Potential Of Cell Cultures Research Articles

Nuclear receptor subfamily 4A2: Novel role and a potential therapeutic lead in cancers.

e18017 Background: Perineural invasion (PNI) is a pathological process where cancer cells infiltrate surrounding nerve tissues, markedly increasing the risk of cancer recurrence, metastasis, post-surgical complications, and associated pain. Alarmingly, PNI is prevalent in 80 - 100% of Head and Neck Squamous Carcinoma (HNSCC) and Pancreatic Ductal Adenocarcinoma (PDAC) cases. In these tumors, nerve invasion often occurs as a distinct route of metastasis, bypassing the usual lymphatic or vascular routes. Despite the rigor of surgical interventions and post-operative radiation, PNI-associated tumors recurrently manifest, emphasizing an urgent need to elucidate the underlying molecular interplay between tumor cells and nerves. Methods: Our study focuses on the Nuclear receptor subfamily 4A2 (NR4A2) transcription factor, known for its pivotal roles in cellular homeostasis and response to pathological conditions. Results: Comprehensive analyses employing tissue microarrays, curated public datasets, and cell lines unveiled a consistent overexpression of NR4A2 in HNSCC and PDAC tumor cells compared to their normal counterparts. Intriguingly, genome-wide chromatin immunoprecipitation (ChIP) sequencing with an NR4A2-specific antibody highlighted an enrichment of genes linked to neuronal guidance, suggesting a probable NR4A2-mediated mechanism underpinning PNI. Further, an in-depth analysis of these ChIP-enriched neuronal genes using The Cancer Genome Atlas (TCGA) confirmed their heightened expression in HNSCC and PDAC tumor tissues relative to healthy tissues. Advancing our research with graph neural networks and sophisticated bioinformatics, we identified two potent lead compounds targeting NR4A2. Preliminary Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) assessments of these compounds indicate favorable safety and efficacy profiles for clinical application. We are currently conducting comprehensive evaluations of these compounds, deciphering their molecular mode of action and therapeutic potential in cell culture and animal models. Conclusions: To sum up, our findings reveal a yet unknown role of NR4A2 in the landscape of PNI in HNSCC and PDAC. The AI-driven discovery and ensuing validation of NR4A2-centric inhibitors present a novel therapeutic avenue to curb cancer progression, prevent metastasis, and potentially improve patient outcomes.

Abstract 4901: The role of protein disulfide isomerase and copper in the paraptotic cell death of clinically investigated anticancer thiosemicarbazones

Abstract α-N-Heterocyclic thiosemicarbazones (TSCs) have long been investigated as anticancer compounds. Triapine is one of the best-known TSCs for anticancer therapy and is currently tested in a clinical phase III trial. To further improve the anticancer activity of TSCs, novel derivatives (such as DpC and COTI-2) have been developed and clinically investigated for their activity against solid tumors. These novel TSCs belong to a subclass (incl. the tetra-methylated Triapine derivative, Me2NNMe2) that are terminally disubstituted and demonstrated enhanced anticancer activity in vitro and in vivo. The improved activity of these TSCs correlated with their ability to form stable copper complexes as well as induce paraptosis, a form of programmed but caspase-independent cell death, that is characterized by the formation of vesicles originating from the endoplasmic reticulum (ER). However, the molecular events behind paraptosis induction are still not fully elucidated. Consequently, the aim of this study was to investigate the molecular signaling behind paraptosis as well as the reasons behind the induction of this quite unknown form of cell death by TSCs. A whole-genome gene expression analysis of cells treated with a terminally disubstituted TSC revealed an upregulation of thiol homeostasis-related genes, copper metabolism, and metallothionein genes as well as ER stress response genes. In addition, we found an upregulation of the copper-sensitive and thiol-containing protein disulfide isomerase (PDI), which was confirmed on protein level. In cell-free activity studies, PDI was indeed inhibited by terminally disubstituted TSCs, although only in form of their copper complexes. In agreement, removal of copper ions by specific chelators greatly reduced their anticancer activity as well as paraptotic potential in cell culture. This inhibition was hypothesized to be induced by a general disruption of the thiol redox homeostasis affecting also other responsive proteins and resulting in a more oxidative environment, especially in the ER. In confirmation, thiol-containing small molecules could reduce the compound’s anticancer activity in viability assays. Furthermore, glutathione as well as PDI were detected predominantly in their oxidized form in cells treated with terminally disubstituted TSCs. Consequently, terminally disubstituted TSCs, which are characterized by higher anticancer activity, induce paraptosis due to their high copper complex stability. The formation of the complex results in the interaction with thiol-containing proteins and subsequently in the disruption of the thiol-redox homeostasis, which leads to paraptotic cell death. Overall this work shed light on the paraptotic cell death and will be of interest for future clinical development of anticancer TSCs, as paraptosis is hypothesized to overcome apoptosis-resistance of cancer cells. Citation Format: Sonja Hager, Walter Berger, Christian R. Kowol, Eva A. Enyedy, Petra Heffeter. The role of protein disulfide isomerase and copper in the paraptotic cell death of clinically investigated anticancer thiosemicarbazones. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4901.

599 Selective inhibition Of fibro-inflammatory kinase TAK1: A potential therapeutic strategy to ameliorate systemic sclerosis

Fibrosis leads to failure of the skin, lungs, and other organs in systemic sclerosis (SSc); accounts for substantial morbidity and mortality; and lacks effective therapy. Recent findings implicated TGFβ-activated kinase 1 (TAK1), triggered by TGF-β and toll-like receptor signaling, in SSc pathogenesis. The objectives were to evaluate the activation of TAK1 signaling axis in SSc patients and to evaluate the antifibrotic ability of pharmacological TAK1 blockade in organ fibrosis. HS-276, a drug-like novel small molecule inhibitor of TAK1 was screened for its anti-fibrotic potential in cell cultures using foreskin, adult and SSc skin fibroblasts, and in bleomycin induced skin and lung fibrosis model.

Anti-Cancer Properties of Theaflavins.

Cancer is a disease characterized by aberrant proliferative and apoptotic signaling pathways, leading to uncontrolled proliferation of cancer cells combined with enhanced survival and evasion of cell death. Current treatment strategies are sometimes ineffective in eradicating more aggressive, metastatic forms of cancer, indicating the need to develop novel therapeutics targeting signaling pathways which are essential for cancer progression. Historically, plant-derived compounds have been utilized in the production of pharmaceuticals and chemotherapeutic compounds for the treatment of cancer, including paclitaxel and docetaxel. Theaflavins, phenolic components present in black tea, have demonstrated anti-cancer potential in cell cultures in vitro and in animal studies in vivo. Theaflavins have been shown to inhibit proliferation, survival, and migration of many cancer cellswhile promoting apoptosis. Treatment with theaflavins has been associated with increased levels of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspases-3, -7, -8, and -9, all markers of apoptosis, and increased expression of the proapoptotic marker Bcl-2-associated X protein (Bax) and concomitant reduction in the antiapoptotic marker B-cell lymphoma 2 (Bcl-2). Additionally, theaflavin treatment reduced phosphorylated Akt, phosphorylated mechanistic target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), and c-Myc levels with increased expression of the tumour suppressor p53. This review summarizes the current in vitro and in vivo evidence available investigating the anti-cancer effects of theaflavins across various cancer cell lines and animal models.

Roles of vitamins in stem cells.

Stem cells can differentiate to diverse cell types in our body, and they hold great promises in both basic research and clinical therapies. For specific stem cell types, distinctive nutritional and signaling components are required to maintain the proliferation capacity and differentiation potential in cell culture. Various vitamins play essential roles in stem cell culture to modulate cell survival, proliferation and differentiation. Besides their common nutritional functions, specific vitamins are recently shown to modulate signal transduction and epigenetics. In this article, we will first review classical vitamin functions in both somatic and stem cell cultures. We will then focus on how stem cells could be modulated by vitamins beyond their nutritional roles. We believe that a better understanding of vitamin functions will significantly benefit stem cell research, and help realize their potentials in regenerative medicine.

Selective Targeting of Myoblast Fusogenic Signaling and Differentiation-Arrest Antagonizes Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue malignancy comprised histologically of skeletal muscle lineage precursors that fail to exit the cell cycle and fuse into differentiated syncytial muscle-for which the underlying pathogenetic mechanisms remain unclear. In contrast to myogenic transcription factor signaling, the molecular machinery that orchestrates the discrete process of myoblast fusion in mammals is poorly understood and unexplored in RMS. The fusogenic machinery in Drosophila, however, is understood in much greater detail, where myoblasts are divided into two distinct pools, founder cells (FC) and fusion competent myoblasts (fcm). Fusion is heterotypic and only occurs between FCs and fcms. Here, we interrogated a comprehensive RNA-sequencing database and found that human RMS diffusely demonstrates an FC lineage gene signature, revealing that RMS is a disease of FC lineage rhabdomyoblasts. We next exploited our Drosophila RMS-related model to isolate druggable FC-specific fusogenic elements underlying RMS, which uncovered the EGFR pathway. Using RMS cells, we showed that EGFR inhibitors successfully antagonized RMS RD cells, whereas other cell lines were resistant. EGFR inhibitor-sensitive cells exhibited decreased activation of the EGFR intracellular effector Akt, whereas Akt activity remained unchanged in inhibitor-resistant cells. We then demonstrated that Akt inhibition antagonizes RMS-including RMS resistant to EGFR inhibition-and that sustained activity of the Akt1 isoform preferentially blocks rhabdomyoblast differentiation potential in cell culture and in vivo. These findings point towards selective targeting of fusion- and differentiation-arrest via Akt as a broad RMS therapeutic vulnerability. SIGNIFICANCE: EGFR and its downstream signaling mediator AKT1 play a role in the fusion and differentiation processes of rhabdomyosarcoma cells, representing a therapeutic vulnerability of rhabdomyosarcoma.

Clinical Variables that Influence Properties of Human Mesenchymal Stromal Cells

Many bone tissue engineering studies use animal or immortalized human mesenchymal stromal cells (hMSCs), which include osteoblast progenitors, because of the abundance of those cells. It is advantageous to study the less plentiful human MSCs in research on bone tissue engineering because of the clinical relevance and because of the importance of discovering how to optimize hMSCs for autogenous use. There is growing evidence that some of the irreproducibility in biological experiments with hMSCs can be attributed to variable clinical characteristics of the subjects from whom they were obtained. Patients who could benefit from bone tissue engineering are likely to have cellular deficits and compromised biochemical milieu, some of which can be managed by customized in vitro treatments. Many donor characteristics are associated with their hMSC in vitro properties. Studies with cohorts with similar characteristics show that some deficits are modifiable in vitro and can be managed with greater understanding of their pathophysiological mechanisms. Even hMSCs from elders can be rejuvenated in vitro with safe agents. Additional value in studying physiology of hMSCs from characterized subjects is to develop rationales for new in vivo therapies to ensure skeletal health throughout the lifespan. During this era of intensive tissue engineering research, it is appealing to use human mesenchymal stromal cells (hMSCs) for bone tissue engineering research because of their clinical relevance. Use of the patient’s own cells for therapeutic applications is called autogenous cell-based therapy. Research shows that properties of hMSCs isolated from a subject’s marrow depend on many clinical characteristics and that, in some cases, their osteoblast differentiation potential in cell culture can be optimized safely. These observations also suggest ways to optimize the functions of the skeleton throughout the lifespan.

Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis.

Effect of doping in carbon nanotubes on the viability of biomimetic chitosan‐carbon nanotubes‐hydroxyapatite scaffolds

This work describes the preparation and characterization of biomimetic chitosan/multiwall carbon nanotubes/nano-hydroxyapatite (CTS/MWCNT/nHAp) scaffolds and their viability for bone tissue engineering applications. The cryogenic process ice segregation-induced self-assembly (ISISA) was used to fabricate 3D biomimetic CTS scaffolds. Proper combination of cryogenics, freeze-drying, nature and molecular ratio of solutes give rise to 3D porous interconnected scaffolds with clusters of nHAp distributed along the scaffold surface. The effect of doping in CNT (e.g. with oxygen and nitrogen atoms) on cell viability was tested. Under the same processing conditions, pore size was in the range of 20-150 μm and irrespective on the type of CNT. Studies on cell viability with scaffolds were carried out using human cells from periosteum biopsy. Prior to cell seeding, the immunophenotype of mesenchymal periosteum or periosteum-derived stem cells (MSCs-PCs) was characterized by flow cytometric analysis using fluorescence-activated and characteristic cell surface markers for MSCs-PCs. The characterized MSCs-PCs maintained their periosteal potential in cell cultures until the 2nd passage from primary cell culture. Thus, the biomimetic CTS/MWCNT/nHAp scaffolds demonstrated good biocompatibility and cell viability in all cases such that it can be considered as promising biomaterials for bone tissue engineering.

In-vitro and in-vivo phenotype of type Asia 1 foot-and-mouth disease viruses utilizing two non-RGD receptor recognition sites

BackgroundFoot-and-mouth disease virus (FMDV) uses a highly conserved Arg-Gly-Asp (RGD) triplet for attachment to host cells and this motif is believed to be essential for virus viability. Previous sequence analyses of the 1D-encoding region of an FMDV field isolate (Asia1/JS/CHA/05) and its two derivatives indicated that two viruses, which contained an Arg-Asp-Asp (RDD) or an Arg-Ser-Asp (RSD) triplet instead of the RGD integrin recognition motif, were generated serendipitously upon short-term evolution of field isolate in different biological environments. To examine the influence of single amino acid substitutions in the receptor binding site of the RDD-containing FMD viral genome on virus viability and the ability of non-RGD FMDVs to cause disease in susceptible animals, we constructed an RDD-containing FMDV full-length cDNA clone and derived mutant molecules with RGD or RSD receptor recognition motifs. Following transfection of BSR cells with the full-length genome plasmids, the genetically engineered viruses were examined for their infectious potential in cell culture and susceptible animals.ResultsAmino acid sequence analysis of the 1D-coding region of different derivatives derived from the Asia1/JS/CHA/05 field isolate revealed that the RDD mutants became dominant or achieved population equilibrium with coexistence of the RGD and RSD subpopulations at an early phase of type Asia1 FMDV quasispecies evolution. Furthermore, the RDD and RSD sequences remained genetically stable for at least 20 passages. Using reverse genetics, the RDD-, RSD-, and RGD-containing FMD viruses were rescued from full-length cDNA clones, and single amino acid substitution in RDD-containing FMD viral genome did not affect virus viability. The genetically engineered viruses replicated stably in BHK-21 cells and had similar growth properties to the parental virus. The RDD parental virus and two non-RGD recombinant viruses were virulent to pigs and bovines that developed typical clinical disease and viremia.ConclusionsFMDV quasispecies evolving in a different biological environment gained the capability of selecting different receptor recognition site. The RDD-containing FMD viral genome can accommodate substitutions in the receptor binding site without additional changes in the capsid. The viruses expressing non-RGD receptor binding sites can replicate stably in vitro and produce typical FMD clinical disease in susceptible animals.

Efficient conversion of tetrapeptide-based TSAO prodrugs to the parent drug by dipeptidyl-peptidase IV (DPPIV/CD26)

A novel prodrug approach has been evaluated using the anti-HIV-active TSAO molecule as the prototype drug to prove the kinetics with purified enzyme and the principles of conversion to the parent compound in sera and cell culture. When a variety of tetrapeptidyl amide prodrugs of NAP-TSAO were synthesized and exposed to purified dipeptidyl-peptidase IV (DPPIV/CD26) as well as human and bovine sera, they are converted to the parent NAP-TSAO drug in two successive steps by both purified CD26 and human and bovine serum. The efficiency of conversion strongly depends on the nature of the amino acid that has to be cleaved-off from the prodrug molecule. The tetrapeptidyl prodrug 20 showed a more than 10-fold improved water-solubility in comparison to that of the parent compound NAP-TSAO. The antiviral activity of the prototype NAP-TSAO could also be modulated by introducing different tetrapeptide moieties on the molecule resulting, in some cases, in a superior antiviral potential in cell culture than the parent drug.

Inhibiting TGF-β signaling restores immune surveillance in the SMA-560 glioma model

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.

Erythropoietin--a novel concept for neuroprotection.

Neuroprotection as a means to prevent or oppose pathological neuronal loss in central nervous system disease of various pathophysiological origins represents a novel therapeutic approach. This approach is supported by extensive experimental evidence on cell culture and animal studies demonstrating beneficial effects of growth factors on neuronal survival and functional recovery. The clinical use of neuroprotective agents has been hampered by the toxicity of many of the compounds that showed promising therapeutic potential in animal studies. The focus of this review is on a novel neuroprotective approach with erythropoietin, a hematopoietic growth factor that: 1) is expressed in the human central nervous system, 2) is hypoxia-inducible, 3) has demonstrated remarkable neuroprotective potential in cell culture and animal models of disease, 4) has multiple protective effects (antiapoptotic, neurotrophic, antioxidant, angiogenic), and 5) is a clinically extremely well tolerated compound.

Characterization of Ngef, a Novel Member of the Dbl Family of Genes Expressed Predominantly in the Caudate Nucleus

We have identified Ngef as a novel member of the family of Dbl genes. Many members of this family have been shown to function as guanine nucleotide exchange factors for the Rho-type GTPases. Ngef is predominantly expressed in brain, with the strongest signal in the caudate nucleus, a region associated with the control of movement. Ngef contains a translated trinucleotide repeat, a polyglutamic acid stretch interrupted by a glycine. We have localized the Ngef gene to mouse chromosome 1 and the human homologue of Ngef to human chromosome 2q37. We have shown in preliminary experiments that Ngef has transforming potential in cell culture and is able to induce tumors in nude mice.

Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus.

The nonsegmented negative-strand RNA viruses (order Mononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3' end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.