-
Experimental Hematology & Oncology Jan 2024Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of...
BACKGROUND
Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis.
METHOD
OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition's mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival.
RESULTS
We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells.
CONCLUSION
These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.
PubMed: 38291540
DOI: 10.1186/s40164-023-00468-1 -
Cureus Dec 2023AMONDYS 45 (casimersen) is an antisense oligonucleotide therapy used to treat Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by a mutation in... (Review)
Review
AMONDYS 45 (casimersen) is an antisense oligonucleotide therapy used to treat Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by a mutation in the DMD gene. Symptoms include progressive muscle weakness, respiratory and cardiac complications, and premature death. Casimersen targets a specific mutation in the DMD gene that results in the absence of dystrophin protein, a key structural component of muscle fibers. While there is currently no cure for DMD, exon-skipping therapy works by restoring the reading frame of the mutated gene, allowing the production of a partially functional dystrophin protein. Clinical trials of casimersen have shown promising results in increasing dystrophin production, as measured by polymerase chain reaction (PCR) droplets when compared to placebo. In a randomized double-blind trial, patients who received casimersen had significantly higher dystrophin levels when compared to those who received placebo. Casimersen therapy is administered through repeated intravenous infusions, although the optimal dosage and duration of treatment are still under investigation. Based on the completed and ongoing clinical trials, casimersen has been well tolerated, with most adverse events being mild and unrelated to casimersen. In 2021, AMONDYS 45 (casimersen) received approval from the US Food and Drug Administration (FDA) for the treatment of Duchene muscular dystrophy in patients with a mutation of the DMD gene that is amenable to exon 45 skipping. These collective findings indicate that casimersen has the potential to elicit functional changes in individuals with DMD, although further studies are necessary to comprehensively evaluate the specific functional improvements. Regardless, the FDA approval and ongoing clinic trials mark a significant milestone in the development of DMD treatments and offer hope for those affected by this debilitating disease.
PubMed: 38283433
DOI: 10.7759/cureus.51237 -
BioRxiv : the Preprint Server For... Jan 2024Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans....
Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, . mRNA and protein was expressed throughout the developing head and was enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, and are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with DYRK1A mutations.
PubMed: 38260562
DOI: 10.1101/2024.01.13.575394 -
RSC Advances Jan 2024Cancer is a life-threatening illness all over the world, and developing anticancer treatments with high efficacy and low side effects remains a challenge. The quinoline...
Cancer is a life-threatening illness all over the world, and developing anticancer treatments with high efficacy and low side effects remains a challenge. The quinoline ring structure has long been recognized as a flexible nucleus in the design and synthesis of physiologically active chemicals. In this study, five new 2-morpholino-4-anilinoquinoline compounds were synthesized and their biological anticancer potential against the HepG2 cell line was assessed. The compounds produced demonstrated varying responses against HepG2 cells, with compounds 3c, 3d, and 3e exhibiting the highest activity, with IC values of 11.42, 8.50, and 12.76 μM, respectively. It is a critical requirement that anticancer medications are able to selectively decrease cancer growth while not causing damage to normal cells. Compound 3e exhibited increased activity while maintaining adequate selectivity. It was also the most effective chemical against cell migration and adhesion, which could play an important role in drug resistance and cell metastasis. In total, the findings revealed good possibilities for anticancer therapy, suggesting a target for future development of anticancer medication.
PubMed: 38249681
DOI: 10.1039/d3ra07495a -
Acta Parasitologica Mar 2024Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a primitive opportunistic protozoon, and one of the earliest differentiated eukaryotes. Despite its primitive...
BACKGROUND
Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a primitive opportunistic protozoon, and one of the earliest differentiated eukaryotes. Despite its primitive nature, G. lamblia has a sophisticated cytoskeleton system, which is closely related to its proliferation and pathogenicity. Meanwhile, α giardin is a G. lamblia-specific cytoskeleton protein, which belongs to the annexin superfamily. Interestingly, G. lamblia has 21 annexin-like α giardins, i.e., more than higher eukaryotes. The functional differences among α giardin members are not fully understood.
METHODS
We took α-4 giardin, a member of α giardin family, as a research object. A morpholino-mediated knockdown experiment was performed to identify the effect of α-4 giardin on G. lamblia trophozoites biological traits. A yeast two-hybrid cDNA library of G. lamblia strain C2 trophozoites was screened for interaction partners of α-4 giardin. Co-immunoprecipitation and fluorescent colocalization confirmed the relationship between G. lamblia EB1 (gEB1) and α-4 giardin.
RESULTS
α-4 Giardin could inhibit the proliferation and adhesion of G. lamblia trophozoites. In addition, it interacted with G. lamblia EB1 (gEB1).
CONCLUSIONS
α-4 Giardin was involved in proliferation and adhesion in G. lamblia trophozoites, and EB1, a crucial roles in mitosis, was an interacting partner of α-4 giardin.
Topics: Giardia lamblia; Protozoan Proteins; Trophozoites; Cytoskeletal Proteins; Protein Binding; Two-Hybrid System Techniques
PubMed: 38206477
DOI: 10.1007/s11686-023-00774-y -
Ecotoxicology and Environmental Safety Jan 2024Iprodione is an effective and broad-spectrum fungicide commonly used for early disease control in fruit trees and vegetables. Due to rainfall, iprodione often finds its...
Iprodione is an effective and broad-spectrum fungicide commonly used for early disease control in fruit trees and vegetables. Due to rainfall, iprodione often finds its way into water bodies, posing toxicity risks to non-target organisms and potentially entering the human food chain. However, there is limited information available regarding the developmental toxicity of iprodione specifically on the liver in existing literature. In this study, we employed larval and adult zebrafish as models to investigate the toxicity of iprodione. Our findings revealed that iprodione exposure led to yolk sac edema and increased mortality in zebrafish. Notably, iprodione exhibited specific effects on zebrafish liver development. Additionally, zebrafish exposed to iprodione experienced an overload of reactive oxygen species, resulting in the upregulation of p53 gene expression. This, in turn, triggered hepatocyte apoptosis and disrupted carbohydrate/lipid metabolism as well as energy demand systems. These results demonstrated the substantial impact of iprodione on zebrafish liver development and function. Furthermore, the application of astaxanthin (an antioxidant) and p53 morpholino partially mitigated the liver toxicity caused by iprodione. To summarize, iprodione induces apoptosis through the upregulation of p53 mediated by oxidative stress signals, leading to liver toxicity in zebrafish. Our study highlights that exposure to iprodione can result in hepatotoxicity in zebrafish, and it may potentially pose toxicity risks to other aquatic organisms and even humans.
Topics: Animals; Humans; Zebrafish; Reactive Oxygen Species; Tumor Suppressor Protein p53; Oxidative Stress; Chemical and Drug Induced Liver Injury; Embryo, Nonmammalian; Apoptosis; Aminoimidazole Carboxamide; Hydantoins
PubMed: 38181604
DOI: 10.1016/j.ecoenv.2023.115911 -
Frontiers in Cellular Neuroscience 2023Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the...
Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive . We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs . Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted.
PubMed: 38179205
DOI: 10.3389/fncel.2023.1292012 -
Cell Proliferation May 2024The study of neurogenesis is essential to understanding fundamental developmental processes and for the development of cell replacement therapies for central nervous...
The study of neurogenesis is essential to understanding fundamental developmental processes and for the development of cell replacement therapies for central nervous system disorders. Here, we designed an in vivo drug screening protocol in developing zebrafish to find new molecules and signalling pathways regulating neurogenesis in the ventral spinal cord. This unbiased drug screen revealed that 4 cyclooxygenase (COX) inhibitors reduced the generation of serotonergic interneurons in the developing spinal cord. These results fitted very nicely with available single-cell RNAseq data revealing that floor plate cells show differential expression of 1 of the 2 COX2 zebrafish genes (ptgs2a). Indeed, several selective COX2 inhibitors and two different morpholinos against ptgs2a reduced the number of serotonergic neurons in the ventral spinal cord and led to locomotor deficits. Single-cell RNAseq data and different pharmacological manipulations further revealed that COX2-floor plate-derived prostaglandin D promotes neurogenesis in the developing spinal cord by promoting mitotic activity in progenitor cells. Rescue experiments using a phosphodiesterase-4 inhibitor suggest that intracellular changes in cAMP levels underlie the effects of COX inhibitors on neurogenesis and locomotion. Our study provides compelling in vivo evidence showing that prostaglandin signalling promotes neurogenesis in the ventral spinal cord.
Topics: Animals; Zebrafish; Neurogenesis; Spinal Cord; Cyclooxygenase 2; Drug Evaluation, Preclinical; Cyclooxygenase 2 Inhibitors; Zebrafish Proteins; Signal Transduction; Cyclooxygenase Inhibitors
PubMed: 38155412
DOI: 10.1111/cpr.13594 -
Frontiers in Oncology 2023Enhanced inflammatory responses promote tumor progression by activating toll-like receptors (TLRs), which in turn are inhibited by C-type lectin like receptors (CTLRs),...
BACKGROUND
Enhanced inflammatory responses promote tumor progression by activating toll-like receptors (TLRs), which in turn are inhibited by C-type lectin like receptors (CTLRs), like CLEC12A. Although the presence of CLEC12A in acute myeloid leukemia is well established, its role in non-hematopoietic tumors is still obscure. In hematopoietic tumors, CLEC12A mostly inhibits TLRs and modulates inflammatory responses via NF-κB signaling. In this study, the fate of tumor progression was determined by modulating CLEC12A using artemisinin (ART), a FDA-approved anti-malarial drug, known for its anti-cancer and immunomodulatory properties with minimal adverse effects on normal cells.
METHOD
Effects of ART were primarily determined on hematological factors and primary metastatic organs, such as lungs, kidney and liver in normal and tumor-bearing BALB/c mice. Tumor-bearing mice were treated with different concentrations of ART and expressions of CLEC12A and associated downstream components were determined. CLEC12A was overexpressed in MDA-MB-231 and 4T1 cells, and the effects of ART were analyzed in the overexpressed cells. Silencing TLR4 using vivo morpholino was performed to elucidate its role in tumor progression in response to ART. Finally, CLEC12A modulation by ART was evaluated in the resident cancer stem cell (CSC) population.
RESULTS
ART did not alter physiology of normal mice, in contrast to tumor-bearing mice, where ART led to tumor regression. In addition, ART reduced expression of CLEC12A. Expectedly, TLR4 expression increased, but surprisingly, that of NF-κB (RelA) and JNK/pJNK decreased, along with reduced inflammation, reduced autophagy and increased apoptosis. All the above observations reverted on overexpression of CLEC12A in MDA-MB-231 and 4T1 cells. Inhibition of TLR4, however, indicated no change in the expressions of CLEC12A, NF-κB, or apoptotic markers. The effect of ART showed a similar trend in the CSC population as in cancer cells.
CONCLUSION
This study, for the first time, confirmed a differential role of CLEC12A in non-hematopoietic tumor and cancer stem cells in response to ART. Subsequent interaction and modulation of CLEC12A with ART induced tumor cell death and abrogation of CSCs, confirming a more comprehensive tumor therapy with reduced risk of recurrence. Therefore, ART may be repurposed as an effective drug for cancer treatment in future.
PubMed: 38144525
DOI: 10.3389/fonc.2023.1242432 -
The Journal of Biological Chemistry Jan 2024Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously...
Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies in vitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation in vivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.
Topics: Animals; Mice; Cell Movement; Inflammation; Leukotriene B4; Macrophages; Receptors, Leukotriene B4; Zebrafish
PubMed: 38097183
DOI: 10.1016/j.jbc.2023.105561