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Discover Oncology May 2024Breast cancer is a prevalent malignant tumor among women with an increasing incidence rate annually. Breast cancer stem cells (BCSCs) are integral in impeding tumor...
Breast cancer is a prevalent malignant tumor among women with an increasing incidence rate annually. Breast cancer stem cells (BCSCs) are integral in impeding tumor advancement and addressing drug resistance. Bestatin serves as an adjuvant chemotherapy, triggering apoptosis in cancer cells. In this study, the effects of bestatin on sorted BCSCs from breast cancer cell lines have been studied. Our results indicated that bestatin inhibits the migration and proliferation of breast cancer cells by reducing the stemness of BCSCs both in vitro and in vivo. Puromycin-sensitive aminopeptidase is implicated in the process through the regulation of cell cycle, resulting in heightened cell apoptosis and diminished cell proliferation of BCSCs. Our study suggest that targeting cancer stem cell may offer a promising approach in breast cancer treatment, presenting noval therapeutic strategies for patients with breast cancer.
PubMed: 38814491
DOI: 10.1007/s12672-024-01063-4 -
Angewandte Chemie (International Ed. in... Jun 2023Puromycin derivatives containing an emissive thieno[3,4-d]-pyrimidine core, modified with azetidine and 3,3-difluoroazetidine as Me N surrogates, exhibit translation...
Puromycin derivatives containing an emissive thieno[3,4-d]-pyrimidine core, modified with azetidine and 3,3-difluoroazetidine as Me N surrogates, exhibit translation inhibition and bactericidal activity similar to the natural antibiotic. The analogues are capable of cellular puromycylation of nascent peptides, generating emissive products without any follow-up chemistry. The 3,3-difluoroazetidine-containing analogue is shown to fluorescently label newly translated peptides and be visualized in both live and fixed HEK293T cells and rat hippocampal neurons.
Topics: Rats; Animals; Humans; Puromycin; HEK293 Cells; Peptides
PubMed: 36973168
DOI: 10.1002/anie.202216784 -
Cold Spring Harbor Protocols Jun 2024The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as...
The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as immunostaining or for the study of promoter activity by cloning a fluorescence reporter gene under the control of that promoter. We previously reported that puromycin selection is efficient in transgenic or larvae expressing an selection marker. A concentration of puromycin of >10 µg/mL is lethal for larvae, unless they carry the resistance gene, conferring them resistance to puromycin concentrations of 25-80 µg/mL. A drawback of this fully dominant selection marker is that, unlike with fluorescence markers, homozygous transgenics cannot be distinguished from heterozygotes. Here, we outline the procedure for selecting puromycin-resistant transgenic larvae.
Topics: Animals; Puromycin; Larva; Animals, Genetically Modified; Anopheles; Selection, Genetic
PubMed: 37696571
DOI: 10.1101/pdb.prot108308 -
Scientific Reports Sep 2022Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes...
Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes mTORC1 activation with Leu via glutaminolysis and Leu absorption via an antiporter. However, Gln has also been shown to inhibit mTORC1 activity. To resolve this paradox, we aimed to elucidate the effects of Gln on Leu-mediated mTORC1 activation. We administered Leu, Gln, tryptophan, Leu + Gln, or Leu + tryptophan to mice after 24-h fasting. The mice were then administered puromycin to evaluate protein synthesis and the gastrocnemius muscle was harvested 30 min later. Phosphorylated eukaryotic initiation factor 4E-binding protein 1, 70-kDa ribosomal protein S6 kinase 1, and Unc-51 like kinase 1 levels were the highest in the Leu + Gln group and significantly increased compared with those in the control group; however, Gln alone did not increase the levels of phosphorylated proteins. No difference in glutamate dehydrogenase activity was observed between the groups. Leu concentrations in the gastrocnemius muscle were similar in the Leu-intake groups. Our study highlights a novel mechanism underlying the promotive effect of Gln on Leu-mediated mTORC1 activation, providing insights into the pathway through which amino acids regulate muscle protein metabolism.
Topics: Amino Acids; Animals; Antiporters; Eating; Eukaryotic Initiation Factor-4E; Glutamate Dehydrogenase; Glutamine; Leucine; Mechanistic Target of Rapamycin Complex 1; Mice; Muscle Proteins; Phosphorylation; Puromycin; Tryptophan
PubMed: 36151270
DOI: 10.1038/s41598-022-20251-2 -
Molecular Biomedicine Jun 2022The 'angiogenic switch' is critical for tumor progression. However, the pathological details and molecular mechanisms remain incompletely characterized. In this study,...
The 'angiogenic switch' is critical for tumor progression. However, the pathological details and molecular mechanisms remain incompletely characterized. In this study, we established mammal xenografts in zebrafish to visually investigate the first vessel growth (angiogenic switch) in real-time, by inoculating tumor cells into the perivitelline space of live optically transparent Transgenic (flk1:EGFP) zebrafish larvae. Using this model, we found that hypoxia and hypoxia-inducible factor (HIF) signaling were unnecessary for the angiogenic switch, whereas vascular endothelial growth factor A gene (Vegfa) played a crucial role. Mechanistically, transcriptome analysis showed that the angiogenic switch was characterized by inhibition of translation, but not hypoxia. Phosphorylation of eukaryotic translation initiation factor 2 alpha (Eif2α) and the expression of Vegfa were increased in the angiogenic switch microtumors, and 3D tumor spheroids, and puromycin-treated tumor cells. Vegfa overexpression promoted early onset of the angiogenic switch, whereas Vegfa knockout prevented the first tumor vessel from sprouting. Pretreatment of tumor cells with puromycin promoted the angiogenic switch in vivo similarly to Vegfa overexpression, whereas Vegfa knockdown suppressed the increase. This study provides direc and dynamic in vivo evidences that inhibition of translation, but not hypoxia or HIF signaling promotes the angiogenic switch in tumor by increasing Vegfa transcription.
PubMed: 35695994
DOI: 10.1186/s43556-022-00081-4 -
Non-coding RNA Jun 2022Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the...
Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the glomerular filtration barrier in the kidney. Therefore, podocyte injury with morphological alteration and detachment from glomerular capillaries leads to severe proteinuria and subsequent renal failure through glomerulosclerosis. Previous RNA sequencing analysis of primary rat podocytes exposed to puromycin aminonucleoside (PAN), a well-known experimental model of injured podocytes, identified several transcripts as being aberrantly expressed. However, how the expression of these transcripts is regulated remains unclear. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally inhibit the expression of their target transcripts. In this study, using small RNA sequencing analysis, was identified as the most upregulated transcript in PAN-treated rat podocytes. overexpression in E11 podocyte cells led to shrunken cells with abnormal actin cytoskeletons. Consistent with these changes in cell morphology, gene ontology (GO) enrichment analysis showed that interactive GO terms related to cell morphogenesis were enriched with the predicted targets of . Of the predicted targets highly downregulated by PAN, () is a nonmuscle myosin predicted to be involved in actin filament organization and thought to play a role in podocyte morphogenesis and injury. We demonstrated that targets Myo1d by luciferase assays, qRT-PCR, and Western blotting. Furthermore, we showed that was present in urine from PAN- but not saline-administrated rats. Taken together, our data suggest that may serve as a therapeutic target and a biomarker for podocyte injury.
PubMed: 35736640
DOI: 10.3390/ncrna8030043 -
Experimental Cell Research Sep 2022Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs...
Mitochondrial cloning is a promising approach to achieve homoplasmic mitochondrial DNA (mtDNA) mutations. We previously developed a microfluidic device that performs single mitochondrion transfer from a mtDNA-intact cell to a mtDNA-less (ρ) cell by promoting cytoplasmic connection through a microtunnel between them. In the present study, we described a method for generating transmitochondrial cybrids using the microfluidic device. After achieving mitochondrial transfer between HeLa cells and thymidine kinase-deficient ρ143B cells using the microfluidic device, selective culture was carried out using a pyruvate and uridine (PU)-absent and 5-bromo-2'-deoxyuridine-supplemented culture medium. The resulting cells contained HeLa mtDNA and 143B nuclei, but both 143B mtDNA and HeLa nuclei were absent in these cells. Additionally, these cells showed lower lactate production than parent ρ143B cells and disappearance of PU auxotrophy for cell growth. These results suggest successful generation of transmitochondrial cybrids using the microfluidic device. Furthermore, we succeeded in selective harvest of generated transmitochondrial cybrids under a PU-supplemented condition by removing unfused ρ cells with puromycin-based selection in the microfluidic device.
Topics: Cytoplasm; DNA, Mitochondrial; HeLa Cells; Humans; Hybrid Cells; Lab-On-A-Chip Devices; Mitochondria
PubMed: 35659971
DOI: 10.1016/j.yexcr.2022.113233 -
Kidney International Sep 2021Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized...
Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphangiogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.
Topics: Animals; Apolipoprotein A-I; Endothelial Cells; Kidney; Lymphangiogenesis; Lymphatic Vessels; Mice; Rats; Vascular Endothelial Growth Factor C
PubMed: 34102217
DOI: 10.1016/j.kint.2021.05.028 -
Puromycin-sensitive aminopeptidase is required for C2C12 myoblast proliferation and differentiation.Journal of Cellular Physiology Jul 2021The ubiquitin-proteasome system is a major protein degradation pathway in the cell. Proteasomes produce several peptides that are rapidly degraded to free amino acids by...
The ubiquitin-proteasome system is a major protein degradation pathway in the cell. Proteasomes produce several peptides that are rapidly degraded to free amino acids by intracellular aminopeptidases. Our previous studies reported that proteolysis via proteasomes and aminopeptidases is required for myoblast proliferation and differentiation. However, the role of intracellular aminopeptidases in myoblast proliferation and differentiation had not been clarified. In this study, we investigated the effects of puromycin-sensitive aminopeptidase (PSA) on C2C12 myoblast proliferation and differentiation by knocking down PSA. Aminopeptidase enzymatic activity was reduced in PSA-knockdown myoblasts. Knockdown of PSA induced impaired cell cycle progression in C2C12 myoblasts and accumulation of cells at the G2/M phase. Additionally, after the induction of myogenic differentiation in PSA-knockdown myoblasts, multinucleated circular-shaped myotubes with impaired cell polarity were frequently identified. Cell division cycle 42 (CDC42) knockdown in myoblasts resulted in a loss of cell polarity and the formation of multinucleated circular-shaped myotubes, which were similar to PSA-knockdown myoblasts. These data suggest that PSA is required for the proliferation of myoblasts in the growth phase and for the determination of cell polarity and elongation of myotubes in the differentiation phase.
Topics: Aminopeptidases; Animals; Cell Differentiation; Cell Line; Cell Proliferation; Mice; Muscle Development; Myoblasts
PubMed: 33378552
DOI: 10.1002/jcp.30237 -
Stem Cell Research & Therapy Mar 2022The liver plays an important role in various metabolic processes, including protein synthesis, lipid and drug metabolisms and detoxifications. Primary culture of...
BACKGROUND
The liver plays an important role in various metabolic processes, including protein synthesis, lipid and drug metabolisms and detoxifications. Primary culture of hepatocytes is used for the understanding of liver physiology as well as for the drug development. Hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. Alternatively, systems using animal models and hepatocellular carcinoma cells have been established, but interspecies differences, variation between human cell sources and limited hepatic functions are among the challenges faced when using these models. Therefore, there is still a need for a highly stable method to purify human hepatocytes with functional sufficiency. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply.
METHODS
We first established a growth culture system for hepatocytes derived from patients with drug-induced liver injury using fetal mouse fibroblasts and EMUKK-05 medium. We then evaluated the morphology, proliferative capacity, chromosome stability, gene and protein expression profiles, and drug metabolic capacity of hepatocytes in early, middle and late passages with and without puromycin. In addition, hepatic maturation in 3D culture was evaluated from morphological and functional aspects.
RESULTS
In our culture system, the stable proliferation of human hepatocytes was achieved by co-culturing with mouse fetal fibroblasts, resulting in dedifferentiation into hepatic progenitor-like cells. We purified human hepatocytes by selection with cytocidal puromycin and cultured them for more than 60 population doublings over a span of more than 350 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytocidal antibiotics because of enhanced drug-metabolizing activity.
CONCLUSIONS
These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.
Topics: Animals; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cytochrome P-450 Enzyme System; Hepatocytes; Humans; Liver; Mice
PubMed: 35279203
DOI: 10.1186/s13287-022-02776-5