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1.
Hum Mol Genet ; 33(11): 1001-1014, 2024 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-38483348

RESUMEN

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.


Asunto(s)
Repeticiones de Minisatélite , Páncreas Exocrino , Humanos , Repeticiones de Minisatélite/genética , Animales , Ratones , Páncreas Exocrino/metabolismo , Páncreas Exocrino/enzimología , Células HEK293 , Mutagénesis Insercional/genética , Alelos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/enzimología , Frecuencia de los Genes , Masculino , Femenino , Lipasa/genética
2.
Anal Chem ; 96(4): 1707-1716, 2024 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-38241523

RESUMEN

Improving the retention of small-molecule-based therapeutic agents in tumors is crucial to achieve precise diagnosis and effective therapy of cancer. Herein, we propose a ß-galactosidase (ß-Gal)-activated and red light-induced RNA modification (GALIRM) strategy for prolonged tumor imaging. A ß-Gal-activatable near-infrared (NIR) fluorescence (FL) and positron emission tomography (PET) bimodal probe 68Ga-NOTA-FCG consists of a triaaza triacetic acid chelator NOTA for 68Ga-labeling, a ß-Gal-activated photosensitizer CyGal, and a singlet oxygen (1O2)-susceptible furan group for RNA modification. Studies have demonstrated that the probe emits an activated NIR FL signal upon cleavage by endogenous ß-Gal overexpressed in the lysosomes, which is combined with the PET imaging signal of 68Ga allowing for highly sensitive imaging of ovarian cancer. Moreover, the capability of 68Ga-NOTA-FCG generating 1O2 under 690 nm illumination could be simultaneously unlocked, which can trigger the covalent cross-linking between furan and nucleotides of cytoplasmic RNAs. The formation of the probe-RNA conjugate can effectively prevent exocytosis and prolong retention of the probe in tumors. We thus believe that this GALIRM strategy may provide entirely new insights into long-term tumor imaging and efficient tumor treatment.


Asunto(s)
Neoplasias Ováricas , Luz Roja , Femenino , Humanos , Fluorescencia , Radioisótopos de Galio , Tomografía de Emisión de Positrones/métodos , beta-Galactosidasa , Furanos
3.
Biochem Biophys Res Commun ; 703: 149646, 2024 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-38350212

RESUMEN

Legumain is overexpressed in diverse tumors, serving as a significant tumor biomarker. Our study aimed to develop a new positron emission tomography (PET) probe [68Ga]Ga-NOTA-SF-AANM for imaging the expression level of legumain in vivo. The radio-labeling of [68Ga]Ga-NOTA-SF-AANM was accomplished within 15 min. The probe has good stability in vitro. NOTA-SF-AANM exhibited rapid response to recombinant human legumain enzyme, enabling intramolecular condensation cyclization. Cellular uptake and lysosomal co-localization experiments demonstrated that the probe was able to differentiate specifically between MDA-MB-468 and PC-3 cancer cells with varying degrees of legumain expression. PET imaging displayed a significant and persistent signal (3.59 ± 0.30 %ID/mL at 60 min) in MDA-MB-468 tumors, while PC-3 tumors exhibited lower radioactivity (1.08 ± 0.35 %ID/mL at 60 min), further validating the specific targeting of [68Ga]Ga-NOTA-SF-AANM towards legumain. [68Ga]Ga-NOTA-SF-AANM is a promising tool for precise diagnosis of legumain-related diseases due to its advantages in radio-labeling and accurate monitoring of legumain expression levels.


Asunto(s)
Cisteína Endopeptidasas , Radioisótopos de Galio , Neoplasias , Humanos , Tomografía de Emisión de Positrones/métodos , Neoplasias/diagnóstico por imagen , Lisosomas , Línea Celular Tumoral
4.
J Transl Med ; 22(1): 404, 2024 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-38689297

RESUMEN

BACKGROUND: Ischemic heart disease is one of the leading causes of mortality worldwide, and thus calls for development of more effective therapeutic strategies. This study aimed to identify potential therapeutic targets for coronary heart disease (CHD) and myocardial infarction (MI) by investigating the causal relationship between plasma proteins and these conditions. METHODS: A two-sample Mendelian randomization (MR) study was performed to evaluate more than 1600 plasma proteins for their causal associations with CHD and MI. The MR findings were further confirmed through Bayesian colocalization, Summary-data-based Mendelian Randomization (SMR), and Transcriptome-Wide Association Studies (TWAS) analyses. Further analyses, including enrichment analysis, single-cell analysis, MR analysis of cardiovascular risk factors, phenome-wide Mendelian Randomization (Phe-MR), and protein-protein interaction (PPI) network construction were conducted to verify the roles of selected causal proteins. RESULTS: Thirteen proteins were causally associated with CHD, seven of which were also causal for MI. Among them, FES and PCSK9 were causal proteins for both diseases as determined by several analytical methods. PCSK9 was a risk factor of CHD (OR = 1.25, 95% CI: 1.13-1.38, P = 7.47E-06) and MI (OR = 1.36, 95% CI: 1.21-1.54, P = 2.30E-07), whereas FES was protective against CHD (OR = 0.68, 95% CI: 0.59-0.79, P = 6.40E-07) and MI (OR = 0.65, 95% CI: 0.54-0.77, P = 5.38E-07). Further validation through enrichment and single-cell analysis confirmed the causal effects of these proteins. Moreover, MR analysis of cardiovascular risk factors, Phe-MR, and PPI network provided insights into the potential drug development based on the proteins. CONCLUSIONS: This study investigated the causal pathways associated with CHD and MI, highlighting the protective and risk roles of FES and PCSK9, respectively. FES. Specifically, the results showed that these proteins are promising therapeutic targets for future drug development.


Asunto(s)
Proteínas Sanguíneas , Enfermedad Coronaria , Análisis de la Aleatorización Mendeliana , Infarto del Miocardio , Proteómica , Humanos , Infarto del Miocardio/sangre , Infarto del Miocardio/genética , Proteómica/métodos , Enfermedad Coronaria/sangre , Enfermedad Coronaria/genética , Proteínas Sanguíneas/metabolismo , Mapas de Interacción de Proteínas/genética , Teorema de Bayes , Terapia Molecular Dirigida , Factores de Riesgo , Estudio de Asociación del Genoma Completo , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/metabolismo
5.
Bioconjug Chem ; 35(9): 1352-1362, 2024 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-39187748

RESUMEN

Prostate cancer is the most prevalent malignant tumor affecting male individuals worldwide. The accurate early detection of prostate cancer is crucial to preventing unnecessary diagnosis and subsequent excessive treatment. Prostate-specific membrane antigen (PSMA) has emerged as a promising biomarker for the diagnosis of prostate cancer. In this study, a dual-modality imaging probe utilizing aptamer technology was developed for positron emission tomography/near-infrared fluorescence (PET/NIRF) imaging, and the specificity and sensitivity of the probe toward PSMA were evaluated both in vitro and in vivo. The probe precursor NOTA-PSMA-Cy5 was synthesized via automated solid-phase oligonucleotide synthesis. Subsequently, the PET/NIRF dual-modality probe [68Ga]Ga-NOTA-PSMA-Cy5 was successfully prepared and exhibited favorable fluorescence properties and stability in vitro. The binding specificity of [68Ga]Ga-NOTA-PSMA-Cy5 to PSMA was assessed through flow cytometry, fluorescence imaging, and cellular uptake experiments in LNCaP cells and PC-3 cells. In vivo PET/NIRF imaging studies demonstrated the sensitive and specific binding of [68Ga]Ga-NOTA-PSMA-Cy5 to PSMA. Overall, the PET/NIRF dual-modality probe [68Ga]Ga-NOTA-PSMA-Cy5 shows promise for the diagnosis of prostate cancer and for the fluorescence-guided identification of PSMA-positive cancer lesions during surgical procedures.


Asunto(s)
Aptámeros de Nucleótidos , Colorantes Fluorescentes , Radioisótopos de Galio , Glutamato Carboxipeptidasa II , Tomografía de Emisión de Positrones , Neoplasias de la Próstata , Tomografía de Emisión de Positrones/métodos , Humanos , Masculino , Colorantes Fluorescentes/química , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/diagnóstico , Aptámeros de Nucleótidos/química , Glutamato Carboxipeptidasa II/metabolismo , Glutamato Carboxipeptidasa II/análisis , Animales , Línea Celular Tumoral , Radioisótopos de Galio/química , Antígenos de Superficie/análisis , Antígenos de Superficie/metabolismo , Ratones , Imagen Óptica/métodos , Células PC-3
6.
Bioconjug Chem ; 35(5): 665-673, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38598424

RESUMEN

Enhancing the accumulation and retention of small-molecule probes in tumors is an important way to achieve accurate cancer diagnosis and therapy. Enzyme-stimulated macrocyclization of small molecules possesses great potential for enhanced positron emission tomography (PET) imaging of tumors. Herein, we reported an 18F-labeled radiotracer [18F]AlF-RSM for legumain detection in vivo. The tracer was prepared by a one-step aluminum-fluoride-restrained complexing agent ([18F]AlF-RESCA) method with high radiochemical yield (RCY) (88.35 ± 3.93%) and radiochemical purity (RCP) (>95%). More notably, the tracer can be transformed into a hydrophobic macrocyclic molecule under the joint action of legumain and reductant. Simultaneously, the tracer could target legumain-positive tumors and enhance accumulation and retention in tumors, resulting in the amplification of PET imaging signals. The enhancement of radioactivity enables PET imaging of legumain activity with high specificity. We envision that, by combining this highly efficient 18F-labeled strategy with our intramolecular macrocyclization reaction, a range of radiofluorinated tracers can be designed for tumor PET imaging and early cancer diagnosis in the future.


Asunto(s)
Cisteína Endopeptidasas , Radioisótopos de Flúor , Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Radioisótopos de Flúor/química , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/análisis , Animales , Ciclización , Ratones , Humanos , Radiofármacos/química , Línea Celular Tumoral , Ratones Endogámicos BALB C , Fluoruros/química , Ratones Desnudos
7.
Eur J Nucl Med Mol Imaging ; 51(3): 625-640, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37878029

RESUMEN

PURPOSE: Due to tumor heterogeneity, immunohistochemistry (IHC) showed poor accuracy in detecting the expression of programmed cell death ligand-1 (PD-L1) in patients. Positron emission tomography (PET) imaging is considered as a non-invasive technique to detect PD-L1 expression at the molecular level visually, real-timely and quantitatively. This study aimed to develop novel peptide-based radiotracers [68Ga]/[18F]AlF-NOTA-IMB for accurately detecting the PD-L1 expression and guiding the cancer immunotherapy. METHODS: NOTA-IMB was prepared by connecting 2,2'-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)- 2-oxoethyl)-1,4,7-triazonane-1,4-diyl) diacetic acid (NOTA-NHS) with PD-L1-targeted peptide IMB, and further radiolabeled with 68Ga or 18F-AlF. In vitro binding assay was conducted to confirm the ability of [68Ga]/[18F]AlF-NOTA-IMB to detect the expression of PD-L1. In vivo PET imaging of [68Ga]NOTA-IMB and [18F]AlF-NOTA-IMB in different tumor-bearing mice was performed, and dynamic changes of PD-L1 expression level induced by immunotherapy were monitored. Radioautography, western blotting, immunofluorescence staining and biodistribution analysis were carried out to further evaluate the specificity of radiotracers and efficacy of PD-L1 antibody immunotherapy. RESULTS: [68Ga]NOTA-IMB and [18F]AlF-NOTA-IMB were both successfully prepared with high radiochemical yield (> 95% and > 60%, n = 5) and radiochemical purity (> 95% and > 98%, n = 5). Both tracers showed high affinity to human and murine PD-L1 with the dissociation constant (Kd) of 1.00 ± 0.16/1.09 ± 0.21 nM (A375-hPD-L1, n = 3) and 1.56 ± 0.58/1.21 ± 0.39 nM (MC38, n = 3), respectively. In vitro cell uptake assay revealed that both tracers can specifically bind to PD-L1 positive cancer cells A375-hPD-L1 and MC38 (5.45 ± 0.33/3.65 ± 0.15%AD and 5.87 ± 0.27/2.78 ± 0.08%AD at 120 min, n = 3). In vivo PET imaging and biodistribution analysis showed that the tracer [68Ga]NOTA-IMB and [18F]AlF-NOTA-IMB had high accumulation in A375-hPD-L1 and MC38 tumors, but low uptake in A375 tumor. Treatment of Atezolizumab induced dynamic changes of PD-L1 expression in MC38 tumor-bearing mice, and the tumor uptake of [68Ga]NOTA-IMB decreased from 3.30 ± 0.29%ID/mL to 1.58 ± 0.29%ID/mL (n = 3, P = 0.026) after five treatments. Similarly, the tumor uptake of [18F]AlF-NOTA-IMB decreased from 3.27 ± 0.63%ID/mL to 0.89 ± 0.18%ID/mL (n = 3, P = 0.0004) after five treatments. However, no significant difference was observed in the tumor uptake before and after PBS treatment. Biodistribution, radioautography, western blotting and immunofluorescence staining analysis further demonstrated that the expression level of PD-L1 in tumor-bearing mice treated with Atezolizumab significantly reduced about 3 times and correlated well with the PET imaging results. CONCLUSION: [68Ga]NOTA-IMB and [18F]AlF-NOTA-IMB were successfully prepared for PET imaging the PD-L1 expression noninvasively and quantitatively. Dynamic changes of PD-L1 expression caused by immunotherapy can be sensitively detected by both tracers. Hence, the peptide-based radiotracers [68Ga]NOTA-IMB and [18F]AlF-NOTA-IMB can be applied for accurately detecting the PD-L1 expression in different tumors and monitoring the efficacy of immunotherapy.


Asunto(s)
Antígeno B7-H1 , Neoplasias , Humanos , Ratones , Animales , Antígeno B7-H1/metabolismo , Distribución Tisular , Radioisótopos de Galio/química , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , Péptidos/metabolismo , Inmunoterapia , Neoplasias/diagnóstico por imagen , Neoplasias/terapia
8.
Eur J Nucl Med Mol Imaging ; 51(7): 1826-1840, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38319321

RESUMEN

PURPOSE: Neuropilin-1 (NRP-1) is a multifunctional protein involved in a variety of biological processes such as angiogenesis, tumorigenesis and immunomodulation. It was usually overexpressed in many cancer cell lines and correlated with poor prognosis of breast cancer. Positron emission tomography (PET) is an advanced imaging technique for detecting the function and metabolism of tumor-associated molecules in real time, dynamically, quantitatively and noninvasively. To improve the level of early diagnosis and evaluate the prognosis of breast cancer, an NRP-1 targeting peptide-based tracer [68 Ga]Ga-NOTA-PEG4-CK2 was designed to sensitively and specifically detect the NRP-1 expression in vivo via PET imaging. METHODS: In silico modeling and microscale thermophoresis (MST) assay were carried out to design the NRP-1 targeting peptide NOTA-PEG4-CK2, and it was further radiolabeled with 68 Ga to prepare the tracer [68 Ga]Ga-NOTA-PEG4-CK2. The radiochemical yield (RCY), radiochemical purity (RCP), molar activity (Am), lipid-water partition coefficient (Log P) and stability of [68 Ga]Ga-NOTA-PEG4-CK2 were assessed. The targeting specificity of the tracer for NRP-1 was investigated by in vitro cellular uptake assay and in vivo PET imaging as well as blocking studies. The sensitivity of the tracer in monitoring the dynamic changes of NRP-1 expression induced by chemical drug was also investigated in vitro and in vivo. Ex vivo biodistribution, autoradiography, western blot, and immunofluorescence staining were also performed to study the specificity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1. RESULTS: [68 Ga]Ga-NOTA-PEG4-CK2 was designed and synthesized with high RCY (> 98%), high stability (RCP > 95%) and high affinity to NRP-1 (KD = 25.39 ± 1.65 nM). In vitro cellular uptake assay showed that the tracer [68 Ga]Ga-NOTA-PEG4-CK2 can specifically bind to NRP-1 positive cancer cells MDA-MB-231 (1.04 ± 0.04% at 2 h) rather than NRP-1 negative cancer cells NCI-H1299 (0.43 ± 0.05%). In vivo PET imaging showed the maximum tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 in MDA-MB-231 xenografts (4.16 ± 0.67%ID/mL) was significantly higher than that in NCI-H1299 xenografts (1.03 ± 0.19%ID/mL) at 10 min post injection, and the former exhibited higher tumor-to-muscle uptake ratio (5.22 ± 0.18) than the latter (1.07 ± 0.27) at 60 min post injection. MDA-MB-231 xenografts pretreated with nonradioactive precursor NOTA-PEG4-CK2 showed little tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 (1.67 ± 0.38%ID/mL at 10 min post injection). Both cellular uptake assay and PET imaging revealed that NRP-1 expression in breast cancer MDA-MB-231 could be effectively suppressed by SB-203580 treatment and can be sensitively detected by [68 Ga]Ga-NOTA-PEG4-CK2. Ex vivo analysis also proved the high specificity and sensitivity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1 expression in MDA-MB-231 xenografts. CONCLUSION: A promising NRP-1 targeting PET tracer [68 Ga]Ga-NOTA-PEG4-CK2 was successfully prepared. It showed remarkable specificity and sensitivity in monitoring the dynamic changes of NRP-1 expression. Hence, it could provide valuable information for early diagnosis of NRP-1 relevant cancers and evaluating the prognosis of cancer patients.


Asunto(s)
Radioisótopos de Galio , Neuropilina-1 , Tomografía de Emisión de Positrones , Neuropilina-1/metabolismo , Tomografía de Emisión de Positrones/métodos , Animales , Ratones , Humanos , Línea Celular Tumoral , Distribución Tisular , Femenino , Compuestos Heterocíclicos con 1 Anillo/química , Marcaje Isotópico , Péptidos/química , Regulación Neoplásica de la Expresión Génica , Radiofármacos/farmacocinética , Radiofármacos/química
9.
Pancreatology ; 24(4): 511-521, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38485544

RESUMEN

BACKGROUND & AIMS: Protease-sensitive PNLIP variants were recently associated with chronic pancreatitis (CP) in European populations. The pathological mechanism yet remains elusive. Herein, we performed a comprehensive genetic and functional analysis of PNLIP variants found in a large Chinese cohort, aiming to further unravel the enigmatic association of PNLIP variants with CP. METHODS: All coding and flanking intronic regions of the PNLIP gene were analyzed for rare variants by targeted next-generation sequencing in 1082 Chinese CP patients and 1196 controls. All novel missense variants were subject to analysis of secretion, lipase activity, and proteolytic degradation. One variant was further analyzed for its potential to misfold and induce endoplasmic reticulum (ER) stress. p.F300L, the most common PNLIP variant associated with CP, was used as a control. RESULTS: We identified 12 rare heterozygous PNLIP variants, with 10 being novel. The variant carrier frequency did not differ between the groups. Of them, only the variant p.A433T found in a single patient was considered pathologically relevant. p.A433T exhibited increased susceptibility to proteolytic degradation, which was much milder than p.F300L. Interestingly, both variants exhibited an increased tendency to misfold, leading to intracellular retention as insoluble aggregates, reduced secretion, and elevated ER stress. CONCLUSIONS: Our genetic and functional analysis of PNLIP variants identified in a Chinese CP cohort suggests that the p.A433T variant and the previously identified p.F300L variant are not only protease-sensitive but also may be potentially proteotoxic. Mouse studies of the PNLIP p.F300L and p.A433T variants are needed to clarify their role in CP.


Asunto(s)
Pueblo Asiatico , Predisposición Genética a la Enfermedad , Pancreatitis Crónica , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pueblo Asiatico/genética , China/epidemiología , Estudios de Cohortes , Pueblos del Este de Asia , Estrés del Retículo Endoplásmico/genética , Variación Genética , Lipasa/genética , Mutación Missense , Pancreatitis Crónica/genética
10.
Mol Pharm ; 21(7): 3513-3524, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38867453

RESUMEN

The estrogen receptor α positive (ERα+) subtype represents nearly 70% of all breast cancers (BCs), which seriously threaten women's health. Positron emission computed tomography (PET) characterizes its superiority in detecting the recurrence and metastasis of BC. In this article, an array of novel PET probes ([18F]R-1, [18F]R-2, [18F]R-3, and [18F]R-4) targeting ERα based on the tetrahydropyridinyl indole scaffold were developed. Among them, [18F]R-3 and [18F]R-4 showed good target specificity toward ERα and could distinguish MCF-7 (ERα+) and MDA-MB-231 (ERα-) tumors efficiently. Especially, [18F]R-3 could differentiate the ERα positive/negative tumors successfully with a higher tumor-to-muscle uptake ratio (T/M) than that of [18F]R-4. The radioactivity of [18F]R-3 in the MCF-7 tumor was 5.24 ± 0.84%ID/mL and its T/M ratio was 2.49 ± 0.62 at 25 min postinjection, which might be the optimal imaging time point in PET scanning. On the contrary, [18F]R-3 did not accumulate in the MDA-MB-231 tumor at all. The autoradiography analysis of [18F]R-3 on the MCF-7 tumor-bearing mice model was consistent with the PET imaging results. [18F]R-3 exhibited the pharmacokinetic property of rapid distribution and slow clearance, making it suitable for use as a diagnostic PET probe. Overall, [18F]R-3 was capable of serving as a PET radiotracer to delineate the ERα+ tumor and was worthy of further exploitation.


Asunto(s)
Neoplasias de la Mama , Receptor alfa de Estrógeno , Radioisótopos de Flúor , Tomografía de Emisión de Positrones , Radiofármacos , Animales , Humanos , Femenino , Receptor alfa de Estrógeno/metabolismo , Radioisótopos de Flúor/farmacocinética , Ratones , Tomografía de Emisión de Positrones/métodos , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Radiofármacos/farmacocinética , Células MCF-7 , Línea Celular Tumoral , Ratones Desnudos , Distribución Tisular , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de Xenoinjerto , Diseño de Fármacos
11.
Mol Pharm ; 21(3): 1382-1389, 2024 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-38372213

RESUMEN

Cathepsin B, a lysosomal protease, is considered as a crucial biomarker for tumor diagnosis and treatment as it is overexpressed in numerous cancers. A stimulus-responsive SF scaffold has been reported to detect the activity of a variety of tumor-associated enzymes. In this work, a small-molecule PET tracer ([68Ga]NOTA-SF-CV) was developed by combining an SF scaffold with a cathepsin B-specific recognition substrate Cit-Val. Upon activation by cathepsin B, [68Ga]NOTA-SF-CV could form the cyclization product in a reduction environment, resulting in reduced hydrophilicity. This unique property could effectively prevent exocytosis of the tracer in cathepsin B-overexpressing tumor cells, leading to prolonged retention and amplified PET imaging signal. Moreover, [68Ga]NOTA-SF-CV had great targeting specificity to cathepsin B. In vivo microPET imaging results showed that [68Ga]NOTA-SF-CV was able to effectively visualize the expression level of cathepsin B in various tumors. Hence, [68Ga]NOTA-SF-CV may be served as a potential tracer for diagnosing cathepsin B-related diseases.


Asunto(s)
Radioisótopos de Galio , Neoplasias , Humanos , Radioisótopos de Galio/química , Catepsina B , Tomografía de Emisión de Positrones/métodos , Neoplasias/diagnóstico por imagen , Radiofármacos/química , Línea Celular Tumoral
12.
Mol Pharm ; 21(1): 255-266, 2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38093483

RESUMEN

Immune checkpoint inhibitors (ICIs) therapy based on programmed cell death ligand 1 (PD-L1) has shown significant development in treating several carcinomas, but not all patients respond to this therapy due to the heterogeneity of PD-L1 expression. The sensitive and accurate quantitative analysis of in vivo PD-L1 expression is critical for treatment decisions and monitoring therapy. In the present study, an aptamer-based dual-modality positron emission tomography/near-infrared fluorescence (PET/NIRF) imaging probe was developed, and its specificity and sensitivity to PD-L1 were assessed in vitro and in vivo. The probe precursor NOTA-Cy5-R1 was prepared by using automated solid-phase oligonucleotide synthesis. PET/NIRF dual-modality probe [68Ga]Ga-NOTA-Cy5-R1 was successfully synthesized and radiolabeled. The binding specificity of [68Ga]Ga-NOTA-Cy5-R1 to PD-L1 was evaluated by flow cytometry, fluorescence imaging, and cellular uptake in A375-hPD-L1 and A375 cells, and it showed good fluorescence properties and stability in vitro. In vivo PET/NIRF imaging studies illustrated that [68Ga]Ga-NOTA-Cy5-R1 can sensitively and specifically bind to PD-L1 positive tumors. Meanwhile, the rapid clearance of probes from nontarget tissues achieved a high signal-to-noise ratio. In addition, changes of PD-L1 expression in NCI-H1299 xenografts treated with cisplatin (CDDP) were sensitivity monitored by [68Ga]Ga-NOTA-Cy5-R1 PET imaging, and ex vivo autoradiography and western blot analyses correlated well with the change of PD-L1 expression in vivo. Overall, [68Ga]Ga-NOTA-Cy5-R1 showed notable potency as a dual-modality PET/NIRF imaging probe for visualizing tumors and monitoring the dynamic changes of PD-L1 expression, which can help to direct and promote the clinical practice of ICIs therapy.


Asunto(s)
Antígeno B7-H1 , Neoplasias , Humanos , Antígeno B7-H1/metabolismo , Radioisótopos de Galio/química , Tomografía de Emisión de Positrones/métodos , Anticuerpos , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Línea Celular Tumoral
13.
Bioorg Chem ; 153: 107810, 2024 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-39276489

RESUMEN

Although antibody-based immune checkpoint blockades have been successfully used in antitumor immunotherapy, the low response rate is currently the main problem. In this work, a small-molecule programmed cell death-ligand (PD-L1) inhibitor, LG-12, was developed and radiolabeled with 131I to obtain the chemically and biologically identical radiopharmaceutical [131I]LG-12, which aimed to improve the antitumor effect by combination of LG-12 and [131I]LG-12. LG-12 showed high inhibitory activity to PD-1/PD-L1 interaction. The results of cell uptake and biodistribution studies indicated that [131I]LG-12 could specifically bind to PD-L1 in B16-F10 tumors. It could induce immunogenic cell death and the release of high mobility group box 1 and calreticulin. The combination of [131I]LG-12 and LG-12 could significantly inhibit tumor growth and resulted in enhanced antitumor immune response. This PD-L1 small-molecule inhibitor based combination strategy has great potential for tumor treatment.

14.
Int J Med Sci ; 21(7): 1194-1203, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38818468

RESUMEN

This study aims to elucidate the roles of Phosphoglycerate Mutase Family Member 5 (Pgam5) and Prohibitin 2 (Phb2) in the context of hyperglycemia-induced myocardial dysfunction, a critical aspect of diabetic cardiomyopathy. The research employed primary cardiomyocytes, which were then subjected to hyperglycemia treatment to mimic diabetic conditions. We used siRNA transfection to knock down Pgam5 and overexpressed Phb2 using adenovirus transfection to assess their individual and combined effects on cardiomyocyte health. Mitochondrial function was evaluated through measurements of mitochondrial membrane potential using the JC-1 probe, and levels of mitochondrial reactive oxygen species (ROS) were assessed. Additionally, the study involved qPCR analysis to quantify the transcriptional changes in genes related to mitochondrial fission and mitophagy. Our findings indicate that hyperglycemia significantly reduces cardiomyocyte viability and impairs mitochondrial function, as evidenced by decreased mitochondrial membrane potential and increased ROS levels. Pgam5 knockdown was observed to mitigate these adverse effects, preserving mitochondrial function and cardiomyocyte viability. On the molecular level, Pgam5 was found to regulate genes associated with mitochondrial fission (such as Drp1, Mff, and Fis1) and mitophagy (including Parkin, Bnip3, and Fundc1). Furthermore, overexpression of Phb2 countered the hyperglycemia-induced mitochondrial dysfunction and normalized the levels of key mitochondrial antioxidant enzymes. The combined data suggest a protective role for both Pgam5 knockdown and Phb2 overexpression against hyperglycemia-induced cellular and mitochondrial damage. The study elucidates the critical roles of Pgam5 and Phb2 in regulating mitochondrial dynamics in the setting of hyperglycemia-induced myocardial dysfunction. By modulating mitochondrial fission and mitophagy, Pgam5 and Phb2 emerge as key players in preserving mitochondrial integrity and cardiomyocyte health under diabetic conditions. These findings contribute significantly to our understanding of the molecular mechanisms underlying diabetic cardiomyopathy and suggest potential therapeutic targets for mitigating myocardial dysfunction in diabetes.


Asunto(s)
Cardiomiopatías Diabéticas , Hiperglucemia , Potencial de la Membrana Mitocondrial , Dinámicas Mitocondriales , Miocitos Cardíacos , Prohibitinas , Animales , Humanos , Ratas , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/patología , Cardiomiopatías Diabéticas/metabolismo , Cardiomiopatías Diabéticas/etiología , Hiperglucemia/metabolismo , Hiperglucemia/complicaciones , Hiperglucemia/genética , Mitocondrias Cardíacas/metabolismo , Dinámicas Mitocondriales/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitofagia/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo
15.
Int J Med Sci ; 21(13): 2464-2479, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39439466

RESUMEN

MAPKK4 has been implicated in the pathological mechanisms underlying myocardial and vascular injury, specifically influencing endothelial cell damage and programmed cell death via subcellular pathways. Nevertheless, the regulatory role of MAPKK4 in coronary microvascular injury following myocardial infarction remains unconfirmed, and the exploration of targeted mitochondrial protective therapeutic agents remains unaddressed. In light of this gap, we established a MAPKK4 gene-modified mouse model of ischemia-reperfusion injury and employed Buyang Huanwu decoction (BYHW), a traditional cardiovascular therapeutic formula, to assess its efficacy in treating coronary microvascular injury post-ischemia-reperfusion. The study aimed to elucidate the mechanism by which BYHW mitigates coronary microvascular injury induced by ischemia-reperfusion through the attenuation of endothelial cell apoptosis. Experimental outcomes revealed that high-dose BYHW significantly ameliorated coronary microvascular injury post-ischemia-reperfusion, restoring the structural integrity of the coronary microvasculature and reducing inflammation and oxidative stress. Contrarily, in transgenic mice overexpressing MAPKK4, BYHW intervention failed to attenuate microvascular inflammation and oxidative stress. To further investigate, we simulated hypoxia/reoxygenation injury in vascular endothelial cells using a MAPKK4-related cellular gene modification model. The results indicated that BYHW attenuates inflammatory damage and enhances the viability of vascular endothelial cells following hypoxic stress, inhibiting apoptosis via the mitochondrial pathway. However, overexpression of MAPKK4/p38 negated the therapeutic effects of BYHW, showing no impact on endothelial cell apoptosis and oxidative stress under hypoxic conditions. Molecular interaction studies confirmed that the active components of BYHW, Astragaloside IV and Ligustrazine, interact with the MAPKK4/P38 axis. In vitro experiments further suggested that the interaction between MAPKK4 and P38 play a crucial role in the ability of BYHW to inhibit apoptosis in coronary microvascular endothelial cells. Therapeutically, MAPKK4 may potentiate the apoptotic pathway in microvascular endothelial cells by modulating downstream P38 expression and phosphorylation, thereby exacerbating ischemia-reperfusion-induced coronary microvascular endothelial injury. From an in vivo perspective, the transgenic overexpression of MAPKK4 and P38 inhibited the microvascular protective effects of BYHW. These findings collectively underscore the significance of the MAPKK4-P38 axis in the protection of coronary microvascular endothelial cells.


Asunto(s)
Apoptosis , Medicamentos Herbarios Chinos , Células Endoteliales , Daño por Reperfusión Miocárdica , Animales , Medicamentos Herbarios Chinos/farmacología , Apoptosis/efectos de los fármacos , Ratones , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Humanos , Microvasos/efectos de los fármacos , Microvasos/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratones Transgénicos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Animales de Enfermedad , Masculino , Transducción de Señal/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/patología
16.
Molecules ; 29(15)2024 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-39124874

RESUMEN

Developing reusable and easy-to-operate biocatalysts is of significant interest in biodiesel production. Here, magnetic whole-cell catalysts constructed through immobilizing recombinant Escherichia coli cells (containing MAS1 lipase) into Fe3O4-chitosan magnetic microspheres (termed MWCC@MAS1) were used for fatty acid methyl ester (FAME) production from waste cooking oil (WCO). During the preparation process of immobilized cells, the effects of chitosan concentration and cell concentration on their activity and activity recovery were investigated. Optimal immobilization was achieved with 3% (w/v) chitosan solution and 10 mg wet cell/mL cell suspension. Magnetic immobilization endowed the whole-cell catalysts with superparamagnetism and improved their methanol tolerance, enhancing the recyclability of the biocatalysts. Additionally, we studied the effects of catalyst loading, water content, methanol content, and reaction temperature on FAME yield, optimizing these parameters using response surface methodology and Box-Behnken design. An experimental FAME yield of 89.19% was gained under the optimized conditions (3.9 wt% catalyst loading, 22.3% (v/w) water content, 23.0% (v/w) methanol content, and 32 °C) for 48 h. MWCC@MAS1 demonstrated superior recyclability compared to its whole-cell form, maintaining about 86% of its initial productivity after 10 cycles, whereas the whole-cell form lost nearly half after just five cycles. These results suggest that MWCC@MAS1 has great potential for the industrial production of biodiesel.


Asunto(s)
Biocombustibles , Quitosano , Escherichia coli , Microesferas , Escherichia coli/genética , Escherichia coli/metabolismo , Quitosano/química , Células Inmovilizadas/metabolismo , Aceites de Plantas/química , Lipasa/metabolismo , Lipasa/genética , Metanol/química , Culinaria
17.
Small ; 19(30): e2300420, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37046177

RESUMEN

Constructing all-solid-state lithium-sulfur batteries (ASSLSBs) cathodes with efficient charge transport and mechanical flexibility is challenging but critical for the practical applications of ASSLSBs. Herein, a multiscale structural engineering of sulfur/carbon composites is reported, where ultrasmall sulfur nanocrystals are homogeneously anchored on the two sides of graphene layers with strong SC bonds (denoted as S@EG) in chunky expanded graphite particles via vapor deposition method. After mixing with Li9.54 Si1.74 P1.44 S11.7 Cl0.3 (LSPSCL) solid electrolytes (SEs), the fabricated S@EG-LSPSCL cathode with interconnected "Bacon and cheese sandwich" feature can simultaneously enhance electrochemical reactivity, charge transport, and chemomechanical stability due to the synergistic atomic, nanoscopic and microscopic structural engineering. The assembled InLi/LSPSCL/S@EG-LSPSCL ASSLSBs demonstrate ultralong cycling stability over 2400 cycles with 100% capacity retention at 1 C, and a record-high areal capacity of 14.0 mAh cm-2 at a record-breaking sulfur loading of 8.9 mg cm-2 at room temperature as well as high capacities with capacity retentions of ≈100% after 600 cycles at 0 and 60 °C. Multiscale structural engineered sulfur/carbon cathode has great potential to enable high-performance ASSLSBs for energy storage applications.

18.
Pancreatology ; 23(8): 1036-1040, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37926600

RESUMEN

BACKGROUND/OBJECTIVES: Studies of a rare homozygous missense mutation identified in two brothers diagnosed with congenital pancreatic lipase deficiency (CPLD) provided the first definitive evidence linking CPLD with missense mutations in the gene of PNLIP. Herein, we investigated the molecular basis for the loss-of-function in the three novel PNLIP variants (c.305G > A, p.(W102∗); c.562C > T, p.(R188C); and c.1257G > A, p.(W419∗)) associated with CPLD. METHODS: We characterized three novel PNLIP variants in transfected cells by assessing their secretion, intracellular distribution, and markers of endoplasmic reticulum (ER) stress. RESULTS: All three variants had secretion defects. Notably, the p.R188C and p.W419∗ variants induced misfolding of PNLIP and accumulated as detergent-insoluble aggregates resulting in elevated BiP at both protein and mRNA levels indicating increased ER stress. CONCLUSIONS: All three novel PNLIP variants cause a loss-of-function through impaired secretion. Additionally, the p.R188C and p.W419∗ variants may induce proteotoxicity through misfolding and potentially increase the risk for pancreatic acinar cell injury.


Asunto(s)
Células Acinares , Lipasa , Enfermedades Pancreáticas , Humanos , Masculino , Células Acinares/enzimología , Lipasa/deficiencia , Lipasa/genética , Mutación Missense , Enfermedades Pancreáticas/congénito , Enfermedades Pancreáticas/enzimología , Células HEK293
19.
Mol Pharm ; 20(8): 4228-4235, 2023 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-37409670

RESUMEN

Nowadays, one of the most effective methods of tumor immunotherapy is blocking programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) immune checkpoints. However, there is still a significant challenge in selecting patients to benefit from immune checkpoint therapies. Positron emission tomography (PET), a noninvasive molecular imaging technique, offers a new approach to accurately detect PD-L1 expression and allows for a better prediction of response to PD-1/PD-L1 target immunotherapy. Here, we designed and synthesized a novel group of aryl fluorosulfate-containing small-molecule compounds (LGSu-1, LGSu-2, LGSu-3, and LGSu-4) based on the phenoxymethyl-biphenyl scaffold. After screening by the time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the most potent compound LGSu-1 (half maximal inhibitory concentration (IC50): 15.53 nM) and the low-affinity compound LGSu-2 (IC50: 189.70 nM) as a control were selected for 18F-radiolabeling by sulfur(VI) fluoride exchange chemistry (SuFEx) to use for PET imaging. [18F]LGSu-1 and [18F]LGSu-2 were prepared by a one-step radiofluorination reaction in over 85% radioconversion and nearly 30% radiochemical yield. In B16-F10 melanoma cell assays, [18F]LGSu-1 (5.00 ± 0.06%AD) showed higher cellular uptake than [18F]LGSu-2 (2.55 ± 0.04%AD), in which cell uptake could be significantly blocked by the nonradioactivity LGSu-1. In vivo experiments, micro-PET imaging of B16-F10 tumor-bearing mice and radiographic autoradiography of tumor sections showed that [18F]LGSu-1 was more effectively accumulated in the tumor due to the higher binding affinity with PD-L1. The above experimental results confirmed the potential of the small-molecule probe LGSu-1 as a targeting PD-L1 imaging tracer in tumor tissues.


Asunto(s)
Antígeno B7-H1 , Neoplasias , Ratones , Animales , Antígeno B7-H1/metabolismo , Fluoruros , Receptor de Muerte Celular Programada 1/metabolismo , Ligandos , Tomografía de Emisión de Positrones/métodos , Azufre , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral
20.
Bioorg Med Chem Lett ; 84: 129196, 2023 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-36828298

RESUMEN

Two neuropilin 1 (NRP1)-targeted near-infrared fluorescence probes for tumor imaging were synthesized via click reaction. These two probes achieve excellent solubility and less aggregation. Importantly, they were able to rapidly target NRP1-overexpressing tumors and had long retention within tumors. Additionally, QS-1 with appropriate hydrophilicity displays higher tumor to muscle (T/M) ratio. And QS-1 can be easily modified with other functional group, and serve as a platform for constructing dual-modal or dual-targeting probes.


Asunto(s)
Colorantes Fluorescentes , Neuropilina-1 , Línea Celular Tumoral , Imagen Óptica
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