Novel Thiosemicarbazones Sensitize Pediatric Solid Tumor Cell-Types to Conventional Chemotherapeutics through Multiple Molecular Mechanisms

Combining low-dose chemotherapies is a strategy for designing less toxic and more potent childhood cancer treatments. We examined the effects of combining the novel thiosemicarbazones, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), or its analog, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), with the standard chemotherapies, celecoxib (CX), etoposide (ETO), or temozolomide (TMZ). These combinations were analyzed for synergism to inhibit proliferation of three pediatric tumor cell-types, namely osteosarcoma (Saos-2), medulloblastoma (Daoy) and neuroblastoma (SH-SY5Y). In terms of mechanistic dissection, this study discovered novel thiosemicarbazone targets not previously identified and which are important for considering possible drug combinations. In this case, DpC and Dp44mT caused: (1) up-regulation of a major protein target of CX, namely cyclooxygenase-2 (COX-2); (2) down-regulation of the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), which is known to affect TMZ resistance; (3) down-regulation of mismatch repair (MMR) proteins, MSH2 and MSH6, in Daoy and SH-SY5Y cells; and (4) down-regulation in all three cell-types of the MMR repair protein, MLH1, and also topoisomerase 2α (Topo2α), the latter of which is an ETO target. While thiosemicarbazones up-regulate the metastasis suppressor, NDRG1, in adult cancers, it is demonstrated herein for the first time that they induce NDRG1 in all three pediatric tumor cell-types, validating its role as a potential target. In fact, siRNA studies indicated that NDRG1 was responsible for MGMT down-regulation that may prevent TMZ resistance. Examining the effects of combining thiosemicarbazones with CX, ETO, or TMZ, the most promising synergism was obtained using CX. Of interest, a positive relationship was observed between NDRG1 expression of the cell-type and the synergistic activity observed in the combination of thiosemicarbazones and CX. These studies identify novel thiosemicarbazone targets relevant to childhood cancer combination chemotherapy.

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Tumor quiescence: elevating SOX2 in diverse tumor cell types downregulates a broad spectrum of the cell cycle machinery and inhibits tumor growth

BMC Cancer ◽

10.1186/s12885-020-07370-7 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Ethan P. Metz ◽

Erin L. Wuebben ◽

Phillip J. Wilder ◽

Jesse L. Cox ◽

Kaustubh Datta ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Tumor Growth ◽

Tumor Cell ◽

Tumor Cells ◽

Molecular Mechanisms ◽

Cell Types ◽

Cell Cycle Machinery

Abstract Background Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. Methods To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. Results Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. Conclusions Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

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Proteomics Analysis of Cellular Network in Human Bone Marrow Reveals Lineage Skewing Towards Megakaryocytes and Decrease in Lymphoid Development upon Aging

Blood ◽

10.1182/blood.v128.22.2658.2658 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2658-2658

Author(s):

Anthony D. Ho ◽

Patrick Horn ◽

Marco Hennrich ◽

Samira Jaeger ◽

Natalie Romanov ◽

...

Keyword(s):

Bone Marrow ◽

Molecular Mechanisms ◽

Cell Types ◽

Cellular Level ◽

Lymphoid Cells ◽

Down Regulation ◽

Proteomics Analysis ◽

Age Related ◽

Erythroid Precursors ◽

Related Proteins

Abstract Background: Whereas proteomics analyses at a cellular level have been performed in murine HPC, most proteomics studies on human material have thus far focused on examining human tissues with a mixture of cell types and not at a specific cellular level. Whole proteomics analyses might reveal genuine molecular alterations und mechanisms of aging on human hematopoietic cells. Aim: To identify the age-related molecular changes in human hematopoietic progenitor cells (HPC) as well as in the cellular elements of the human bone marrow niche, we have applied mass spectrometry-based platform for proteomics analysis of this network of bone marrow (BM) cells at a cellular level. The overarching goal is to acquire a systems understanding of the molecular mechanisms involved in aging of human HPC as well as other cellular constituents of the marrow niche, and to combine the cell biological, proteomic, and genetic studies for a better understanding of age-related diseases such as MDS. Methods: In this study, we have simultaneously characterized at the proteomics level HPC as well as the cellular microenvironment in BM from each individual human subject. Cells were harvested from the BM of 69 healthy human subjects [Age Group (AG) 20-29 years: n=19; AG 30-39: n=16; AG 40-49: n=9; AG 50+: N=15]. BM cells were separated for nucleated cells using Ficoll, followed by sorting with specific marker constellations, i.e. HPC (CD34+), lymphoid cells (CD34-/CD45+/SSClow), myeloid precursors (CD34-/CD45med/SSChigh), monocyte-macrophages (CD34-/CD45+/CD14+), erythroid precursors (CD34-/CD45-), and mesenchymal stromal cells (MSC) based on plastic adherence upon culture. Results: In 60 of these 69 healthy subjects we were able to harvest an adequate amount of material from 5 to 6 the aforementioned subsets from the respective individual, yielding altogether 342 samples for proteomics analysis. After digestion (trypsinization), isobaric labelling of peptides (TMT) of appropriate quality was successfully performed in 270 of these 342 samples derived from 69 healthy human subjects. These 270 samples underwent tandem mass-spectrometry analyses. The number of proteins identified were >12,000, covering 65% of the estimated human proteome (HPC: >7,500; Lymphoid cells: >8,500; Myeloid Precursors: >7,500; Mono/Macro: >8,500; Erythroid Precursors: >6,500; and in MSC: >9,000 proteins). We have then focused on the molecular mechanisms, specifically proteomic alterations involved in aging of these 6 cell types. Gene Set Enrichment Analysis (GSEA) using previously defined gene sets specific for common lymphoid progenitors (CLP), bi-potent granulocyte/macrophage progenitors (GMP), and megakaryocyte progenitors (MKP) revealed significant down-regulation of CLP related proteins and significant up-regulation of MKP related proteins in HPC upon aging. The same down-regulation of CLP proteins and up-regulation of MKP proteins were also found in the BM lymphoid cells. Further significant alterations of note are a decrease in cell cycle related proteins upon aging in HPC, lymphoid cells, as well as in MSC, and an increase in GMP related proteins upon aging in lymphoid cells, erythroid precursors and in MSC. Extensive pathway and network analyses are underway. Conclusions: We have for the first time accomplished a multiplex and comprehensive analysis of a community of cells in human BM, comprising HPC and 5 other cell types that constitute the niche and have identified >12,000 proteins in this cellular network. Proteomics analyses have demonstrated a significant down-regulation of CLP related proteins in human HPC and in marrow lymphoid cells, as well as an up-regulation of MKP in all the cell types tested upon aging. Of note is also a decrease in cell cycle related proteins with age. This study has thus provided evidence that lineage skewing towards megakaryocytic and decrease in lymphoid development upon aging occur in human HPC as well as in other human BM cells. Disclosures No relevant conflicts of interest to declare.

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Anaphylactic Degranulation of Mast Cells: Focus on Compound Exocytosis

Journal of Immunology Research ◽

10.1155/2019/9542656 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 9

Author(s):

Ofir Klein ◽

Ronit Sagi-Eisenberg

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Molecular Mechanisms ◽

Secretory Granules ◽

Cell Types ◽

Secretory Cells ◽

Regulated Exocytosis ◽

Open Questions ◽

Conflicting Evidence ◽

Compound Exocytosis

Anaphylaxis is a notorious type 2 immune response which may result in a systemic response and lead to death. A precondition for the unfolding of the anaphylactic shock is the secretion of inflammatory mediators from mast cells in response to an allergen, mostly through activation of the cells via the IgE-dependent pathway. While mast cells are specialized secretory cells that can secrete through a variety of exocytic modes, the most predominant mode exerted by the mast cell during anaphylaxis is compound exocytosis—a specialized form of regulated exocytosis where secretory granules fuse to one another. Here, we review the modes of regulated exocytosis in the mast cell and focus on compound exocytosis. We review historical landmarks in the research of compound exocytosis in mast cells and the methods available for investigating compound exocytosis. We also review the molecular mechanisms reported to underlie compound exocytosis in mast cells and expand further with reviewing key findings from other cell types. Finally, we discuss the possible reasons for the mast cell to utilize compound exocytosis during anaphylaxis, the conflicting evidence in different mast cell models, and the open questions in the field which remain to be answered.

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Genetic analysis of amyotrophic lateral sclerosis identifies contributing pathways and cell types

Science Advances ◽

10.1126/sciadv.abd9036 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabd9036

Author(s):

Sara Saez-Atienzar ◽

Sara Bandres-Ciga ◽

Rebekah G. Langston ◽

Jonggeol J. Kim ◽

Shing Wan Choi ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Membrane Trafficking ◽

Molecular Mechanisms ◽

Cell Types ◽

Polygenic Risk Score ◽

Genome Wide ◽

Genome Wide Data ◽

Data Driven Approach ◽

Single Nucleus ◽

Lateral Sclerosis

Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely, neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated six differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, MAPKAPK3, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

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In-depth transcriptomic analysis of human retina reveals molecular mechanisms underlying diabetic retinopathy

Scientific Reports ◽

10.1038/s41598-021-88698-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kolja Becker ◽

Holger Klein ◽

Eric Simon ◽

Coralie Viollet ◽

Christian Haslinger ◽

...

Keyword(s):

Diabetic Retinopathy ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Vision Loss ◽

Ganglion Cells ◽

Expression Profiles ◽

Cell Types ◽

Sequencing Data ◽

Disease Stages ◽

Post Transcriptional Regulation

AbstractDiabetic Retinopathy (DR) is among the major global causes for vision loss. With the rise in diabetes prevalence, an increase in DR incidence is expected. Current understanding of both the molecular etiology and pathways involved in the initiation and progression of DR is limited. Via RNA-Sequencing, we analyzed mRNA and miRNA expression profiles of 80 human post-mortem retinal samples from 43 patients diagnosed with various stages of DR. We found differentially expressed transcripts to be predominantly associated with late stage DR and pathways such as hippo and gap junction signaling. A multivariate regression model identified transcripts with progressive changes throughout disease stages, which in turn displayed significant overlap with sphingolipid and cGMP–PKG signaling. Combined analysis of miRNA and mRNA expression further uncovered disease-relevant miRNA/mRNA associations as potential mechanisms of post-transcriptional regulation. Finally, integrating human retinal single cell RNA-Sequencing data revealed a continuous loss of retinal ganglion cells, and Müller cell mediated changes in histidine and β-alanine signaling. While previously considered primarily a vascular disease, attention in DR has shifted to additional mechanisms and cell-types. Our findings offer an unprecedented and unbiased insight into molecular pathways and cell-specific changes in the development of DR, and provide potential avenues for future therapeutic intervention.

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MicroRNAs and Stem-like Properties: The Complex Regulation Underlying Stemness Maintenance and Cancer Development

Biomolecules ◽

10.3390/biom11081074 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1074

Author(s):

Giuseppina Divisato ◽

Silvia Piscitelli ◽

Mariantonietta Elia ◽

Emanuela Cascone ◽

Silvia Parisi

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Epithelial Mesenchymal Transition ◽

Embryonic Stem ◽

Cell Types ◽

Cancer Development ◽

Special Focus ◽

Self Renewal ◽

Mesenchymal Transition ◽

Different Cell Types

Embryonic stem cells (ESCs) have the extraordinary properties to indefinitely proliferate and self-renew in culture to produce different cell progeny through differentiation. This latter process recapitulates embryonic development and requires rounds of the epithelial–mesenchymal transition (EMT). EMT is characterized by the loss of the epithelial features and the acquisition of the typical phenotype of the mesenchymal cells. In pathological conditions, EMT can confer stemness or stem-like phenotypes, playing a role in the tumorigenic process. Cancer stem cells (CSCs) represent a subpopulation, found in the tumor tissues, with stem-like properties such as uncontrolled proliferation, self-renewal, and ability to differentiate into different cell types. ESCs and CSCs share numerous features (pluripotency, self-renewal, expression of stemness genes, and acquisition of epithelial–mesenchymal features), and most of them are under the control of microRNAs (miRNAs). These small molecules have relevant roles during both embryogenesis and cancer development. The aim of this review was to recapitulate molecular mechanisms shared by ESCs and CSCs, with a special focus on the recently identified classes of microRNAs (noncanonical miRNAs, mirtrons, isomiRs, and competitive endogenous miRNAs) and their complex functions during embryogenesis and cancer development.

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Shisa6 mediates cell-type specific regulation of depression in the nucleus accumbens

Molecular Psychiatry ◽

10.1038/s41380-021-01217-8 ◽

2021 ◽

Author(s):

Hee-Dae Kim ◽

Jing Wei ◽

Tanessa Call ◽

Nicole Teru Quintus ◽

Alexander J. Summers ◽

...

Keyword(s):

Nucleus Accumbens ◽

Molecular Mechanisms ◽

Cell Types ◽

Current Treatment ◽

Specific Cell ◽

Excitatory Synapses ◽

Cell Type ◽

Cell Type Specific ◽

Specific Regulation ◽

Circuit Function

AbstractDepression is the leading cause of disability and produces enormous health and economic burdens. Current treatment approaches for depression are largely ineffective and leave more than 50% of patients symptomatic, mainly because of non-selective and broad action of antidepressants. Thus, there is an urgent need to design and develop novel therapeutics to treat depression. Given the heterogeneity and complexity of the brain, identification of molecular mechanisms within specific cell-types responsible for producing depression-like behaviors will advance development of therapies. In the reward circuitry, the nucleus accumbens (NAc) is a key brain region of depression pathophysiology, possibly based on differential activity of D1- or D2- medium spiny neurons (MSNs). Here we report a circuit- and cell-type specific molecular target for depression, Shisa6, recently defined as an AMPAR component, which is increased only in D1-MSNs in the NAc of susceptible mice. Using the Ribotag approach, we dissected the transcriptional profile of D1- and D2-MSNs by RNA sequencing following a mouse model of depression, chronic social defeat stress (CSDS). Bioinformatic analyses identified cell-type specific genes that may contribute to the pathogenesis of depression, including Shisa6. We found selective optogenetic activation of the ventral tegmental area (VTA) to NAc circuit increases Shisa6 expression in D1-MSNs. Shisa6 is specifically located in excitatory synapses of D1-MSNs and increases excitability of neurons, which promotes anxiety- and depression-like behaviors in mice. Cell-type and circuit-specific action of Shisa6, which directly modulates excitatory synapses that convey aversive information, identifies the protein as a potential rapid-antidepressant target for aberrant circuit function in depression.

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Exploring the Intracrine Functions of VEGF-A

Biomolecules ◽

10.3390/biom11010128 ◽

2021 ◽

Vol 11 (1) ◽

pp. 128

Author(s):

Sophie Wiszniak ◽

Quenten Schwarz

Keyword(s):

Cell Growth ◽

Molecular Mechanisms ◽

Direct Action ◽

Cancer Therapeutics ◽

Cell Types ◽

Vascular Endothelial ◽

Signalling Molecule ◽

Endothelial Growth Factor ◽

Important Signalling Molecule ◽

Vegf Signalling

Vascular endothelial growth factor A (VEGF-A or VEGF) is a highly conserved secreted signalling protein best known for its roles in vascular development and angiogenesis. Many non-endothelial roles for VEGF are now established, with the discovery that VEGF and its receptors VEGFR1 and VEGFR2 are expressed in many non-vascular cell-types, as well as various cancers. In addition to secreted VEGF binding to its receptors in the extracellular space at the cell membrane (i.e., in a paracrine or autocrine mode), intracellularly localised VEGF is emerging as an important signalling molecule regulating cell growth, survival, and metabolism. This intracellular mode of signalling has been termed “intracrine”, and refers to the direct action of a signalling molecule within the cell without being secreted. In this review, we describe examples of intracrine VEGF signalling in regulating cell growth, differentiation and survival, both in normal cell homeostasis and development, as well as in cancer. We further discuss emerging evidence for the molecular mechanisms underpinning VEGF intracrine function, as well as the implications this intracellular mode of VEGF signalling may have for use and design of anti-VEGF cancer therapeutics.

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Molecular Alterations in Sporadic and SOD1-ALS Immortalized Lymphocytes: Towards a Personalized Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms22063007 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3007

Author(s):

Isabel Lastres-Becker ◽

Gracia Porras ◽

Marina Arribas-Blázquez ◽

Inés Maestro ◽

Daniel Borrego-Hernández ◽

...

Keyword(s):

Motor Neurons ◽

Molecular Mechanisms ◽

Cell Types ◽

Therapeutic Interventions ◽

Autophagic Flux ◽

Metabolic Disturbances ◽

Molecular Alterations ◽

Healthy Donors ◽

Inflammatory Profile ◽

Als Patients

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.

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First blood: the endothelial origins of hematopoietic progenitors

Angiogenesis ◽

10.1007/s10456-021-09783-9 ◽

2021 ◽

Author(s):

Giovanni Canu ◽

Christiana Ruhrberg

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Cell Types ◽

Yolk Sac ◽

Vascular Endothelial ◽

Hematopoietic Stem ◽

Close Proximity ◽

Clinical Interest

AbstractHematopoiesis in vertebrate embryos occurs in temporally and spatially overlapping waves in close proximity to blood vascular endothelial cells. Initially, yolk sac hematopoiesis produces primitive erythrocytes, megakaryocytes, and macrophages. Thereafter, sequential waves of definitive hematopoiesis arise from yolk sac and intraembryonic hemogenic endothelia through an endothelial-to-hematopoietic transition (EHT). During EHT, the endothelial and hematopoietic transcriptional programs are tightly co-regulated to orchestrate a shift in cell identity. In the yolk sac, EHT generates erythro-myeloid progenitors, which upon migration to the liver differentiate into fetal blood cells, including erythrocytes and tissue-resident macrophages. In the dorsal aorta, EHT produces hematopoietic stem cells, which engraft the fetal liver and then the bone marrow to sustain adult hematopoiesis. Recent studies have defined the relationship between the developing vascular and hematopoietic systems in animal models, including molecular mechanisms that drive the hemato-endothelial transcription program for EHT. Moreover, human pluripotent stem cells have enabled modeling of fetal human hematopoiesis and have begun to generate cell types of clinical interest for regenerative medicine.

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