Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS.

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

Potential of Novel Methyl Jasmonate Analogs as Anticancer Agents to Metabolically Target HK-2 Activity in Glioblastoma Cells.

Change in the energy metabolism of cancer cells, which display significant differences compared to normal cells, is a rising phenomenon in developing new therapeutic approaches against cancers. One of the metabolic enzymes, hexokinase-II (HK-II) is involved in glycolysis, and inhibiting the HK-II activity may be a potential metabolic target for cancer therapy as most of the drugs in clinical use act on DNA damage. Methyl jasmonate (MJ) is one of the compounds blocking HK-II activity in cancer cells. In a previous study, we showed that the novel MJ analogs inhibit HK-II activity through VDAC detachment from the mitochondria. In this study, to evaluate the potential of targeting HK-2 activity, through patient cohort analysis, we first determined HK-2 expression levels and prognostic significance in highly lethal glioblastoma (GBM) brain tumor. We then examined the in vitro therapeutic effects of the novel analogs in the GBM cells. Here, we report that, among all, compound-10 (C-10) showed significant in vitro therapeutic efficacy as compared to MJ which is in use for preclinical and clinical studies. Afterward, we analyzed cell death triggered by C-10 in two different GBM cell lines. We found that C-10 treatment increased the apoptotic/necrotic cells and autophagy in GBM cells. The newly developed analog, C-10, was found to be lethal against GBM by the activation of cell death authorities, mostly in a necrotic and autophagic fashion at the early stages of the treatment. Considering that possibly decreased intracellular ATP levels by C-10 mediated inhibition of HK-2 activity and disabled VDAC interaction, a more detailed analysis of HK-2 inhibition-mediated cell death can provide a deep understanding of the mechanism of action on the oncosis/necroptosis axis. These findings provide an option to design clinically relevant and effective novel HK-II inhibitors and suggest novel MJ analogs to further study them as potential anticancer agents against GBM.

Design and synthesis of novel caffeic acid phenethyl ester (CAPE) derivatives and their biological activity studies in glioblastoma multiforme (GBM) cancer cell lines.

Glioblastoma Multiforme (GBM) is the most aggressive brain tumor and classified as one of the deadliest cancers. The current treatment plans for GBM remains to be ineffective because of its rapid progress and inability of the drugs used to cross the blood-brain barrier (BBB). Thus, developing more effective and potent medicines for GBM are needed. There have been several reports demonstrating that CAPE presents reasonably good anti-cancer activity in certain cancer cell lines and can penetrate the blood-brain barrier. Accordingly, in this study we synthesized several novel CAPE analogs with the addition of more druggable handles and solubilizing entities and subsequently evaluated their in vitro therapeutic efficacies in GBM cell lines (T98G and LN229). The most potent compound was then examined extensively and results showed that the 50 µM novel CAPE analog (compound 10) significantly decreases the viability of both T98G and LN229 GBM cells as compared to CAPE itself. Moreover, the compound 10 was not cytotoxic to healthy human cells (fibroblast-like mesenchymal stem cells) at the same concentration. Apoptotic (32.8%, and 44.6%) cell populations were detected in the compound 10 treated groups for LN229 and T98G, respectively. As an indication of apotosis, significantly increased PARP cleavage was detected in compound 10 versus CAPE treated LN229. In addition, we conducted molecular docking and molecular dynamics (MD) simulations studies on certain targets playing roles on GBM disease pathway such as NF-κB, EGFR, TNF-α, ERK2, PAPR1, hCA IX and hCA XII. Our findings demonstrated that designed CAPE analogs have anti-cancer activity on GBM cells and in silico studies also demonstrate the inhibitory ability of suggested compounds via interactions with critical residues in binding pockets of studied targets. Here, we suggest the novel CAPE analog to study further against GBM. Therefore, identification of the compound related molecular signature may provide more to understand the mechanism of action.

Mesenchymal Stem Cell-Based COVID-19 Therapy: Bioengineering Perspectives.

The novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Mesenchymal stem cells (MSCs) are currently utilized in clinics for pulmonary inflammatory diseases, including acute respiratory distress syndrome and acute lung injury. Given that MSCs offer a promising treatment against COVID-19, they are being used against COVID-19 in more than 70 clinical trials with promising findings. Genetically engineered MSCs offer promising therapeutic options in pulmonary diseases. However, their potential has not been explored yet. In this review, we provide perspectives on the functionally modified MSCs that can be developed and harnessed for COVID-19 therapy. Options to manage the SARS-CoV-2 infection and its variants using various bioengineering tools to increase the therapeutic efficacy of MSCs are highlighted.

IL13 Fused Pseudomonas Exotoxin Targets Various Cancers In Vitro.

BACKGROUND/AIM: Pseudomonas exotoxin (PE) is one of the most widely used toxins in the construction of therapeutic fusion proteins in pre-clinical studies followed by phase trials. In principle, PE acts by blocking protein synthesis through catalyzing the inactivation of elongation factor-2 (EF-2). The interleukin-13 fused PE (IL13-PE) cytotoxin was previously designed to target GBM cells. In this study, the cytotoxic effects of IL13-PE were evaluated in 5 different types of cancers and the therapeutic effects were further analyzed in a lung cancer cell line, NCI-H460. Conceptually, in another lung cancer cell line (A549), IL13Rα2 was overexpressed by lentiviruses (A549-IL13Rα2) and evaluated for cytotoxic efficacy of IL13-PE. MATERIALS AND METHODS: The expression profile of IL13Rα2 in different cancer cell lines was determined by RT-PCR. Secretable toxin fusion was expressed in the toxin resistant HEK-293T cell line (293T-TxR) by using a plasmid coding for IL13-PE and IRES-GFP (LV-IL13-PE-IRES/GFP). Next, the cells were shown to produce and secrete functional IL13-PE by dot blot analysis, followed by cell viability assays and cell death analysis. RESULTS: Upon treatment with IL13-PE, a significant decrease in cell viability was selectively demonstrated in cancer cells with cognate receptor expression. IL13-PE treatment increased the apoptotic/necrotic cell populations in the NCI-H460 cell line. CONCLUSION: Our results demonstrate that IL13-PE can be a therapeutic target for tumors bearing mostly IL13Rα2 positive cell populations. Our findings also suggest a cell-based delivery option for the recombinant toxins in the treatment of different cancers which can provide a solution for the clinical use of toxin therapy.

IL13Rα2­ and EGFR­targeted pseudomonas exotoxin potentiates the TRAIL­mediated death of GBM cells.

Glioblastomas (GBMs) are refractory to current treatments and novel therapeutic approaches need to be explored. Pro­apoptotic tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) is tumor­specific and has been shown to induce apoptosis and subsequently kill GBM cells. However, approximately 50% of GBM cells are resistant to TRAIL and a combination of TRAIL with other therapeutics is necessary to induce mechanism­based cell death in TRAIL­resistant GBMs. The present study examined the ability of the tumor cell surface receptor, interleukin (IL)­13 receptor α2 (IL13Rα2)­ and epidermal growth factor receptor (EGFR)­targeted pseudomonas exotoxin (PE) to sensitize TRAIL­resistant GBM cells and assessed the dual effects of interleukin 13­PE (IL13­PE) or EGFR nanobody­PE (ENb­PE) and TRAIL for the treatment of a broad range of brain tumors with a distinct TRAIL therapeutic response. Receptor targeted toxins upregulated TRAIL death receptors (DR4 and DR5) and suppressed the expression of anti­apoptotic FLICE­inhibitory protein (FLIP) and X­linked inhibitor of apoptosis protein (XIAP). This also led to the induction of the cleavage of caspase­8 and caspase­9 and resulted in the sensitization of highly resistant established GBM and patient­derived GBM stem cell (GSC) lines to TRAIL­mediated apoptosis. These findings provide a mechanism­based strategy that may provide options for the cell­mediated delivery of bi­functional therapeutics to target a wide spectrum of TRAIL­resistant GBMs.

Integrin α5ß1 Mediated Cellular Reorganization in Human Mesenchymal Stem Cells During Neuronal Differentiation.

BACKGROUND/AIM: Mesenchymal stem cells (MSCs) have been widely used for yielding neurons in culture to study nervous system pathologies and develop regenerative approaches. In this study, cellular rearrangements of human MSCs related to the expression of the fibronectin common receptor integrin α5ß1 and its cell surface localization during neuronal differentiation, were examined. MATERIALS AND METHODS: Proliferation kinetics of neuronal induced hMSCs (hMd-Neurons) were quantified by BrdU assay, and hMd-Neurons were immunostained for neuronal marker expression. Additionally, cDNA and protein samples were collected at different time points for integrin α5ß1 expression analysis. RESULTS: Endogenous integrin α5ß1 expression was significantly upregulated by day 6 and maintained until day 12. Cell surface localization of α5ß1 integrin was increased by day 6; the integrin was internalized into the cytosol by day 12. CONCLUSION: Integrin dynamics around day 6 of differentiation might be involved in neuronal differentiation and maturation or specification of hMd-Neurons.

Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity.

Mesenchymal stem cells have the ability to transdifferentiate into neurons and therefore one of the potential adult stem cell source for neuronal tissue regeneration applications and understanding neurodevelopmental processes. In many studies on human mesenchymal stem cell (hMSC) derived neurons, success in neuronal differentiation was limited to neuronal protein expressions which is not statisfactory in terms of neuronal activity. Established neuronal networks seen in culture have to be investigated in terms of synaptic signal transmission ability to develop a culture model for human neurons and further studying the mechanism of neuronal differentiation and neurological pathologies. Accordingly, in this study, we analysed the functionality of bone marrow hMSCs differentiated into neurons by a single step cytokine-based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (≥75%) as demonstrated by Ca++ imaging. Furthermore, when electrically stimulated, hMSC derived neurons (hMd-Neurons) matched the response of a typical neuron in the process of maturation. Our results reveal that a combination of neuronal inducers enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. Conceptually, we suggest these functional hMd-Neurons to be used as a tool for disease modelling of neuropathologies and neuronal differentiation studies.

Synthesis of novel methyl jasmonate derivatives and evaluation of their biological activity in various cancer cell lines.

Warburg hypothesized that the energy consumption of cancer cells is different than the normal cells. When compared to normal conditions, cancer cells do not undergo tricarboxylic acid (TCA) cycle therefore resulting in more lactate in the cells. Glycolysis pathway is a way of cancer cells to provide energy. The first step in glycolysis is the phosphorylation of glucose to glucose-6-phosphate. This reaction is catalyzed by the hexokinase-II enzyme (HK-II) which is known to be overexpressed in tumor cells. The feeding of cancer cells can be prevented by inhibiting the hexokinase-II enzyme in the first step of aerobic glycolysis. In literature, Methyl Jasmonate (MJ) is known as a Hexokinase-II inhibitor since it disposes VDAC and HK-II interaction on mitochondrial membrane. In our study, we aimed to increase the activity by synthesizing the novel MJ analogues with appropriate modifications. Here we report Hexokinase-2 enzyme and cell viability study results in different cancer cells. Based on the three different cancer cell lines we investigated, our novel MJ analogues proved to be more potent than the original molecule. Thus this research may provide more efficacious/novel HK-II inhibitors and may shed light to develop new anti-cancer agents.

Antioxidant Activity of Blackthorn (Prunus spinosa L.) Fruit Extract and Cytotoxic Effects on Various Cancer Cell Lines.

OBJECTIVE: Blackthorn (Prunus spinosa L. (Rosaceae) is a shrup whose fruits are consumed as food in Turkey. This study was aimed to evaluate antioxidant activity of methanol extract of P. spinosa and its cytotoxic effects on cancer cell lines. METHOD: Methanol extract of P. spinosa fruit was evaluated for its in vitro cytotoxic activity on multiform (GBM) brain cancer (LN229, U87 and T98G) and pancreas cancer (PANC-1 and AsPC-1) cell lines. Cell viability assays were performed by calculating the percentage of viable cells using a luminescence system, and spectrophotometrically. measuring its antioxidant ABTS and DPPH radical scavenging activities. Differences were considered as statistically significant at p*<0.001 and p**<0.0005 according to unpaired student t-test. RESULTS: Methanol extract of P. spinosa fruit showed 2548±18 mg GAE/100 g correspon-ding to the total phenolic content, and moderate antioxidant activity (0.1896±0.1143 and 0.0729±0.0348) by ABTS� and DPPH� assays. CONCLUSION: To the best of our knowledge, after evaluating the results of brain and pancreas cancer cell lines, significant cytotoxic activities with 50-63% cell viability of GBM brain cancer cells were determined while no cytotoxicity was observed on pancreas cancer cell lines, PANC-1; and AsPC-1. The results of this study showed that the methanol extract of P. spinosa fruit has significant antioxidant capacity and leads to statistically significant decreased viability on glioblastoma brain cancer cells.

DNA topoisomerase IIß as a molecular switch in neural differentiation of mesenchymal stem cells.

Two isoforms of DNA topoisomerase II (topo II) have been identified in mammalian cells, named topo IIα and topo IIß. Topo IIα plays an essential role in segregation of daughter chromosomes and thus for cell proliferation in mammalian cells. Unlike its isozyme topo IIα, topo IIß is greatly expressed upon terminal differentiation of neuronal cells. Although there have been accumulating evidence about the crucial role of topo IIß in neural development through activation or repression of developmentally regulated genes at late stages of neuronal differentiation, there have been no reports that analyzed the roles of topo IIß in the neural trans differentiation process of multipotent stem cells. Terminal differentiation of neurons and transdifferentiation of Mesenchymal Stem Cells (MSCs) are two distinct processes. Therefore, the functional significance of topo IIß may also be different in these differentiation systems. MSC transdifferentiation into neuron-like cells represents an useful model to further validate the role of topo IIß in neuronal differentiation. The aim of this study is to evaluate the subset of genes that are regulated in neural transdifferentiation of bone marrow-derived human MSCs (BM-hMSCs) in vitro and find genes related with topo IIß. For this purpose, topo IIß was silenced by specific small interfering RNAs in hMSCs and cells were induced to differentiate into neuron-like cells. Differentiation and silencing of topo IIß were monitored by real-time cell analysis and also expressions of topo II isoforms were analyzed. Change in transcription patterns of genes upon topo IIß silencing was identified by DNA microarray analysis, and apparently genes involved in regulation of several ion channels and transporters, vesicle function, and cell calcium metabolism were particularly affected by topo IIß silencing suggesting that topoIIß silencing can significantly alter the gene expression pattern of genes involved in variety of biological processes and signal transduction pathways including transcription, translation, cell trafficking, vesicle function, transport, cell morphology, neuron guidance, growth, polarity, and axonal growth. It appears that the deregulation of these pathways may contribute to clarify the further role of topo IIß in neural differentiation.

Engineering toxin-resistant therapeutic stem cells to treat brain tumors.

Pseudomonas exotoxin (PE) potently blocks protein synthesis by catalyzing the inactivation of elongation factor-2 (EF-2). Targeted PE-cytotoxins have been used as antitumor agents, although their effective clinical translation in solid tumors has been confounded by off-target delivery, systemic toxicity, and short chemotherapeutic half-life. To overcome these limitations, we have created toxin-resistant stem cells by modifying endogenous EF-2, and engineered them to secrete PE-cytotoxins that target specifically expressed (interleukin-13 receptor subunit alpha-2) or overexpressed (epidermal growth factor receptor) in glioblastomas (GBM). Molecular analysis correlated efficacy of PE-targeted cytotoxins with levels of cognate receptor expression, and optical imaging was applied to simultaneously track the kinetics of protein synthesis inhibition and GBM cell viability in vivo. The release of IL13-PE from biodegradable synthetic extracellular matrix (sECM) encapsulated stem cells in a clinically relevant GBM resection model led to increased long-term survival of mice compared to IL13-PE protein infusion. Moreover, multiple patient-derived GBM lines responded to treatment, underscoring its clinical relevance. In sum, integrating stem cell-based engineering, multimodal imaging, and delivery of PE-cytotoxins in a clinically relevant GBM model represents a novel strategy and a potential advancement in GBM therapy.

Comparison of long-term retinoic acid-based neural induction methods of bone marrow human mesenchymal stem cells.

The differentiation of human mesenchymal stem cells (hMSCs) into neural cells in vitro provides a potential tool to be utilized for cell therapy of neurodegenerative disorders. Although previous studies repeated different protocols for the induction of neural cells from hMSCs in vitro, the results were not in complete agreement. In this study, we have attempted to compare three of these neural induction methods; retinoic acid (RA) treatment, RA treatment in serum reduced conditions, and treatment using other chemical compounds (dimethyl sulfoxide and potassium chloride) along with RA by real-time cell analysis and immunofluorescent staining of neural markers. RA treatment led to a slow progression of cells into neural-like morphology with the expression of neural protein neurofilament whereas reducing serum during RA treatment caused a much more extended differentiation process. Additionally, neural-like morphology was persistent in the later periods of differentiation in RA treatment. On the other hand, chemical induction caused cell shrinkages mimicking neural-like morphology in a short time and loss of this morphology along with increased cell death in later periods. Among the three methods compared, RA treatment was the most reliable one in terms of stability of differentiation and neural protein expressions.

Selective silencing of DNA topoisomerase IIß in human mesenchymal stem cells by siRNAs (small interfering RNAs).

hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIß is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIß selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIß-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.