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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 -
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 -
Stem Cell Research & Therapy Sep 2023To explore whether local transplantation of mesenchymal stem cells (MSCs) in temporal muscle can promote collateral angiogenesis and to analyze its main mechanisms of...
BACKGROUND
To explore whether local transplantation of mesenchymal stem cells (MSCs) in temporal muscle can promote collateral angiogenesis and to analyze its main mechanisms of promoting angiogenesis.
METHODS
Bilateral carotid artery stenosis (BCAS) treated mice were administrated with encephalo-myo-synangiosis (EMS), and bone marrow mesenchymal stem cells (BMSCs) were transplanted into the temporal muscle near the cerebral cortex. On the 30th day after EMS, the Morris water maze, immunofluorescence, laser speckle imaging, and light sheet microscopy were performed to evaluate angiogenesis; In addition, rats with bilateral common carotid artery occlusion were also followed by EMS surgery, and BMSCs from GFP reporter rats were transplanted into the temporal muscle to observe the survival time of BMSCs. Then, the concentrated BMSC-derived conditioned medium (BMSC-CM) was used to stimulate HUVECs and BMECs for ki-67 immunocytochemistry, CCK-8, transwell and chick chorioallantoic membrane assays. Finally, the cortical tissue near the temporal muscle was extracted after EMS, and proteome profiler (angiogenesis array) as well as RT-qPCR of mRNA or miRNA was performed.
RESULTS
The results of the Morris water maze 30 days after BMSC transplantation in BCAS mice during the EMS operation, showed that the cognitive impairment in the BCAS + EMS + BMSC group was alleviated (P < 0.05). The results of immunofluorescence, laser speckle imaging, and light sheet microscopy showed that the number of blood vessels, blood flow and astrocytes increased in the BCAS + EMS + BMSC group (P < 0.05). The BMSCs of GFP reporter rats were applied to EMS and showed that the transplanted BMSCs could survive for up to 14 days. Then, the results of ki-67 immunocytochemistry, CCK-8 and transwell assays showed that the concentrated BMSC-CM could promote the proliferation and migration of HUVECs and BMECs (P < 0.05). Finally, the results of proteome profiler (angiogenesis array) in the cerebral cortex showed that the several pro-angiogenesis factors (such as MMP-3, MMP-9, IGFBP-2 or IGFBP-3) were notably highly expressed in MSC transplantation group compared to others.
CONCLUSIONS
Local MSCs transplantation together with EMS surgery can promote angiogenesis and cognitive behavior in chronic brain ischemia mice. Our study illustrated that MSC local transplantation can be the potential therapeutical option for improving EMS treatment efficiency which might be translated into clinical application.
Topics: Mice; Rats; Animals; Ki-67 Antigen; Proteome; Sincalide; Neovascularization, Pathologic; Mesenchymal Stem Cells; Brain Ischemia
PubMed: 37667370
DOI: 10.1186/s13287-023-03465-7 -
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 -
Journal of Ethnopharmacology Mar 2024Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its...
ETHNOPHARMACOLOGICAL RELEVANCE
Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its underlying functional mechanism still remains unknown.
AIM OF THE STUDY
The study aimed to explore the anti-hepatocarcinogenic effects of M-BJJW, specifically its influence on PD-L1-mediated immune evasion in hypoxic conditions, and elucidate the related molecular mechanisms in HCC.
MATERIALS AND METHODS
To investigate the therapeutic efficacy and mechanisms underlying M-BJJW's effects on HCC, we employed a diethylnitrosamine (DEN)-induced rat model maintained for 120 days. Following model establishment, flow cytometry was utilized to assess the distribution of immune cell populations in peripheral blood, spleens, and tumor tissues after M-BJJW administration. Simultaneously, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze cytokine profiles in serum samples. Immunohistochemistry was employed to determine the expression levels of crucial proteins within tumor tissues. Furthermore, HCC cells exposed to CoCl underwent Western blot analysis to validate the expression levels of HIF-1α, PD-L1, STAT3, and nuclear factor kappa B (NF-κB) p65. The modulatory effects of STAT3 and NF-κB p65 were investigated using specific inhibitors and activators in wild-type cell lines. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was utilized to identify the chemical constituents present in M-BJJW-medicated serum. The immunomodulatory properties and the anti-tumor activities of M-BJJW were evaluated by co-culturing with peripheral blood mononuclear cells (PBMC) and the CCK-8 assay. Additionally, we assessed M-BJJW's impact on hypoxia-induced alterations in HCC cell lines using immunofluorescence and Western blot assessments.
RESULTS
M-BJJW exhibited substantial therapeutic advantages by effectively alleviating pathological deterioration within the HCC microenvironment. In the DEN-induced rat model, M-BJJW administration notably reduced tumor growth. Flow cytometry analyses revealed an increased proportion of Cytotoxic T lymphocytes (CTLs) accompanied by a simultaneous decrease in regulatory T cells (Tregs). ELISA data supported a marked decrease in pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α). Immunohistochemistry confirmed the suppressive effect of M-BJJW on the expression of HIF-1α and PD-L1. Notably, western blotting unveiled the role of HIF-1α in regulating PD-L1 expression via the STAT3 and NF-κB signaling pathways in HCC cell lines, which was validated using activators and inhibitors of STAT3 and NF-κB. The CCK-8 assay and co-culture techniques demonstrated the anti-tumor activity of M-BJJW. Immunofluorescence and western blotting further confirmed that M-BJJW-containing serum dose-dependently inhibited HIF-1α, PD-L1, p-STAT3, and p-p65 in hypoxic HCC cell lines.
CONCLUSIONS
M-BJJW demonstrates significant therapeutic potential against HCC by influencing the hypoxic microenvironment, thereby regulating the immunosuppressive milieu. Specifically, M-BJJW modulates the HIF-1α/STAT3/NF-κB signaling pathway, leading to reduced PD-L1 expression and an elevated ratio of cytotoxic T lymphocytes (CTLs), while concurrently decreasing T regulatory cells (Tregs) and immunosuppressive factors. These synergistic effects aid in countering PD-L1-mediated immune evasion, presenting compelling pharmacological evidence supporting the clinical application of M-BJJW as a therapeutic approach for HCC.
Topics: Rats; Animals; NF-kappa B; Carcinoma, Hepatocellular; Leukocytes, Mononuclear; Liver Neoplasms; B7-H1 Antigen; Immune Evasion; Sincalide; Signal Transduction; Tumor Microenvironment
PubMed: 38104877
DOI: 10.1016/j.jep.2023.117577 -
Adipocyte Dec 2024Platinum is a commonly used drug for ovarian cancer (OvCa) treatment, but drug resistance limits its clinical application. This study intended to delineate the effects...
BACKGROUND
Platinum is a commonly used drug for ovarian cancer (OvCa) treatment, but drug resistance limits its clinical application. This study intended to delineate the effects of adipocytes on platinum resistance in OvCa.
METHODS
OvCa cells were maintained in the adipocyte-conditioned medium. Cell viability and apoptosis were detected by CCK-8 and flow cytometry, separately. Proliferation and apoptosis-related protein expression were assayed by western blot. The IC values of cisplatin and carboplatin were determined using CCK-8. IGF1 secretion and expression were assayed via ELISA and western blot, respectively. A xenograft model was established, and pathological changes were detected by H&E staining. Proliferation and apoptosis-associated protein expression was assessed via IHC.
RESULTS
Adipocytes promoted the viability and repressed cell apoptosis in OvCa, as well as enhancing platinum resistance, while the addition of IGF-1 R inhibitor reversed the effects of adipocytes on proliferation, apoptosis, and drug resistance of OvCa cells. Treatment with different concentrations of Ojeok-san (OJS) inhibited the adipocyte-induced platinum resistance in OvCa cells by suppressing IGF1. The combined treatment of OJS and cisplatin significantly inhibited tumour growth with good mouse tolerance.
CONCLUSION
In summary, OJS inhibited OvCa proliferation and platinum resistance by suppressing adipocyte paracrine IGF1 secretion.
Topics: Humans; Female; Animals; Mice; Cisplatin; Insulin-Like Growth Factor I; Sincalide; Drug Resistance, Neoplasm; Ovarian Neoplasms; Adipocytes
PubMed: 37993991
DOI: 10.1080/21623945.2023.2282566 -
Aging Aug 2019In this study, expression of the SPC25 gene was characterized in breast cancer (BC), and its effects on BC development and progression, functions in BC cells, and...
In this study, expression of the SPC25 gene was characterized in breast cancer (BC), and its effects on BC development and progression, functions in BC cells, and potential underlying mechanisms were examined. Data from TCGAportal and FIREBROWSE indicated that SPC25 was upregulated in BC tissues compared to normal tissues, and CANCERTOOL indicated that higher SPC25 mRNA levels were associated with increased probability of recurrence and poorer survival in BC patients. BC patients with higher SPC25 expression displayed shorter distant metastasis-free survival, relapse-free survival, and overall survival. Colony formation and CCK-8 experiments confirmed that SPC25 promoted proliferation of BC cells. Single-cell analysis indicated that SPC25 is associated with cell cycle regulation, DNA damage and repair, and BC cell proliferation. SPC25 knockdown suppressed proliferation of BC cells. MiRNAs, circRNAs, RNA-binding proteins, transcription factors, and immune factors that might interact with SPC25 mRNA to promote BC were also identified. These findings suggest that SPC25 levels are higher in more malignant BC subtypes and are associated with poor prognosis in BC patients. In addition, DNA methyltransferase inhibitor and transcription factors inhibitor treatments targeting SPC25 might improve survival in BC patients.
Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA Damage; DNA Repair; Female; Genes, cdc; Humans; Microtubule-Associated Proteins; Neoplasm Recurrence, Local; Prognosis; Progression-Free Survival; RNA, Messenger; Sincalide; Survival Analysis; Treatment Outcome; Tumor Stem Cell Assay; Up-Regulation
PubMed: 31400751
DOI: 10.18632/aging.102153