U937 Cells Research Articles

Investigation of Anticancer Properties of Cinnamon Phytochemicals on Protein expression in Glioblastoma Multiforme Cell Lines (U87-MG)

Natural products are appealing as anticancer agents because of their low toxicity profiles. Cinnamon is a naturally occurring component with a variety of pharmacological properties, including anti-oxidant, anti-microbial, and anti-cancer properties for cancer cell apoptosis. Therefore, the current studies were performed to investigate Anticancer Properties of Cinnamon Phytochemicals on Protein expression in Glioblastoma Multiforme Cell Lines (U87-MG) since the proteins are directly involved in cell survival and function.Hot and cold extraction for Cinnamon performed from Cinnamon verum followed by phytochemicals tests. The Glioblastoma Multiforme Cell Lines U87 was subjected to cell viability by MTT Assay with Addition of cinnamon extract. The protein content was determined by spectrometric assay and its expression was investigated by SDS PAGE.The aqueous Cinnamon extract found to be positive for Alkaloids, Tannins, Saponins, Flavonoids, Terpenoids (only for Hot extract), Phenols and Reducing sugars (only for Cold extract). The cell viability at cinnamon extract concentration of 0.051 mg/100μl is shown to be highest and cell death at maximum cinnamon extract concentration of 1.65 mg/100μl is highest. Further these phytochemicals together showed less protein content on cultured U-87 cells and displayed lower protein expression in gel band pattern when compared to their control. The Cinnamon extract phytochemicals showed significant anticancer properties for reduced U87 cell viability with IC50 0.729. Thus positive phytochemicals result has shown to play an important role in inhibiting cancer properties.by showing less protein content and its expression for cinnamon phytochemicals treated U87 cell lines.

BPTF promotes glioma development through USP34-mediated de-ubiquitination of FOXC1.

Glioma is the most prevalent malignant tumor of the brain, and the study of the molecular mechanisms associated with its development has important clinical significance. Our previous study found that BPTF promotes the malignant phenotype of glioma and is significantly associated with poor prognosis; the downstream regulatory mechanisms are explored in this study. Western blot and immunohistochemical staining were used to detect protein expression in cells or tissues. BPTF knockdown as well as FOXC1-overexpressing lentiviruses were used in combination for the construction of the U251 cell model, leading to functional rescue experiments. CCK8 assay, flow cytometry, scratch assay, and Transwell assay were used to detect cell proliferation, apoptosis, and migration, respectively. Finally, immunoprecipitation assays, combined with western blot (WB), were used to detect the interaction between proteins as well as the level of ubiquitination modification. The obtained results suggested that BPTF knockdown may inhibit the malignant behavior of glioma cells by downregulating FOXC1 expression. Moreover, FOXC1 expression was significantly higher in glioma tissues than in normal brain tissues and was significantly associated with higher tumor stage and worse patient prognosis. Finally, the mechanism of FOXC1 regulation by BPTF was found to result from the affected protein stability of FOXC1 through USP34-mediated de-ubiquitylation. In conclusion, the BPTF/FOXC1 axis was identified as a key promotor in glioma development and may be a potential target in the inhibition of glioma development.

Effects of polyphyllin Ⅶ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

Effect of Regulation of Chemerin/Chemokine-like Receptor 1/Stimulator of Interferon Genes Pathway on Astrocyte Recruitment to Aβ Plaques.

Recruitment and accumulation of reactive astrocytes around senile plaques are common pathological features of Alzheimer's disease (AD), with unclear mechanisms. Chemerin, an adipokine implicated in neuroinflammation, acts through its receptor, chemokine-like receptor 1 (CMKLR1), which also functions as a receptor for amyloid β (Aβ). The impact of the chemerin/CMKLR1 axis on astrocyte migration towards Aβ plaques is unknown. Here we investigated the effect of CMKLR1 on astrocyte migration around Aβ deposition in APP/PS1 mice with Cmklr1 knockout (APP/PS1-Cmklr1-/-). CMKLR1-expressed astrocytes were upregulated in the cortices and hippocampi of 9-month-old APP/PS1 mice. Chemerin mainly co-localized with neurons, and its expression was reduced in the brains of APP/PS1 mice, compared to WT mice. CMKLR1 deficiency decreased astrocyte colocalization with Aβ plaques in APP/PS1-Cmklr1-/- mice, compared to APP/PS1 mice. Activation of the chemerin/CMKLR1 axis promoted the migration of primary cultured astrocytes and U251 cells, and reduced astrocyte clustering induced by Aβ42. Mechanistic studies revealed that chemerin/CMKLR1 activation induced STING phosphorylation. Deletion of STING attenuated the promotion of the chemerin/CMKLR1 axis relative to astrocyte migration and abolished the inhibitory effect of chemerin on Aβ42-induced astrocyte clustering. These findings suggest the involvement of the chemerin/CMKLR1/STING pathway in the regulation of astrocyte migration and recruitment to Aβ plaques/Aβ42.

Fluorine-18 labeling PEGylated 6-boronotryptophan for PET scanning of mice for assessing the pharmacokinetics for boron neutron capture therapy of brain tumors

Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15–50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.

Exploring the role of GHRH antagonist MIA-602 in overcoming Doxorubicin-resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the rapid proliferation of mutagenic hematopoietic progenitors in the bone marrow. Conventional therapies include chemotherapy and bone marrow stem cell transplantation; however, they are often associated with poor prognosis. Notably, growth hormone-releasing hormone (GHRH) receptor antagonist MIA-602 has been shown to impede the growth of various human cancer cell lines, including AML. This investigation examined the impact of MIA-602 as monotherapy and in combination with Doxorubicin on three Doxorubicin-resistant AML cell lines, KG-1A, U-937, and K-562. The in vitro results revealed a significant reduction in cell viability for all treated wild-type cells. Doxorubicin-resistant clones were similarly susceptible to MIA-602 as the wild-type counterpart. Our in vivo experiment of xenografted nude mice with Doxorubicin-resistant K-562 revealed a reduction in tumor volume with MIA-602 treatment compared to control. Our study demonstrates that these three AML cell lines, and their Doxorubicin-resistant clones, are susceptible to GHRH antagonist MIA-602.

Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model.

Glioblastoma multiforme (GBM) is a malignant tumor with a higher prevalence in men and a higher survival rate in transmenopausal women. It exhibits distinct areas influenced by changing environmental conditions. This study examines how these areas differ in the levels of estrogen receptors (ERs) which play an important role in the development and progression of many cancers, and whose expression levels are often correlated with patient survival. This study utilized two research models: an in vitro model employing the U87 cell line and a second model involving tumors resected from patients (including tumor core, enhancing tumor region, and peritumoral area). ER expression was assessed at both gene and protein levels, with the results validated using confocal microscopy and immunohistochemistry. Under hypoxic conditions, the U87 line displayed a decrease in ERβ mRNA expression and an increase in ERα mRNA expression. In patient samples, ERβ mRNA expression was lower in the tumor core compared to the enhancing tumor region (only in males when the study group was divided by sex). In addition, ERβ protein expression was lower in the tumor core than in the peritumoral area (only in women when the study group was divided by sex). Immunohistochemical analysis indicated the highest ERβ protein expression in the enhancing tumor area, followed by the peritumoral area, and the lowest in the tumor core. The findings suggest that ER expression may significantly influence the development of GBM, exhibiting variability under the influence of conditions present in different tumor areas.

PTPN7 mediates macrophage-polarization and determines immunotherapy in gliomas: A single-cell sequencing analysis.

Protein tyrosine phosphatase non-receptor type 7 (PTPN7) is a signaling molecule that regulates a multitude of cellular processes, spanning cell proliferation, cellular differentiation, the mitotic cycle, and oncogenic metamorphosis. However, the characteristic of PTPN7 in the glioma microenvironment has yet to be elucidated. The prognostic value, genomic features, immune characteristics, chemotherapy prediction, and immunotherapy prediction of PTPN7 were systematically explored at the bulk sequencing level. The cell evolution trajectory, cell communication pattern, and cell metabolic activity related to PTPN7 were systematically explored at the single-cell sequencing level. HMC3 and M0 cells were cocultured with U251 and T98G cells, and flow cytometry was carried out to investigate the polarization of HMC3 and M0. Transwell assay and CCK-8 assay were performed to explore the migration and proliferation activity of U251 and T98G. The expression level of PTPN7 is significantly elevated in glioma and indicates malignant features. PTPN7 expression predicts worse prognosis of glioma patients. PTPN7 is associated with genome alteration and immune infiltration. Besides, PTPN7 plays a crucial role in modulating metabolic and immunogenic processes, particularly by influencing the activity of microglia and macrophages through multiple signaling pathways involved in cellular communication. Specifically, PTPN7 actively mediates inflammation-resolving-polarization of macrophages and microglia and protects glioma from immune attack. PTPN7 could also predict the response of immunotherapy. PTPN7 is critically involved in inflammation-resolving-polarization mediated by macrophage and microglia and promotes the immune escape of glioma cells.

Urease and human glioblastoma (U87) cells growth inhibitory iridoid glycoside acetates from Nyctanthes arbor-tristis Linn.

The present study was undertaken on the chemical constituents of ethanol extract of aerial parts of Nyctanthes arbor-tristis Linn., and their determination of growth inhibitory activity against glioblastoma multiforme cell line (U87) and urease inhibitory activity. Six constituents were isolated including two new arbortristoside F tetraacetate (1) and arbortristoside G heptaacetate (2) and four known arborside A tetraacetate (3), arborside C pentaacetate (4), 6,7-di-O-acetyl-6β-hydroxyloganin hexaacetate (5) and nyctanthoside heptaacetate (6) iridoid glycoside acetates. Their structures were elucidated using spectroscopic methods, including 1D and 2D NMR and mass analyses. Compounds 3 and 6 showed significant growth inhibition of U87 cell line (76.41 and 87.62% inhibition) respectively while 2, 4 and 5 showed moderate inhibition. 3 and 6 showed notable urease inhibition (IC50 = 17.2 ± 0.4 and 17.2 ± 0.7 µM) respectively, and IC50 of 2, 4 and 5 were 23.8 − 56.3 µM. Compound 1 was inactive.

Abstract LB444: Identification of a novel and selective Transglutaminase 2 (TGM2) inhibitors modulate tumor microenvironment in Glioblastoma

Abstract Background: TGM (Transglutaminase) family enzymes are Ca2+ dependent and engaged in specific posttranslational modifications by cross-linking extracellular matrix (ECM) proteins. ECM protein complex is more stable and resistant to enzymatic degradation. These enzymes modify proteins by cross-linking epsilon-(gamma-glutamyl), lysine, or (gamma-glutamyl) polyamine bonds. TGM2 (Transglutaminase 2) is a ubiquitous enzyme, and its expression is identified in the cytoplasm, plasma membrane, and the nucleus of various cells. The membrane-bound form of TGM2 binds GTP and functions as a G protein. TGM2 regulates cell survival, proliferation, migration, and modulation through ECM organization. We explored the role of TGM2 in Glioblastoma cells and its role in modulating the tumor microenvironment, cell proliferation, and survival. In addition to TGM2's role in GBM tumors, its expression has been linked to resistance to certain therapies in glioblastoma was investigated. Targeted modulation of macrophages with TGM2 inhibitors impacts the immune response against GBM, potentially enhancing the efficacy. Understanding the intricate interplay between TGM2, and GBM-associated macrophages is a key component of the tumor microenvironment (TME) and crucial for the development of targeted TGM2 inhibitors. Materials and Methods: Human Transglutaminase 2 (hTGM2) activity of inhibitors was assayed using the fluorescent transamination assay incorporating dansylcadaverine into glutamine-donor substrate N, N-dimethyl casein, and DCC. Cellular efficacies of selected TGM2 inhibitors evaluated in U138 and U87 glioblastoma cells in CellTiter-Glo Luminescent cell viability assay. Results: We have identified a series of novel small molecule TGM2 inhibitors based on our initial lead fragment hits with an IC50s 6-29 uM from our in-house MolecuLern fragment library. Fragments2Lead (F2L) optimizations strategies, synthesis, and SAR studies provided a lead SLX-9029 and SLX-9031 inhibitors with IC50s of 1.2, 1.5 uM in inhibiting TGM2 activity in our established cell-free fluorescent transamination assay. The initial TMG2 inhibitor leads further screening for TGM2-expressed U138 and U87 glioblastoma cells demonstrating promising cellular efficacies as singles agents. Additional GBM cell profiling, combination studies along with safety secondary pharmacology, cellular toxicities, and PK results will be discussed. Conclusion: In summary, we identified promising lead TGM2 inhibitors that show activity in both cell-free and cell-based TGM2-expressed GMB cells. The TGM family members, selectivity, additional GBM cellular profiling, ADME, cellular toxicity, and in vivo PK studies are underway, and these results will be presented. Citation Format: Hariprasad Vankayalapati, Chenyu Lin, Zhaoang li, Kyle Medley, David J. Bearss. Identification of a novel and selective Transglutaminase 2 (TGM2) inhibitors modulate tumor microenvironment in Glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB444.

Cannabidiol induces ERK activation and ROS production to promote autophagy and ferroptosis in glioblastoma cells

Small molecule-driven ERK activation is known to induce autophagy and ferroptosis in cancer cells. Herein the effect of cannabidiol (CBD), a phytochemical derived from Cannabis sativa, on ERK-driven autophagy and ferroptosis has been demonstrated in glioblastoma (GBM) cells (U87 and U373 cells). CBD imparted significant cytotoxicity in GBM cells, induced activation of ERK (not JNK and p38), and increased intracellular reactive oxygen species (ROS) levels. It increased the autophagy-related proteins such as LC3 II, Atg7, and Beclin-1 and modulated the expression of ferroptosis-related proteins such as glutathione peroxidase 4 (GPX4), SLC7A11, and TFRC. CBD significantly elevated the endoplasmic reticulum stress, ROS, and iron load, and decreased GSH levels. Inhibitors of autophagy (3-MA) and ferroptosis (Fer-1) had a marginal effect on CBD-induced autophagy/ferroptosis. Treatment with N-acetyl-cysteine (antioxidant) or PD98059 (ERK inhibitor) partly reverted the CBD-induced autophagy/ferroptosis by decreasing the activation of ERK and the production of ROS. Overall, CBD induced autophagy and ferroptosis through the activation of ERK and generation of ROS in GBM cells.

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

Has-miR-134-5p inhibits the proliferation and migration of glioma cells by regulating the BDNF/ERK signaling pathway.

Our research investigated the effects of hsa-miR-134-5p on glioma progression, focusing on its interaction with the BDNF/ERK signaling pathway. U251 and U87 cell lines were analyzed post-transfection with hsa-miR-134-5p mimics and inhibitors, confirming the miRNA's binding to BDNF using dual luciferase assays. Q-PCR was employed to measure expression changes, revealing that hsa-miR-134-5p markedly inhibited glioma cell proliferation, migration, and invasion, as evidenced by CCK8, monoclonal formation, and Transwell assays. Scratch tests and Western blotting demonstrated hsa-miR-134-5p's modulation of the BDNF/ERK pathway and associated decrease in MMP2/9 protein levels. Flow cytometry suggested that hsa-miR-134-5p might also block the G0/S phase transition. In vivo studies using nude mice corroborated the tumor-suppressing effects of hsa-miR-134-5p, which were negated by elevated BDNF levels. Comparative protein analysis across groups confirmed the pathway's significance in tumorigenesis. Our findings identify hsa-miR-134-5p as a key molecule impeding glioma cell growth by curtailing the BDNF/ERK pathway, with the reversal by BDNF upregulation pointing to the potential of therapeutically exploiting the hsa-miR-134-5p/BDNF axis in glioma care.

511 Iron Oxide Nanoplatform HMC-FMX for Targeted Chemotherapeutic Treatment of Glioblastoma

INTRODUCTION: Glioblastoma multiforme (GBM), is the most common and aggressive form of primary brain tumors in adults with a high mortality rate. Despite the emergence of potent chemotherapies, clinical efficacy has been limited in part due to the blood-brain barrier (BBB). Therefore, recurrence rate is high, limiting life expectancy. Our nanoplatform is based on ferumoxytol (FMX), an FDA-approved MRI-sensitive supramagnetic iron oxide nanoparticle conjugated to heptamethine cyanine (HMC), a near infrared-flourescent ligand that specifically targets organic anion transporter polypeptides (OATP) expressed in GBM and endothelial cells. METHODS: To show the specificity of OATP-targeting by HMC-FMX nanoplatform, cells were pre-incubated with OATP inhibitors and then incubated with HMC-FMX. Fluorescent intensity and HMC uptake were assesed by flow cytometry. Cytotoxicity of the drug-loaded nanoparticles, HMC-FMX(SN38) and HMC-FMX(CPT) compared to SN38 and Camptothecin (CPT) in U87 cells was assessed by flow cytometry following cell staining with Annexin and 7-AAD. RESULTS: The HMC-FMX nanoplatform is successfully taken up by both U87 and endothelial cells. OATP inhibition significantly decreased HMC uptake (10%-30%) in comparison to control in U87 and endothelial cells. In addition, HMC-FMX(SN38) and HMC-FMX(CPT) exerted increased cytotoxic effect on U87 cells compared with SN38 and CPT alone. Furthermore, majority of apoptotic cells were observed in the HMC-positive cells in contrast to HMC-negative cells. CONCLUSIONS: HMC-FMX can encapsulate chemotherapeutic drugs, such as SN38 and CPT. HMC-FMX as a nanoplatform demonstrates specific, safe and efficient drug delivery through the BBB into GBM cells in vitro.

Mirna-383-5p Functions as an Anti-oncogene in Glioma through the Akt/mTOR Signaling Pathway by Targeting VEGFA.

Previously, we have screened 59 differentially expressed miRNAs and 419 mRNAs in the glioblastoma samples that have been compared to the peritumoral tissues using bioinformatics analyses, which included miRNA-383-5p and vascular endothelial growth factor A (VEGFA). miRNA-383-5p and VEGFA/Akt/mTOR pathway play important regulatory roles in the malignant biological behavior of glioma. Glioma cell lines, U87 and U251, were collected for in vitro experiments. miRNA-383-5p and VEGFA expression levels were detected with qRT-PCR and WB. The protein expressions of Akt, mTOR, and VEGFR in U87 and U251 were detected with WB. The effect of miRNA-383-5p on the VEGFA activity was verified by dual-luciferase reporter assay. CCK-8 was used to examine the U87 and U251 cells' inhibition. Flow cytometry and transwell assays were used to detect cell apoptosis and invasion, respectively. Our research data indicated overexpression of miRNA-383-5p to suppress malignant biological behavior, which was manifested as promoting the apoptosis of U87 and U251 cells and inhibiting invasion, proliferation, and metastasis. VEGFA is one of the downstream target genes of miRNA-383- 5p. miRNA-383-5p could inhibit the expression of VEGFA and Akt/mTOR signaling pathways. Overexpression of VEGFA can reverse the inhibitory effect of miRNA-383-5p and reactivate the Akt/mTOR signaling pathway. Our results indicate that miRNA-383-5p functions as an anti-oncogene by inhibiting the VEGFA/Akt/mTOR signaling pathway in glioma cells. These data provide potential therapeutic targets for glioblastoma.

503 Engineering GBM-specific T Cells Using Photothermal Prussian Blue Nanoparticles

INTRODUCTION: Adoptive T cell therapy (ATCT) has successfully treated hematological malignancies and is currently under investigation for solid tumor therapy. In contrast to existing chimeric antigen receptor (CAR) T cell and/or antigen-specific T cell approaches, which target defined antigens, our PBNP-based approach can target a broad repertoire of antigens in GBM in an antigen-agnostic manner. METHODS: We treated GBM cells with PBNP-based photothermal therapy (PBNP-PTT) prior to culturing with dendritic cells (DCs), and subsequent stimulation of T cells. RESULTS: When PBNP-PTT was administered to U87 cells at an immunogenic thermal dose, we effectively expanded U87-specific T cells. DCs cultured ex vivo with PBNP-PTT-treated U87 cells enabled 15- 30-fold expansion of CD4+ and CD8+ T cells. Upon co-culture with target U87 cells, these T cells secreted IFN-G in a specific and dose-dependent manner (up to 647-fold over controls). T cells manufactured using PBNP-PTT elicited specific cytolytic activity against target U87 cells (33-93% killing at an E:T ratio of 20:1), while sparing normal human astrocytes. Similarly, PBNP-PTT on SNB19 cells resulted in a 7-39-fold expansion of T cells, which elicited 25-66% killing of SNB19 cells at an E:T ratio of 20:1. U87-luc cells were then orthotopically inoculated into NSG mice to establish xenograft GBM, and U87-specific T cells developed via PBNP-PTT were intracranially injected. These T cells eliminated U87 tumors in vivo and significantly improved mouse survival compared to mice treated with PBS (p = 0.0047) or non-specific T cells (p = 0.0123) from the same donor. CONCLUSIONS: These findings provide proof-of-concept data supporting the use of PBNP-PTT to stimulate and expand tumor-specific T cells ex vivo for potential use as an ATCT approach for patients with GBM. We are currently using patient-derived tumor samples to generate IND-enabling data.

Piperine, quercetin, and curcumin identified as promising natural products for topical treatment of cutaneous leishmaniasis

Leishmania braziliensis (L. braziliensis) causes cutaneous leishmaniasis (CL) in the New World. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administer. Topical treatment would be the ideal option for the treatment of CL. This underscores the urgent need for affordable and effective treatments, with natural compounds being explored as potential solutions. The alkaloid piperine (PIP), the polyphenol curcumin (CUR), and the flavonoid quercetin (QUE), known for their diverse biological properties, are promising candidates to address these parasitic diseases. Initially, the in vitro cytotoxicity activity of the compounds was evaluated using U-937 cells, followed by the assessment of the leishmanicidal activity of these compounds against amastigotes of L. braziliensis. Subsequently, a golden hamster model with stationary-phase L. braziliensis promastigote infections was employed. Once the ulcer appeared, hamsters were treated with QUE, PIP, or CUR formulations and compared to the control group treated with meglumine antimoniate administered intralesionally. We observed that the three organic compounds showed high in vitro leishmanicidal activity with effective concentrations of less than 50 mM, with PIP having the highest activity at a concentration of 8 mM. None of the compounds showed cytotoxic activity for U937 macrophages with values between 500 and 700 mM. In vivo, topical treatment with QUE daily for 15 days produced cured in 100% of hamsters while the effectiveness of CUR and PIP was 83% and 67%, respectively. No failures were observed with QUE. Collectively, our data suggest that topical formulations mainly for QUE but also for CUR and PIP could be a promising topical treatment for CL. Not only the ease of obtaining or synthesizing the organic compounds evaluated in this work but also their commercial availability eliminates one of the most important barriers or bottlenecks in drug development, thus facilitating the roadmap for the development of a topical drug for the management of CL caused by L. braziliensis.

Folic Acid Functionalized AQ4N/Gd@PDA Nanoplatform with Real-Time Monitoring of Hypoxia Relief and Enhanced Synergistic Chemo/Photothermal Therapy in Glioma.

Hypoxia is often associated with glioma chemoresistance, and alleviating hypoxia is also crucial for improving treatment efficacy. However, although there are already some methods that can improve efficacy by alleviating hypoxia, real-time monitoring that can truly achieve hypoxia relief and efficacy feedback still needs to be explored. AQ4N/Gd@PDA-FA nanoparticles (AGPF NPs) were synthesized using a one-pot method and were characterized. The effects of AGPF NPs on cell viability, cellular uptake, and apoptosis were investigated using the U87 cell line. Moreover, the effectiveness of AGPF NPs in alleviating hypoxia was explored in tumor-bearing mice through photoacoustic imaging. In addition, the diagnosis and treatment effect of AGPF NPs were evaluated by magnetic resonance imaging (MRI) and bioluminescent imaging (BLI) on orthotopic glioma mice respectively. In vitro experiments showed that AGPF NPs had good dispersion, stability, and controlled release. AGPF NPs were internalized by cells through endocytosis, and could significantly reduce the survival rate of U87 cells and increase apoptosis under irradiation. In addition, we monitored blood oxygen saturation at the tumor site in real-time through photoacoustic imaging (PAI), and the results showed that synergistic mild-photothermal therapy/chemotherapy effectively alleviated tumor hypoxia. Finally, in vivo anti-tumor experiments have shown that synergistic therapy can effectively alleviate hypoxia and inhibit the growth of orthotopic gliomas. This work not only provides an effective means for real-time monitoring of the dynamic feedback between tumor hypoxia relief and therapeutic efficacy, but also offers a potential approach for the clinical treatment of gliomas.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.