Cell Line U937 Research Articles

Electrophysiological Impact of SARS-CoV-2 Envelope Protein in U251 Human Glioblastoma Cells: Possible Implications in Gliomagenesis?

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, the acute respiratory disease which, so far, has led to over 7 million deaths. There are several symptoms associated with SARS-CoV-2 infections which include neurological and psychiatric disorders, at least in the case of pre-Omicron variants. SARS-CoV-2 infection can also promote the onset of glioblastoma in patients without prior malignancies. In this study, we focused on the Envelope protein codified by the virus genome, which acts as viroporin and that is reported to be central for virus propagation. In particular, we characterized the electrophysiological profile of E-protein transfected U251 and HEK293 cells through the patch-clamp technique and FURA-2 measurements. Specifically, we observed an increase in the voltage-dependent (Kv) and calcium-dependent (KCa) potassium currents in HEK293 and U251 cell lines, respectively. Interestingly, in both cellular models, we observed a depolarization of the mitochondrial membrane potential in accordance with an alteration of U251 cell growth. We, therefore, investigated the transcriptional effect of E protein on the signaling pathways and found several gene alterations associated with apoptosis, cytokines and WNT pathways. The electrophysiological and transcriptional changes observed after E protein expression could explain the impact of SARS-CoV-2 infection on gliomagenesis.

The combination therapy using tyrosine kinase receptors inhibitors and repurposed drugs to target patient-derived glioblastoma stem cells

The lesson from many studies investigating the efficacy of targeted therapy in glioblastoma (GBM) showed that a future perspective should be focused on combining multiple target treatments. Our research aimed to assess the efficacy of drug combinations against glioblastoma stem cells (GSCs). Patient-derived cells U3042, U3009, and U3039 were obtained from the Human Glioblastoma Cell Culture resource. Additionally, the study was conducted on a GBM commercial U251 cell line. Gene expression analysis related to receptor tyrosine kinases (RTKs), stem cell markers and genes associated with significant molecular targets was performed, and selected proteins encoded by these genes were assessed using the immunofluorescence and flow cytometry methods. The cytotoxicity studies were preceded by analyzing the expression of specific proteins that serve as targets for selected drugs. The cytotoxicity study using the MTS assay was conducted to evaluate the effects of selected drugs/candidates in monotherapy and combinations. The most cytotoxic compounds for U3042 cells were Disulfiram combined with Copper gluconate (DSF/Cu), Dacomitinib, and Foretinib with IC50 values of 52.37 nM, 4.38 µM, and 4.54 µM after 24 h incubation, respectively. Interactions were assessed using SynergyFinder Plus software. The analysis enabled the identification of the most effective drug combinations against patient-derived GSCs. Our findings indicate that the most promising drug combinations are Dacomitinib and Foretinib, Dacomitinib and DSF/Cu, and Foretinib and AZD3759. Since most tested combinations have not been previously examined against glioblastoma stem-like cells, these results can shed new light on designing the therapeutic approach to target the GSC population.

HNRNPA2B1 stabilizes NFATC3 levels to potentiate its combined actions with FOSL1 to mediate vasculogenic mimicry in GBM cells

BackgroundVasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM.AimsThe aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM.MethodsWe have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein–protein interactions, ChIP was used to detect DNA–protein complexes, and RIP was used to detect RNA–protein complexes. Histochemical staining was used to detect VM tube formation in vivo.ResultsFocusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo.ConclusionTaken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.Graphical 1. NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression which leads to an increase in VM of GBM.2. FOSL1 interacts with NFATC3 to further facilitate VEGFR2 gene expression and VM.3. HNRNPA2B1 enhances NFATC3 mRNA stability to increase VEGFR2 expression and VM.

TRIP13 Plays an Important Role in the Sensitivity of Leukemia Cell Response to Sulforaphane Therapy.

Sulforaphane is one of the most characterized isothiocyanate compounds in cruciferous vegetables and shows anticancer effects, especially antileukemia properties. However, the molecular mechanism of the growth inhibition effect of sulforaphane in acute myeloid leukemia (AML) has not been fully explored. In the present study, a proteomic analysis was performed on the AML cell line U937 responding to sulforaphane treatment to identify novel and efficient therapeutic targets of sulforaphane on AML cells. Key driver analysis was run on the leukemia network, and TRIP13 was identified as a key regulatory factor in sulforaphane-induced growth inhibition in U937 cells. Pretreatment with DCZ0415, an inhibitor of TRIP13, could significantly attenuate sulforaphane-induced cell apoptosis and cell cycle arrest in vitro through the PI3K/Akt/mTOR signaling pathway. In addition, the inhibitory effect of sulforaphane on the tumor volume could also be obviously attenuated by the pretreatment of DCZ0415 in vivo. These results indicate that TRIP13 plays an important role in the sensitivity of leukemia cell response to sulforaphane treatment, and these findings expand the understanding of the mechanism of the antileukemic effect of sulforaphane and provide a new target for the treatment of AML.

A Phyto-mycotherapeutic Supplement, Namely Ganostile, as Effective Adjuvant in Brain Cancer Management: An In Vitro Study Using U251 Human Glioblastoma Cell Line.

The current standard oncotherapy for glioblastoma is limited by several adverse side effects, leading to a short-term patient survival rate paralleled by a worsening quality of life (QoL). Recently, Complementary and Integrative Medicine's (CIM) innovative approaches have shown positive impacts in terms of better response to treatment, side effect reduction, and QoL improvement. In particular, promising potential in cancer therapy has been found in compounds coming from phyto- and mycotherapy. The objective of this study was to demonstrate the beneficial effects of a new phyto-mycotherapy supplement, named Ganostile, in the human glioblastoma cell line U251, in combination with chemotherapeutic agents, i.e., Cisplatin and a new platinum-based prodrug. Choosing a supplement dosage that mimicked oral supplementation in humans (about 1 g/day), through in vitro assays, microscopy, and cytometric analysis, it has emerged that the cells, after 48hr continuous exposure to Ganostile in combination with the chemical compounds, showed a higher mortality and a lower proliferation rate than the samples subjected to the different treatments administered individually. In conclusion, our data support the use of Ganostile in integrative oncology protocols as a promising adjuvant able to amplify conventional and new drug effects and also reducing resistance mechanisms often observed in brain tumors.

Targeting androgen receptor signaling in glioblastoma: A novel biomarker and therapeutic approach.

e14016 Background: Glioblastoma is a lethal brain cancer. Clinical patterns of disease suggest that androgens influence glioblastoma risk and prognosis, and androgen receptor (AR) transcript levels and protein expression are upregulated in glioblastoma compared to normal brain. Thus, potent brain-penetrant anti-androgen therapies may be an effective treatment strategy in glioblastoma. Methods: Clinical samples and patient-derived glioma xenografts (PDXs) were assessed for AR expression by immunohistochemistry. Glioblastoma cell lines and patient-derived models maintained under stem cell conditions were tested in vitro for their response to anti-androgen therapies (abiraterone, enzalutamide and seviteronel). Change in plasticity marker ZEB1 was measured using immunofluorescence. Stem cell function was assessed using a tumoursphere assay. In an intracranial AR positive glioblastoma PDX model (RN1) in an immunocompromised mouse, the most effective drug in vitro, seviteronel, was tested alone and in combination with temozolomide or radiation. Tumor growth was monitored with weekly bioluminescent imaging, with survival as the primary endpoint. Results: Cytoplasmic AR staining was present in ~55% of glioblastoma samples using immunohistochemistry; this biomarker has shown to be predictive in triple-negative breast cancer. AR positive model RN1 was inhibited by low concentrations of anti-androgen agents, with seviteronel having the lowest half maximal inhibitory concentration (IC50) after 96 h (enzalutamide 52 µM, abiraterone 12 µM, seviteronel 7 µM). AR negative model WK1 was inhibited though with higher IC50 values after 96 h (enzalutamide 63 µM, seviteronel 21 µM). Cell lines U87 and U251 were also inhibited by anti-androgen monotherapy. Immunofluorescence analysis showed downregulation of plasticity marker ZEB1 with anti-androgens (normalized mean fluorescent intensity: abiraterone 0.84, enzalutamide 0.91, seviteronel 0.74). Tumoursphere assays demonstrated that anti-androgens inhibit the tumor-forming ability of cells, with seviteronel and abiraterone showing more inhibition than enzalutamide. In mice intracranially implanted with RN1 cells, seviteronel improved overall survival compared to vehicle alone (36 vs 27.5 days, p=0.03). There was no observed benefit when seviteronel was added to radiotherapy (33 vs 39 days, p=0.19). Adding seviteronel to temozolomide limited tumor growth by IVIS but did not significantly improve overall survival compared to temozolomide alone (63.5 vs 56.5 days, p=0.37). Conclusions: Targeting AR with brain penetrant anti-androgen drugs may be a promising biomarker-directed therapeutic strategy for glioblastoma. To address the limitations of our study, further experiments to optimize translational treatment protocols, address the role of biological sex and examine the role of the immune microenvironment are underway.

Co-treatment of human GBM cell lines with imipridone ONC206 and tumor treating fields: Effect on on cell death and p-AKT.

e14033 Background: Glioblastoma (GBM) is the most common primary brain malignancy with dismal prognosis. Tumor Treating Fields (TTFields) therapy is available for the treatment of both newly diagnosed and recurrent glioblastoma and represents a new category of treatment modalities in oncologic therapy. Despite decades of study, few FDA approved modalities are available for GBM and provide limited survival improvements for patients. AKT functions as a key serine/threonine kinase in the RTK/PTEN/PI3K pathway, and extensive genomic analysis of GBM has revealed that this pathway is mutated in a significant majority of GBM cases. Activation of this pathway ultimately leads to the activation of AKT, and phospho-AKT levels are elevated in the majority of GBM tumor samples and cell lines. Studies have shown that increased p-AKT levels contribute to uncontrolled growth, resistance to apoptosis, and enhanced invasive capabilities of glioma cells, which are characteristics of tumor progression in GBM. AKT serves as a critical hub in this pathway, enabling the amplification of growth signals, thereby making the inhibition of AKT an appealing and promising therapeutic target for treating GBM. Imipridone ONC206 is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). Here we show inhibition of p-AKT as well as upregulation of cleaved PARP following cotreatment of ONC206 and TTFields. Methods: We investigated the effect of ONC206 at IC50s plus TTFields in LN229, U138, U251 and T98G human GBM cell lines at different time points. Effect of treatment on cPARP, BCL-2, CHOP, caspase-10, and p-AKT were investigated using western blot. Results: Treatment with ONC206 at the IC50 in T98G cells with TTFields at 24h showed synergistic upregulation of cPARP as well as inhibition of BCL-2. At 96hour time point, inhibition of p-AKT was observed following all treatments (ONC206, TTFields, and co-application compared to control. In U251 cells, following treatment of ONC206 and TTFields, inhibition of BCL-2 was observed at 24h time point. Upregulation of CHOP and Casapse-10 at 96h time point was seen, as well as inhibition of p-AKT. Spheroid models showed structure disruption and growth arrest following combination treatment. At 72 h time point co treatment with ONC206 and TTFields resulted in synergistic upregulation of cleaved PARP. Conclusions: Here we showed cotreatment with TTFields and ONC206 results in inhibition of p-AKT, the key node in GBM growth pathway. This is the first report of cleaved PARP upregulation following TTFields and ONC206 in Human GBM cell line. Further studies to investigate the in vivo efficacy of TTField treatment in GBM with ONC206 are warranted. More studies are needed to understand the exact mechanism of synergy.

Effects of PIM1 Gene on Proliferation, Apoptosis and JAK2/STAT3 Signaling Pathway of Acute Myeloid Leukemia U937 Cells

To investigate the effects of the serine/threonine kinase family member 1 (PIM1) gene on the proliferation and apoptosis of acute myeloid leukemia (AML) U937 cells, and the regulation effect on Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Bone marrow mononuclear cells from newly diagnosed adult AML patients and patients with iron deficiency anemia were collected and PIM1 mRNA expression was detected by RT-qPCR. AML cell line U937 cells were divided into U937 group (U937 cells were cultured normally), Si-PIM1 group (U937 cells were transfected with low expression adenovirus vector containing PIM1 mRNA), Si-NC group (U937 cells were transfected with low expression adenovirus vector without PIM1 mRNA), coumermycin A1 (CoA1) group (JAK2 activator CoA1 was added to U937 cells at a concentration of 20 μmol/L), and Si-PIM1+CoA1 group (U937 cells were transfected with adenoviral vector containing low expression of PIM1 mRNA and added with CoA1 at a concentration of 20 μmol/L). After culture for 24 h, the expressions of PIM1 mRNA and protein, JAK2/STAT3 pathway, cell cycle and apoptosis-related proteins in U937 cells were detected by RT-qPCR and Western blot, the cell proliferation activity was detected by MTT assay, and flow cytometry was used to detect cell cycle changes and apoptosis rate. The PIM1 mRNA expression level in bone marrow mononuclear cells in AML patients was higher than that in patients with iron deficiency anemia (P < 0.05). Compared with U937 group, PIM1 mRNA and protein, phosphorylated JAK2 (p-JAK2)/JAK2, phosphorylated STAT3 (p-STAT3)/STAT3, Cyclin D1, cyclin-dependent kinase 2 (CDK2) protein, cell proliferation activity, S phase and G 2/M phase proportions were decreased in Si-PIM1 group (all P < 0.05), while p27, Caspase-3 protein, G0/G1 phase proportion and apoptosis rate were increased (all P < 0.05). However, the changes of above indicators in CoA1 group were just opposite to those in Si-PIM1 group, indicating that CoA1 could reverse the effect of Si-PIM1 on U937 cells. There were no significant differences in above indexes of U937 cells between U937 group, Si-PIM1+CoA1 group and Si-NC group (P >0.05). Knockdown of PIM1 gene expression can inhibit U937 cell proliferation and promote apoptosis, in order to alleviate ALM process, which may be related to the inhibition of JAK2/STAT3 pathway activation.

Synthesis, structure and in vitro anticancer activity of copper(II) and platinum(II) complexes with pyrrol-appended dipicolylamine ligand

Copper complexes exhibit important potential applications as non-platinum based metallodrugs particularly in the field of anticancer research. In this paper, mononuclear copper(II) and platinum(II) complexes containing pyrrole-linked dipicolylamine, namely PPMdpa-Cu and PPMdpa-Pt, were synthesized and characterized. Among these complexes, PPMdpa-Cu exhibited a stronger ability to bind to CT-DNA (calf-thymus DNA) compared to PPMdpa-Pt. In addition, an MTT assay performed throughout our studies demonstrated that PPMdpa-Cu featured significant antitumor activity against the three cancer cell lines U87, AsPC-1 and HepG2, superior to that of PPMdpa-Pt as well as the ligand, PPMdpa. Particularly, the IC50 of PPMdpa-Cu against HepG2 was found to be as low as 27.3 μM. Finally, anticancer mechanism studies showed that PPMdpa-Cu could induce apoptosis in HepG2 cells associated with mitochondrial damage and oxidative stress caused by the generation of reactive oxygen species (ROS).

Effect of gold-conjugated resveratrol nanoparticles on glioma cells and its underlying mechanism.

Glioblastoma is the most aggressive brain tumor with poor prognosis. Although Resveratrol (Rsv) is known to have therapeutic effects on glioma, the effects of gold-conjugated resveratrol nanoparticles (Rsv-AuNPs) on glioma cells are rarely reported. We aimed to investigate the effects of Rsv-AuNPs on glioma cells and its underlying mechanism. Human glioma cell line U87 was treated with different concentrations of Rsv-AuNPs. CCK-8, transwell, and wound healing assay were performed to measure the effects of Rsv-AuNPs on cell proliferation, invasion, and migration ability, respectively. Flow cytometry assay was used to detect the effects of Rsv-AuNPs on apoptosis. Changes of protein expressions related to proliferation, invasion, migration, and apoptosis were measured by Western blot assay. In addition, the inhibitory role of Rsv-AuNPs in the PI3K/AKT/mTOR signaling pathway was verified by using PI3K inhibitor LY294002. Rsv-AuNPs treatment significantly suppressed proliferation, migration, and invasion of U87 cells (allP <0.05) and increased the apoptosis rate (P <0.05). The changes of proteins related to proliferation, migration, invasion and apoptosis were consistent (allP <0.05). Moreover, Rsv-AuNPs treatment significantly inhibited the phosphorylation of PI3K, AKT and mTOR proteins in U87 cells (P <0.05). The present study found that Rsv-AuNPs inhibited the proliferation, migration, and invasion of U87 cells and induced apoptosis by inhibiting the activation of PI3K/AKT/mTOR signaling pathway. In the future, Rsv-AuNPs might be applied to the clinical treatment of glioma through more in-depth animal and clinical research.

ITRAQ-based proteomic study on monocyte cell model discovered an association of LAMP2 downregulation with HIV-1 latency

BackgroundPatients with immunodeficiency virus-1 (HIV-1) infection are challenging to be cured completely due to the existence of HIV-1 latency reservoirs. However, the knowledge of the mechanisms and biomarkers associated with HIV-1 latency is limited. Therefore, identifying proteins related to HIV-1 latency could provide new insights into the underlying mechanisms of HIV-1 latency, and ultimately contribute to the eradication of HIV reservoirs.MethodsAn Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-labeled subcellular proteomic study was performed on an HIV-1 latently infected cell model (U1, a HIV-1-integrated U937 cell line) and its control (U937). Differentially expressed proteins (DEPs) were analyzed using STRING-DB. Selected DEPs were further evaluated by western blotting and multiple reaction monitoring technology in both cell model and patient-derived cluster of differentiation 4 (CD4)+ T cells. Finally, we investigated the relationship between a specific DEP lysosome-associated membrane glycoprotein 2 (LAMP2) and HIV-1 reactivation by panobinostat or lysosome regulation by a lysosomotropic agent hydroxychloroquine in U1 and U937 cells.ResultsIn total, 110 DEPs were identified in U1 cells comparing to U937 control cells. Bioinformatics analysis suggested associations of the altered proteins with the immune response and endosomal/lysosomal pathway. LAMP2, leukocyte surface antigen CD47, CD55, and ITGA6 were downregulated in HIV-1 latent cells. Downregulated LAMP2 was further confirmed in resting CD4+ T cells from patients with latent HIV-1 infection. Furthermore, both HIV-1 reactivation by panobinostat and stimulation with hydroxychloroquine upregulated LAMP2 expression.ConclusionsOur results indicated the involvement of the endosomal/lysosomal pathway in HIV-1 latency in macrophage cell model. The down-modulation of LAMP2 was associated with HIV latency, and the restoration of LAMP2 expression accompanied the transition of viral latency to active infection. This study provides new insights into the mechanism of HIV-1 latency and potential strategies for eradicating HIV-1 reservoirs by targeting LAMP2 expression.

Senotherapeutic Peptide 14 Suppresses Th1 and M1 Human T Cell and Monocyte Subsets In Vitro.

Inflammation contributes to the onset and exacerbation of numerous age-related diseases, often manifesting as a chronic condition during aging. Given that cellular senescence fosters local and systemic inflammation, senotherapeutic interventions could potentially aid in managing or even reducing inflammation. Here, we investigated the immunomodulatory effects of the senotherapeutic Peptide 14 (Pep 14) in human peripheral blood mononuclear cells (PBMCs), monocytes, and macrophages. We found that, despite failing to significantly influence T cell activation and proliferation, the peptide promoted a Th2/Treg gene expression and cytokine signature in PBMCs, characterized by increased expression of the transcription factors GATA3 and FOXP3, as well as the cytokines IL-4 and IL-10. These observations were partially confirmed through ELISA, in which we observed increased IL-10 release by resting and PHA-stimulated PBMCs. In monocytes from the U-937 cell line, Pep 14 induced apoptosis in lipopolysaccharide (LPS)-stimulated cells and upregulated IL-10 expression. Furthermore, Pep 14 prevented LPS-induced activation and promoted an M2-like polarization in U-937-derived macrophages, evidenced by decreased expression of M1 markers and increased expression of M2 markers. We also showed that the conditioned media from Pep 14-treated macrophages enhanced fibroblast migration, indicative of a functional M2 phenotype. Taken together, our findings suggest that Pep 14 modulates immune cell function towards an anti-inflammatory and regenerative phenotype, highlighting its potential as a therapeutic intervention to alleviate immunosenescence-associated dysregulation.

Anticancer Activity of Aaptos suberitoides on Glioblastoma Multiforme Cell Line.

Glioblastoma Multiforme (GBM) poses a significant challenge due to its high aggressiveness and unfavorable prognosis, with existing treatments demonstrating limited efficacy in prolonging survival rates. This study aimed to assess the anticancer properties of Aaptos suberitoides extracts and fraction on the U87 cell line, serving as a representative model for GBM. U87 cells were treated with ethanol extracts derived from Aaptos suberitoides, specifically two extracts (OAA-1 and OAA-2) and one ethyl acetate fraction (EA) isolated from specimens collected on Pramuka Island and Tinjil Island. The evaluation encompased microscopic observation and MTT assay to determine the IC50. Subsequently, antiproliferative effects were investigated through apoptosis and cell cycle assays. The extract demonstrated cytotoxic activity against U87 cells, with OAA-1 and OAA-2 exhibiting IC50 values of 35.78 μg/mL and 25.38 μg/mL, respectively. OAA-1 notably induced apoptosis at 50 μg/mL and induced cell cycle arrest. On other hand, OAA-2, while also inducing apoptosis significantly, had a lesser impact on cell cycle arrest. In contrast, EA induced significant apoptosis at a concentration of 100 μg/mL. The ethanol extracts and the ethyl acetate fraction of Aaptos suberitoides emerged as a promising candidate for Glioblastoma Multiforme cancer therapy, showing potential in inhibiting cell proliferation and inducing apoptosis.

Immunogenic cell death mediated TLR3/4-activated MSCs in U87 GBM cell line

Background and aimsGlioblastoma (GBM) is an aggressive primary brain cancer with no promising curative therapies. It has been indicated that MSCs can interact with the tumour microenvironment (TME) through the secretion of soluble mediators regulating intercellular signalling within the TME. TLRs are a multigene family of pattern recognition receptors with evolutionarily conserved regions and are widely expressed in immune and other body cells. MSCs by TLRs can recognize conserved molecular components (DAPMPs and PAPMPs) and activate signalling pathways, which regulate immune and inflammatory responses. MSCs may exert immunomodulatory functions through interaction with their expressed toll-like receptors (TLRs) and exert a protective effect against tumour antigens. As an emerging approach, we aimed to monitor the U87 cell line growth, migration and death markers following specific TLR3/4-primed-MSCs-CMs treatment. Methods and resultsWe investigated the phenotypic and functional outcomes of primed-CMs and glioma cell line co-culture following short-term, low-dose TLR3/4 priming. The gene expression profile of target genes, including apoptotic markers and related genes, was analyzed by qRT-PCR. MicroRNA-Seq examined the miRNA expression patterns, and flow cytometry evaluated the cell viability and cycle stages. The results showed significant changes in apoptosis and likely necroptosis-related markers following TLR3/4-primed-MSCs-CMs exposure in the glioma cell line. Notably, we observed a considerable induction of selective pro-apoptotic markers and both the early and late stages of apoptosis in treated U87 cell lines. Additionally, the migration rate of glioma cells significantly decreased following MSCs-CM treatment. ConclusionOur findings confirmed that the exposure of TLR3/4-activated-MSCs-CMs with glioma tumour cells possibly changes the immunogenicity of the tumour microenvironment and induces immunogenic programmed cell death. Our results can support the idea that TLR3/4-primed-MSCs can lead to innate immune-mediated cell death and modify tumour cell biology in invasive and metastatic cancers.

AQP8 Modulates Mitochondrial H2O2 Transport to Influence Glioma Proliferation

Background Aquaporin-8 (AQP8) is involved in impacting glioma proliferation and can effect tumour growth by regulating Intracellular reactive oxygen species (ROS) signalling levels. In addition to transporting H2O2, AQP8 has been shown to affect ROS signaling, but evidence is lacking in gliomas. In this study, we aimed to investigate how AQP8 affects ROS signaling in gliomas. Materials and methods We constructed A172 and U251 cell lines with AQP8 knockdown and AQP8 rescue by CRISPR/Cas9 technology and overexpression of lentiviral vectors. We used CCK-8 and flow cytometry to test cell proliferation and cycle, immunofluorescence and Mito-Tracker CMXRos to observe the distribution of AQP8 expression in glioma cells, Amplex and DHE to study mitochondria release of H2O2, mitochondrial membrane potential (MMP) and NAD+/NADH ratio to assess mitochondrial function and protein blotting to detect p53 and p21 expression. Result We found that AQP8 co-localised with mitochondria and that knockdown of AQP8 inhibited the release of H2O2 from mitochondria and led to increased levels of ROS in mitochondria, thereby impairing mitochondrial function. We also discovered that AQP8 knockdown resulted in suppression of cell proliferation and was blocked at the G0/G1 phase with increased expression of mitochondrial ROS signalling-related p53/p21. Conclusions This finding provides further evidence for mechanistic studies of AQP8 as a prospective target for the treatment of gliomas.

Optimization of Epigenetic Modifier Drug Combination for Synergistic Effect against Glioblastoma Multiform Cancer Cell Lines

Glioblastoma multiforme (GBM), is a frequent class of malignant brain tumors. Epigenetic therapy, especially with synergistic combinations is highly paid attention for aggressive solid tumors like GBM. Here, RSM optimization has been used to increase the efficient arrest of U87 and U251 cell lines due to synergistic effects. Cell lines were treated with SAHA, 5-Azacytidine, GSK-126, and PTC-209 individually and then RSM was used to find most effective combinations. Results showed that optimized combinations significantly reduce cell survival and induce cell cycle arrest and apoptosis in both cell lines. Expression of cyclin B1 and cyclin D1 were decreased while caspase3 increased expression.

The Effect of Melatonin on the Expression of H19, MALAT, HOTAIR, THRIL, and NOS Activity in Leishmania major-Infected Macrophages

Background: Leishmania spp. protozoa cause leishmaniasis by infecting macrophages. Long non-coding RNAs (lncRNAs), such as H19, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT), HOX Antisense Intergenic RNA (HOTAIR), and TNF and HNRNPL Related Immuno-regulatory lncRNA (THRIL), play a role in macrophage polarization and gene regulation. Additionally, leukocytes can synthesize and respond to melatonin, yet the regulatory role of melatonin in Leishmania major-infected macrophages is not well understood. Objectives: This study aimed to assess the impact of melatonin on the expression of lncRNAs like H19, MALAT, HOTAIR, and THRIL, as well as on nitric oxide synthase (NOS) activity in L. major-infected macrophages. Methods: Leishmania major promastigotes and U937 cell lines were cultured. Macrophages were infected with the promastigotes and subsequently treated with melatonin at concentrations of 3, 10, 30, and 100 nM for durations of 4, 24, and 48 hours. The expression levels of the lncRNAs and NOS activity were measured using quantitative Polymerase Chain Reaction (q-PCR) and spectrophotometry, respectively. Results: Melatonin treatment (100 nM) significantly increased the expression of H19 compared to the control after 48 hours (P = 0.002). There was also a significant upregulation of MALAT and HOTAIR in macrophages treated with 3 nM melatonin compared to controls after 48 hours (P = 0.02 and P = 0.003, respectively). Additionally, THRIL expression significantly increased in the melatonin group (10 nM) compared to the control after 4 hours of treatment (P = 0.003). An increase in NOS activity was observed in the melatonin group (100 nM) compared to the control at 4 hours, 24 hours, and 48 hours (P = 0.034, P = 0.011, and P = 0.014, respectively). Conclusions: The findings suggest that melatonin may enhance the expression of H19, THRIL, MALAT, and HOTAIR, as well as NOS activity in macrophages infected with L. major. The upregulation of these lncRNAs by melatonin could potentially improve the macrophages' ability to combat L. major infection.

Phytochemical profiling, antioxidant potential and cytotoxic activity of Sargassum prismaticum extracts: implications for therapeutic applications

Seaweeds are sources of bioactive compounds with medicinal properties, which make them attractive candidates for natural therapeutic agents. Marine brown algae are known to possess anti-inflammatory, hepatoprotective, anticancer properties, etc. Present study was carried out to identify the phytochemical constituents, antioxidant and cytotoxic activities of Sargassum prismaticum in two different solvents viz., chloroform and methanol. Chloroform was found to be the superior solvent for phenol and flavonoid extraction. Antioxidant activity was determined using DPPH and ABTS assays; however, the methanolic extract demonstrated better antioxidant potential. The highest cell cytotoxicity with an IC50 value of 7.6 ± 0.02 μg/mL was observed in methanolic extract, while the chloroform extract had an IC50 value of 9.6 ± 0.03 μg/mL against U937 cell line. These finding suggest that Sargassum prismaticum possesses potent antioxidant and cytotoxic properties, making it a potential candidate for further study as a novel antioxidant drug source.

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.

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.