Human Acute Leukemia Cell Line Research Articles

Ethnobotanical survey and biological activities of plants used for cancer treatment in traditional Senegalese medicine

Female breast cancer is known to be one of the leading causes of death in Senegal. In Senegal, the absence of a national cancer control program, the lack of specialized infrastructure and qualified human resources and the exorbitant cost of care have contributed to the extensive use of traditional medecine, particularly in rural areas. This study aims to inventory the medicinal plants used by these healers and to assess the cytotoxic and antioxidant activities of the most widely used one. Data on healers and their use practices and information on plants were collected through the administration of a structured questionnaire. Based on their citation frequencies during the survey, Antiaris Africana Engler, Hymenocardia Acida Tul. and Halouf Halal (local name) were selected for chemical and biological studies. Their hydroalcoholic extracts were analyzed in terms of antioxidant capacity and cytotoxic effects, again, in the human cancer cell line. The study revealed a total of 65 medicinal plants belonging to 35 different families. The plant parts used by traditional healers are leaves (63.89%), roots (11.11%), bark (15.28%), fruits (2.78%), and others (6.94%). Generally, herbal medicine is prepared as a powder and mixed with water by maceration (55.38%) and administered orally. A. Africana ranked first with a citation frequency of 5.7% and its hydroalcoholic extract had the highest antioxidant activity in TEAC (6533.64±7 μmol ET/g dry plant) and in ORAC (3745.17±4.8 μmol ET/g dry plant) followed by H. Acida in TEAC (3115.6±145 μmol ET/g dry plant) and in ORAC (4105.29±872 μmol ET/g dry plant). The hydroalcoholic extract of A. Africana exhibited the highest cytotoxic activity in MCF-7 (Human mammary) and THP-1 (Human acute monocytic leukemia cell line) but had low activity against HTC-116 (Human carcinoma colorectal) and A-375 (Human skin malignant melanoma). The percentages of proapoptotic cells were, respectively, 68.85±6.22, 58.1±1.90 and 48.58±1.4%. These results provide scientific support for the traditional use of medicinal plants in cancer treatment and constitute a database for biological screening to isolate cytotoxic plant-based molecules.

Rapamycin increases leukemia cell sensitivity to chemotherapy by regulating mTORC1 pathway-mediated apoptosis and autophagy.

This study investigated the effect of rapamycin alone and in combination with chemotherapy (doxorubicin and cytarabine) on AML. Human acute monocytic leukemia cell line SHI-1 and NPG AML model mice created by intravenous injection of SHI-1 cell were treated with rapamycin, chemotherapy, or rapamycin plus chemotherapy. Analysis by cell counting kit-8, western blot, flow cytometry, and immunohistochemistry was performed, and results suggested that both rapamycin and chemotherapy inhibited proliferation of SHI-1 cells both in vitro and in vivo, suppressed neoplasm growth in vivo, and promoted survival of NPG AML mice. The antitumor effect of rapamycin plus chemotherapy was better than that of rapamycin alone and chemotherapy alone. In addition, western blot results demonstrated that rapamycin inhibited the phosphorylation of mTOR downstream targets 4EBP1 and S6K1 in SHI-1 cells, and increased the pro-apoptosis-related protein Bax and autophagy-associated proteins Beclin-1, LC3B-II, and ATG5 while reducing the anti-apoptosis-related protein Bcl-2. In conclusion, the results of this study indicate that rapamycin acts synergistically with doxorubicin and cytarabine in AML treatment, and its underlying mechanism might be associated with mTORC1 pathway-mediated apoptosis and autophagy.

[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.

Ribonucleic Acid Sequencing Reveals the Upregulation and Resolution of Inflammation and Extracellular Matrix Remodeling in Lidocaine-Treated Human Acute Monocytic Leukemia Cell Line THP-1.

Lidocaine, a local anesthetic widely used in dentistry, is esteemed for its efficacy and safety. Recent research reveals its additional role in modulating the immune system, and particularly in reducing inflammation crucial for protecting tooth-supporting tissues. Notably, monocytes and macrophages, essential cellular components overseeing various physiological and pathological processes, stand as potential mediators of lidocaine's effects. Therefore, this study aimed to investigate how lidocaine influences cell behavior using RNA sequencing. To investigate the effect of lidocaine on THP-1 cells' behavior, we performed an MTT assay and RNA-Seq along with qPCR analyses to evaluate the transcriptomic and proteomic changes in THP-1 cells. Our results showed that a high dose of lidocaine (>1 mM) had a significant cytotoxic effect on THP-1 cells. However, a lidocaine dose lower than 0.5 mM induced a mixed anti-inflammatory profile by significantly upregulating tissue remodeling (GDF15, FGF7, HGF, COL4A3, COL8A2, LAMB2, LAMC2, PDGFRA, and VEGFA) and through the resolution of inflammation (Cpeb4, Socs1, Socs2, Socs3, Dusp1, Tnfaip3, and Gata3) gene cassettes. This study explores the effect of lidocaine on the THP-1 in the M2-like healing phenotype and provides potential applications of lidocaine's therapeutic effectiveness in dental tissue repair.

Formononetin and Dihydroartemisinin Act Synergistically to Induce Apoptosis in Human Acute Myeloid Leukemia Cell Lines.

Enhanced cell survival and drug resistance in tumor cells have been linked to the overexpression of antiapoptotic members of the Bcl-2 family proteins, including Bcl-2 and Mcl-1. The aim of this study was to explore the impact of formononetin and dihydroartemisinin combination on the growth and apoptosis of acute myeloid leukemia (AML) cells. In this experimental study, the cell survival and cell proliferation were tested by MTT assay and trypan blue staining. The evaluation of cell apoptosis was conducted using Hoechst 33342 staining and a colorimetric assay to measure caspase-3 activity. To determine the mRNA levels of Mcl-1, Bcl-2, Bax, and Cyclin D1, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed. We showed that treatment with either formononetin or dihydroartemisinin alone, led to significant decrease in the cell survival and growth, and triggered apoptosis in U937 and KG-1 AML cell lines. Moreover, treatment with each of the compounds alone significantly decreased the mRNA levels of Mcl-1, Bcl-2 and Cyclin D1 mRNA, while, the expression level of Bax mRNA was enhanced. Combination of two compounds showed a synergistic anti-cancer effect. The anti-leukemic potential of formononetin and dihydroartemisinin is exerted through the effect on cell cycle progression and intrinsic pathway of apoptosis. Therefore, they can be considered as a potential anti-leukemic agent alone or along with existing chemotherapeutic drugs.

Biological Effects of Small Sized Graphene Oxide Nanosheets on Human Leukocytes.

Since the discovery of graphene, there has been a wide range of the literature dealing with its versatile structure and easy binding of biomolecules as well as its large loading capacity. In the emerging field of immunotherapy, graphene and its derivatives have potential uses as drug delivery platforms directly into tumour sites or as adjuvants in cancer vaccines, as they are internalized by monocytes which in turn may activate adaptive anti-tumoral immune responses. In this study, we expose cells of the innate immune system and a human acute monocytic leukemia cell line (THP-1) to low doses of small-sized GO nanosheets functionalized with bovine serum albumin (BSA) and fluorescein isothiocyanate (FITC), to study their acute response after internalization. We show by flow cytometry, uptake in cells of GO-BSA-FITC reaches 80% and cell viability and ROS production are both unaffected by exposure to nanoparticles. On the contrary, GO-BSA nanosheets seem to have an inhibitory effect on ROS production, probably due to their antioxidant properties. We also provided results on chemotaxis of macrophages derived from peripheral blood monocytes treated with GO-BSA. In conclusion, we showed the size of nanosheets, the concentration used and the degree of functionalization were important factors for biocompatibility of GO in immune cells. Its low cytotoxicity and high adaptability to the cells of the innate immune system make it a good candidate for deployment in immunotherapy, in particular for delivering protein antigens to monocytes which activate adaptive immunity.

Discovery and Optimization of WDR5 Inhibitors via Cascade Deoxyribonucleic Acid-Encoded Library Selection Approach.

The DNA-encoded library (DEL) is a powerful hit generation tool for chemical biology and drug discovery; however, the optimization of DEL hits remained a daunting challenge for the medicinal chemistry community. In this study, hit compounds targeting the WIN binding domain of WDR5 were discovered by the initial three-cycle linear DEL selection, and their potency was further enhanced by a cascade DEL selection from the focused DEL designed based on the original first run DEL hits. As expected, these new compounds from the second run of focused DEL were more potent WDR5 inhibitors in the protein binding assay confirmed by the off-DNA synthesis. Interestingly, selected inhibitors exhibited good antiproliferative activity in two human acute leukemia cell lines. Taken together, this new cascade DEL selection strategy may have tremendous potential for finding high-affinity leads against WDR5 and provide opportunities to explore and optimize inhibitors for other targets.

Ubiquitin-Mediated Degradation of MORF4L1 By FBXO11 Suppresses Cholesterol Biosynthesis in T Cell Acute Lymphoblastic Leukemia

The substrate receptor of SKP1-Cullin1-FBOX complex, FBXO11, is frequently mutated in both diffuse large B cell lymphoma and acute myeloid leukemia, and typically functions as a tumor suppressor by targeting substrates for ubiquitin-mediated degradation. Although FBXO11 is also mutated in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines, the underlying mechanism in T-ALL development remains to be established. Here, we identified that both endogenous activation and exogenous overexpression of FBXO11 could suppress the growth of T-ALL cell in vitro and in vivo. While conversely, loss of FBXO11 accelerates the T-ALL cell proliferation. Transcriptomic and total quantitative proteomic analyses in FBXO11-depleted T-ALL cells reveal activation of the cholesterol biosynthesis pathway with accumulation of intracellular cholesterol levels. Importantly, we identified a chromatin regulator MORF4L1, mortality factor 4 like protein 1, as a functional substrate of FBXO11 in T-ALL cells. We first observed that FBXO11 exhibited frequent copy number alterations in T-ALL cell lines and in patient cohorts from public datasets. Moreover, by CRISPR SAM (Synergistic Activation Mediator) endogenous activation and lentiviral mediated gene overexpression, we validated that FBXO11 played a role in inhibiting tumor cell proliferation in T-ALL cells both in vitro and in vivo. Conversely, loss of FBXO11 accelerated T-ALL cell proliferation. Taken together, our results suggest that FBXO11 functions as a tumor suppressor in T-ALL. Mechanistically, we uncovered that the absence of FBXO11 could activate the cholesterol synthesis signaling pathway from both the RNA-seq and the TMT proteomic analysis in FBXO11-depleted MOLT4 cells compared to control cells. Concordantly, loss of FBO11 resulted in the accumulation of intracellular cholesterol level, suggesting that FBXO11 may suppress cholesterol biosynthesis in T-ALL cells. To understand how FBXO11 controls cholesterol biosynthesis, we further identified the potential substrates from the combined quantitative transcriptome and proteome analysis. MORF4L1, a chromatin modifier involved in histone acetylation, was identified as a key substrate of FBXO11 based on its upregulation at the protein level in FBXO11-depleted cells. Moreover, our data suggest MORF4L1 is a regulator of cholesterol biosynthesis in T-ALL. The inhibition of MORF4L1 suppressed the cellular growth of T-ALL cell lines, and led to the downregulation of the expression of cholesterol-related genes, such as gene HMGCR and HMGCS1. Of note, suppression of MORF4L1 and its cholesterol biosynthesis target genes such as HMGCR, HMGCS1 could rescue the accelerated growth effect after loss of FBXO11, indicating a functional role for MORF4L1 in T-ALL development. In summary, our data support a model wherein the degradation of MORF4L1 by FBXO11 suppresses leukemia cell growth by controlling cholesterol biosynthesis. The identification of this regulatory mechanism is required for T-ALL and provides valuable insights into the metabolic vulnerabilities of leukemia.

NAMPT Haploinsufficiency Is a Therapeutic Vulnerability in High-Risk Myeloid Malignancies

Monosomy 7 (−7) and del7q are the most common high-risk cytogenetic abnormalities and occur across the spectrum of myeloid disorders. -7/del(7q) also co-occur with other high-risk factors such as mutations or deletion of TP53. Standard, non-specific chemotherapy drugs are less effective in these high-risk patients, resulting in a median survival rate of approximately one year. To identify novel, targeted agents for these neoplasms, we sought “collateral lethal” genes, i.e. essential, druggable genes encoded on chromosome 7 that are haploinsufficient upon their loss of heterozygosity. To this end, we first identified essential genes located on chromosome 7. We mined genome-wide CRISPR/Cas9 screen data from 27 human acute myeloid leukemia (AML) cell lines within the DepMap database and identified 65 chromosome 7-encoded, candidate essential genes in AML. To determine those essential genes with known inhibitors, we intersected these 65 candidates with the Drug-Gene Interaction and canSAR.ai databases. We found that nicotinamide phosphoribosyltransferase ( NAMPT), the rate-limiting enzyme that catalyzes the first step of NAD + biosynthesis from nicotinamide, was one of four essential and druggable targets on chromosome 7. Notably, NAMPT was the only essential gene on chromosome 7 with a first-in-class orally bioavailable inhibitor, KPT 9274, currently in clinical trials (NCT04914845). Moreover, NAMPT DNA copy number, mRNA, and protein levels all negatively correlated with cell line sensitivity to NAMPT loss, implicating NAMPT as a collateral lethal candidate gene. We confirmed that isogenic human AML cell lines CRISPR-engineered to partially reduce NAMPT DNA copy led to reduced NAMPT protein levels, indicating NAMPT is a haploinsufficient gene. Cells with ~50% NAMPT levels were significantly more sensitive to the KPT 9274 treatment, and became less viable and more apoptotic, compared to control locus edited cells. To determine whether TP53 status could alter this vulnerability, we generated cell lines with both TP53 loss and 50% NAMPT loss. We found TP53 mutational status did not attenuate the observed effects of NAMPT inhibition on cell survival. Viability assays with primary patient AML samples confirmed that -7/del(7q) status imparts significantly elevated sensitivity to KPT 9274 treatment than samples with diploid chromosome 7 alleles. In summary, our findings reveal NAMPT heterozygosity as a therapeutic vulnerability in high-risk myeloid neoplasms that warrants clinical follow-up. Further, we provide a framework centered on data-mining for uncovering collateral lethal genes in other cancers with recurrent chromosomal arm level deletions.

Targeting IGF2BP3 Enhances Anti-Leukemic Effects of Menin-MLL Inhibition in MLL-AF4 Leukemia

Background: MLL-rearranged leukemias are a clinically challenging and biologically unique subtype of leukemias associated with poor prognosis. While novel therapeutic strategies have been primarily directed at epigenetic dysregulation, post-transcriptional gene regulatory mechanisms have emerged as important mediators in leukemogenesis and have the unexplored potential to be potent combinatorial therapeutic targets. Previously, we found that the RNA-binding protein IGF2BP3 is a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Methods: We studied the combined effects of targeting IGF2BP3 and the Menin-MLL interaction in MLL-AF4 driven leukemia in vitro and in vivo, using genetic inhibition through CRISPR-Cas9 mediated deletion of Igf2bp3 and pharmacologic inhibition of the Menin-MLL interaction with commercially available inhibitors MI-503, MI-463, and MI-538. In vitro, we tested the human B-cell acute lymphoblastic leukemia cell lines, SEM, RS4;11 and NALM6, and MLL-Af4-transformed murine hematopoietic stem and progenitor cells (herein referred to as MLL-Af4 Lin-), derived from bone marrow of Cas9 mice. Results: Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the negative effects of Menin-MLL inhibition on leukemic cell growth, colony formation and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and Menin-MLL inhibition led to increased differentiation in vitro and in vivo in functional readouts and by gene expression analyses. Both MI-503 treatment and IGF2BP3 knockdown in MLL-Af4 Lin- cells showed a shift towards more differentiated colony morphologies in colony formation assays, decreased expression of c-Kit and increased expression of maturation markers by flow cytometry, and morphologic changes consistent with increased differentiation. To gain insight into these phenotypic findings, we examined gene expression from MLL-Af4 Lin- cells with IGF2BP3 knockdown and treated with MI-503. Metascape analysis revealed significant enrichment in pathways involved in cell differentiation and activation, particularly in leukocytes. This was observed in both I3KO and MI-503 treated cells, with significant overlap in shared differentially expressed genes in both conditions. Next, we looked at the overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 knockdown with targets identified from MLL-AF4 ChIP and IGF2BP3 CLIP in CD11b+ and MLL-Af4 Lin- cells. We found significant overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 CLIP targets, suggesting that IGF2BP3 directly regulates genes that are affected by Menin-MLL inhibition. Furthermore, we saw overlap between differentially expressed genes with IGF2BP3 knockdown and MLL-AF4 ChIP targets identified in SEM and RS4;11 cells. These patterns highlight the interaction between the MLL-AF4 and IGF2BP3 transcriptomes and suggest a mechanism for the synergistic inhibition of leukemia by targeting both MLL-AF4 and IGF2BP3. To examine the combined effects of IGF2BP3 knockdown and Menin-MLL inhibition on leukemic engraftment and survival in vivo, we transplanted MLL-Af4 Lin- cells, depleted (or non-depleted) for IGF2BP3 and treated with MI-503 at 0.5 μM for 5 days, to initiate leukemia. We observed a significant decrease in peripheral blood leukemic engraftment with MI-503 treatment and IGF2BP3 knockdown. IGF2BP3 knockdown had a greater effect on survival and attenuating disease than MI-503 alone and showed enhanced anti-leukemic effects in combination. Conclusions: Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4 mediated leukemogenesis and is a potent therapeutic target and suggests a novel combinatorial approach to targeting leukemia at both the transcriptional and post-transcriptional level.

Influence of Microbiota-Related Metabolites Associated with Inflammation and Sepsis on the Peroxidase Activity of Cyclooxygenase in Healthy Human Monocytes and Acute Monocytic Leukemia Cells.

The human microbiota produces metabolites that can enter the bloodstream and exert systemic effects on various functions in both healthy and pathological states. We have studied the participation of microbiota-related metabolites in bacterial infection by examining their influence on the activity of cyclooxygenase (COX) as a key enzyme of inflammation. The influence of aromatic microbial metabolites, derivatives of phenylalanine (phenylpropionic acid, PPA), tyrosine (4-hydroxyphenyllactic acid, HPLA), and tryptophan (indolacetic acids, IAA), the concentrations of which in the blood change notably during sepsis, was evaluated. Also, the effect of itaconic acid (ITA) was studied, which is formed in macrophages under the action of bacterial lipopolysaccharides (LPS) and appears in the blood in the early stages of infection. Metabiotic acetyl phosphate (AcP) as a strong acetylating agent was also tested. The activity of COX was measured via the TMPD oxidation colorimetric assay using the commercial pure enzyme, cultured healthy monocytes, and the human acute monocytic leukemia cell line THP-1. All metabolites in the concentration range of 100-500 μM lowered the activity of COX. The most pronounced inhibition was observed on the commercial pure enzyme, reaching up to 40% in the presence of AcP and 20-30% in the presence of the other metabolites. On cell lysates, the effect of metabolites was preserved, although it significantly decreased, probably due to their interaction with other targets subject to redox-dependent and acetylation processes. The possible contribution of the redox-dependent action of microbial metabolites was confirmed by assessing the activity of the enzyme in the presence of thiol reagents and in model conditions, when the COX-formed peroxy intermediate was replaced with tert-butyl hydroperoxide (TBH). The data show the involvement of the microbial metabolites in the regulation of COX activity, probably due to their influence on the peroxidase activity of the enzyme.

Asteriquinones from Aspergillus sp. GZWMJZ-258 and Their Derivatives.

A new asteriquinone, ochrindole F (1), and five previously reported analogues (2-6) were isolated from the culture of the fungus Aspergillus sp. GZWMJZ-258, an endophyte of Garcinia multiflora. The structure of compound 1 was determined by a spectroscopic analysis. Furthermore, eight new derivatives (7-14) were synthesized from major metabolites 2 and 3. These compounds showed selective antiproliferative activity against the human acute myeloid leukemia (AML) cell line MV4-11, among which compound 12 showed the strongest activity with an IC50 value of 0.14 μM and the highest selectivity with a selectivity index greater than 710. An initial probe of the mechanism of action showed that compounds 12 and 14 could inhibit the expression of FLT-3 in the MV4-11 cell line.

Anti-inflammatory effects of cold atmospheric plasma irradiation on the THP-1 human acute monocytic leukemia cell line.

Cold atmospheric plasma (CAP) has been studied and clinically applied to treat chronic wounds, cancer, periodontitis, and other diseases. CAP exerts cytotoxic, bactericidal, cell-proliferative, and anti-inflammatory effects on living tissues by generating reactive species. Therefore, CAP holds promise as a treatment for diseases involving chronic inflammation and bacterial infections. However, the cellular mechanisms underlying these anti-inflammatory effects of CAP are still unclear. Thus, this study aimed to elucidate the anti-inflammatory mechanisms of CAP in vitro. The human acute monocytic leukemia cell line, THP-1, was stimulated with lipopolysaccharide and irradiated with CAP, and the cytotoxic effects of CAP were evaluated. Time-course differentiation of gene expression was analyzed, and key transcription factors were identified via transcriptome analysis. Additionally, the nuclear localization of the CAP-induced transcription factor was examined using western blotting. The results indicated that CAP showed no cytotoxic effects after less than 70 s of irradiation and significantly inhibited interleukin 6 (IL6) expression after more than 40 s of irradiation. Transcriptome analysis revealed many differentially expressed genes (DEGs) following CAP irradiation at all time points. Cluster analysis classified the DEGs into four distinct groups, each with time-dependent characteristics. Gene ontology and gene set enrichment analyses revealed CAP-induced suppression of IL6 production, other inflammatory responses, and the expression of genes related to major histocompatibility complex (MHC) class II. Transcription factor analysis suggested that nuclear factor erythroid 2-related factor 2 (NRF2), which suppresses intracellular oxidative stress, is the most activated transcription factor. Contrarily, regulatory factor X5, which regulates MHC class II expression, is the most suppressed transcription factor. Western blotting revealed the nuclear localization of NRF2 following CAP irradiation. These data suggest that CAP suppresses the inflammatory response, possibly by promoting NRF2 nuclear translocation.

Biological activities and GC–MS based chemical profiling of polymolecular methanol extract of Alternaria alternata KUDB15

ObjectivesThe ecological niche of soil for microorganisms that produce beneficial naturally occurring and biologically active products suitable for pharmaceutical industries is widely recognized. The purpose of the current study is to isolate fungus from soil samples of Dandeli, Karnataka and its metabolic profiling and investigating biological activities. MethodsSoil fungal isolates were screened for their antimicrobial potency against pathogens. The selected fungal isolate was morphologically characterized and ascertained by 18S rRNA gene sequencing, and the extract was investigated by gas chromatography-mass spectrometry (GC–MS) to identify the bio-molecules. Later the fungal extract was evaluated for antioxidant, antimicrobial, anti-mycobacterial and anti-proliferative activity against Human acute leukemia (K562) cell line. ResultsThe potential fungal isolate, Alternaria sp. KUDB15 showed a number of bioactive compounds through GC–MS analysis. A significant antioxidant activity (IC50 = 37.60 µg/ml) was also reported for this organism. A significant inhibitory activity against tested pathogens was shown by Alternaria sp. KUDB15 along with a strong anti-mycobacterial activity with an IC50 value of 3.12 μg/ml. During anticancer assay, this organism showed a concentration dependent reduction in cell viability having IC50 of 112.09 µg/ml. Further purification of the crude extract with column chromatography and thin layer chromatography (TLC) recorded several bands and the particular band having antibacterial activity was determined by bioautography. ConclusionsIn the present research, the in vitro evaluation of biological activities of the fungal extract suggested that it might serve as a useful foundation for the discovery of compounds that have potential for medical applications. Additional research is required to identify and understand the structure of bio-molecules.

Celastrol attenuates human parvovirus B19 NS1‑induced NLRP3 inflammasome activation in macrophages.

Human parvovirus B19 (B19V) has been strongly associated with a variety of inflammatory disorders, such as rheumatoid arthritis (RA), inflammatory bowel disease and systemic lupus erythematosus. Non‑structural protein 1 (NS1) of B19V has been demonstrated to play essential roles in the pathological processes of B19V infection due to its regulatory properties on inflammatory cytokines. Celastrol, a quinone methide isolated from Tripterygiumwilfordii, has displayed substantial potential in treating inflammatory diseases, such as psoriasis and RA. However, little is known about the effects of celastrol on B19V NS1‑induced inflammation. Therefore, cell viability assay, migration assay, phagocytosis analysis, zymography assay, ELISA and immunoblotting were conducted to verify the influences of celastrol on macrophages. The present study reported the attenuating effects of celastrol on B19V NS1‑induced inflammatory responses in macrophages derived from human acute monocytic leukemia cell lines, U937 and THP‑1. Although the migration was not significantly decreased by celastrol in both U937 and THP‑1 macrophages, significantly decreased viability, migration and phagocytosis were detected in both B19V NS1‑activated U937 and THP‑1 macrophages in the presence of celastrol. Additionally, celastrol significantly decreased MMP‑9 activity and the levels of inflammatory cytokines, including IL‑6, TNF‑α and IL‑1β, in B19V NS1‑activated U937 and THP‑1 cells. Notably, significantly decreased levels of NLR family pyrin domain‑containing 3, apoptosis‑associated speck‑like, caspase‑1 and IL‑18 proteins were observed in both B19V NS1‑activated U937 and THP‑1 cells in the presence of celastrol, indicating the involvement of the inflammasome pathway. To the best of our knowledge, the present study is the first to report on the attenuating effects of celastrol on B19V NS1‑induced inflammatory responses in macrophages, suggesting a therapeutic role for celastrol in B19V NS1‑related inflammatory diseases.

2-Substituted 1,5-benzothiazepine-based HDAC inhibitors exert anticancer activities on human solid and acute myeloid leukemia cell lines

Herein, we report the development of a new series of histone deacetylase inhibitors (HDACi) containing a 2-substituted 1,5-benzothiazepine scaffold. First, a virtual combinatorial library (∼1.6 × 103 items) was built according to a convenient synthetic route, and then it was submitted to molecular docking experiments on seven HDACs isoforms belonging to classes I and II. Integrated computational filters were used to select the most promising ones that were synthesized through an optimized approach, also amenable to generating both racemic and enantioenriched benzothiazepine-based derivatives. The obtained compounds showed potent HDAC inhibitory activity, especially those containing the sulphone moiety, endowed with IC50 in the nanomolar range. In addition, in vitro outcomes of our synthesized compounds demonstrated a cytotoxic effect on U937 and HCT116 cell lines and an arrest in the G2/M phase (13 ≤ IC50 ≤ 18 µM). Finally, Western blot analyses outlined the modulation of the histone acetyl markers such as H3K9/14, acetyl-tubulin, and the apoptotic indicator p21 in both cancer cell lines, disclosing a good HDAC inhibitor activity exerted by the designed items. Given the key role of HDACs in many cellular pathways, which makes these enzymes appealing and “hot” drug targets, our findings highlighted the importance of these 2-substituted 1,5-benzothiazepine-based compounds (both in the reduced and oxidized version) for the development of novel epidrugs.

Evaluating the Antioxidant Properties of the Ancient-Crop Tef (Eragrostis tef) Grain Extracts in THP-1 Monocytes.

Tef (Eragrostis tef) is an orphan crop that is widely grown in East Africa, primarily in Ethiopia as a staple crop. It is becoming popular in the Western world owing to its nutritious and gluten-free grains and the forage quality of its biomass. Tef is also considered to have a high antioxidant capacity based on cell-free studies. However, the antioxidant activity of tef has never been validated using a physiologically relevant cell model. The purpose of this study was to investigate the antioxidant capacity of tef grain extracts using a mammalian cell model. We hypothesized that the tef grain extracts are capable of modulating the cellular antioxidant response via the modulation of glutathione (GSH) biosynthetic pathways. Therefore, we evaluated the antioxidant activity of purified tef grain extracts in the human acute monocytic leukemia (THP-1) cell line. Our findings revealed that the organic fraction of grain extracts increased the cellular GSH level, which was more evident for brown-colored tef than the ivory variety. Moreover, a brown-tef fraction increased the expressions of GSH-pathway genes, including γ-glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits and glutathione reductase (GR), an enzyme that plays a key role in GSH biosynthesis, suggesting that tef extracts may modulate GSH metabolism. Several compounds were uniquely identified via mass spectrometry (MS) in GSH-modulating brown-tef samples, including 4-oxo-β-apo-13-carotenone, γ-linolenic acid (methyl ester), 4,4'-(2,3-dimethyl-1,4-butanediyl)bis-phenol (also referred to as 8,8'-lignan-4,4'-diol), and (3β)-3-[[2-[4-(Acetylamino)phenoxy]acetyl]oxy]olean-12-en-28-oic acid. Tef possesses antioxidant activity due to the presence of phytochemicals that can act as direct antioxidants, as well as modulators of antioxidant-response genes, indicating its potential role in alleviating diseases triggered by oxidative stresses. To the best of our knowledge, this is the first report revealing the antioxidant ability of tef extracts in a physiologically relevant human cell model.

Ruxolitinib inhibits the proliferation and induces the apoptosis of MLL-r ALL cells through inactivating JAK/STAT signaling pathway.

The childhood patients with mixed-lineage leukemia rearrangement (MLL-r) gene have worse outcome than non-MLL, and thus often treated with high-risk chemotherapy regimens, so targeted therapy is important for this type of leukemia. This purpose of study was to explore the effects of ruxolitinib on the proliferation, apoptosis, and cell cycle of Nalm-6 cells. In this study, human acute lymphoblastic leukemia (ALL) cell line Nalm-6 was used as the research object. By constructing an MLL overexpression vector to transfect Nalm-6 cells, exogenous JAK2/STAT3 signal pathway inhibitor ruxolitinib was applied to observe the proliferation, apoptosis, and cell cycle changes of the transfected Nalm-6 cells. Western blot was performed to determine the proteins (MLL-BP, JAK, STAT) involved in the mechanism of action of MLL-r leukemia. CCK8 assay and flow cytometry (FCM) were used for testing the proliferation and apoptosis among MLL-BP transfected Nalm-6 cells. Firstly, we determine the IC50 of ruxolitinib on Nalm-6 cells. Secondly, FCM and CCK8 showed that ruxolitinib dosedependentlyinhibits proliferation of Nalm-6 cells by blocking the cell cycle at G0/G1 phase. In addition, FCM showed that ruxolitinib promoted the apoptosis of MLL-BP transfected Nalm-6 cells. Mechanistically, ruxolitinib inactivated JAK/STAT signaling pathway in MLL-BP transfected Nalm-6 cells, mediating ruxolitinib's inhibition of cell proliferation, and inducing apoptosis. Finally, ruxolitinib significantly inhibited the proliferation of MLL-r ALL cells and promoted their apoptosis. These data provide compelling evidence that ruxolitinib is a promising agent against MLL-r leukemia cell line. However, it needs going through multiple more steps to confirm before it can be an option in clinical practice.

Insights into the Hippocampus Proteomics Reveal Epigenetic Properties of Walnut-Derived Peptides in a Low-Grade Neuroinflammation Model.

Epigenetic mechanisms that dysregulate gene expressions may play a significant role in the development of neurological disorders. However, whether peptides can modulate epigenetic mechanisms remains elusive. This work aimed to investigate the impact of pretreatment with walnut-derived peptides─WHP and YVLLPSPK─on DNA methylation in a low-grade neuroinflammation model. The enriched KEGG pathways included oxidative phosphorylation, riboflavin metabolism, ribosome, and pyrimidine metabolism, which are associated with methylation modification by oral administration of YVLLPSPK in mice with scopolamine-induced cognitive deficits. Furthermore, when THP-1 cells (human acute monocytic leukemia cell line) were exposed to lipopolysaccharide (LPS)-induced inflammation responses, both WHP and YVLLPSPK markedly inhibited the level of Il-6 to 2.05 ± 0.76 and 1.29 ± 0.19 (p < 0.05) and also declined the mRNA expression of Mcp-1 to 1.64 ± 0.02 and 3.29 ± 1.21 (p < 0.01), respectively. Meanwhile, YVLLPSPK decreased the activities of DNA methyltransferases (DNMTs) to 1.03 ± 0.02 and 1.20 ± 0.31 (p < 0.05) based on Dnmt3b and Tet2, respectively. The results indicated that YVLLPSPK modulated DNA methylation in embryonic and neural precursor cells in creating new methylation patterns. Further trials are needed to assess the mechanisms underlying DNA methylation changes through peptides in the pathophysiology of neurological disorders.

Scaffold-hopping of Linifanib to Design 6-Phenylisoxazolo[3,4-b]pyridin-3-amine Derivatives as FLT3 Inhibitors for Treating Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the most common type of blood cancer. Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinase family. Overexpression of FLT3 was found in 70-100% of patients with acute myeloid leukaemia. FLT3 internal tandem duplication alteration (ITD) and the tyrosine kinase domain (TKD) are the most common molecular alteration in AML, and FLT3 has become a promising drug target for AML. Objective: A series of 6-phenylisoxazolo[3,4-b]pyridin-3-amine derivatives F1–F15 with amide bonds as FLT3 inhibitors were designed and synthesized in order to find a new lead compound to treat AML. Methods: We designed an original scaffold-hopping protocol by combing the RECAP tool with the Gilde-Based Core-Hopping tool to design novel FLT3 inhibitors based on Linifanib. Inhibitors assembled were ranked by the docking scores generated by Glide. Compounds undisclosed among the top 10 were selected to design a series of 6-phenylisoxazolo[3,4-b]pyridin-3-amine derivatives as FLT3 inhibitors. The kinase inhibitory activities of the fifteen compounds were assayed on FLT3 and FLT3-ITD. The antitumor activities of the structurally modified compounds F1–F15 were evaluated against MOLM-13 and MV4-11, typical FLT3-dependent human AML cells carrying FLT3-ITD mutants and the FLT3-independent human cervical carcinoma cell line HL-60 (harboring wide-type FLT3). Results: Structure–activity relationship (SAR) analysis showed that F14 could inhibit FLT3 and FLT3-ITD by 52% and 45.55%, respectively, at the concentration of 1 ΜF14 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines, MOLM-13, and MV4-11 (harboring FLT3-ITD mutant) with IC50 values of 2.558 μM and 1.785 μM, respectively. Conclusion: F14 could be used as a novel lead compound to further develop FLT3 inhibitors against AML with FLT3-ITD mutant