Acid Phosphatase Assay Research Articles

L-cysteine contributes to destructive activities of odontogenic cysts/tumor.

Odontogenic cysts/tumor can cause severe bone destruction, which affects maxillofacial function and aesthetics. Meanwhile, metabolic reprogramming is an important hallmark of diseases. Changes in metabolic flow affect all aspects of disease, especially bone-related diseases. At present, the researches on pathogenesis of odontogenic cysts/tumor are mainly focused on the level of gene regulation, but the effects of metabolic alterations on odontogenic cysts/tumor have still underexplored. Imaging analysis was used to evaluate the lesion size of different odontogenic lesions. Tartrate resistant acid phosphatase (TRAP) and immunohistochemistry (IHC) assays were utilized to detect the differences in bone destruction activity in odontogenic cysts and tumors. Furthermore, metabolomics and weighted gene co-expression network analysis (WGCNA) were conducted for the metabolomic features and key metabolite screening, respectively. The effect of ferroptosis inhibition on bone destruction was confirmed by IHC, immunofluorescence, and malondialdehyde colorimetric assay. The bone destruction activity of ameloblastoma (AM) was the strongest and the weakest in odontogenic cysts (OC). High-throughput targeted metabolomics was used to map the metabolomic profiles of OC, odontogenic keratocyst (OKC) and AM. WGCNA and differential analysis identified L-cysteine in OKC and AM. Cystathionine γ-lyase (CTH) was further screened by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The functions of L-cysteine were further validated. Finally, we confirmed that CTH affected destructive activities by regulating the sensitivity of epithelial cells to ferroptosis. High-throughput targeted metabolomics performed on diseased tissue confirmed the unique alteration of metabolic profiles in OKC and AM. CTH and its metabolite L-cysteine are the key factors regulating destructive activities.

An off-on fluorescence method for acid phosphatase assay based on the inner filter effect of MnO2 nanosheets on vitamin B2

Fluorescence analysis has attracted much attention due to its rapidity and sensitivity. The present work describes a novel fluorescence detection method for acid phosphatase (ACP) on the basis of inner-filter effect (IFE), where MnO2 nanosheets (MnO2 NSs) and vitamin B2 (VB2) are served as absorbers and fluorophores, respectively. In the absence of ACP, the absorption band of MnO2 NSs overlaps well with the excitation band of VB2, resulting in effective IFE and inhibition of VB2 fluorescence. In the presence of ACP, 2-phospho-L-ascorbic acid trisodium salt (AAP) is hydrolyzed to generate ascorbic acid (AA), which efficiently trigger the reduction of MnO2 NSs into Mn2+ ions, causing the weakening of the MnO2 NSs absorption band and the recovery of VB2 fluorescence. Further investigation indicates that the fluorescence recovery degree of VB2 increases with the increase of ACP concentration. Under selected experimental conditions, the proposed method can achieve sensitive detection of ACP in the ranges of 0.5–4.0 mU/mL and 4.0–15 mU/mL along with a limit of detection (LOD) as low as 0.14 mU/mL. Finally, this method was successfully applied for the detection of ACP in human serum samples with satisfactory recoveries in the range of 95.0 %-108 %.

Abstract 2269: Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer

Abstract PIK3CA mutations occur in ~15% of triple-negative breast cancers (TNBCs) and aberrantly activate the PI3K-AKT pathway, resulting in uncontrolled cell proliferation. Alpelisib is approved for hormone receptor-positive (HR+), HER2-negative BC patients with a PIK3CA mutation; however, not all PIK3CA-mutated (MUT), HR+ patients respond to alpelisib and others acquire resistance following exposure, suggesting adaptive mechanisms of PI3K activation. It has been proven that in TNBC, there exists a cohort of mutated genes which activate PI3K-AKT signaling. We hypothesize that testing alpelisib in combination with repurposed approved drugs, targeting alternative mechanisms of PI3K-AKT pathway activation, may be used to treat a cohort of TNBC patients. TNBC patient genetic data from the MSK (N=168), METABRIC (N=209), and TCGA (N=171) datasets were analyzed using cBioPortal to identify frequently occurring mutations. Differential expression analyses based on mutational status were performed on CCLE cell line (N=705) microarray and TCGA BC patient (N=981) RNA-sequencing data using R. Dysregulated genes were functionally analyzed using DAVID Ontology and those associated with poorer survival in TNBC were identified using KM-Plotter. STRING database was used to investigate protein interactions. The antiproliferative effects of targeted drugs tested alone and in combination in a panel of 6 TNBC cell lines were determined using acid phosphatase assays. Reverse-phase protein array analysis was carried out on protein lysates from cells following 24 hour treatment with drugs used alone and in combination.TP53 is the most frequently mutated gene in TNBC and mutations were associated with significant genetic dysregulation. A comprehensive analysis of upregulated genes in TP53-MUT TNBC led to the identification of 5 potential therapeutic targets - MET, S100A9, LCN2, CA9, TACSTD2, and KLK5 - based on their association with poorer survival in TNBC, protein interactions with TP53 and PIK3CA, and availability of targeted drugs. MET was selected for in vitro analysis based on its strong correlation with PI3K-AKT signaling. The presence of a TP53 mutation was associated with decreased antiproliferative effects in TNBC cell lines treated with alpelisib. Treatment with alpelisib in combination with a MET inhibitor, crizotinib, demonstrated greater antiproliferative effects in TP53-MUT/PIK3CA-MUT cell lines when compared to TP53 or PIK3CA MUT only models.TP53 mutations are associated with genetic dysregulation in TNBC and may confer resistance to alpelisib. Targeting upregulated proteins in a cohort of TP53-MUT/PIK3CA-MUT patients may improve sensitivity to alpelisib via dual inhibition of PI3K-AKT pathway activation. Protein analysis will uncover the mechanisms of synergy observed between alpelisib and crizotinib, and the therapeutic potential of the combination will be experimentally investigated in vivo. Citation Format: Grace Colley, Alexander Eustace, Dalal AlSultan. Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2269.

Abstract 1978: The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer

Abstract Introduction: Constitutive activation of KRAS through gain-of-function mutations, such as KRASG12C, is thought to reduce sensitivity to HER-targeted TKIs like neratinib (N), lapatinib (L) and tucatinib (T). Sotorasib (S) is a KRASG12C specific inhibitor clinically approved for the treatment of advanced KRASG12C-mutated non-small cell lung cancer. One identified resistance mechanism to sotorasib is the increase of multiple phosphorylated receptor tyrosine kinases, including EGFR and HER2. The objective of this study was to assess the in vitro efficacy of the single agents N, L, T and S, and the combination of N and S in HER2-amplified, KRASG12C-mutated cancer cell lines. Methods: The antiproliferative effects of N, L, T and S were assessed in the HER2-amplified and KRASG12C-mutated KYSE-410 (esophageal) and NCI-H2030 (lung) cancer cell lines, and KRASG12C-mutated MiaPaca-2 (pancreatic) cell line by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between N and S, matrix assays were performed and analyzed using Combenefit software. To further determine the efficacy of the drugs, changes in signaling pathways were assessed by Western blotting. Results: N displayed nanomolar IC50 values (< 100 nM) in all three cell lines, while nanomolar IC50 values with L and T were only achieved in KYSE-410 (IC50 = 426.84 nM and IC50 = 363.38 nM respectively). S was most potent in MiaPaca-2 (IC50 = 6.04 nM), followed by NCI-H2030 (IC50 = 270.60 nM) and KYSE-410 (IC50 = 5.46 µM). Matrix assays showed that NS was broadly additive in MiaPaca-2 cells, and synergistic in both NCI-H2030 and KYSE-410 cell lines. N and NS numerically decreased HER2 levels compared to control in NCI-H2030 after 24h of treatment (6.4-fold, p = 0.58 and 2.5-fold, p = 0.79 respectively) but not in KYSE-410. KRAS levels were significantly increased (1.89-fold, p ≤ 0.05) after treatment with NS in NCI-H2030. No significant effects were observed on the activation of HER2 and EGFR in either cell line at 24h. In NCI-H2030, NS had a significantly stronger inhibitory effect on pERK compared to N (p ≤ 0.0001) and S (p = 0.012) alone. In KYSE-410, pERK levels were also slightly decreased with NS, but not significantly different from N (p = 0.12). However, pAkt levels in KYSE-410 were significantly reduced by N (1.3-fold, p = 0.009) and NS (1.7-fold, p ≤ 0.0001) compared to control, with NS showing a significantly greater decrease than N (p = 0.026). It thus appears that the synergy between N and S occurs through a stronger inhibition of ERK activity in NCI-H2030, while it occurs through a stronger inhibition of Akt activity in KYSE-410. Conclusion: NS is additive or synergistic in the three KRASG12C-mutated cancer cell lines examined. Further work is planned to investigate the basis of the synergy observed. Citation Format: Myra E. Castel, Neil T. Conlon, Naomi Walsh, Georg F. Bischof, John P. Crown, Denis M. Collins. The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1978.

Repurposing of Loperamide as a New Drug With Anticancer Activity for Human Osteosarcoma.

Osteosarcoma is an aggressive malignant bone tumor, with unfavorable outcomes in patients with metastatic and recurrent disease. To improve patient survival new treatment options are needed. By using the drug repurposing approach, which takes advantage of already approved drugs with non-oncology primary use, we investigated the activity of loperamide, a peripheral opiate receptor agonist, a drug widely used in clinical practice to treat acute non-specific and chronic diarrhea, on human osteosarcoma. Human osteosarcoma cell lines (143B, Saos-2, HOS and MG-63) and multidrug-resistant MG-63DXR30 cells were treated with loperamide. Proliferation and cell viability were determined by viable cell count and acid phosphatase assay. Loperamide activity on cell cycle and apoptosis induction were evaluated by flow cytometry and a luminescence assay testing caspase 3/7 activity, respectively. Loperamide significantly inhibited cell proliferation, through alteration of cell cycle profile at G0/G1 phase and apoptotic death in human osteosarcoma cells. Furthermore, loperamide significantly inhibited the growth of multidrug-resistant osteosarcoma cells. Our findings provide new perspectives for loperamide and its therapeutic repositioning for the treatment of osteosarcoma.

Atp6v1h Deficiency Blocks Bone Loss in Simulated Microgravity Mice through the Fos-Jun-Src-Integrin Pathway.

The microgravity conditions in outer space are widely acknowledged to induce significant bone loss. Recent studies have implicated the close relationship between Atp6v1h gene and bone loss. Despite this, the role of Atp6v1h in bone remodeling and its molecular mechanisms in microgravity have not been fully elucidated. To address this, we used a mouse tail suspension model to simulate microgravity. We categorized both wild-type and Atp6v1h knockout (Atp6v1h+/-) mice into two groups: regular feeding and tail-suspension feeding, ensuring uniform feeding conditions across all cohorts. Analysis via micro-CT scanning, hematoxylin-eosin staining, and tartrate-resistant acid phosphatase assays indicated that wild-type mice underwent bone loss under simulated microgravity. Atp6v1h+/- mice exhibited bone loss due to Atp6v1h deficiency but did not present aggravated bone loss under the same simulated microgravity. Transcriptomic sequencing revealed the upregulation of genes, such as Fos, Src, Jun, and various integrin subunits in the context of simulated microgravity and Atp6v1h knockout. Real-time quantitative polymerase chain reaction (RT-qPCR) further validated the modulation of downstream osteoclast-related genes in response to interactions with ATP6V1H overexpression cell lines. Co-immunoprecipitation indicated potential interactions between ATP6V1H and integrin beta 1, beta 3, beta 5, alpha 2b, and alpha 5. Our results indicate that Atp6v1h level influences bone loss in simulated microgravity by modulating the Fos-Jun-Src-Integrin pathway, which, in turn, affects osteoclast activity and bone resorption, with implications for osteoporosis. Therefore, modulating Atp6v1h expression could mitigate bone loss in microgravity conditions. This study elucidates the molecular mechanism of Atp6v1h's role in osteoporosis and positions it as a potential therapeutic target against environmental bone loss. These findings open new possibilities for the treatment of multifactorial osteoporosis.

Development of a fluorometric and colorimetric dual-mode sensing platform for acid phosphatase assay based on Fe3+ functionalized CuInS2/ZnS quantum dots

BackgroundThe spectral dual-mode response towards analyte has been attracted much attention, benefiting from the higher detection accuracy of such strategy in comparison to single signal readout. However, the currently reported dual-mode sensors for acid phosphatase (ACP) activity are still limited, and most of them more or less exist some deficiencies, such as complicated construction procedure, high-cost, poor biocompatibility, aggregation-caused quenching and limited emission capacity. ResultsHerein, we employed Fe3+ functionalized CuInS2/ZnS quantum dots (CIS/ZnS QDs) as nanosensor to develop a novel fluorometric and colorimetric dual-mode assay for ACP activity, combing with ACP-triggered hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA). The Fe3+ binding to CIS/ZnS QDs can be reduced into Fe2+ during the determination, resulting in the dramatically weakened photoinduced electron transfer (PET) effect and the disappearance of competition absorption. Thus, a highly sensitive ACP assay in the range of 0.22–12.5 U L−1 through fluorescence “turn-on” mode has been achieved with a detection of limit (LOD) of 0.064 U L−1. Meanwhile, the ACP activity can also be quantified by spectrophotometry based on the chromogenic reaction of the formed Fe2+ with 1,10-phenanthroline (Phen). Moreover, the designed nanosensor with good biocompatibility was successfully applied to image and monitor the ACP levels in living cells. SignificanceWe believe that the proposed method has remarkable advantages and potential application for ACP assay in terms of the high accuracy, simplicity, low cost, as well as its adequate sensitivity.

Polysaccharides Derived from Drynaria fortunei Attenuated Osteoclast Differentiation Induced by Receptor Activator of Nuclear Factor-κB Ligand by Modulating NFATc1 and c-Fos

The rhizome of Drynaria fortunei (Kunze ex Mett.) J. Sm., which is known as “Golsebo” in Korea, traditionally has been used to heal various inflammatory conditions, including bone metabolism disorders. It relieves blood extravasation, stops bleeding, repairs broken bone tissue, treats bone fractures, and kills bacteria. In this study, we evaluated the modulatory effects of DFP on the differentiation of bone-marrow-derived macrophages into osteoclasts. We performed tartrate-resistant acid phosphatase assays using DFP (at different concentrations and molecular weights) to evaluate the degree of bone resorption in the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis of bone-marrow-derived macrophages. TRAP activity increased with increasing DFP concentrations (0–200 µg/mL). Additionally, DFP significantly inhibited RANKL-induced osteoclastogenesis and controlled RANKL-mediated overexpression of c-Fos and nuclear factor of activated T cells 1, thereby downregulating osteoclast-specific gene (Atp6v0d2, cathepsin K, and DC-STAMP) expression. DFP thus has potential as a nutraceutical candidate for treating bone loss diseases, including osteoporosis in postmenopausal women.

Multifunctional electrospun nanofibrous scaffold enriched with alendronate and hydroxyapatite for balancing osteogenic and osteoclast activity to promote bone regeneration.

Electrospun composite nanofiber scaffolds are well known for their bone and tissue regeneration applications. This research is focused on the development of PVP and PVA nanofiber composite scaffolds enriched with hydroxyapatite (HA) nanoparticles and alendronate (ALN) using the electrospinning technique. The developed nanofiber scaffolds were investigated for their physicochemical as well as bone regeneration potential. The results obtained from particle size, zeta potential, SEM and EDX analysis of HA nanoparticles confirmed their successful fabrication. Further, SEM analysis verified nanofiber's diameters within 200-250nm, while EDX analysis confirmed the successful incorporation of HA and ALN into the scaffolds. XRD and TGA analysis revealed the amorphous and thermally stable nature of the nanofiber composite scaffolds. Contact angle, FTIR analysis, Swelling and biodegradability studies revealed the hydrophilicity, chemical compatibility, suitable water uptake capacity and increased in-vitro degradation making it appropriate for tissue regeneration. The addition of HA into nanofiber scaffolds enhanced the physiochemical properties. Additionally, hemolysis cell viability, cell adhesion and proliferation by SEM as well as confocal microscopy and live/dead assay results demonstrated the non-toxic and biocompatibility behavior of nanofiber scaffolds. Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) assays demonstrated osteoblast promotion and osteoclast inhibition, respectively. These findings suggest that developed HA and ALN-loaded PVP/PVA-ALN-HA nanofiber composite scaffolds hold significant promise for bone regeneration applications.

Phloridzin Docosahexaenoate Inhibits Spheroid Formation by Breast Cancer Stem Cells and Exhibits Cytotoxic Effects against Paclitaxel-Resistant Triple Negative Breast Cancer Cells.

The eradication of cancer stem cells (CSCs) is vital to successful cancer treatment and overall disease-free survival. CSCs are a sub-population of cells within a tumor that are defined by their capacity for continuous self-renewal and recapitulation of new tumors, demonstrated in vitro through spheroid formation. Flavonoids are a group of phytochemicals with potent anti-oxidant and anti-cancer properties. This paper explores the impact of the flavonoid precursor phloridzin (PZ) linked to the ω-3 fatty acid docosahexaenoate (DHA) on the growth of MCF-7 and paclitaxel-resistant MDA-MB-231-TXL breast cancer cell lines. Spheroid formation assays, acid phosphatase assays, and Western blotting were performed using MCF-7 cells, and the cell viability assays, Annexin-V-488/propidium iodide (PI) staining, and 7-aminoactinomycin D (7-AAD) assays were performed using MDA-MB-231-TXL cells. PZ-DHA significantly reduced spheroid formation, as well as the metabolic activity of MCF-7 breast cancer cells in vitro. Treatment with PZ-DHA also suppressed the metabolic activity of MDA-MB-231-TXL cells and led to apoptosis. PZ-DHA did not have an observable effect on the expression of the drug efflux transporters ATP-binding cassette super-family G member 2 (ABCG2) and multidrug resistance-associated protein 1 (MRP1). PZ-DHA is a potential treatment avenue for chemo-resistant breast cancer and a possible novel CSC therapy. Future pre-clinical studies should explore PZ-DHA as a chemo-preventative agent.

Novel quinazoline-1,2,3-triazole hybrids with anticancer and MET kinase targeting properties

Oncogenic activation of receptor tyrosine kinases (RTKs) such as MET is associated with cancer initiation and progression. We designed and synthesized a new series of quinazoline derivatives bearing 1,2,3-triazole moiety as targeted anticancer agents. The MET inhibitory effect of synthesized compounds was assessed by homogeneous time-resolved fluorescence (HTRF) assay and western blot analysis. Sulforhodamine B assay was conducted to examine the antiproliferative effects of synthetic compounds against 6 cancer cell lines from different origins including MET-dependent AsPC-1, EBC-1 and MKN-45 cells and also Mia-Paca-2, HT-29 and K562 cells. The growth inhibitory effect of compounds in a three-dimensional spheroid culture was examined by acid phosphatase (APH) assay, while apoptosis induction was evaluated by Annexin V/propidium iodide method. Compound 8c bearing p-methyl benzyl moiety on the triazole ring exhibited the highest MET inhibitory capacity among tested agents that was further confirmed by western blot findings. Derivatives 8c and 8h exhibited considerable antiproliferative effects against all tested cell lines, with more inhibitory effects against MET-positive cells with IC50 values as low as 6.1 μM. These two agents also significantly suppressed cell growth in spheroid cultures and induced apoptosis in MET overexpressing AsPC-1 cells. Moreover, among a panel of 24 major oncogenic kinases, the PDGFRA kinase was identified as a target of 8c and 8h compounds. The docking study results of compounds 8c and 8h were in agreement with experimental findings. The results of the present study suggest that quinazoline derivatives bearing 1,2,3-triazole moiety may represent promising targeted anticancer agents.

Abstract 4041: The EML4-ALK fusion protein mediates reduced sensitivity to the combination of neratinib and dasatinib

Abstract Introduction: The anaplastic lymphoma kinase (ALK) gene encodes a receptor tyrosine kinase involved in cellular proliferation, differentiation, and cell death. ALK becomes oncogenic when it forms a fusion gene. The EML4-ALK fusion gene has been identified mainly in non-small cell lung cancer (NSCLC). It is unknown whether neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI) targeting EGFR, HER2 and HER4, is effective in cancers harboring EML4-ALK gene fusion. The objective of this study was to assess in vitro efficacy of neratinib in combination with dasatinib (Src/Abl TKI) or crizotinib (ALK TKI) in EML4-ALK+ NSCLC. Methods: The antiproliferative effects of neratinib, dasatinib and crizotinib were assessed in the CRISPR/Cas9-modified EML4-ALK fusion-A549 (EML4-ALK+) and parental A459 (A549-Par) NSCLC cell lines by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between neratinib and dasatinib, and neratinib and crizotinib, matrix assays were performed and analyzed using Combenefit software. To further assess the efficacy of the TKIs, changes in signaling pathways were assessed by Western blotting, and apoptosis induction and cell migration were measured with the Incucyte® S3 imaging system. Results: Neratinib, crizotinib, and dasatinib displayed nanomolar IC50 values in both cell lines. As expected, EML4-ALK+ cells were more sensitive to crizotinib than A549-Par cells (IC50 = 595 nM vs 1 µM, p ≤ 0.05). EML4-ALK expression led to numerical increases in neratinib IC50 value (326.37 ± 44.34 nM in EML4-ALK+ vs 247 ± 32.65 nM in A549-Par) and dasatinib IC50 value (39.95 ± 5.67 nM in EML4-ALK+ vs 27.75 ± 18.53 nM in A549-Par) but these changes were not statistically significant. Matrix assays showed that neratinib-crizotinib (NC) was more effective than crizotinib alone in both cell lines, with more potent sensitivity in EML4-ALK+ cells. The neratinib-dasatinib (ND) combination was synergistic in the A549-Par cell line but only additive in the EML4-ALK+ cell line, suggesting a reduced sensitivity to the ND combination. EML4-ALK+ cells had sustained pERK1/2 levels after treatment with all TKIs, except NC, compared to A549-Par cells. Neratinib alone was significantly less effective at inhibiting pAkt (99.2 ± 5.3 % in EML4-ALK+ vs 64.9 ± 19.0 %, p ≤ 0.05) and pERK1/2 (91.3 ± 4.6 % in EML4-ALK+ vs 54.9 ± 15.9 %, p ≤ 0.01) in EML4-ALK+ cells than in A549-Par cells at 24h. In A549-Par cells, ND caused the highest apoptotic induction, followed by NC. Apoptosis levels induced by ND in EML4-ALK+ cells were 4-fold lower at 72hr than in A549-Par cells (p ≤ 0.0001). Dasatinib and ND were more effective than crizotinib or NC at preventing cell migration in both cell lines. Conclusion: EML4-ALK potentially decreases sensitivity to the neratinib-dasatinib combination. This warrants further investigation in other EML4-ALK+ models. Citation Format: Myra E. Castel, Neil T. Conlon, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. The EML4-ALK fusion protein mediates reduced sensitivity to the combination of neratinib and dasatinib. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4041.

In Vitro Analysis of Cytotoxic Activities of Monotheca buxifolia Targeting WNT/β-Catenin Genes in Breast Cancer Cells.

Breast cancer (BC) is known to be the most common malignancy among women throughout the world. Plant-derived natural products have been recognized as a great source of anticancer drugs. In this study, the efficacy and anticancer potential of the methanolic extract of Monotheca buxifolia leaves using human breast cancer cells targeting WNT/β-catenin signaling was evaluated. We used methanolic and other (chloroform, ethyl acetate, butanol, and aqueous) extracts to discover their potential cytotoxicity on breast cancer cells (MCF-7). Among these, the methanol showed significant activity in the inhibition of the proliferation of cancer cells because of the presence of bioactive compounds, including phenols and flavonoids, detected by a Fourier transform infrared spectrophotometer and by gas chromatography mass spectrometry. The cytotoxic effect of the plant extract on the MCF-7 cells was examined by MTT and acid phosphatase assays. Real-time PCR analysis was performed to measure the mRNA expression of WNT-3a and β-catenin, along with Caspase-1,-3,-7, and -9 in MCF-7 cells. The IC50 value of the extract was found to be 232 μg/mL and 173 μg/mL in the MTT and acid phosphatase assays, respectively. Dose selection (100 and 300 μg/mL) was performed for real-time PCR, Annexin V/PI analysis, and Western blotting using Doxorubicin as a positive control. The extract at 100 μg/mL significantly upregulated caspases and downregulated the WNT-3a and β-catenin gene in MCF-7 cells. Western blot analysis further confirmed the dysregulations of the WNT signaling component (*** p< 0.0001). The results showed an increase in the number of dead cells in methanolic extract-treated cells in the Annexin V/PI analysis. Our study concludes that M. buxifolia may serve as an effective anticancer mediator through gene modulation that targets WNT/β-catenin signaling, and it can be further characterized using more powerful experimental and computational tools.

MXene-derived Ti3C2 quantum dots-based ratiometric fluorescence probe for ascorbic acid and acid phosphatase determination

As a burgeoning zero-dimensional nanomaterial, MXene quantum dots which are derived from two-dimensional transition-metal carbides have attracted a lot of interest lately. The transformation of two-dimensional MXene into zero-dimensional structures of a few nanometers enables these recently discovered materials to possess an exceptional quantum size effect and remarkable photoluminescence characteristics. Hence, we synthesized N doped Ti3C2 MXene quantum dots (N-Ti3C2 MQDs) exhibiting bright blue fluorescence through small-molecule amine-assisted solvothermal strategy. In this work, we constructed a novel ratiometric fluorescence biosensor for the determination of ascorbic acid (AA) and acid phosphatase (ACP) based on the excellent optical performance of N-Ti3C2 MQDs and combined o-phenylenediamine (OPD). This ratiometric strategy was established based on the inner filter effect between N-Ti3C2 MQDs and the oxide of OPD. Sensitive fluorometric analyses of AA and ACP were achieved successfully in the respective ranges of 2–240 μM and 0.15–3.75 U/L with respective limits of detection down to 0.82 μM and 0.02 U/L. Moreover, the response time of this fluorescence sensor for AA and ACP determination was less than 1.5 h. The intraday and interday relative standard deviations for AA and ACP assay were all below 8.3 %, suggesting the good reproducibility of this sensor. In addition, the profuse color changed under UV light with analytes concentrations increased and exhibited great potential for visual detection and on-site application. The proposed method was further utilized to screen of potential ACP inhibitors, indicating that this probe has great potential in clinical practice.

Evaluating methods to create protein functionalized catanionic vesicles.

Catanionic surfactant vesicles (SVs) composed of sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT) have potential applications as targeted drug delivery systems, vaccine platforms, and diagnostic tools. To facilitate these applications, we evaluated various methods to attach proteins to the surface of SDBS/CTAT vesicles. Acid phosphatase from wheat germ was used as a model protein. Acid phosphatase was successfully conjugated to vesicles enriched with a Triton-X 100 derivative containing an unsaturated ester. Enzymatic activity of acid phosphatase attached to vesicles was assessed using an acid phosphatase assay. Results from the acid phosphatase assay indicated that 15 ± 3% of the attached protein remained functional but the presence of vesicles interferes with the assay. DLS and zeta potential results correlated with the protein functionalization studies. Acid phosphatase functionalized vesicles had an average diameter of 175 ± 85 nm and an average zeta potential of -61 ± 5 mV in PBS. As a control, vesicles enriched with Triton-X 100 were prepared and analyzed by DLS and zeta potential measurements. Triton X-100 enriched vesicles had an average diameter of 140 ± 67 nm and an average zeta potential of -49 ± 2 mV in PBS. Functionalizing the surface of SVs with proteins may be a key step in developing vesicle-based technologies. For drug delivery, antibodies could be used as targeting molecules; for vaccine formulation, functionalizing the surface with spike proteins may produce novel vaccine platforms.

Galectin-3 interacts with PD-1 and counteracts the PD-1 pathway-driven regulation of T cell and osteoclast activity in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and bone erosions. The glycosylated programmed death-1 (PD-1) receptor plays an important role in regulating immune responses and maintaining tolerance. In this study, we focus on two features observed in RA: impaired PD-1 signalling and Galectin-3 (Gal-3) upregulation. We hypothesize that Gal-3 binds PD-1 and PD-1 ligands, potentially contributing to impaired PD-1 signalling. PD-1 and Gal-3 levels in RA synovial fluid (SF) and plasma were evaluated by ELISA. PD-1 and Gal-3 interaction was examined by Surface Plasmon Resonance and ELISA. PD-1, PD-L1 and Gal-3 expression on mononuclear cells from SF and peripheral blood as well as fibroblast-like synoviocytes were examined by flow cytometry. Effects of Gal-3 and PD-L1 on osteoclast formation was evaluated by tartrate-resistant acid phosphatase assay. We show that Gal-3 binds PD-1 and PD-L1. Results demonstrated high expression of PD-1 and Gal-3 on mononuclear cells, especially from SF. Gal-3 inhibited PD-1 signalling when PD-L1 was present. Furthermore, a role of Gal-3 in osteoclast formation was observed in vitro, both directly but also through PD-1:PD-L1 inhibition. Effects of Gal-3 on the PD-1 signalling axis are proposed to be inhibitory, meaning high Gal-3 levels in the complex synovial microenvironment are not desirable in RA. Preventing Gal-3's inhibitory role on PD-1 signalling could, therefore, be a therapeutic target in RA by affecting inflammatory T cell responses and osteoclasts.

Sensitive acid phosphatase assay based on light-activated specific oxidase mimic activity

Acid phosphatase (ACP) is a key marker in the diagnosis of many diseases. However, exploiting a simple and sensitive sensor for the real-time quantitative analysis of ACP is still challenging. Herein, we attempted to develop a sensitive colorimetric sensing strategy for the detection of ACP based on light-activated oxidase mimic property of carbon dots (CDs). The synthesized CDs were proved to be capable of intrinsic light-activated oxidase mimic activity, which could generate reactive oxygen species to oxidize chromogenic substrate under ultraviolet light stimulation. Interestingly, this light-activated oxidase mimic behavior would be effectively suppressed by the antioxidant ascorbic acid (AA), a product from the hydrolysis of 2-phospho-L-ascorbic acid trisodium (AAP) mediated by ACP. Based on the above property, a facile and sensitive colorimetric sensing method for ACP was developed. Under the optimal conditions, the linear range for ACP 0.1–5.5 U/L, and the detection limit was 0.056 U/L. Compared with conventional nanozyme based ACP assay systems, the catalytic activity of light-activated nanozyme could be conveniently regulated by switching the light on and off, which made it easier to precisely control the extent of the reaction and ensured the accuracy of the assay. In addition, the proposed sensing system would be readout directly by the naked eye or smartphone-based RGB analysis system, and have been successfully applied to analyze diluted in diluted fetal bovine serum and urine samples spiked with ACP. All these results indicated that this approach holds good promise for future applications in clinical analysis and point-of-care (POC) biosensor platforms.

Third-generation sequencing-selected Scardovia wiggsiae promotes periodontitis progression in mice.

Periodontitis is an oral-bacteria-directed disease that occurs worldwide. Currently, periodontal pathogens are mostly determined using traditional culture techniques, next-generation sequencing, and microbiological screening system. In addition to the well-known and cultivatable periodontal bacteria, we aimed to discover a novel periodontal pathogen by using DNA sequencing and investigate its role in the progression of periodontitis. This study identified pathogens from subgingival dental plaque in patients with periodontitis by using the Oxford Nanopore Technology (ONT) third-generation sequencing system and validated the impact of selected pathogen in periodontitis progression by ligature-implanted mice. Twenty-five patients with periodontitis and 25 healthy controls were recruited in this study. Subgingival plaque samples were collected for metagenomic analysis. The ONT third-generation sequencing system was used to confirm the dominant bacteria. A mouse model with ligature implantation and bacterial injection verified the pathogenesis of periodontitis. Neutrophil infiltration and osteoclast activity were evaluated using immunohistochemistry and tartrate-resistant acid phosphatase assays in periodontal tissue. Gingival inflammation was evaluated using pro-inflammatory cytokines in gingival crevicular fluids. Alveolar bone destruction in the mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. Scardovia wiggsiae (S.wiggsiae) was dominant in the subgingival plaque of the patients with periodontitis. S.wiggsiae significantly deteriorated ligature-induced neutrophil infiltration, osteoclast activation, alveolar bone destruction, and the secretion of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α in the mouse model. Our metagenome results suggested that S.wiggsiae is a dominant flora in patients with periodontitis. In mice, the induction of neutrophil infiltration, proinflammatory cytokine secretion, osteoclast activation, and alveolar bone destruction further verified the pathogenic role of S.wiggsiae in the progress of periodontitis. Future studies investigating the metabolic interactions between S.wiggsiae and other periodontopathic bacteria are warranted.

Assessment of Basic Biological Functions Exerted by miRNAs.

To evaluate basic miRNA function, it is vital to assess various cellular processes, such as cell viability and proliferation, under different miRNA levels. Here we describe the process of overexpression of miRNA in vitro via transfection and subsequent downstream evaluation using the acid phosphatase assay.

Study of the anticancer effect of new quinazolinone hydrazine derivatives as receptor tyrosine kinase inhibitors.

The advent of novel receptor tyrosine kinase inhibitors has provided an important therapeutic tool for cancer patients. In this study, a series of quinazolinone hydrazide triazole derivatives were designed and synthesized as novel MET (c-MET) receptor tyrosine kinase inhibitors. The antiproliferative effect of the synthesized compounds was examined against EBC-1, A549, HT-29 and U-87MGcells by MTT assay. MET kinase inhibitory effect was tested by a Homogenous Time Resolved Fluorescence (HTRF) assay. The antiproliferative effect of compounds in a three-dimensional spheroid culture was studied by acid phosphatase (APH) assay, while apoptosis induction was examined by Hoechst 33258 staining. We found that compound CM9 bearing p-bromo benzyl pendant inhibited MET kinase activity at the concentrations of 10-50μM (% Inhibition = 37.1-66.3%). Compound CM9 showed antiproliferative effect against cancer cells, in particular lung cancer cells with MET amplification (EBC-1) with an IC50 value of 8.6μM. Moreover, this derivative inhibited cell growth in spheroid cultures in a dose-dependent manner and induced apoptosis in cancer cells. Assessment of inhibitory effect of CM9 against a panel of 18 different protein kinases demonstrated that this compound also inhibits ALK, AXL, FGFR1, FLT1 (VEGFR1) and FLT4 (VEGFR3) more than 50% at 25μM. Finally, molecular docking and dynamics simulation corroborated the experimental findings and showed critical structural features for the interactions between CM9 and target kinases. The findings of this study present quinazolinone hydrazide triazole derivatives as kinase inhibitors with considerable anticancer effects.