A novel α,ß-unsaturated ketone inhibits leukemia cell growth as PARP1 inhibitor.

Leukemia is a malignant disease of the hematopoietic system, in which clonal leukemia cells accumulate and inhibit normal hematopoiesis in the bone marrow and other hematopoietic tissues as a result of uncontrolled proliferation and impaired apoptosis, among other mechanisms. In this study, the anti-leukemic effect of a compound (SGP-17-S) extracted from Chloranthus multistachys, a plant with anti-inflammatory, antibacterial and anti-tumor effects, was evaluated. The effect of SGP-17-S on the viability of leukemic cell was demonstrated by MTT assay, cell cycle, and apoptosis were assessed by flow cytometry using PI staining and Annexin V/PI double staining. Combinations of network pharmacology and cellular thermal shift assay (CETSA) with western blot were used to validate agents that act on leukemia targets. The results showed that SGP-17-S inhibited the growth of leukemia cells in a time- and dose-dependent manner. SGP-17-S blocked HEL cells in the G2 phase, induced apoptosis, decreased Bcl-2 and caspase-8 protein expression, and increased Bax and caspase-3 expression. In addition, CETSA revealed that PARP1 is an important target gene for the inhibition of HEL cell growth, and SGP-17-S exerted its action on leukemia cells by targeting PARP1. Therefore, this study might provide new solutions and ideas for the treatment of leukemia.

Design and Development of Gold/reduced Graphene Oxide Nanocomposite loaded with Cyclophosphamide: Potential Application in Treatment of Breast Cancer.

OBJECTIVE: Cyclophosphamide (CP) is a widely used anti-cancer drug. It works by alkylation and is commonly used in cancer treatment. In this study, the goal was to create biodegradable drug delivery carriers with minimal side effects for breast cancer treatment by developing gold nanoparticles/reduced graphene oxide (Au-rGO) nanocomposites using a sustainable synthesis method and loading them with cyclophosphamide. METHODS: Cyclophosphamide-loaded gold/reduced graphene oxide nanocomposites (Au-rGOCP) were synthesized and evaluated using FT-IR, XRD, release pattern, and FE-SEM techniques. Furthermore, the anticancer effect against breast cancer cells was evaluated through MTT and Annexin V assays. CAT, SOD, and GPx biomarkers were used to assess the antioxidant effect of the free and nano-formulated cyclophosphamide. RESULTS: The characterization results showed the effective loading of cyclophosphamide in the nanocarriers. Additionally, Au-rGO had a higher drug loading capacity for cyclophosphamide during a 24-hour contact period (92.34%). The pH value affected the amount of cyclophosphamide released from the nanocarriers. Au-rGO/CP displayed significant in vitro anti-cancer activity against MCF-7 cancer cells relative to free CP and rGO/CP. According to Annexin V assay results, Au-rGO/CP induced a higher apoptosis rate in MCF-7 breast cancer cells than other forms. CONCLUSION: In conclusion, our findings demonstrate that the gold-decorated reduced graphene oxide nanocomposite enhances treatment efficacy and significantly increases apoptosis and cell death induction. As a result, CP-loaded Au-rGO-based compounds could be a promising treatment for breast cancer.

The Relevance, Predictability, and Utility of Annexin A5 for Human Physiopathology.

As an important functional protein molecule in the human body, human annexin A5 (hAnxA5) is widely found in human cells and body fluids. hAnxA5, the smallest type of annexin, performs a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calcium-dependent manner and plays an important role in many human physiological and pathological processes. The free state hAnxA5 exists in the form of monomers and usually forms a polymer in a specific self-assembly manner when exerting biological activity. This review systematically discusses the current knowledge and understanding of hAnxA5 from three perspectives: physiopathological relevance, diagnostic value, and therapeutic utility. hAnxA5 affects the occurrence and development of many physiopathological processes. Moreover, hAnxA5 can be used independently or in combination as a biomarker of physiopathological phenomena for the diagnosis of certain diseases. Importantly, based on the properties of hAnxA5, many novel drug candidates have been designed and prepared for application in actual medical practice. However, there are also some gaps and shortcomings in hAnxA5 research. This in-depth study will not only expand the understanding of structural and functional relationships but also promote the application of hAnxA5 in the field of biomedicine.

Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells.

OBJECTIVE: In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma (HEp-2) (ATCC® CCL-23™) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 µM were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse™ MitoPotential kit manufacturer instructions. RESULTS: MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 µM). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. CONCLUSIONS: Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.

[Effect of plasminogen activator urokinase receptor gene on the activation and apoptosis of neutrophil].

Objective: To investigate the effect of plasminogen activator urokinase receptor (PLAUR) gene on neutrophil activation and apoptosis in neutrophil-like cell model. Methods: Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid (ATRA). Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells (siRNA group). The phosphate buffer saline (PBS) group (untransfected neutrophil-like cells) and normal blank control group (NC group) (neutrophil-like cells transfected with blank plasmid) were used as controls (n=3). After starvation culture and addition of interleukin-17 afterwards in these 3 groups, the expression of CD11b on the cell membrane was detected by flow cytometry, and the levels of myeloperoxide (MPO) and extracellular neutrophil traps (NETs) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) to investigate the activation of neutrophil-like cells. The apoptosis was detected by flow cytometry with annexin V/propidium iodide (PI) double staining and the expressions of apoptosis-related proteins caspase-3, bax and bcl-2 were detected by Western blotting. Results: The expression of CD11b in siRNA group (32.37±8.17) was lower than that in PBS group (46.27±1.54) and NC group (53.07±8.14) (P<0.05) by flow cytometry. The levels of MPO and NETs (33.37±1.11, 57.69±3.03) in the supernatant of siRNA group were significantly lower than those in PBS group (41.64±2.20, 77.60±4.33) and NC group (40.84±5.11, 76.15±2.10) (P<0.05). Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group (20.42%±2.45%) was significantly higher than that in PBS group (11.91%±2.23%) and NC group (11.13%±2.56%) (P<0.05). The relative expression of caspase-3 protein and bax protein (0.84±0.05, 0.83±0.04) in siRNA group was significantly higher than that in PBS group (0.68±0.02, 0.63±0.08) and NC group (0.71±0.01, 0.66±0.10) (P<0.05), and the relative expression of anti-apoptosis protein bcl-2 decreased in siRNA group (0.38±0.02) than in PBS group (0.73±0.05) and NC group (0.69±0.06) (P<0.05). Conclusion: PLAUR promotes the activation of neutrophil-like cells and inhibits the apoptosis.

Multiparametric Analysis of Apoptosis by Flow Cytometry.

Flow cytometry remains the most widely used method for detecting and quantifying apoptosis and related forms of cell death in mammalian cells. The multiparametric nature of flow cytometry allows multiple apoptotic characteristics to be labeled and analyzed in a single sample, making it a powerful tool for analyzing the complex progression of apoptotic death. This chapter provides methods for combining assays for single apoptotic characteristics like caspase activation, annexin V binding, and cell membrane permeability into multiparametric assays that provide deeper insights into the cell death process. This approach to analyzing multiple apoptotic characteristics simultaneously yields far more information than single-parameter assays. While more informative than single-parameter assays, these multicolor methods can still be analyzed on relatively simple flow cytometers, making them widely accessible.

Fludioxonil induces cardiotoxicity via mitochondrial dysfunction and oxidative stress in two cardiomyocyte models.

Fludioxonil (Flu) is a phenylpyrrole fungicide and is currently used in over 900 agricultural products globally. Flu possesses endocrine-disrupting chemical-like properties and has been shown to mediate various physiological and pathological changes, such as apoptosis and differentiation, in diverse cell lines. However, the effects of Flu on cardiomyocytes have not been studied so far. The present study investigated the effects of Flu on mitochondria in AC16 human cardiomyocytes and H9c2 rat cardiomyoblasts. Flu decreased cell viability in a water-soluble tetrazolium assay and mediated morphological changes suggestive of apoptosis in AC16 and H9c2 cells. We confirmed that annexin V positive cells were increased by Flu through annexin V/propidium iodide staining. This suggests that the decrease in cell viability due to Flu may be associated with increased apoptotic changes. Flu consistently increased the expression of pro-apoptotic markers such as Bcl-2-associated X protein (Bax) and cleaved-caspase 3. Further, Flu reduced the oxygen consumption rate (OCR) in AC16 and H9c2 cells, which is associated with decreased mitochondrial membrane potential (MMP) as observed through JC-1 staining. In addition, Flu augmented the production of mitochondrial reactive oxygen species, which can trigger oxidative stress in cardiomyocytes. Taken together, these results indicate that Flu induces mitochondrial dysregulation in cardiomyocytes via the downregulation of the OCR and MMP and upregulation of the oxidative stress, consequently resulting in the apoptosis of cardiomyocytes. This study provides evidence of the risk of Flu toxicity on cardiomyocytes leading to the development of cardiovascular diseases and suggests that the use of Flu in agriculture should be done with caution and awareness of the probable health consequences of exposure to Flu.

Preconception anti-annexin A5 antibodies are associated with subsequent live birth in women with recurrent miscarriage: A retrospective study from China.

PROBLEM: To evaluate the correlation between the antiannexin A5 antibodies (aAnxA5) multiples of median (MOM) and subsequent pregnancy outcomes in women with recurrent miscarriage (RM). METHODS: Totally, 310 RM women were included in this study and grouped into tertiles according to their MOM of preconception aAnxA5 circulating levels determined by ELISA. The effect of aAnxA5 on the pregnancy outcomes was performed using multiple logistic regression. The outcomes included early miscarriage (before 10 weeks of gestation), late miscarriage (between 10 and 24 weeks), ongoing pregnancy (beyond 10 weeks), and live birth (after 24 weeks) characterized by pregnancy with fetal heartbeat. RESULTS: For each unit increase in aAnxA5 MOM, the odds of live birth after 24 weeks and ongoing pregnancy were reduced by 40.2% (OR = .598; 95%CI 0.406-0.882, P = .010) and 38.1% (OR = .619; 95%CI 0.424-0.904, P = .013), respectively, after adjusting for demographic and clinical characteristics. The rise in aAnxA5 MOM was associated with an increased risk of early miscarriage (OR = 1.616; 95%CI 1.106-2.361, P = .013) and miscarriage (early + late miscarriage) (OR = 1.671; 95%CI 1.134-2.464, P = .010). Further subgroup analyses showed a decreased risk of live birth rates after 24 weeks of gestation in the two subgroups: maternal age ≥35 years (OR = .131; 95%CI 0.026-0.652), and previous pregnancy loss ≥ 3 (OR = .381; 95%CI 0.173-0.837). CONCLUSIONS: Higher preconception aAnxA5 MOM levels in women with RM may be linked with a decreased risk of live birth after 24 weeks and an increased risk of early miscarriage, especially in individuals aged ≥35 years or with previous pregnancy losses ≥3.

The reduction of microglial efferocytosis is concomitant with depressive-like behavior in CUMS-treated mice.

BACKGROUND: Microglial efferocytosis plays a crucial role in facilitating and sustaining homeostasis in the central nervous system, and it is involved in neuropsychiatric disorders. How microglial efferocytosis is affected under the condition of major depressive disorder (MDD) remains elusive. In this study, we hypothesized that microglial efferocytosis in the hippocampus is impaired in the chronic unpredicted mild stress (CUMS) model of MDD, which is involved in the development of MDD. METHOD: Depressive-like behavior in adult male mice was induced by CUMS and confirmed by behavioral tests. Microglial efferocytosis was evaluated using immunofluorescence staining of hippocampal slices and primary microglia co-cultured with apoptotic cells. The protein and mRNA levels of phagocytosis-related molecules and inflammation-related cytokines were detected using western blotting and RT-qPCR, respectively. Annexin V was injected to mimic impairment of microglial efferocytosis. TREM2-siRNA was further used on primary microglia to examine efferocytosis-related signaling pathways. RESULTS: Microglia were activated and the expression of proinflammatory cytokines was increased in CUMS mice, while microglial efferocytosis and efferocytosis-related molecules were decreased. Inhibition of the TREM2/Rac1 pathway impaired microglial efferocytosis. Annexin V injection inhibited microglial efferocytosis, increased inflammation in the hippocampus and depressive-like behavior. LIMITATIONS: The potential antidepressant effect of the upregulation of the TREM2/Rac1 pathway was not evaluated. CONCLUSIONS: Impairment of microglial efferocytosis is involved in the development of depressive-like behavior, with downregulation of the TREM2/Rac1 pathway and increased inflammation. These results may increase our understanding of the pathophysiological mechanisms associated with MDD and provide novel targets for therapeutic interventions.

The investigation of cytotoxic and apoptotic activity of Cl-amidine on the human U-87 MG glioma cell line.

BACKGROUND: Peptidyl (protein) arginine deiminases (PADs) provide the transformation of peptidyl arginine to peptidyl citrulline in the presence of calcium with posttranslational modification. The dysregulated PAD activity plays an important role on too many diseases including also the cancer. In this study, it has been aimed to determine the potential cytotoxic and apoptotic activity of chlorine-amidine (Cl-amidine) which is a PAD inhibitor and whose effectiveness has been shown in vitro and in vivo studies recently on human glioblastoma cell line Uppsala 87 malignant glioma (U-87 MG) forming an in vitro model for the glioblastoma multiforme (GBM) which is the most aggressive and has the highest mortality among the brain tumors. METHODS: In the study, the antiproliferative and apoptotic effects of Cl-amidine on GBM cancer model were investigated. The antiproliferative effects of Cl-amidine on U-87 MG cells were determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate method at the 24th and 48th hours. The apoptotic effects were analyzed by Annexin V and Propidium iodide staining, caspase-3 activation, and mitochondrial membrane polarization (5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide) methods in the flow cytometry. RESULTS: It has been determined that Cl-amidine exhibits notable antiproliferative properties on U-87 MG cell line in a time and concentration-dependent manner, as determined through the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Assessment of apoptotic effects via Annexin V and Propidium iodide staining and 5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide methods has revealed significant efficacy, particularly following a 24-hour exposure period. It has been observed that Cl-amidine induces apoptosis in cells by enhancing mitochondrial depolarization, independently of caspase-3 activation. Furthermore, regarding its impact on healthy cells, it has been demonstrated that Cl-amidine shows lower cytotoxic effects when compared to carmustine, an important therapeutic agent for glioblastoma. CONCLUSION: The findings of this study have shown that Cl-amidine exhibits significant potential as an anticancer agent in the treatment of GBM. This conclusion is based on its noteworthy antiproliferative and apoptotic effects observed in U-87 MG cells, as well as its reduced cytotoxicity toward healthy cells in comparison to existing treatments. We propose that the antineoplastic properties of Cl-amidine should be further investigated through a broader spectrum of cancer cell types. Moreover, we believe that investigating the synergistic interactions of Cl-amidine with single or combination therapies holds promise for the discovery of novel anticancer agents.

Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase.

Ample evidence indicates that bufalin (BFN), a cardiotonic steroid in Bufo toad toxin, possesses a potent anticancer activity mainly by stimulating apoptosis in cancer cells. Human red blood cells (RBCs) undergo eryptosis which contributes to a plethora of pathological conditions. No reports, however, have examined the potential toxicity of BFN to RBCs. This study aims to characterize the biochemical mechanisms governing the influence of BFN on the physiology and lifespan of RBCs. Isolated RBCs from healthy volunteers were exposed to anticancer concentrations of commercially available BFN from the skin of Bufo gargarizans (10-200 µM) for 24 h at 37 °C. Photometric assays were used to estimate hemolysis and hemolytic markers, and flow cytometry was used to detect eryptotic markers. Phosphatidylserine externalization was captured by fluorescein isothiocyante-labeled annexin V, cellular dimensions by light scatter patterns, and intracellular Ca2+ and reactive oxygen species (ROS) by fluorogenic dyes Fluo4/AM and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. BFN caused Ca2+-independent hemolysis and release of LDH, AST, CK, and K+, and increased annexin V-bound cells, cytosolic Ca2+, cell shrinkage, and ROS levels. BFN also disrupted Na+ and Mg2+ trafficking, and was sensitive to PEG 8000, sucrose, SB203580, and NSC 23766. In whole blood, BFN depleted hemoglobin stores, increased fragmented RBCs, and was selectively toxic to reticulocytes, lymphocytes, and platelets. In conclusion, BFN elicits premature RBC death, subject to regulation by p38 MAPK and Rac1 GTPase, and is detrimental to other peripheral blood cells. Altogether, these novel findings prompt cautious consideration of the toxin in anticancer therapy.

CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.

BACKGROUND: The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. METHOD AND RESULTS: The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. CONCLUSION: The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

The macrophage-associated prognostic gene ANXA5 promotes immunotherapy resistance in gastric cancer through angiogenesis.

Gastric cancer (GC) remains a predominant form of malignant tumor globally, necessitating innovative non-surgical therapeutic approaches. This investigation aimed to delineate the expression landscape of macrophage-associated genes in GC and to evaluate their prognostic significance and influence on immunotherapeutic responsiveness. Utilizing the CellMarker2.0 database, we identified 69 immune cell markers with prognostic relevance in GC, including 12 macrophage-specific genes. A Weighted Gene Co-Expression Network Analysis (WGCNA) isolated 3,181 genes correlated with these macrophage markers. The Cancer Genome Atlas (TCGA-STAD) dataset was employed as the training set, while data from the GSE62254 served as the validation cohort. 13 genes were shortlisted through LASSO-Cox regression to formulate a prognostic model. Multivariable Cox regression substantiated that the calculated risk score serves as an imperative independent predictor of overall survival (OS). Distinct macrophage infiltration profiles, pathway associations, treatment susceptibilities, and drug sensitivities were observed between high- and low-risk groups. The preliminary validation of ANXA5 in predicting the survival rates of GC patients at 1 year, 3 years, and 5 years, as well as its expression levels were higher and role in promoting tumor angiogenesis in GC through immunohistochemistry and angiogenesis experiments. In summary, macrophage-related genes were potentially a novel crosstalk mechanism between macrophages and endothelial cells in the tumor microenvironment, and the interplay between inflammation and angiogenesis might have also offered new therapeutic targets, providing a new avenue for personalized treatment interventions.

Indigofera suffruticosa aerial parts extract induce G2/M arrest and ATR/CHK1 pathway in Jurkat cells.

BACKGROUND: Indigofera suffruticosa Mill. is used as a folk medicine for treating patients with leukemia, however very little is known regarding the molecular mechanism of its anti-leukemic activity and the chemical profile of the active extract. The present study aimed to reveal the molecular effect of I. suffruticosa aerial parts extract (ISAE) on leukemia cells and its chemical constituents. METHODS: Cytotoxicity of ISAE were determined by resazurin viability assay, multitox - Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach. RESULTS: We showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine. CONCLUSIONS: ISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.

Intratumor injected gold molecular clusters for NIR-II imaging and cancer therapy.

Surgical resections of solid tumors guided by visual inspection of tumor margins have been performed for over a century to treat cancer. Near-infrared (NIR) fluorescence labeling/imaging of tumor in the NIR-I (800 to 900 nm) range with systemically administrated fluorophore/tumor-targeting antibody conjugates have been introduced to improve tumor margin delineation, tumor removal accuracy, and patient survival. Here, we show Au25 molecular clusters functionalized with phosphorylcholine ligands (AuPC, ~2 nm in size) as a preclinical intratumorally injectable agent for NIR-II/SWIR (1,000 to 3,000 nm) fluorescence imaging-guided tumor resection. The AuPC clusters were found to be uniformly distributed in the 4T1 murine breast cancer tumor upon intratumor (i.t.) injection. The phosphocholine coating afforded highly stealth clusters, allowing a high percentage of AuPC to fill the tumor interstitial fluid space homogeneously. Intra-operative surgical navigation guided by imaging of the NIR-II fluorescence of AuPC allowed for complete and non-excessive tumor resection. The AuPC in tumors were also employed as a photothermal therapy (PTT) agent to uniformly heat up and eradicate tumors. Further, we performed in vivo NIR-IIb (1,500 to 1,700 nm) molecular imaging of the treated tumor using a quantum dot-Annexin V (QD-P3-Anx V) conjugate, revealing cancer cell apoptosis following PTT. The therapeutic functionalities of AuPC clusters combined with rapid renal excretion, high biocompatibility, and safety make them promising for clinical translation.

Real-Time Imaging of Single Retinal Cell Apoptosis in a Non-Human Primate Ocular Hypertension Model.

Purpose: To evaluate the feasibility of using DARC (detection of apoptosing retinal cells) technology as a biomarker for preclinical assessment of glaucomatous damage in a non-human primate (NHP) model of ocular hypertension (OHT). Methods: Elevated intraocular pressure (IOP) was induced by applying a laser to the trabecular meshwork in each eye of NHPs. Changes in DARC counts in the retina, identified as fluorescent-tagged annexin V (ANX776)-positive cells, were evaluated together with optic nerve damage, assessed using spectral domain-optical coherence tomography. The pharmacokinetic properties of ANX776 in both healthy and OHT model monkeys were also examined. Results: Sustained elevation of IOP and subsequent thinning of the retinal nerve fiber layer thickness (RNFLT) around the optic nerve head were confirmed in the OHT model. Increases in DARC counts were also detected after IOP elevation. We identified a statistically significant relationship between cumulative DARC counts and reductions in RNFLT both globally and in each peripapillary sector. Intravenous administration of ANX776 increased blood annexin V in a dose-dependent manner, which was subsequently eliminated. Conclusions: This study revealed that DARC technology can effectively assess glaucomatous damage in an NHP OHT model. We obtained the fundamental data that could serve as a reference for developing preclinical models to evaluate the pharmacodynamics of neuroprotective agents using DARC technology in NHP OHT models. Translational Relevance: Our basic data in a monkey OHT model could be useful for future preclinical studies using DARC technology to estimate the pharmacodynamic response of neuroprotective agents.

Unraveling Interaction of Rhenium-188 Microspheres with Primary Hepatic Cancer Cell: A Breakthrough Study.

Introduction: Hepatocellular carcinoma is a prevalent contributor to global mortality rates. The main palliative treatments are trans-arterial chemoembolization and selective intra-arterial radionuclide therapy. Methods: A novel freeze-dried nonradioactive microsphere kit formulation has been developed, and the behavior and therapeutic potential of 188Re microspheres have been assessed. The microspheres were labeled with fluorescein isothiocyanate (FITC) and 188ReO4-. The uptake of FITC microspheres by HepG2 cells was examined at various time intervals. The impact of 188Re microspheres on cell viability and the mode of cell death were investigated with HepG2 cells using MTT and Annexin FITC-V/propidium iodide (PI) apoptosis assay. Results: The labeling efficiency of microspheres was more than 99% with FITC and 188ReO4-. The maximum uptake of FITC microspheres by HepG2 cells was achieved at 6 h. The exposure to 188Re microspheres has shown a decrease in cellular viability from 77.81% ± 0.015% to 42.03% ± 0.148% at 192 h of incubation (∼11 half-lives). The cellular uptake of 188Re microspheres was 0.255-0.901 MBq. These values were concordant with Annexin FITC-V/PI apoptosis assay. At 192 h, 53.28% ± 0.01% of cells entered the apoptotic phase after treatment with 188Re microspheres, and only 39.34% ± 0.02% of cells remained viable. However, in the cells treated with 188ReO4- alone, 74.86% ± 0.005% of cells were viable, and only 24.75% ± 0.577% of cells were in the early apoptotic phase at 192 h. Conclusion: The data revealed that 188Re microspheres treatment led to significant growth inhibition in HepG2 cells compared with 188ReO4-.

Infra-patellar fat pad-derived mesenchymal stem cells maintain their chondrogenic differentiation potential after arthroscopic harvest with blood-product supplementation.

PURPOSE: Mesenchymal stem cells/medicinal signaling cells (MSCs) possess therapeutic potential and are used in regenerative orthopaedics. The infra-patellar fat pad (IFP) is partially resected during knee arthroscopy (KASC) and contains MSCs. Heat, irrigation, and mechanical stress during KASC may decrease MSC's therapeutic potential. This study assessed MSCs' regenerative potential after arthroscopic IFP harvest and potential effects of two blood products (BP) (platelet-rich plasma (PRP), hyperacute serum (HAS)) on MSCs' viability and chondrogenic differentiation capacity. METHODS: IFP was arthroscopically harvested, isolated, and counted (n = 5). Flow cytometry was used to assess cell viability via staining with annexin V/7-AAD and stemness markers via staining for CD90, CD73, and CD105. MSCs were incubated with blood products, and metabolic activity was determined via an XTT assay. Deposition of cartilage extracellular matrix was determined in histologic sections of chondrogenically differentiated 3D pellet cultures via staining with Alcian Blue. Expression of cartilage-specific genes (SOX9, MMP3/13, ACAN, COL1/2) was analyzed via quantitative PCR. RESULTS: MSC isolation from IFP yielded 2.66*106 ± 1.49*106 viable cells from 2.7 (0.748) g of tissue. MSC markers (CD 90/105/73) were successfully detected and annexin V staining showed 81.5% viable cells. XTT showed increased metabolic activity. Within the BP groups, this increase was significant (days 0-14, p < 0.05). PCR showed expression of cartilage-specific genes in each group. COL2 (p < 0.01) as well as ACAN (p < 0.001) expression levels were significantly higher in the HAS group. Histology showed successful differentiation. CONCLUSION: Arthroscopic harvest of IFP-MSCs yields sufficient cells with maintained regenerative potential and viability. Blood products further enhance MSCs' viability.

Identification of a Novel CD8+ T cell exhaustion-related gene signature for predicting survival in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major health concern, necessitating a deeper understanding of its prognosis and underlying mechanisms. This study aimed to investigate the mechanism and prognostic value of CD8+ T Cell exhaustion (CD8+ TEX)-related genes in HCC and construct a survival prognosis prediction model for patients with HCC. METHODS: CD8+ TEX-related genes associated with HCC prognosis were analysed and identified, and a prognostic prediction model was constructed using the 'least absolute shrinkage and selection operator' Cox regression model. Immunohistochemistry was used to verify the expression of the model genes in HCC tissues. A nomogram was constructed based on risk scores and clinical features, and its predictive efficacy was verified. The expression of STAM, ANXA5, and MAD2L2 in HCC cell lines was detected by western blotting; subsequently, these genes were knocked down in HCC cell lines by small interfering RNA, and their effects on the proliferation and migration of HCC cell lines were detected by colony formation assay, cck8, wound healing, and transwell assays. RESULTS: Six genes related to CD8+ TEX were included in the risk-prediction model. The prognosis of patients with HCC in the low-risk group was significantly better than that of those in the high-risk group. Cox regression analysis revealed that the risk score was an independent risk factor for the prognosis of patients with HCC. The differentially expressed genes in patients with high-risk HCC were mainly enriched in the nucleotide-binding oligomerization domain-containing protein-like receptor, hypoxia-inducible factor-1, and tumour programmed cell death protein (PD)-1/PD-L1 immune checkpoint pathways. The CD8+ TEX-related genes STAM, ANXA5, and MAD2L2 were knocked down in HCC cell lines to significantly inhibit cell proliferation and migration. The prediction results of the nomogram based on the risk score showed a good fit and application value. CONCLUSION: The prediction model based on CD8+ TEX-related genes can predict the prognosis of HCC and provide a theoretical basis for the early identification of patients with poor HCC prognosis.

Enhanced CD1d phosphatidylserine presentation using a single-domain antibody promotes immunomodulatory CD1d-TIM-3 interactions.

BACKGROUND: CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. METHODS: To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. RESULTS: Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. CONCLUSION: Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.