Transcriptomic analysis of glucosidase II beta subunit (GluIIß) knockout A549 cells reveals its roles in regulation of cell adhesion molecules (CAMs) and anti-tumor immunity.

Glucosidase II beta subunit (GluIIß), encoded from PRKCSH, is a subunit of the glucosidase II enzyme responsible for quality control of N-linked glycoprotein folding and suppression of GluIIß led to inhibitory effect of the receptor tyrosine kinase (RTKs) activities known to be critical for survival and development of cancer. In this study, we investigated the effect of GluIIß knockout on the global gene expression of cancer cells and its impact on functions of immune cells. GluIIß knockout lung adenocarcinoma A549 cell line was generated using CRISPR/Cas9-based genome editing system and subjected to transcriptomic analysis. Among 23,502 expressed transcripts, 1068 genes were significantly up-regulated and 807 genes greatly down-regulated. The KEGG enrichment analysis showed significant down-regulation of genes related extracellular matrix (ECM), ECM-receptor interaction, cytokine-cytokine receptor interaction and cell adhesion molecules (CAMs) in GluIIß knockout cells. Of 9 CAMs encoded DEG identified by KEGG enrichment analysis, real time RT-PCR confirmed 8 genes to be significantly down-regulated in all 3 different GluIIß knockout clones, which includes cadherin 4 (CDH4), cadherin 2 (CDH2), versican (VCAN), integrin subunit alpha 4 (ITGA4), endothelial cell-selective adhesion molecule (ESAM), CD274 (program death ligand-1 (PD-L1)), Cell Adhesion Molecule 1 (CADM1), and Nectin Cell Adhesion Molecule 3 (NECTIN3). Whereas PTPRF (Protein Tyrosine Phosphatase Receptor Type F) was significantly decreased only in 1 out of 3 knockout clones. Microscopic analysis revealed distinctively different cell morphology of GluIIß knockout cells with lesser cytoplasmic and cell surface area compared to parental A549 cells and non-targeted transfected cells.Further investigations revealed that Jurkat E6.1 T cells or human peripheral blood mononuclear cells (PBMCs) co-cultured with GluIIß knockout A549 exhibited significantly increased viability and tumor cell killing activity compared to those co-cultured with non-target transfected cells. Analysis of cytokine released from Jurkat E6.1 T cells co-cultured with GluIIß knockout A549 cells showed significant increased level of angiogenin and significant decreased level of ENA-78. In conclusion, knockout of GluIIß from cancer cells induced altered gene expression profile that improved anti-tumor activities of co-cultured T lymphocytes and PBMCs thus suppression of GluIIß may represent a novel approach of boosting anti-tumor immunity.

Targeting CD38/ ADP-ribosyl cyclase as a novel therapeutic strategy for identification of three potent agonists for leukopenia treatment.

Leukopenia is the most common side effect of chemotherapy and radiotherapy. It potentially deteriorates into a life-threatening complication in cancer patients. Despite several agents being approved for clinical administration, there are still high incidences of pathogen-related disease due to a lack of functional immune cells. ADP-ribosyl cyclase of CD38 displays a regulatory effect on leukopoiesis and the immune system. To explore whether the ADP-ribosyl cyclase was a potential therapeutic target of leukopenia. We established a drug screening model based on an ADP-ribosyl cyclase-based pharmacophore generation algorithm and discovered three novel ADP-ribosyl cyclase agonists: ziyuglycoside II (ZGSII), brevifolincarboxylic acid (BA), and 3,4-dihydroxy-5-methoxybenzoic acid (DMA). Then, in vitro experiments demonstrated that these three natural compounds significantly promoted myeloid differentiation and antibacterial activity in NB4 cells. In vivo, experiments confirmed that the compounds also stimulated the recovery of leukocytes in irradiation-induced mice and zebrafish. The mechanism was investigated by network pharmacology, and the top 12 biological processes and the top 20 signaling pathways were obtained by intersecting target genes among ZGSII, BA, DMA, and leukopenia. The potential signaling molecules involved were further explored through experiments. Finally, the ADP-ribosyl cyclase agonists (ZGSII, BA, and DMA) has been found to regenerate microbicidal myeloid cells to effectively ameliorate leukopenia-associated infection by activating CD38/ADP-ribosyl cyclase-Ca2+-NFAT. In summary, this study constructs a drug screening model to discover active compounds against leukopenia, reveals the critical roles of ADP-ribosyl cyclase in promoting myeloid differentiation and the immune response, and provides a promising strategy for the treatment of radiation-induced leukopenia.

Optimization of Hydrolysis Conditions, Isolation, and Identification of Biologically Active Peptides Derived from Acheta domesticus for Antioxidant and Collagenase Inhibition.

The study aimed to optimize hydrolysis conditions and isolate and identify bioactive peptides with anti-skin aging effects from Acheta domesticus (house cricket). A. domesticus proteins underwent hydrolysis using Alcalase® and optimized conditions using response surface methodology through a face-centered central composite design. Variable controls (enzyme-substrate concentration (E/S), time, and temperature) were assessed for their impact on activities against collagenase, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH●), and degree of hydrolysis of protein hydrolysate (PH). PH was also investigated for composition, anti-skin aging, and anti-inflammatory effects. Amino acid sequences with potent anti-skin aging activity were isolated and identified using ultrafiltration, gel filtration chromatography, and liquid chromatography coupled with tandem mass spectrometry, employing de novo sequencing. Optimal conditions for producing PH with maximum anti-skin aging activity were an E/S concentration of 2.1% (w/w), 227 min, and 61.5 °C. Glutamic acid was a predominant amino acid and PH exhibited a molecular weight below 15 kDa. Additionally, PH displayed significant activities against collagenase, hyaluronidase, DPPH●, lipid peroxidation, and NF-κB-mediated inflammation (p < 0.05). Three novel anti-skin aging peptides were identified-Ala-Val-Thr-Lys-Ala-Asp-Pro-Tyr-Thr-Asp-Gln, Thr-Val-Met-Glu-Leu-Asn-Asp-Leu-Val-Lys-Ala-Phe, and Val-Pro-Leu-Leu-Glu-Pro-Trp-exhibiting the most potent collagenase and DPPH● inhibition. Therefore, this study proposed that PH, produced with Alcalase® under optimal conditions, emerges as a promising substance with potent anti-skin aging activity for the cosmeceutical industry.

Acheta domesticus: A Natural Source of Anti-Skin-Aging Ingredients for Cosmetic Applications.

Acheta domesticus is an edible insect, rich in nutritional value and considered a sustainable protein source. This study aimed to investigate the potential application of A. domesticus extracts for anti-skin-aging purposes. The extracts were prepared by maceration at ambient temperature with 95% ethanol or hexane and maceration in gentle heat (45 °C) with 95% v/v ethanol or DI water. The extracts were examined for total protein, phenolic, and flavonoid contents. Protein molecular weight distribution was analyzed. The safety of the extracts was investigated in terms of irritation and cytotoxicity. Biological activities relevant to the inhibition of skin aging were evaluated, including increasing transforming growth factor-beta 1 (TGF-ß1) expression and inhibitory activities on collagenase and hyaluronidase. The aqueous extract from maceration in gentle heat had the highest total protein content (63 ± 1% w/w), total phenolic content (0.48 ± 0.03 mg GAE/g extract), TGF-ß1 stimulating activities (33 ± 2 pg/mL), and collagenase inhibition (with a half maximal inhibitory concentration of 26 ± 1 µg/mL) among various extracts investigated. It caused no irritation to the hen's egg chorioallantoic membrane and showed no cytotoxicity to human dermal fibroblasts and peripheral blood mononuclear cells. Therefore, aqueous A. domesticus extract is proposed as an innovative natural anti-skin-aging ingredient.

MicroRNA­223 overexpression suppresses protein kinase C ε expression in human leukemia stem cell­like KG­1a cells.

MicroRNA-223 (miR-223) is dysregulated in various cancer types, including acute myeloid leukemia (AML). Despite this, there has been a lack of studies exploring the role of miR-223 in leukemic stem cells, particularly those involved in drug resistance, a major cause of chemotherapy failure in AML. The present study aimed to elucidate the impact of miR-223 on drug resistance in the leukemic stem-cell line, KG-1a. Two AML cell lines, KG-1 and KG-1a, differing in the proportion of CD34+CD38- cells, were assessed for doxorubicin (DOX) sensitivity using the Cell Counting Kit-8 assay. The expression levels of miR-223 and protein kinase C ε (PKCε) were evaluated via reverse transcription-quantitative PCR and western blot analysis. The association between miR-223 and its target, PKCε, was confirmed by luciferase activity assay. The effects of miR-223 overexpression and PKCε inhibition were also evaluated in KG-1a cells using miR-223 mimic and small interfering (si)RNA transfection, respectively. Daunorubicin was then used to assess drug sensitivity in the siRNA-transfected KG-1a cells. Compared with KG-1 cells, KG-1a cells displayed greater resistance to DOX, and had increased PKCε levels and decreased miR-223 expression. Overexpression of miR-223 led to PKCε protein downregulation in KG-1a cells, which was further confirmed by a luciferase assay demonstrating miR-223 targeting of PKCε. However, despite these effects, miR-223 overexpression and PKCε inhibition did not change drug sensitivity in KG-1a cells compared with negative control cells. In summary, the present study demonstrated that miR-223 could target and silence PKCε expression in KG-1a cells; however, the chemoresistance of KG-1a cells to anthracycline drugs may not be directly associated with the low expression of miR-223.

Antioxidant activity of Sophora exigua and liposome development of its powerful extract.

Sophora exigua (SE) was sequentially extracted using hexane, ethyl acetate, and ethanol. The obtained extracts were tested for antioxidant activity. Among them, the fractionated ethyl acetate extract (SE-EA) showed the highest potential in free radical scavenging and ferric-reducing properties. The chemical analysis identified sophoraflavanone G as one of the active ingredients in SE-EA. According to SE-EA solubility, SE-EA liposomes were developed using a sonication-assisted thin film method. Cholesterol and phospholipids were used as the main compositions of the liposomes. The obtained liposomes were spherical with different nano-size ranges, size distribution, and zeta potential depending on SE-EA and total lipid concentrations. SE-EA liposomes were slightly bigger than their empty liposomes. All liposomes exhibited a phospholipid crystalline structure. Cholesterol and SE-EA existed in the liposomes as an amorphous state. SE-EA liposomes with high total lipid content exhibited high entrapment efficiency and sustained release behavior. Whereas liposomes with low total lipid content showed low entrapment efficiency and fast-release behavior. All SE-EA liposomes showed stronger antioxidant activity than the non-entrapped SE-EA. In conclusion, SE-EA is a natural source of potent antioxidants. The developed SE-EA liposomes are a promising pharmaceutical formulation to efficiently deliver the active ingredients of SE-EA and are suitable for further study in vivo.

Effects of Salts and Other Contaminants on Ciprofloxacin Removal Efficiency of Green Synthesized Copper Nanoparticles.

Ciprofloxacin (CIP), a broad-spectrum fluoroquinolone antibiotic, is commonly used in aquaculture to prevent and treat bacterial infections in aquatic animals. For this reason, aquatic environments contain CIP and its derivatives, which lead to the development of drug-resistant bacteria. In the present study, copper nanoparticles were prepared using Garcinia mangostana extract (GME-CuNPs) as a reducing agent and evaluated for their CIP removal efficiency (CRE). The results demonstrate that within 20 min, GME-CuNPs at 25 mM possess a CRE of 92.02 ± 0.09% from CIP-containing aqueous media with pH 6-7. The CRE is influenced by both monovalent and divalent salts. A high salt concentration significantly reduces the CRE. Contaminants in fish wastewater can reduce the CRE, but phenolics, flavonoids, tannins, and ammonia do not affect the CRE. Our results reveal that the CRE is controlled by electrostatic attraction between the negatively charged GME-CuNPs and the cationic species of CIP. The CRE is reduced by wastewater with a pH higher than 8.0, in which the CIP molecules have a negative charge, resulting in a repulsive force due to the negative charge of GME-CuNPs. In fish wastewater with a pH lower than 7.0, GME-CuNPs show the potential to achieve a CRE above 80%. Therefore, pH adjustment to a range of 6-7 in fish wastewater before treatment is deemed imperative. It is concluded that the newly developed GME-CuNPs possess excellent activity in CIP elimination from actual fish wastewater samples. Our findings suggest that GME-CuNPs can be a promising tool to effectively eliminate antibiotics from the environment.

Chitosan Alginate Nanoparticles of Protein Hydrolysate from Acheta domesticus with Enhanced Stability for Skin Delivery.

This study aimed to develop chitosan alginate nanoparticles (CANPs) for enhanced stability for dermal delivery of protein hydrolysate from Acheta domesticus (PH). CANPs, developed using ionotropic pre-gelation followed by the polyelectrolyte complex technique, were characterized for particle size, polydispersity index (PDI), and zeta potential. After the incorporation of PH into CANPs, a comprehensive assessment included encapsulation efficiency, loading capacity, morphology, chemical analyses, physical and chemical stability, irritation potential, release profile, skin permeation, and skin retention. The most optimal CANPs, comprising 0.6 mg/mL sodium alginate, 1.8 mg/mL calcium chloride, and 0.1 mg/mL chitosan, exhibited the smallest particle size (309 ± 0 nm), the narrowest PDI (0.39 ± 0.01), and pronounced negative zeta potential (-26.0 ± 0.9 mV), along with an encapsulation efficiency of 56 ± 2%, loading capacity of 2.4 ± 0.1%, release of 40 ± 2% after 48 h, and the highest skin retention of 12 ± 1%. The CANPs induced no irritation and effectively enhanced the stability of PH from 44 ± 5% of PH remaining in a solution to 74 ± 4% after three-month storage. Therefore, the findings revealed the considerable potential of CANPs in improving PH stability and skin delivery, with promising applications in cosmetics and related fields.

Anticancer activity of Curcuma aeroginosa essential oil and its nano-formulations: cytotoxicity, apoptosis and cell migration effects.

BACKGROUND AND AIMS: Curcuma aeruginosa, commonly known as "kha-min-dam" in Thai, holds significance in Asian traditional medicine due to its potential in treating various diseases, having properties such as anti-HIV, hepatoprotective, antimicrobial and anti-androgenic activities. This study explores the anticancer activity of C. aeruginosa essential oil (CAEO) and its nano-formulations. METHODS: CAEO obtained from hydrodistillation of C. aeruginosa fresh rhizomes was examined by gas chromatography mass spectroscopy. Cytotoxicity of CAEO was determined in leukaemic K562 and breast cancer MCF-7 cell lines using an MTT assay. Cell cycle analysis and cell apoptosis were determined by flow cytometry. Cell migration was studied through a wound-healing assay. RESULTS: Benzofuran (33.20%) emerged as the major compound of CAEO, followed by Germacrene B (19.12%) and Germacrone (13.60%). Two types of CAEO loaded nano-formulations, nanoemulsion (NE) and microemulsion (ME) were developed. The average droplet sizes of NE and ME were 13.8 ± 0.2 and 21.2 ± 0.2 nm, respectively. In a comparison with other essential oils from the fresh rhizomes of potential plants from the same family (Curcuma longa, Curcuma mangga and Zingiber officinale) on anticancer activity against K562 and MCF-7 cell lines, CAEO exhibited the highest cytotoxicity with IC50 of 13.43 ± 1.09 and 20.18 ± 1.20 µg/mL, respectively. Flow cytometry analysis revealed that CAEO significantly increased cell death, evidenced from the sub-G1 populations in the cell cycle assay and triggered apoptosis. Additionally, CAEO effectively inhibited cell migration in MCF-7 cells after incubation for 12 and 24 h. The developed NE and ME formulations significantly enhanced the cytotoxicity of CAEO against K562 cells with an IC50 of 45.30 ± 1.49 and 41.98 ± 0.96 µg/mL, respectively. CONCLUSION: This study's finding suggest that both nano-formulations, NE and ME, effectively facilitated the delivery of CAEO into cancer cells.

Hyaluronic acid-modified liposomes Potentiated in-vivo anti-hepatocellular carcinoma of icaritin.

Introduction: Icaritin (ICT), a promising anti-hepatocellular carcinoma (HCC) prenylated flavonoid, is hindered from being applied due to its low water solubility and high lipophilicity in poorly differentiated HCC which is associated with upregulation of CD44 isoforms. Thus, hyaluronic acid (HA), a natural polysaccharide with high binding ability to CD44 receptors, was used to formulate a modified liposome as a novel targeted ICT-delivery system for HCC treatment. Methods: The ICT-Liposomes (Lip-ICT) with and without HA were prepared by a combined method of thin-film dispersion and post-insertion. The particle size, polydispersity (PDI), zeta potential, encapsulation efficacy (%EE), drug loading content (%DLC), and in vitro drug release profiles were investigated for physicochemical properties, whereas MTT assay was used to assess cytotoxic effects on HCC cells, HepG2, and Huh7 cells. Tumor bearing nude mice were used to evaluate the inhibitory effect of HA-Lip-ICT and Lip-ICT in vivo. Results: Lip-ICT and HA-Lip-ICT had an average particle size of 171.2 ± 1.2 nm and 208.0 ± 3.2 nm, with a zeta potential of -13.9 ± 0.83 and -24.8 ± 0.36, respectively. The PDI resulted from Lip-ICT and HA-Lip-ICT was 0.28 ± 0.02 and 0.26 ± 0.02, respectively. HA-Lip-ICT demonstrated higher in vitro drug release when pH was dropped from 7.4 to 5.5, The 12-h release rate of ICT from liposomes increased from 30% at pH7.4 to more than 60% at pH5.5. HA-Lip-ICT displayed higher toxicity than Lip-ICT in both HCC cells, especially Huh7with an IC50 of 34.15 ± 2.11 µM. The in vivo tissue distribution and anti-tumor experiments carried on tumor bearing nude mice indicated that HA-Lip- ICT exhibited higher tumor accumulation and achieved a tumor growth inhibition rate of 63.4%. Discussion: The nano-sized Lip-ICT was able to prolong the drug release time and showed long-term killing HCC cells ability. Following conjugation with HA, HA-Lip-ICT exhibited higher cytotoxicity, stronger tumor targeting, and tumor suppression abilities than Lip-ICT attributed to HA-CD44 ligand-receptor interaction, increasing the potential of ICT to treat HCC.