksyguanozine, myeloperoxidase, total antioxidant capacity, SOD2 and CAT activity and expression, and glutathione and glutathione peroxidase activity. Results: The application of immobilized SOD and CAT on UV-irradiated skin reduced malondialdehyde and 8-hydroksy-2'deoksyguanozine levels also SOD and CAT activity and expression increased. The tested enzymes influence glutathione peroxidase activity and level of total antioxidant capacity and glutathione. Conclusion: Immobilized enzymes increased the antioxidative potential of skin following UV irradiation.

A Study of the Activity of Recombinant Mn-Superoxide Dismutase in the Presence of Gold and Silver Nanoparticles.

Superoxide dismutase (SOD) is one of the best characterized enzyme maintaining the redox state in the cell. A bacterial expression system was used to produce human recombinant manganese SOD with a His-tag on the C-end of the protein for better purification. In addition, gold and silver nanoparticles were chemically synthesized in a variety of sizes, and then mixed with the enzyme for immobilization. Analysis by dynamic light scattering and scanning transmission electron microscopy revealed no aggregates or agglomerates of the obtained colloids. After immobilization of the protein on AuNPs and AgNPs, the conjugates were analyzed by SDS-PAGE. It was determined that SOD was adsorbed only on the gold nanoparticles. Enzyme activity was analyzed in colloids of the gold and silver nanoparticles bearing SOD. The presence of a nanoparticle did not affect enzyme activity; however, the amount of protein and size of the gold nanoparticle did influence the enzymatic activity of the conjugate. Our findings confirm that active recombinant human superoxide dismutase can be produced using a bacterial expression system, and that the enzyme can be immobilized on metal nanoparticles. The interaction between enzymes and metal nanoparticles requires further investigation.

Comparison of the antioxidant activity of catalase immobilized on gold nanoparticles via specific and non-specific adsorption.

In this study, we present a comparison of the antioxidant activity of catalase immobilized on gold nanoparticles (AuNPs) by two methods: i) directly on the surface of AuNPs (non-specific immobilization), and ii) via chemical bonding using a linker (specific immobilization). Quantification of the enzyme amount adsorbed on the nanoparticle surface was determined by native-polyacrylamide gel electrophoresis (native-PAGE). Colloidal stability of AuNPs before and after the enzyme immobilization was monitored with dynamic light scattering (DLS) and UV-vis spectroscopy. The size of the metallic core was determined by scanning-transmission electron microscopy (STEM). The enzymatic activity of catalase immobilized on AuNPs was investigated by antioxidant tests and compared with free (non-immobilized) catalase. It was found that the activity of catalase immobilized on AuNPs is affected by the immobilization method. Moreover, it was found that the non-specific immobilization decreased the antioxidant activity while the specific immobilization of catalase allowed the catalase activity to remain at the same level as that of free catalase.

Immobilization of Recombinant Human Catalase on Gold and Silver Nanoparticles.

Human catalase cDNA was cloned into a pEX-C-His vector. Purified recombinant catalase was immobilized on nanoparticles. Gold and silver nanoparticles were synthesized in a variety of sizes by chemical reduction; no agglomerates or aggregates were observed in any of the colloids during dynamic light scattering or scanning transmission electron microscopy analysis. After immobilization on gold nanoparticles, recombinant catalase activity was found to be lower than that of the same amount of enzyme in aqueous solution. However, after 10 days of storage at room temperature, the activity of catalase immobilized on gold nanoparticles (AuNPs) of 13 and 20 nm and coverage of 133% was 68 and 83% greater than catalase in aqueous solution, respectively. During 10 days of experiment, percentage activity of catalase immobilized on those gold nanoparticles was higher in comparison to CAT in aqueous solution. Catalase immobilized on silver nanoparticles did not lose activity as significantly as catalase immobilized on AuNPs. Those results confirm the ability to produce recombinant human enzymes in a bacterial expression system and its potential use while immobilized on silver or gold nanoparticles.

Nanoparticles as Carriers of Proteins, Peptides and Other Therapeutic Molecules.

Nanoparticles have many applications both in industry and medicine. Depending upon their physical and chemical properties, they can be used as carriers of therapeutic molecules or as therapeutics. Nanoparticles are made of synthetic or natural polymers, lipids or metals. Their use allows for faster transport to the place of action, thus prolonging its presence in the body and limiting side effects. In addition, the use of such a drug delivery system protects the drug from rapid disintegration and elimination from the body. In recent years, the use of proteins and peptides as therapeutic molecules has grown significantly. Unfortunately, proteins are subject to enzymatic digestion and can cause unwanted immune response beyond therapeutic action. The use of drug carriers can minimize undesirable side effects and reduce the dose of medication needed to achieve the therapeutic effect. The current study presents the use of several selected drug delivery systems for the delivery of proteins, peptides and other therapeutic molecules.