It is known that prolonged cell passaging may affect the outcomes of cell-based experiments. This study aimed to comprehensively investigate the consequences of long-term culture of U87MG glioblastoma cells, focusing on cell metabolism, morphology, tumorigenicity and drug response. U87MG (L) cells subjected to long-term culturing (20 passages) displayed altered morphology and significantly enhanced autofluorescence of lipofuscin, a well-established marker of oxidative stress and cellular senescence, compared with control U87MG cells maintained for only 5 passages. Fluorescence-lifetime imaging microscopy (FLIM) of NADH revealed the metabolic shift likely associated with glycolysis in long-term cultured U87MG (L) cells. Using the genetically encoded sensor HyPer7, the increase in basal intracellular levels of hydrogen peroxide in U87MG (L) cells was detected. In vivo studies employing orthotopic intracranial xenotransplantation in immunodeficient NSG mice revealed that, unlike short-term cultured U87MG cells, which formed visible tumor nodules with clearly delineated margins, long-term cultured U87MG (L) cells infiltrated diffusely into brain tissues, invaded bone tissue, and exhibited perineural growth. Such an aggressive behavior resulted in worse survival outcomes of xenograft-bearing mice. Notably, U87MG (L) cells became less susceptible to temozolomide, but acquired sensitivity to death receptor 5 (DR5)-selective variant of cytokine TRAIL in vitro and in vivo due to increased DR5 expression on the cell surface and downregulation of cFLIP expression. Our findings with the U87MG cell line indicate that culture duration can alter cellular responses, thereby impacting experimental outcomes. It should be carefully considered when establishing tumor models and evaluating efficacy of potential drug candidates.

Tumor angiogenesis promotes tumor growth, metastasis and disease progression. Different cancer types vary in their angiogenic potential, which may influence prognosis and response to therapy. In the present work, we established xenograft models of three of the most aggressive types of human cancers: glioblastoma U87MG, gastric cancer MKN-45, and pancreatic cancer MIA PaCa-2, in immunodeficient mice. The study of vascular network by optoacoustic microangiography revealed the highest degree of vascularization in U87MG xenografts, and the lowest in MIA PaCa-2 xenografts. As shown by PAS-CD31 dual staining, U87MG-derived tumors also showed the highest expression of the endothelial marker CD31 as well as the highest vasculogenic mimicry capacity. In line with this, metabolic imaging by fluorescence Lifetime Imaging Microscopy (FLIM) of nicotinamide adenine dinucleotide (NADH) revealed that MIA PaCa-2 xenografts were the most glycolytic, whereas U87MG had higher levels of oxidative phosphorylation, and MKN-45 showed intermediate values. Therefore, when creating animal models with xenografted tumors, it is important to understand the angiogenic potential of cancer cells, especially for studying drug candidates with an antiangiogenic effect. Also, the combination of optoacoustics and immunohistochemical analysis with FLIM imaging allows for a comprehensive assessment of both vascularization and the metabolic state of the tumor, which can help predict the therapeutic response.

Anti-angiogenic therapy is a clinically validated method for cancer treatment. It was previously revealed that concurrent targeting of angiogenic and death receptor signaling pathways by a multivalent DR5-specific cytokine TRAIL variant DR5-B genetically fused with the effector peptides, SRH-DR5-B-iRGD, enhances solid tumor suppression and prolongs survival. The SRH peptide is aimed at blocking the tumor neoangiogenesis by preventing activation of the VEGFR2 receptor, while the iRGD peptide interferes with the activation of integrin αvβ3, and enhances the tumor penetration. Here, we investigated how the antiangiogenic activity of the SRH-DR5-B-iRGD fusion protein contributes to its antitumor effects. An integrated approach has been applied involving molecular modeling of SRH-DR5-B-iRGD binding to DR5 receptor, optoacoustic (OA) and optical coherence tomography-based microangiography (OCT-MA) imaging of the vessel networks in xenografts of human glioblastoma and pancreatic adenocarcinoma in nude mice, supported by immunohistochemical (IHC) staining for vascularization marker CD31, and in vitro and in vivo bioactivity studies. Molecular modeling has demonstrated that genetic fusion of DR5-B with the SRH and iRGD peptides not only enables the engagement of additional tumor targets VEGFR2 and integrin αvβ3/NRP-1, but also improves the interaction with DR5 receptor. OA imaging of the vessel network in xenograft tumor nodes of human glioblastoma and pancreatic adenocarcinoma displayed a decrease in the vessel fraction in DR5-B-treated xenograft tumors, with the effect being even more pronounced in SRH-DR5-B-iRGD-treated tumor nodes. This data was consistent with the reduction in the number of perfused vessels in DR5-B and SRH-DR5-B-iRGD-treated tumors as quantified by OCT-MA, and also correlate well with the data obtained by IHC staining and tumor growth inhibition. Ameliorated interaction with the DR5 receptor and imparting antiangiogenic properties to the multivalent fusion protein SRH-DR5-B-iRGD resulted in improved antitumor activity compared to DR5-B. Thereby, SRH-DR5-B-iRGD can be considered as a promising candidate for the treatment of vascularized solid tumors.

The cytokine TRAIL is distinguished by its remarkable ability to preferentially induce apoptosis in transformed, but not in normal, cells. The recombinant TRAIL extracellular domain and other first-generation agonists of DR4 and DR5 death receptors (DRs) have shown very limited antitumor activity in clinical trials. To enhance the antitumor effect, we developed the multitarget recombinant fusion protein SRH-DR5-B-p48 based on the DR5-selective TRAIL variant DR5-B to simultaneously affect tumor cells (DR5-B-mediated apoptosis) and tumor microenvironment, in particular, to suppress angiogenesis. For this purpose, we modeled and produced the recombinant SRH-DR5-B-p48 fusion protein containing antagonistic synthetic peptides (SRH and p48) to VEGFR2 and FGFR1 receptors, respectively. Analysis of molecular trajectories using molecular dynamics methods showed that the SRH and p48 peptides form non-specific temporary contacts with the DR5-B domain. Using enzyme-linked immunosorbent assay, we showed that SRH-DR5-B-p48 was similar to DR5-B in its affinity for the death receptor DR5 and demonstrated a high affinity for VEGFR2 and FGFR1 with nanomolar dissociation constants. SRH-DR5-B-p48 killed tumor cells of various origin more efficiently than DR5-B and destroyed tumor-like structures in 3D cell models, as well as inhibited FGF2-mediated stimulation of fibroblast proliferation. Therefore, the SRH-DR5-B-p48 fusion protein can be considered as a promising agent for the therapy of solid tumors of various origin.

Nanosized carriers based on amphiphilic poly(N-vinylpyrrolidone) (Amph-PVP) are a versatile delivery system for various therapeutic agents such as anti-inflammatory drugs and plasmid DNA, as well as targeted antitumor drugs and proteins. Earlier, we developed Amph-PVP-based nanoparticles decorated by a modified DR5-specific TRAIL variant DR5-B (PVP-DR5-B) or containing the proteasomal inhibitor bortezomib (PVP-BTZ). Both DR5-B and BTZ have antitumor properties and, when combined, act synergistically on tumor cells. In the present study, Amph-PVP nanoparticles were loaded with BTZ and subsequently decorated with the TRAIL variant DR5-B, producing a dual polymeric bionanocomposite system PVP-BTZ-DR5-B. Using 2D and 3D in vitro cultures of human glioblastoma cell lines U87MG and T98G, it was demonstrated that PVP-BTZ-DR5-B nanoparticles were internalized and accumulated in cells more efficiently, demonstrating significantly enhanced cytotoxicity compared to free DR5-B or PVP-BTZ nanoparticles loaded with bortezomib alone. PVP-BTZ-DR5-B nanoparticles also penetrated the blood-brain barrier more efficiently than DR5-B in an in vitro model. Finally, the enhanced antitumor effect of PVP-BTZ-DR5-B was demonstrated in a xenograft model of U87MG glioblastoma cells in zebrafish embryos in vivo. Thereby, coloading of BTZ and DR5-B into the Amph-PVP nanoparticles is a promising approach to enhance the antitumor efficacy of free drugs and overcome glioblastoma resistance.

TRAIL (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand) receptor pathway is an important component of the immune system participating in surveillance and selective elimination of transformed cells. Therefore, TRAIL-based proteins and other agonists of TRAIL death receptors are promising drug candidates for the treatment of malignant tumors and autoimmune diseases. Initially, in the first series of clinical trials, drugs targeting the death receptors DR4 or DR5, did not reveal significant anticancer activity. The reasons for this were multiple TRAIL resistance mechanisms that tumors evolve to evade the efficient induction of apoptotic signaling. However, a wide range of novel TRAIL death receptor-targeted formulations are currently being developed, mainly aimed at improving stability, enhancing death receptor clustering and engagement of additional tumor targets. Over the past two decades, several dozens of multitargeted fusion proteins with either TRAIL protein or DR5-specific agonistic monoclonal antibodies have been created to improve therapeutic efficacy. These include fusions with either short peptide tags or large functional proteins, as well as antibody fragments targeting molecular pathways involved in angiogenesis or proliferative signaling such as EGFR, VEGFR, PD-L1, etc. Collectively, these multimodal proteins deal with enhancing the activation of extrinsic and intrinsic apoptotic pathways in transformed cells, as well as affecting the tumor microenvironment. This comprehensive review aims to systematize the bispecific and multivalent fusion proteins and conjugates targeting TRAIL death receptors, analyze the molecular mechanisms by which they overcome tumor resistance to TRAIL, and assess clinical prospects. Bibliography — 242 references.

Therapy-induced senescence plays a crucial role in cancer treatment, evolving the resistance of malignant cells to therapeutic interventions. Therefore, the discovery of safe and effective senolytics may serve to develop new promising therapeutic options. Here we have revealed the enhanced apoptotic potential of DR5 receptor agonist, receptor-selective TRAIL variant DR5-B in temozolomide-induced senescent glioblastoma cell lines U87MG and T98G and primary tumor samples from patients with diagnosed glioblastoma. Senescence features were most pronounced in p53-proficient, MGMT-deficient U87MG cells, as demonstrated by enlarged cell and nuclei size, increased β-galactosidase activity, p21 expression and lipofuscin autofluorescence. This was accompanied by a metabolic shift towards glycolysis measured by fluorescence lifetime imaging (FLIM) of NADH, and upregulation of DR5, DcR1, DcR2 and cFLIP. As a result, a strong sensitization of U87MG cells to DR5-B-mediated apoptosis was observed after TMZ pre-treatment. However, neither was observed in p53-mutated, MGMT-proficient T98G cells. Differential gene expression analysis in TMZ-treated U87MG cells showed the activation of proinflammatory and proapoptotic signaling and downregulation of genes related to DNA metabolism and cell cycle. Two of three primary patient-derived glioblastoma samples tested acquired similar senescence features and were sensitized to DR5-B-mediated apoptosis by TMZ pre-treatment. These findings suggest that TMZ-induced senescence enhances glioblastoma cell sensitivity to DR5 receptor agonists. However, when developing strategies for senolytic antitumor therapy, the heterogeneous response of tumor cells to senescence induction should be taken into account.

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 αvβ3 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.

In the original publication [...].

Glutamine plays an important role in tumor metabolism. It is known that the core region of solid tumors is deprived of glutamine, which affects tumor growth and spread. Here we investigated the effect of glutamine deprivation on cellular metabolism and sensitivity of human glioblastoma cells U87MG and T98G to drugs of various origin: alkylating cytostatic agent temozolomide; cytokine TRAIL DR5-B – agonist of the DR5 receptor; and GMX1778 – a targeted inhibitor of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), limiting NAD biosynthesis. Bioinformatics analysis of the cell transcriptomes showed that U87MG cells have a more differentiated phenotype than T98G, and also differ in the expression profile of the genes associated with glutamine metabolism. Upon glutamine deprivation, growth rate of the U87MG and T98G cells decreased. Analysis of cellular metabolism by FLIM microscopy of NADH as well as assessment of lactate content in the medium showed that glutamine deprivation shifted metabolic status of the U87MG cells towards glycolysis. This was accompanied by the increase in expression of the stemness marker CD133, which collectively could indicate de-differentiation of these cells. At the same time, we observed increase in both expression of the DR5 receptor and sensitivity of the U87MG cells to DR5-B. On the contrary, glutamine deprivation of T98G cells induced metabolic shift towards oxidative phosphorylation, decrease in the DR5 expression and resistance to DR5-B. The effects of NAMPT inhibition also differed between the two cell lines and were opposite to the effects of DR5-B: upon glutamine deprivation, U87MG cells acquired resistance, while T98G cells were sensitized to GMX1778. Thus, phenotypic and metabolic differences between the two human glioblastoma cell lines caused divergent metabolic changes and contrasting responses to different targeted drugs during glutamine deprivation. These data should be considered when developing treatment strategies for glioblastoma via drug-mediated deprivation of amino acids, as well as when exploring novel therapeutic targets.

Proteasome inhibitor bortezomib is an anticancer agent approved for treatment of multiple myeloma and mantle cell lymphoma. However, its application in other types of cancer, primarily in solid tumors, is limited due to poor pharmacokinetics, inefficient tissue penetration, low stability and frequent adverse effects. In the present study, a novel micellar nano-scaled delivery system was manufactured, composed of amphiphilic poly(N-vinylpyrrolidone) nanoparticles loaded with bortezomib. Similar nanoparticles loaded with prothionamide, a drug without anticancer effect, were used as control. The size and zeta potential of the obtained polymeric micelles were measured by dynamic light scattering. Bortezomib-loaded micelles exhibited significant cytotoxic activity in vitro in monolayer tumor cell cultures (IC50 similar to 6.5 mu g/ml) and in 3D multicellular tumor spheroids (IC50 similar to 8.5 mu g/ml) of human glioblastoma cell lines U87 and T98G. Additionally, the toxic effects in vivo were studied in zebrafish Danio rerio embryos, with an estimated 50% lethal concentration of 0.1 mg/ml. Considering that bortezomib and other molecules from the class of proteasome inhibitors are potent antitumor agents, nanodelivery approach can help reduce adverse effects and expand the range of its applications for treatment of various oncological diseases.

Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was −4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 μM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.

Recently, biodegradable polyelectrolyte multilayer capsules (PMC) have been proposed for anticancer drug delivery. In many cases, microencapsulation allows to concentrate the substance locally and prolong its flow to the cells. To reduce systemic toxicity when delivering highly toxic drugs, such as doxorubicin (DOX), the development of a combined delivery system is of paramount importance. Many efforts have been made to exploit the DR5-dependent apoptosis induction for cancer treatment. However, despite having a high antitumor efficacy of the targeted tumor-specific DR5-B ligand, a DR5-specific TRAIL variant, its fast elimination from a body limits its potential use in a clinic. A combination of an antitumor effect of the DR5-B protein with DOX loaded in the capsules could allow to design a novel targeted drug delivery system. The aim of the study was to fabricate PMC loaded with a subtoxic concentration of DOX and functionalized with the DR5-B ligand and to evaluate a combined antitumor effect of this targeted drug delivery system in vitro. In this study, the effects of PMC surface modification with the DR5-B ligand on cell uptake both in 2D (monolayer culture) and 3D (tumor spheroids) were studied by confocal microscopy, flow cytometry and fluorimetry. Cytotoxicity of the capsules was evaluated using an MTT test. The capsules loaded with DOX and modified with DR5-B demonstrated synergistically enhanced cytotoxicity in both in vitro models. Thus, the use of the DR5-B-modified capsules loaded with DOX at a subtoxic concentration could provide both targeted drug delivery and a synergistic antitumor effect.

The TRAIL (TNF-related apoptosis-inducing ligand) apoptotic pathway is extensively exploited in the development of targeted antitumor therapy due to TRAIL specificity towards its cognate receptors, namely death receptors DR4 and DR5. Although therapies targeting the TRAIL pathway have encountered many obstacles in attempts at clinical implementation for cancer treatment, the unique features of the TRAIL signaling pathway continue to attract the attention of researchers. Special attention is paid to the design of novel nanoscaled delivery systems, primarily aimed at increasing the valency of the ligand for improved death receptor clustering that enhances apoptotic signaling. Optionally, complex nanoformulations can allow the encapsulation of several therapeutic molecules for a combined synergistic effect, for example, chemotherapeutic agents or photosensitizers. Scaffolds for the developed nanodelivery systems are fabricated by a wide range of conventional clinically approved materials and innovative ones, including metals, carbon, lipids, polymers, nanogels, protein nanocages, virus-based nanoparticles, dendrimers, DNA origami nanostructures, and their complex combinations. Most nanotherapeutics targeting the TRAIL pathway are aimed at tumor therapy and theranostics. However, given the wide spectrum of action of TRAIL due to its natural role in immune system homeostasis, other therapeutic areas are also involved, such as liver fibrosis, rheumatoid arthritis, Alzheimer’s disease, and inflammatory diseases caused by bacterial infections. This review summarizes the recent innovative developments in the design of nanodelivery systems modified with TRAIL pathway-targeting ligands.

Autoinduction is a simple approach for heterologous protein expression that helps to achieve the high-level production of recombinant proteins in soluble form. In this work, we investigated if the application of an autoinduction strategy could help to optimize the production of bifunctional protein SRH-DR5-B, the DR5-specific TRAIL variant DR5-B fused to a VEGFR2-specific peptide SRHTKQRHTALH for dual antitumor and antiangiogenic activity. The protein was expressed in Escherichia coli SHuffle B T7, BL21(DE3), and BL21(DE3)pLysS strains. By IPTG induction, the highest expression level was in SHuffle B T7, while by autoinduction, the similar expression level was achieved in BL21(DE3)pLysS. However, in SHuffle B T7, only 45

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

[This corrects the article DOI: 10.3389/fcell.2021.733688.].

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH–DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH–DR5-B can be considered as a promising candidate for therapeutic applications.