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Cell Proliferation Oct 2022The main target of current drugs for alleviating bone loss is osteoclasts. However, the long-term application of such drugs will also cause side effects. Therefore, it...
OBJECTIVES
The main target of current drugs for alleviating bone loss is osteoclasts. However, the long-term application of such drugs will also cause side effects. Therefore, it is of great need to develop new and safer therapeutics for osteoporosis. In recent years, drug development based on gut microbiota has gradually attracted attention. This manuscript investigates the inhibitory effect of urolithin B (UB) on osteoclastogenesis and differentiation in vitro and in ovariectomized (OVX) mice.
MATERIALS AND METHODS
CCK-8 was used to analyse the cytotoxicity of UB; BMMs cells were differentiated into osteoclasts by RANKL, and respectively treated with 1, 5, and 25 μmol/L UB during this process. After one week of intervention, tartrate-resistant acid phosphatase (TRAP) staining was used to analyse the number and average area of osteoclasts. F-actin staining and immunofluorescence staining were conducted to evaluate the morphology and function of osteoclasts. Bone resorption function of osteoclasts was detected by Pit Formation Assay. The expression of osteoclast-related protein genes in RAW264.7 cells were investigated via western blot and RT-PCR assays. Western blot analysis of RANKL-mediated activation of MAPK/NF-κB pathway after 0, 5, 15, 30, 60 min of intervention. For in vivo experiments, OVX mice received intraperitoneal injection of 10, 50 mg/kg every two days, 8 weeks later, the femurs of mice were taken for morphological analysis, and the serum content of CTX-1, a bone metabolism index, was analysed.
RESULTS
UB could inhibit the osteoclast differentiation of rankl-induced bone marrow macrophages (BMMs) and RAW264.7 cells in vitro, suppress the uptake activity of hydroxyapatite and expression of osteoclast-related gene MMP9, CTSK, NFATc1 and c-fos. Furthermore, UB repressed the rankl-induced phosphorylation and degradation of IκB and the phosphorylation of P65 in the NF-κB pathway of RAW264.7 cells, and also down-regulated the phosphorylation level of ERK in the MAPK pathway. For in vivo studies, UB-treated OVX mice showed more significant improved various parameters of distal femur compared with the control group, with fewer NFATc1, MMP9 and TRAP-positive osteoclasts in bone tissues, and less serum content of CTX-1.
CONCLUSION
Urolithin B attenuated bone loss in OVX mice by inhibiting the formation and activation of osteoclasts via down-regulation of the ERK/NF-κB signalling pathway.
Topics: Actins; Animals; Cell Differentiation; Coumarins; Hydroxyapatites; Matrix Metalloproteinase 9; Mice; NF-kappa B; NFATC Transcription Factors; Osteoclasts; Osteogenesis; Osteoporosis; RANK Ligand; Sincalide; Tartrate-Resistant Acid Phosphatase
PubMed: 35708050
DOI: 10.1111/cpr.13291 -
Theranostics 2022The limited effect of adjuvant therapy for advanced bladder cancer (BCa) leads to a poor prognosis. Increasing evidence has shown that RNA N6-methyladenosine (mA)...
The limited effect of adjuvant therapy for advanced bladder cancer (BCa) leads to a poor prognosis. Increasing evidence has shown that RNA N6-methyladenosine (mA) modification plays important functional roles in tumorigenesis. Nevertheless, the role and mechanism of m6A-modified noncoding RNAs (ncRNAs) in BCa remain largely unknown. RT-PCR, western blotting and ONCOMINE dataset were used to determine the dominant mA-related enzyme in BCa. MA-lncRNA epitranscriptomic microarray was used to screen candidate targets of METTL14. RT-PCR, MeRIP and TCGA dataset were carried out to confirm the downstream target of METTL14. CHIRP/MS was conducted to identify the candidate proteins binding to lncDBET. RT-PCR, western blotting, RIP and KEGG analysis were used to confirm the target of lncDBET. The levels of METTL14, lncDBET and FABP5 were tested and . CCK-8, EdU, transwell and flow cytometry assays were performed to determine the oncogenic function of METTL14, lncDBET and FABP5, and their regulatory networks. : We identified that the mA level of total RNA was elevated and that METTL14 was the dominant m6A-related enzyme in BCa. mA modification mediated by METTL14 promoted the malignant progression of BCa by promoting the expression of lncDBET. Upregulated lncDBET activated the PPAR signalling pathway to promote the lipid metabolism of cancer cells through direct interaction with FABP5, thus promoting the malignant progression of BCa and . : Our study establishes METTL14/lncDBET/FABP5 as a critical oncogenic axis in BCa.
Topics: Carcinogenesis; Fatty Acid-Binding Proteins; Humans; Lipid Metabolism; Methyltransferases; Peroxisome Proliferator-Activated Receptors; RNA, Long Noncoding; Sincalide; Urinary Bladder Neoplasms
PubMed: 36168624
DOI: 10.7150/thno.71456 -
International Journal of Molecular... Dec 2019Hyperoside (quercetin 3--β-d-galactopyranoside) is one of the flavonoid glycosides with anti-inflammatory, antidepressant, and anti-cancer effects. But it remains...
Hyperoside (quercetin 3--β-d-galactopyranoside) is one of the flavonoid glycosides with anti-inflammatory, antidepressant, and anti-cancer effects. But it remains unknown whether it had effects on breast cancer. Here, different concentrations of hyperoside were used to explore its therapeutic potential in both breast cancer cells and subcutaneous homotransplant mouse model. CCK-8 and wound healing assays showed that the viability and migration capability of Michigan Cancer Foundation-7 (MCF-7) and 4T1 cells were inhibited by hyperoside, while the apoptosis of cells were increased. Real-time quantitative PCR (qRT-PCR) and western blot analysis were used to detect mRNA and the protein level, respectively, which showed decreased levels of B cell lymphoma-2 (Bcl-2) and X-linked inhibitor of apoptosis (XIAP), and increased levels of Bax and cleaved caspase-3. After exploration of the potential mechanism, we found that reactive oxygen species (ROS) production was reduced by the administration of hyperoside, which subsequently inhibited the activation of NF-κB signaling pathway. Tumor volume was significantly decreased in subcutaneous homotransplant mouse model in hyperoside-treated group, which was consistent with our study in vitro. These results indicated that hyperoside acted as an anticancer drug through ROS-related apoptosis and its mechanism included activation of the Bax-caspase-3 axis and the inhibition of the NF-κB signaling pathway.
Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Survival; Female; Humans; MCF-7 Cells; Mice; NF-kappa B; Quercetin; Reactive Oxygen Species; Signal Transduction; Sincalide; Wound Healing
PubMed: 31878204
DOI: 10.3390/ijms21010131 -
Journal of Translational Medicine Sep 2022Glioblastoma (GBM) is the most common primary malignant brain tumor that leads to lethality. Several studies have demonstrated that mitochondria play an important role...
BACKGROUND
Glioblastoma (GBM) is the most common primary malignant brain tumor that leads to lethality. Several studies have demonstrated that mitochondria play an important role in GBM and that mitochondria-related genes (MRGs) are potential therapeutic targets. However, the role of MRGs in GBM remains unclear.
METHODS
Differential expression and univariate Cox regression analyses were combined to screen for prognostic differentially-expressed (DE)-MRGs in GBM. Based on LASSO Cox analysis, 12 DE-MRGs were selected to construct a risk score model. Survival, time dependent ROC, and stratified analyses were performed to evaluate the performance of this risk model. Mutation and functional enrichment analyses were performed to determine the potential mechanism of the risk score. Immune cell infiltration analysis was used to determine the association between the risk score and immune cell infiltration levels. CCK-8 and transwell assays were performed to evaluate cell proliferation and migration, respectively. Mitochondrial reactive oxygen species (ROS) levels and morphology were measured using a confocal laser scanning microscope. Genes and proteins expression levels were investigated by quantitative PCR and western blotting, respectively.
RESULTS
We identified 21 prognostic DE-MRGs, of which 12 DE-MRGs were selected to construct a prognostic risk score model for GBM. This model presented excellent performance in predicting the prognosis of patients with GBM and acted as an independent predictive factor. Functional enrichment analysis revealed that the risk score was enriched in the inflammatory response, extracellular matrix, and pro-cancer-related and immune related pathways. Additionally, the risk score was significantly associated with gene mutations and immune cell infiltration in GBM. Single-stranded DNA-binding protein 1 (SSBP1) was considerably upregulated in GBM and associated with poor prognosis. Furthermore, SSBP1 knockdown inhibited GBM cell progression and migration. Mechanistically, SSBP1 knockdown resulted in mitochondrial dysfunction and increased ROS levels, which, in turn, increased temozolomide (TMZ) sensitivity in GBM cells by enhancing ferroptosis.
CONCLUSION
Our 12 DE-MRGs-based prognostic model can predict the GBM patients prognosis and 12 MRGs are potential targets for the treatment of GBM. SSBP1 was significantly upregulated in GBM and protected U87 cells from TMZ-induced ferroptosis, which could serve as a prognostic and therapeutic target/biomarker for GBM.
Topics: Biomarkers; DNA-Binding Proteins; Ferroptosis; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mitochondria; Mitochondrial Proteins; Reactive Oxygen Species; Sincalide; Temozolomide
PubMed: 36180956
DOI: 10.1186/s12967-022-03657-4 -
Journal of Nuclear Medicine Technology Sep 2019Sincalide (Kinevac) is widely used in conjunction with cholescintigraphy for a variety of clinical indications. Over the years, numerous publications have verified the... (Review)
Review
Sincalide (Kinevac) is widely used in conjunction with cholescintigraphy for a variety of clinical indications. Over the years, numerous publications have verified the optimal infusion methodology. Published data and consensus recommendations emphasize that sincalide, 0.02 μg/kg, should be infused over 60 min. Production problems sometimes limit the availability of sincalide. In that case, non-Food and Drug Administration pharmacy-compounded sincalide may serve as an alternative. Fatty meals have also been used. Various illnesses and drugs may inhibit gallbladder contraction. Thus, these drugs should be withheld for 48 h before the study. Sincalide cholescintigraphy is most commonly used to diagnose or exclude chronic acalculous gallbladder disease. The study should preferably be performed as an outpatient procedure.
Topics: Drug Administration Routes; Gallbladder; Humans; Radionuclide Imaging; Sincalide
PubMed: 31019045
DOI: 10.2967/jnmt.119.226019 -
Molecular Metabolism Nov 2022Oral squamous cell carcinoma (OSCC) is characterized by high recurrence and metastasis and places a heavy burden on societies worldwide. Cancer cells thrive in a...
OBJECTIVE
Oral squamous cell carcinoma (OSCC) is characterized by high recurrence and metastasis and places a heavy burden on societies worldwide. Cancer cells thrive in a changing microenvironment by reprogramming lipidomic metabolic processes to provide nutrients and energy, activate oncogenic signaling pathways, and manage redox homeostasis to avoid lipotoxicity. The mechanism by which OSCC cells maintain lipid homeostasis during malignant progression is unclear.
METHODS
The altered expression of fatty acid (FA) metabolism genes in OSCC, compared with that in normal tissues, and in OSCC patients with or without recurrence or metastasis were determined using public data from the TCGA and GEO databases. Immunohistochemistry was performed to examine the carboxylesterase 2 (CES2) protein level in our own cohort. CCK-8 and Transwell assays and an in vivo xenograft model were used to evaluate the biological functions of CES2. Mass spectrometry and RNA sequencing were performed to determine the lipidome and transcriptome alterations induced by CES2. Mitochondrial mass, mtDNA content, mitochondrial membrane potential, ROS levels, and oxygen consumption and apoptosis rates were evaluated to determine the effects of CES2 on mitochondrial function in OSCC.
RESULTS
CES2 was downregulated in OSCC patients, especially those with recurrence or metastasis. CES2 OSCC patients showed better overall survival than CES2 OSCC patients. Restoring CES2 expression reduced OSCC cell viability and suppressed their migration and invasion in vitro, and it inhibited OSCC tumor growth in vivo. CES2 reprogrammed lipid metabolism in OSCC cells by hydrolyzing neutral lipid diacylglycerols (DGs) to release free fatty acids and reduce the membrane structure lipid phospholipids (PLs) synthesis. Free FAs were converted to acyl-carnitines (CARs) and transferred to mitochondria for oxidation, which induced reactive oxygen species (ROS) accumulation, mitochondrial damage, and apoptosis activation. Furthermore, the reduction in signaling lipids, e.g., DGs, PLs and substrates, suppressed PI3K/AKT/MYC signaling pathways. Restoring MYC rescued the diminished cell viability, suppressed migratory and invasive abilities, damaged mitochondria and reduced apoptosis rate induced by CES2.
CONCLUSIONS
We demonstrated that CES2 downregulation plays an important role in OSCC by maintaining lipid homeostasis and reducing lipotoxicity during tumor progression and may provide a potential therapeutic target for OSCC.
Topics: Carboxylesterase; Carboxylic Ester Hydrolases; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; DNA, Mitochondrial; Diglycerides; Fatty Acids, Nonesterified; Head and Neck Neoplasms; Homeostasis; Humans; Mitochondria; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Reactive Oxygen Species; Signal Transduction; Sincalide; Squamous Cell Carcinoma of Head and Neck
PubMed: 36113774
DOI: 10.1016/j.molmet.2022.101600 -
Chemical Stability of Reconstituted Sincalide in Sterile Water Under 2 Different Storage Conditions.Journal of Nuclear Medicine Technology Jun 2020This study aimed to evaluate the chemical stability of sincalide at 2 common storage conditions-room temperature and refrigeration-in an attempt to simulate the...
This study aimed to evaluate the chemical stability of sincalide at 2 common storage conditions-room temperature and refrigeration-in an attempt to simulate the conditions faced during centralized reconstitution and subsequent distribution to regional clinical facilities. Sincalide is a peptide hormone product administered parenterally as an aid for diagnostic imaging of hepatobiliary conditions. With an estimated postreconstitution shelf-life of 8 h (updated by the manufacturer in 2014 with limited supporting data) and frequent shortages due to an intermittent supply, there is both clinical and economic value in the experimental determination of the true chemical stability of this agent. Sincalide was reconstituted and stored at both temperatures ( = 4 each), and samples were collected at predetermined time points. A validated high-performance liquid chromatography analytic method was used for quantification of the active ingredient in these samples. Little to no chemical degradation of sincalide was observed for the duration of study, over 8 d, after reconstitution and storage at room temperature. A trend toward a cyclic fluctuation in concentration was also shared among all samples. A similar trend toward little to no chemical degradation and cyclic pattern was observed for the duration of study, over 8 d, after reconstitution and storage in refrigeration. This study supports that from a chemical standpoint, sincalide may potentially be used up to at least 8 d after reconstitution with sterile water, thus providing convenience and cost-saving benefits to medical institutions using the product. The findings of this study, however, warrant microbial testing over this storage duration before any recommendations for extended use can be made.
Topics: Chromatography, High Pressure Liquid; Drug Stability; Drug Storage; Sincalide; Time Factors; Water
PubMed: 32111658
DOI: 10.2967/jnmt.119.230441 -
Toxins Sep 2022Pyrrolizidine alkaloids (PAs) are common constituents of plants and have serious hepatotoxicity. Intermedine (Im) and lycopsamine (La) are two monoesters of PAs that...
Pyrrolizidine alkaloids (PAs) are common constituents of plants and have serious hepatotoxicity. Intermedine (Im) and lycopsamine (La) are two monoesters of PAs that frequently coexist in the PA-containing plants (e.g., and tea). The present study aimed to explore the combined hepatotoxicity and toxicity mechanism of the Im and La mixture. In vitro, the combined cytotoxicity of the Im and La mixture on human hepatocytes (HepD) was examined by CCK-8, colony formation, wound healing, and Annexin V/PI staining assays. The combination of Im and La inhibited the ability of HepD cells to proliferate, colonize, and migrate and induced hepatocytes apoptosis in a dose-dependent manner. In addition to significantly causing a burst of intracellular reactive oxygen species (ROS), mitochondrial apoptosis, and endoplasmic reticulum (ER) stress, the Im and La mixture can also cause an increase in intracellular Ca, triggering the PERK/eIF2α/ATF4/CHOP apoptosis pathway. This study provided the first direct evidence that the combined PAs induced hepatotoxicity through ER-mediated apoptosis. These results supplemented the basic toxicity data for the combined PAs and provided a new perspective for the risk assessment of combined PA toxicity.
Topics: Annexin A5; Apoptosis; Chemical and Drug Induced Liver Injury; Endoplasmic Reticulum Stress; Humans; Pyrrolizidine Alkaloids; Reactive Oxygen Species; Sincalide; Tea
PubMed: 36136571
DOI: 10.3390/toxins14090633 -
Zhongguo Yao Li Xue Bao = Acta... Sep 1994Sincalide (CCK-8) is an antiopioid substance. In this study, we investigated the effects of sincalide and opioids on c-fos expression and their interaction in brain and...
Sincalide (CCK-8) is an antiopioid substance. In this study, we investigated the effects of sincalide and opioids on c-fos expression and their interaction in brain and spinal dorsal horn in vitro. Immunoprecipitation was used for detection of c-fos protein. The results indicated that 0.1 mumol.L-1 sincalide induced c-fos expression markedly in both brain (a 3.8-fold increase in c-fos protein level) and in spinal dorsal horn (a 3.6-fold increase). NDAP (a kappa receptor agonist) 0.1 mumol.L-1 showed some activating effects on c-fos expression, the c-fos protein level increased 2.7 and 2.6 times respectively in brain and spinal dorsal horn. Ohmefentanyl (Ohm, a mu receptor agonist) 0.1 mumol.L-1 also exhibited an inducing effect on c-fos protein production. Sincalide and NDAP exerted some additive effects on c-fos protein production in spinal cord. In contrast, the effect of sincalide on c-fos protein production is antagonistic to that of Ohm. The results suggested that there were changing patterns of interaction on c-fos expression between sincalide and opioids.
Topics: Animals; Brain; Dynorphins; Fentanyl; Peptide Fragments; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sincalide; Spinal Cord
PubMed: 7717068
DOI: No ID Found -
Cell Communication and Signaling : CCS Dec 2022The treatment of chronic myeloid leukemia (CML) is facing the dilemma of tyrosine kinase inhibitors (TKIs) resistance and disease recurrence. The dysfunctional DNA...
BACKGROUND
The treatment of chronic myeloid leukemia (CML) is facing the dilemma of tyrosine kinase inhibitors (TKIs) resistance and disease recurrence. The dysfunctional DNA damage repair mechanism plays an essential role not only in the initiation and progression of hematological malignancies but also links to the development of TKI resistance. Deciphering the abnormally regulated DNA damage repair and proteins involved brings new insights into the therapy of leukemias. As a G2/M phase checkpoint kinase and a DNA damage repair checkpoint kinase engaged in the DNA damage response (DDR), along with an oncogenic driver present in various cancers, the particular involvement of Wee1 in DNA damage is far from clear. Deciphering its function and targeting it via modulating DNA repair pathways is important for improving our understanding of cancer treatment.
METHODS
Wee1 expression was assessed in cell lines using RT-qPCR and western blot, and Wee1 knockdown efficacy was validated using RT-qPCR, western blot, and immunofluorescence. Wee1 function was investigated by CCK-8, colony formation, and flow cytometry assay in vitro. Wee1 role in DNA repair and its interactions with other proteins were then studied using western blot, immunofluorescence, and double plasmid-repair studies. Finally, the CCK-8 and flow cytometry assay was utilized to investigate Wee1 and imatinib's synergistic effect, and a CML mouse model was constructed to study Wee1's role in carcinogenesis in vivo.
RESULTS
Wee1 was reported to respond quickly to DDR in an ATM-γH2AX-MDC1-dependent way upon DNA double-strand breaks (DSBs) occurrence, and it regulated homologous recombination by stimulating the recruitment of critical proteins RAD51/BRCA1 upon DSB sites. Wee1 was also revealed to be abnormally upregulated in CML cells. Further suppression of Wee1 not only causes cell cycle arrest and inhibits the proliferation of cancer cells but also enhances CML cell sensitivity to Imatinib in vitro and in vivo, possibly through an excessive accumulation of overall DSBs.
CONCLUSION
Wee1 is extensively involved in the DRR signaling and DSB repair pathway. Inhibiting abnormally elevated Wee1 benefits CML therapy in both IM-resistant and IM-sensitive cells. Our data demonstrated that Wee1 participated in promoting cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1. In the fight against CML, Wee1's dysregulation in the DNA damage repair mechanism of CML pathogenesis makes it a viable therapeutic target in clinical applications.
Topics: Animals; Mice; Cell Proliferation; DNA Damage; DNA Repair; Drug Resistance, Neoplasm; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Sincalide; Humans
PubMed: 36575478
DOI: 10.1186/s12964-022-01021-z