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Adaptive DNA amplification of synthetic gene circuit opens a way to overcome cancer chemoresistance.Proceedings of the National Academy of... Dec 2023Drug resistance continues to impede the success of cancer treatments, creating a need for experimental model systems that are broad, yet simple, to allow the...
Drug resistance continues to impede the success of cancer treatments, creating a need for experimental model systems that are broad, yet simple, to allow the identification of mechanisms and novel countermeasures applicable to many cancer types. To address these needs, we investigated a set of engineered mammalian cell lines with synthetic gene circuits integrated into their genome that evolved resistance to Puromycin. We identified DNA amplification as the mechanism underlying drug resistance in 4 out of 6 replicate populations. Triplex-forming oligonucleotide (TFO) treatment combined with Puromycin could efficiently suppress the growth of cell populations with DNA amplification. Similar observations in human cancer cell lines suggest that TFOs could be broadly applicable to mitigate drug resistance, one of the major difficulties in treating cancer.
Topics: Animals; Humans; DNA; Drug Resistance, Neoplasm; Genes, Synthetic; Oligonucleotides; Puromycin; Mammals; Neoplasms
PubMed: 38019857
DOI: 10.1073/pnas.2303114120 -
Discover Oncology Nov 2023To investigate the influence of β-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms.
PURPOSE
To investigate the influence of β-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms.
METHODS
PC3 and DU145 cells with stable β-arrestin2 overexpression and C4-2 cells with stable β-arrestin2 knockdown, were constructed via using lentivirus and puromycin selection. MTT and colony formation assays were carried out to investigate the effect of β-arrestin2 expression on the docetaxel resistance of CRPC cells. Glycolysis analysis was used to assess the glycolytic capacity modulated by β-arrestin2. GO enrichment analysis, gene set enrichment analysis and Spearman correlation test were carried out to explore the potential biological function and mechanism via using public data from GEO and TCGA. The expressions of PKM2, Phospho-PKM2, Phospho-ERK1/2 and hnRNP A1 were detected by western blot. Functional blocking experiments were carried out to confirm the roles of PKM2 and hnRNP A1 in the regulation of β-arrestin2's biological functions via silencing PKM2 or hnRNP A1 expression in cells with stable β-arrestin2 overexpression. Finally, nude mice xenograft models were established to confirm the experimental results of cell experiments.
RESULTS
β-Arrestin2 significantly decreased the sensitivity of CRPC cells to docetaxel stimulation, through enhancing the phosphorylation and expression of PKM2. Additionally, β-arrestin2 increased PKM2 phosphorylation via the ERK1/2 signaling pathway and induced PKM2 expression in a post-transcriptional manner through an hnRNP A1-dependent PKM alternative splicing mechanism, rather than by inhibiting its ubiquitination degradation.
CONCLUSION
Our findings indicate that the β-arrestin2/hnRNP A1/PKM2 pathway could be a promising target for treating docetaxel-resistant CRPC.
PubMed: 38019357
DOI: 10.1007/s12672-023-00740-0 -
Biomedicines Nov 2023Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless,...
Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.
PubMed: 38002039
DOI: 10.3390/biomedicines11113039 -
Human Cell Jan 2024Nucleoporin 93 (NUP93) is an important component of the nuclear pore complex, exhibiting pro-tumorigenic properties in some cancers. However, its function in esophageal...
Nucleoporin 93 (NUP93) is an important component of the nuclear pore complex, exhibiting pro-tumorigenic properties in some cancers. However, its function in esophageal squamous cell carcinoma (ESCC) has not been elucidated. This study aimed to investigate the effects of NUP93 in ESCC and the underlying mechanisms involved. Through analysis of public human cancer datasets, we observed higher expression of NUP93 in esophageal cancer tissues than in normal tissues. Stable ESCC cell lines with NUP93 overexpression or knockdown were established by lentiviral vector transduction and puromycin selection. NUP93 knockdown suppressed the proliferation, colony formation, cell cycle transition, migration, and invasion of ESCC cells, while the overexpression of NUP93 displayed opposite effects. NUP93 positively regulated epithelial-mesenchymal transition and AKT signaling transduction in ESCC cells. In addition, NUP93 increased the expression of programmed death ligand 1 (PD-L1) in ESCC cells and attenuated NK cell-mediated lysis of ESCC cells. In vivo experiments demonstrated that NUP93 promotes the growth of ESCC in nude mice, enhances Ki67 and PD-L1 expression, and promotes AKT signaling transduction in xenografts. Mechanistically, we demonstrated that the HECT domain E3 ubiquitin protein ligase 1 (HECTD1) contributes to the ubiquitination and degradation of NUP93 and acts as a tumor suppressor in ESCC. To conclude, this study has shown that NUP93 has pro-tumor properties in ESCC and that HECTD1 functions as an upstream regulator of NUP93 in ESCC. These findings may contribute to the investigation of potential therapeutic targets in ESCC.
Topics: Animals; Humans; Mice; B7-H1 Antigen; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Mice, Nude; Nuclear Pore Complex Proteins; Proto-Oncogene Proteins c-akt; Ubiquitin-Protein Ligases
PubMed: 37993750
DOI: 10.1007/s13577-023-01005-2 -
Environmental Toxicology and... Nov 2023Our study aimed to verify the hypothesis concerning low-frequency magnetic fields (LF-MFs)-related changes in cell viability through the biomechanism(s) based on...
Our study aimed to verify the hypothesis concerning low-frequency magnetic fields (LF-MFs)-related changes in cell viability through the biomechanism(s) based on calcineurin (CaN)-mediated signaling pathways triggered via ROS-like molecules. For experiments, Mono Mac 6 and U937 leukocytic cell lines were chosen and exposed to various LF-MFs and/or puromycin (PMC). The protein expression level of key regulatory proteins of calcium metabolism was examined by Western Blot analysis. In turn, the reactive oxygen species (ROS) and cell viability parameters were evaluated by cytochrome C reduction assay and flow cytometry, respectively. The simultaneous action of applied MF and PMC influenced cell viability in a MF-dependent manner. The changes in cell viability were correlated with protein expression and ROS levels. It was verified experimentally that applied stress stimuli influence cell susceptibility to undergo cell death. Moreover, the evoked bioeffects might be recognized as specific to both types of leukocyte populations.
Topics: Electromagnetic Fields; Reactive Oxygen Species; Calcium; Cell Line; Puromycin; Leukocytes
PubMed: 37984675
DOI: 10.1016/j.etap.2023.104320 -
Clinical and Translational Medicine Nov 2023Translation dysregulation plays a crucial role in tumourigenesis and cancer progression. Oncogenic translation relies on the stability and availability of tRNAs for...
BACKGROUND
Translation dysregulation plays a crucial role in tumourigenesis and cancer progression. Oncogenic translation relies on the stability and availability of tRNAs for protein synthesis, making them potential targets for cancer therapy.
METHODS
This study performed immunohistochemistry analysis to assess NSUN2 levels in thyroid cancer. Furthermore, to elucidate the impact of NSUN2 on anaplastic thyroid cancer (ATC) malignancy, phenotypic assays were conducted. Drug inhibition and time-dependent plots were employed to analyse drug resistance. Liquid chromatography-mass spectrometry and bisulphite sequencing were used to investigate the m C methylation of tRNA at both global and single-base levels. Puromycin intake and high-frequency codon reporter assays verified the protein translation level. By combining mRNA and ribosome profiling, a series of downstream proteins and codon usage bias were identified. The acquired data were further validated by tRNA sequencing.
RESULTS
This study observed that the tRNA m C methyltransferase NSUN2 was up-regulated in ATC and is associated with dedifferentiation. Furthermore, NSUN2 knockdown repressed ATC formation, proliferation, invasion and migration both in vivo and in vitro. Moreover, NSUN2 repression enhanced the sensitivity of ATC to genotoxic drugs. Mechanically, NSUN2 catalyses tRNA structure-related m C modification, stabilising tRNA that maintains homeostasis and rapidly transports amino acids, particularly leucine. This stable tRNA has a substantially increased efficiency necessary to support a pro-cancer translation program including c-Myc, BCL2, RAB31, JUNB and TRAF2. Additionally, the NSUN2-mediated variations in m5C levels and different tRNA Leu iso-decoder families, partially contribute to a codon-dependent translation bias. Surprisingly, targeting NSUN2 disrupted the c-Myc to NSUN2 cycle in ATC.
CONCLUSIONS
This research revealed that a pro-tumour m5C methyltransferase, dynamic tRNA stability regulation and downstream oncogenes, c-Myc, elicits a codon-dependent oncogenic translation network that enhances ATC growth and formation. Furthermore, it provides new opportunities for targeting translation reprogramming in cancer cells.
Topics: Humans; Thyroid Carcinoma, Anaplastic; Methyltransferases; Codon; Thyroid Neoplasms; RNA, Transfer; rab GTP-Binding Proteins
PubMed: 37983928
DOI: 10.1002/ctm2.1466 -
Pharmacology & Therapeutics Dec 2023During carcinogenesis, neoplastic cells accumulate mutations in genes important for cellular homeostasis, producing defective proteins. Viral infections occur when viral... (Review)
Review
During carcinogenesis, neoplastic cells accumulate mutations in genes important for cellular homeostasis, producing defective proteins. Viral infections occur when viral capsid proteins bind to the host cell receptor, allowing the virus to enter the cells. In both cases, proteins play important roles in cancer development and viral infection, so these targets can be exploited to develop alternative treatments. mRNA display technology is a very powerful tool for the development of peptides capable of acting on specific targets in neoplastic cells or on viral capsid proteins. mRNA display technology allows the selection and evolution of peptides with desired functional properties from libraries of many nucleic acid variants. Among other advantages of this technology, the use of flexizymes allows the production of peptides with unnatural amino acid residues, which can enhance the activity of these molecules. From target immobilization, peptides with greater specificity for the targets of interest are generated during the selection rounds. Herein, we will explore the use of mRNA display technology for the development of active peptides after successive rounds of selection, using proteins present in neoplastic cells and viral particles as targets.
Topics: Humans; Capsid Proteins; RNA, Messenger; Peptides; Mutation; Neoplasms
PubMed: 37952905
DOI: 10.1016/j.pharmthera.2023.108559 -
Planta Medica Feb 2024Astragaloside IV (AS-IV) has been shown to provide renal protection in various kidney injury models. However, the metabolic profile variation of AS-IV in pathological...
Astragaloside IV (AS-IV) has been shown to provide renal protection in various kidney injury models. However, the metabolic profile variation of AS-IV in pathological models is not well established. This study aims to explore the metabolic pathway of AS-IV in the classical puromycin aminonucleoside (PAN)-induced kidney injury in a rat model. Twelve Wistar rats were randomly divided into the AS-IV (CA) and the PAN+AS-IV (PA) treatment groups. PAN was injected by a single tail intravenous (i. v.) injection at 5 mg/100 g body weight, and AS-IV was administered intragastrically (i. g.) at 40 mg/kg for 10 days. Fecal samples of these rats were collected, and metabolites of AS-IV were detected by ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) to explore the AS-IV metabolic pathway. The metabolic differences between the AS-IV and PAN+AS-IV groups were compared. A total of 25 metabolites were detected, and deglycosylation, deoxygenation, and methyl oxidation were found to be the main metabolic pathways of AS-IV . The abundance of most of these metabolites in the PAN+AS-IV group was lower than that in the AS-IV treatment group, and differences for seven of them were statistically significant. Our study indicates that AS-IV metabolism is affected in the PAN-induced kidney injury rat model.
Topics: Rats; Animals; Tandem Mass Spectrometry; Chromatography, High Pressure Liquid; Rats, Wistar; Puromycin; Saponins; Triterpenes
PubMed: 37931776
DOI: 10.1055/a-2186-3182 -
BMC Nephrology Oct 2023Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated.
INTRODUCTION
Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated.
METHODS
The NS rat model was established by single intraperitoneal injection of 100 mg/kg puromycin aminonucleoside (PAN). The plasma electrolyte level and urinary sodium excretion were monitored dynamically. The changes of some sodium transporters, including epithelial Na channel (ENaC), Na/H exchanger 3 (NHE3), Na-K-2Cl cotransporter 2 (NKCC2) and Na-Cl cotransporter (NCC) in renal cortex at different time points and the level of peripheral circulation factors were detected.
RESULTS
The urinary sodium excretion of the model group increased significantly on the first day, then decreased compared with the control group, and there was no significant difference between the model group and the control group on the 12th day. The changes of peripheral circulation factors were not obvious. Some sodium transporters in renal cortex increased in varying degrees, while NKCC2 decreased significantly compared with the control group.
CONCLUSIONS
The occurrence of NS edema may not be related to the angiotensin system. The decrease of urinary sodium excretion is independent of the development of albuminuria. During the 18 days of observation, it can be divided into three stages: sodium retention, sodium compensation, and simple water retention. The mechanism is related to the increased expression of α-ENaC, γ-ENaC, NHE3 and NCC in a certain period of time, the compensatory decrease of NKCC2 expression and the continuous increase of aquaporin 2 (AQP2) expression.
Topics: Rats; Animals; Nephrotic Syndrome; Puromycin Aminonucleoside; Sodium; Sodium-Hydrogen Exchanger 3; Aquaporin 2; Epithelial Sodium Channels; Kidney; Membrane Transport Proteins; Solute Carrier Family 12, Member 3; Water
PubMed: 37880610
DOI: 10.1186/s12882-023-03367-z -
Sheng Wu Gong Cheng Xue Bao = Chinese... Oct 2023Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue,...
Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 () gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.
Topics: Humans; Induced Pluripotent Stem Cells; Sirolimus; Caspase 9; RNA, Guide, CRISPR-Cas Systems; Pluripotent Stem Cells; Cell Differentiation; Puromycin
PubMed: 37877393
DOI: 10.13345/j.cjb.230092