Correction: Yagolovich et al. DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma. Int. J. Mol. Sci. 2022, 23, 12687.

In the original publication [...].

Investigation of Inter- and Intratumoral Heterogeneity of Glioblastoma Using TOF-SIMS.

Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity because of the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

The Action of the Pulsed Electric Field of the Subnanosecond Range on Human Tumor Cells.

The action of the pulsed electric field of the subnanosecond range on Jurkat, HEK 293, and U-87 MG human cell lines was studied. The cells were treated in a waveguide in 0.18 ml electrodeless Teflon cuvettes. The electric field strength in the cell culture medium was ~2 kV/cm, the pulse duration was ~1 ns, the leading edge was 150 ps, the frequency was 100 Hz, and the treatment time was 5 min. According to estimates, the change of the transmembrane potential during the pulse was ~20 mV and we assume that it was insufficient for electroporation. Jurkat and HEK 293 cells appeared to be more resistant to the treatment than U-87 MG cells. We have observed that the impulses with the above-mentioned parameters can cause a noticeable change in the mitochondrial activity of U-87 MG cells. © 2022 Bioelectromagnetics Society.

Liposomal Formulation of a PLA2-Sensitive Phospholipid-Allocolchicinoid Conjugate: Stability and Activity Studies In Vitro.

To assess the stability and efficiency of liposomes carrying a phospholipase A2-sensitive phospholipid-allocolchicinoid conjugate (aC-PC) in the bilayer, egg phosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylglycerol-based formulations were tested in plasma protein binding, tubulin polymerization inhibition, and cytotoxicity assays. Liposomes L-aC-PC10 containing 10 mol. % aC-PC in the bilayer bound less plasma proteins and were more stable in 50% plasma within 4 h incubation, according to calcein release and FRET-based assays. Liposomes with 25 mol. % of the prodrug (L-aC-PC25) were characterized by higher storage stability judged by their hydrodynamic radius evolution yet enhanced deposition of blood plasma opsonins on their surface according to SDS-PAGE and immunoblotting. Notably, inhibition of tubulin polymerization was found to require that the prodrug should be hydrolyzed to the parent allocolchicinoid. The L-aC-PC10 and L-aC-PC25 formulations demonstrated similar tubulin polymerization inhibition and cytotoxic activities. The L-aC-PC10 formulation should be beneficial for applications requiring liposome accumulation at tumor or inflammation sites.

DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma.

TRAIL (TNF-related apoptosis-inducing ligand) and its derivatives are potentials for anticancer therapy due to the selective induction of apoptosis in tumor cells upon binding to death receptors DR4 or DR5. Previously, we generated a DR5-selective TRAIL mutant variant DR5-B overcoming receptor-dependent resistance of tumor cells to TRAIL. In the current study, we improved the antitumor activity of DR5-B by fusion with a tumor-homing iRGD peptide, which is known to enhance the drug penetration into tumor tissues. The obtained bispecific fusion protein DR5-B-iRGD exhibited dual affinity for DR5 and integrin αvß3 receptors. DR5-B-iRGD penetrated into U-87 tumor spheroids faster than DR5-B and demonstrated an enhanced antitumor effect in human glioblastoma cell lines T98G and U-87, as well as in primary patient-derived glioblastoma neurospheres in vitro. Additionally, DR5-B-iRGD was highly effective in a xenograft mouse model of the U-87 human glioblastoma cell line in vivo. We suggest that DR5-B-iRGD may become a promising candidate for targeted therapy for glioblastoma.

Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2.

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients' tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.

Dual targeting of DR5 and VEGFR2 molecular pathways by multivalent fusion protein significantly suppresses tumor growth and angiogenesis.

Destroying tumor vasculature is a relevant therapeutic strategy due to its involvement in tumor progression. However, adaptive resistance to approved antiangiogenic drugs targeting VEGF/VEGFR pathway requires the recruitment of additional targets. In this aspect, targeting TRAIL pathway is promising as it is an important component of the immune system involved in tumor immunosurveillance. For dual targeting of malignant cells and tumor vascular microenvironment, we designed a multivalent fusion protein SRH-DR5-B-iRGD with antiangiogenic VEGFR2-specific peptide SRH at the N-terminus and a tumor-targeting and -penetrating peptide iRGD at the C-terminus of receptor-selective TRAIL variant DR5-B. SRH-DR5-B-iRGD obtained high affinity for DR5, VEGFR2 and αvß3 integrin in nanomolar range. Fusion of DR5-B with effector peptides accelerated DR5 receptor internalization rate upon ligand binding. Antitumor efficacy was evaluated in vitro in human tumor cell lines and primary patient-derived glioblastoma neurospheres, and in vivo in xenograft mouse model of human glioblastoma. Multivalent binding of SRH-DR5-B-iRGD fusion efficiently stimulated DR5-mediated tumor cell death via caspase-dependent mechanism, suppressed xenograft tumor growth by >80 %, doubled the lifespan of xenograft animals, and inhibited tumor vascularization. Therefore, targeting DR5 and VEGFR2 molecular pathways with SRH-DR5-B-iRGD protein may provide a novel therapeutic approach for treatment of solid tumors.

The Nitro Group Reshapes the Effects of Pyrido[3,4-g]quinazoline Derivatives on DYRK/CLK Activity and RNA Splicing in Glioblastoma Cells.

Serine-threonine protein kinases of the DYRK and CLK families regulate a variety of vital cellular functions. In particular, these enzymes phosphorylate proteins involved in pre-mRNA splicing. Targeting splicing with pharmacological DYRK/CLK inhibitors emerged as a promising anticancer strategy. Investigation of the pyrido[3,4-g]quinazoline scaffold led to the discovery of DYRK/CLK binders with differential potency against individual enzyme isoforms. Exploring the structure-activity relationship within this chemotype, we demonstrated that two structurally close compounds, pyrido[3,4-g]quinazoline-2,10-diamine 1 and 10-nitro pyrido[3,4-g]quinazoline-2-amine 2, differentially inhibited DYRK1-4 and CLK1-3 protein kinases in vitro. Unlike compound 1, compound 2 efficiently inhibited DYRK3 and CLK4 isoenzymes at nanomolar concentrations. Quantum chemical calculations, docking and molecular dynamic simulations of complexes of 1 and 2 with DYRK3 and CLK4 identified a dramatic difference in electron donor-acceptor properties critical for preferential interaction of 2 with these targets. Subsequent transcriptome and proteome analyses of patient-derived glioblastoma (GBM) neurospheres treated with 2 revealed that this compound impaired CLK4 interactions with spliceosomal proteins, thereby altering RNA splicing. Importantly, 2 affected the genes that perform critical functions for cancer cells including DNA damage response, p53 signaling and transcription. Altogether, these results provide a mechanistic basis for the therapeutic efficacy of 2 previously demonstrated in in vivo GBM models.

Survivin monomer plays an essential role in apoptosis regulation.

Survivin was initially described as an inhibitor of apoptosis and attracted growing attention as one of the most tumor-specific genes in the human genome and a promising target for cancer therapy. Lately, it has been shown that survivin is a multifunctional protein that takes part in several crucial cell processes. At first, it was supposed that survivin functions only as a homodimer, but now data indicate that many processes require monomeric survivin. Moreover, recent studies reveal a special mechanism regulating the balance between monomeric and dimeric forms of the protein. In this paper we studied the mutant form of survivin that was unable to dimerize and investigated its role in apoptosis. We showed that survivin monomer interacts with Smac/DIABLO and X-linked inhibitor of apoptosis protein (XIAP) both in vitro and in vivo. Due to this feature, it protects cells from caspase-dependent apoptosis even more efficiently than the wild-type survivin. We also identified that mutant monomeric survivin prevents apoptosis-inducing factor release from the mitochondrial intermembrane space, protecting human fibrosarcoma HT1080 cells from caspase-independent apoptosis. On the other hand, our results indicate that only wild-type survivin, but not the monomer mutant form, enhances tubulin stability in cells. These findings suggest that survivin partly performs its functions as a monomer and partly as a dimer. The mechanism of dimer-monomer balance regulation may also work as a "switcher" between survivin functions and thereby explain remarkable functional diversities of this protein.

Detection of Transglutaminase 2 conformational changes in living cell.

Transglutaminase 2 (TG2) is a ubiquitous Ca(2+)-dependent protein cross-linking enzyme that is implicated in a variety of biological disorders. In in vitro experiments when Ca(2+) concentration was increased TG2 changed its conformation and was able to cross-link other proteins via formation of an isopeptide bond. However the mechanisms that regulate TG2 transamidation activity in cells are still unknown. In this study we have developed FRET-based method for monitoring TG2 conformation changes and, probably, cross-linking activity in living cells. Using this approach we have showed that a significant amount of TG2 within the cell is accumulated in perinuclear endosomes and has a cross-linking inactive conformation, while TG2 that is located beneath the cell membrane has a transamidation active conformation. After the induction of apoptosis cytoplasmic TG2 changed its conformation and activates while, TG2 in endosomes retained transamidation inactive conformation even at late stages of apoptosis.

Chelation of Zinc with Biogenic Amino Acids: Description of Properties Using Balaban Index, Assessment of Biological Activity on Spirostomum Ambiguum Cellular Biosensor, Influence on Biofilms and Direct Antibacterial Action.

The complexation of biogenic molecules with metals is the widespread strategy in screening for new pharmaceuticals with improved therapeutic and physicochemical properties. This paper demonstrates the possibility of using simple QSAR modeling based on topological descriptors for chelates study. The presence of a relationship between the structure (J) and lipophilic properties (logP) of zinc complexes with amino acids, where two molecules coordinate the central atom through carboxyl oxygen and amino group nitrogen, and thus form a double ring structure, was predicted. Using a cellular biosensor model for Gly, Ala, Met, Val, Phe and their complexes Zn(AA)2, we experimentally confirmed the existence of a direct relationship between logP and biological activity (Ea). The results obtained using topological analysis, Spirotox method and microbiological testing allowed us to assume and prove that the chelate complex of zinc with methionine has the highest activity of inhibiting bacterial biofilms, while in aqueous solutions it does not reveal direct antibacterial effect.

Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors.

Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, we demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, we identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.