Molecular Mechanisms Responsible for In Vitro Cytotoxic Attributes of Conyza bonariensis Extract against Lymphoblastic Leukaemia Jurkat Cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Mohammad Saleem ◽  
Valerie B Schini-Kerth ◽  
Khalid Hussain ◽  
Syed Haroon Khalid ◽  
Muhammad Asif ◽  
...  
Keyword(s):  
Western Blot ◽  
Cytotoxic Activity ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Human Leukemia ◽  
Normal Peripheral Blood ◽  
Conyza Bonariensis

Background: Conyza bonariensis is known to have anti-cancer properties. Objective: The study investigated the in vitro pro-apoptotic properties of Conyza bonariensis (C. bonariensis) towards human lymphoblastic leukemia Jurkat cells. Methods: C. bonariensis are extracted with non-polar solvent by maceration. MTS cell viability assay was employed to determine the cytotoxic activity of the extract towards human leukemia Jurket cells and normal Peripheral Blood Mononuclear Cells (PBMCs) cells. The phytochemical composition of the extract was chemically characterized using HPLC. Flow cytometric studies (FACS) were conducted to explore the pro-apoptotic potential of the extract. Western blot studies were employed to identify the molecular targets involved in the induction of apoptosis. Results: The n-hexane extract showed selective cytotoxic activity towards Jurkat cells. FACS analysis indicated that the extract induced early and late apoptosis in Jurkat cells. Western blot studies revealed that the extract down-regulated the expression of DNMT1, SIRT1, and UHRF1 with a simultaneous up-regulation of the expression of p73 and caspases-3 proteins. HPLC characterization of the extract revealed the presence of phenolic compounds. Conclusion: Overall these findings demonstrate that the anticancer effects of a Conyza bonariensis extract towards human lymphoblastic leukemiais due to the modulation of the activity of multiple oncogenic and tumor suppressor proteins and that its phenolic content is involved are proposed to be responsible for these activities.

scholarly journals Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
M. R. Ricciardi ◽  
R. Licchetta ◽  
S. Mirabilii ◽  
M. Scarpari ◽  
A. Parroni ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Leukemic Cell ◽  
Bioactive Compound ◽  
Primary Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Leukemic Cell Lines

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

scholarly journals NET1 Enhances Proliferation and Chemoresistance in Acute Lymphoblastic Leukemia Cells

2019 ◽  
Vol 27 (8) ◽  
pp. 935-944 ◽  
Author(s):  
Hongbo Sun ◽  
Zhifu Zhang ◽  
Wei Luo ◽  
Junmin Liu ◽  
Ye Lou ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Western Blot ◽  
Colony Formation ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Luciferase Reporter ◽  
Tunel Staining

Acute lymphoblastic leukemia (ALL) is the most prevalent of pediatric cancers. Neuroepithelial cell-transforming 1 (NET1) has been associated with malignancy in a number of cancers, but the role of NET1 in ALL development is unclear. In the present study, we investigated the effect of NET1 gene in ALL cell proliferation and chemoresistance. We analyzed GEO microarray data comparing bone marrow expression profiles of pediatric B-cell ALL samples and those of age-matched controls. MTT and colony formation assays were performed to analyze cell proliferation. ELISA assays, Western blot analyses, and TUNEL staining were used to detect chemoresistance. We confirmed that NET1 was targeted by miR-206 using Western blot and luciferase reporter assays. We identified NET1 gene as one of the most significantly elevated genes in pediatric B-ALL. MTT and colony formation assays demonstrated that NET1 overexpression increases B-ALL cell proliferation in Nalm-6 cells. ELISA assays, Western blot analyses, and TUNEL staining showed that NET1 contributes to ALL cell doxorubicin resistance, whereas NET1 inhibition reduces resistance. Using the TargetScan database, we found that several microRNAs (miRNAs) were predicted to target NET1, including microRNA-206 (miR-206), which has been shown to regulate cancer development. To determine whether miR-206 targets NET1 in vitro, we transfected Nalm-6 cells with miR-206 or its inhibitor miR-206-in. Western blot assays showed that miR-206 inhibits NET1 expression and miR-206-in increases NET1 expression. Luciferase assays using wild-type or mutant 3′-untranslated region (3′-UTR) of NET1 confirmed these findings. We ultimately found that miR-206 inhibits B-ALL cell proliferation and chemoresistance induced by NET1. Taken together, our results provide the first evidence that NET1 enhances proliferation and chemoresistance in B-ALL cells and that miR-206 regulates these effects by targeting NET1. This study therefore not only contributes to a greater understanding of the molecular mechanisms underlying B-ALL progression but also opens the possibility for developing curative interventions.

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

scholarly journals The effect of serum on monocyte tissue factor generation

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 707-714 ◽  
Author(s):  
RL Edwards ◽  
D Perla
Keyword(s):  
T Cells ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
High Serum ◽  
Peripheral Blood Mononuclear ◽  
Serum Factors ◽  
Normal Peripheral Blood ◽  
Stimulatory Activity ◽  
Normal Hemostasis

Abstract Human monocytes generate the procoagulant tissue factor (MTF) following exposure to a variety of immune stimuli in vitro. The generation of MTF is modified by T cells, lymphokines, and immunoregulatory lipoproteins, and recent studies have shown that MTF can be activated in an immune- specific manner following exposure to antigen. We have examined the role of serum factors in the regulation of MTF generation. Low concentrations (less than 1%) of heat-inactivated normal human serum greatly enhanced MTF generation in cultures of normal peripheral blood mononuclear cells. The stimulatory effect was observed in cultures of both unstimulated cells and cells exposed to bacterial lipopolysaccharide. Stimulation was not observed at high serum concentrations (greater than 10%) and could not be explained by endotoxin contamination or activation of the assay system. Stimulatory activity was present in plasma and BaSO4-adsorbed plasma as well as autologous and allogeneic serum, was not abolished by removal of serum lipoproteins, and did not require the presence of T cells for its expression. Sera from 28 different normal volunteers were screened for stimulatory activity and demonstrated a wide variation in potency. These results suggest that a potent factor is present in sera that enhances the expression of MTF activity in vitro. This factor is distinct from previously described lipoprotein regulators and may play a role in the initiation of coagulation in both normal hemostasis and pathologic states.

scholarly journals Identification and characterization of a unique subpopulation (CALLA/CD10/negative) of human neutrophils manifesting a heightened chemotactic response to activated complement

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1624-1629
Author(s):  
RT McCormack ◽  
RD Nelson ◽  
DE Chenoweth ◽  
TW LeBien
Keyword(s):  
Lymphoblastic Leukemia ◽  
Low Frequency ◽  
Chemotactic Response ◽  
Human Neutrophils ◽  
Normal Peripheral Blood ◽  
Cd10 Expression ◽  
Blood Neutrophils

We have previously demonstrated that human neutrophils synthesize the common acute lymphoblastic leukemia antigen (CALLA/CD10). To determine whether CALLA/CD10-positive and -negative neutrophils have similar or distinct functional attributes, we sorted normal peripheral blood neutrophils for CALLA/CD10 expression and compared their chemotactic ability. Surprisingly, the low-frequency (approximately 5%), CALLA/CD10- negative neutrophils displayed a dramatically heightened chemotactic response to activated complement (C') that was (a) specific for C', (b) not observed with other minor subpopulations of neutrophils, (c) not due to previous activation in vivo or in vitro, and (d) apparently not due to an increase in C5a receptors. These results underscore the concept of neutrophil heterogeneity and prompt the hypothesis that CALLA/CD10-negative neutrophils may participate in an inflammatory response to trauma involving complement activation.

scholarly journals Phloridzin Docosahexaenoate (PZ-DHA) : A Novel Naturally Derived Drug Active in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5197-5197
Author(s):  
Niroshaathevi Arumuggam ◽  
Nicole Melong ◽  
Catherine K.L. Too ◽  
Jason N. Berman ◽  
H.P. Vasantha Rupasinghe
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Jurkat Cells ◽  
Interferon Α ◽  
Omega 3 ◽  
Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease that accounts for about 15% of pediatric and 25% of adult ALL. Although risk stratification has provided more tailored therapy and improved the overall survival of T-ALL patients, clinical challenges such as suboptimal drug responses, morbidity from drug toxicities, and drug resistance still exist. Plant polyphenols have therapeutic efficacy as pharmacological adjuvants to help overcome these challenges. They can be acylated with fatty acids to overcome issues concerning bioavailability, such as poor intestinal absorption and low metabolic stability. Phloridzin (PZ), a flavonoid found in apple peels, was acylated with an omega-3 fatty acid, docosahexaenoic acid (DHA), to generate a novel ester called phloridzin docosahexaenoate (PZ-DHA). The cytotoxic effect of PZ-DHA was studied in the human Jurkat T-ALL cell line. PZ-DHA significantly reduced the viability and cellular ATP levels of treated cells. PZ-DHA was found to selectively induce apoptosis in Jurkat cells, while sparing normal murine T-cells. Apoptosis was further confirmed by demonstrating the ability of PZ-DHA to induce morphological alterations, DNA fragmentation, caspase activation, and the release of intracellular lactate dehydrogenase. PZ-DHA also significantly inhibited cell division in Jurkat cells. Furthermore, interferon-α-induced phosphorylation of the transcription factor, STAT3, was downregulated following PZ-DHA treatment. The in vitro efficacy of PZ-DHA was recapitulated in vivo in an established zebrafish xenograft model, where the proliferation of transplanted Jurkat cells was inhibited when PZ-DHA was added to the embryo water. Overall, these findings provide evidence for PZ-DHA as a novel therapeutic agent with activity in T-ALL. Studies examining the effect of PZ-DHA on patient-derived ALL cells engrafted in zebrafish are currently underway. Disclosures No relevant conflicts of interest to declare.

BTK Inhibitors, Irrespective of ITK Inhibition, Increase Efficacy of a CD19/CD3 Bispecific Antibody in CLL

Blood ◽  
2021 ◽  
Author(s):  
Maissa Mhibik ◽  
Erika M. Gaglione ◽  
David Eik ◽  
Ellen K Kendall ◽  
Amy Blackburn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic Activity ◽  
Kinase Inhibitors ◽  
Bispecific Antibody ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Peripheral Blood Mononuclear

Bruton Tyrosine Kinase inhibitors (BTKis) are a preferred treatment for patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, while effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3 bispecific antibody (bsAb) that recruits autologous T cell cytotoxicity against CLL cells in vitro. Compared to observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits IL2 inducible T cell Kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive, and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared to that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including CTLA-4 and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

scholarly journals Identification of Core Gene Biomarkers in Patients with Diabetic Cardiomyopathy

2018 ◽  
Vol 2018 ◽  
pp. 1-15 ◽  
Author(s):  
Ning Li ◽  
Haiming Wu ◽  
Rongxin Geng ◽  
Qizhu Tang
Keyword(s):  
Western Blot ◽  
Diabetic Cardiomyopathy ◽  
Protein Level ◽  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Core Gene ◽  
Cardiomyocyte Hypertrophy ◽  
Diabetic Patients ◽  
Ppi Network

Diabetic cardiomyopathy (DCM) is a disorder of the myocardium in diabetic patients, which is one of the critical complications of diabetes giving rise to an increased mortality. However, the underlying mechanisms of DCM remain incompletely understood presently. This study was designed to screen the potential molecules and pathways implicated with DCM. GSE26887 involving 5 control individuals and 7 DCM patients was selected from the GEO database to identify the differentially expressed genes (DEGs). DAVID was applied to perform gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A protein-protein interaction (PPI) network was also constructed to visualize the interactions among these DEGs. To further validate significant genes and pathways, quantitative real-time PCR (qPCR) and Western blot were performed. A total of 236 DEGs were captured, including 134 upregulated and 102 downregulated genes. GO, KEGG, and the PPI network disclosed that inflammation, immune disorders, metabolic disturbance, and mitochondrial dysfunction were significantly enriched in the development of DCM. Notably, IL6 was an upregulated hub gene with the highest connectivity degree, suggesting that it may interact with a great many molecules and pathways. Meanwhile, SOCS3 was also one of the top 15 hub genes in the PPI network. Herein, we detected the protein level of STAT3 and SOCS3 in a mouse model with DCM. Western blot results showed that the protein level of SOCS3 was significantly lower while phosphorylated-STAT3 (P-STAT3) was activated in mice with DCM. In vitro results also uncovered the similar alterations of SOCS3 and P-STAT3 in cardiomyocytes and cardiac fibroblasts induced by high glucose (HG). However, overexpression of SOCS3 could significantly reverse HG-induced cardiomyocyte hypertrophy and collagen synthesis of cardiac fibroblasts. Taken together, our analysis unveiled potential biomarkers and molecular mechanisms in DCM, which could be helpful to the diagnosis and treatment of DCM.

scholarly journals In vitro and in vivo activity of topotecan against human B-lineage acute lymphoblastic leukemia cells

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2817-2828 ◽  
Author(s):  
FM Uckun ◽  
CF Stewart ◽  
G Reaman ◽  
LM Chelstrom ◽  
J Jin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Topoisomerase I ◽  
Drug Exposure ◽  
Lymphoblastic Leukemia ◽  
Severe Combined Immunodeficiency ◽  
Water Soluble ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
B Lineage

Topotecan [(S)-9-dimethylaminomethyl-10-hydroxycamptothecin hydrochloride; SK&F 104864-A, NSC 609699], a water soluble semisynthetic analogue of the alkaloid camptothecin, is a potent topoisomerase I inhibitor. Here we show that topotecan stabilizes topoisomerase I/DNA cleavable complexes in radiation-resistant human B-lineage acute lymphoblastic leukemia (ALL) cells, causes rapid apoptotic cell death despite high-level expression of bcl-2 protein, and inhibits ALL cell in vitro clonogenic growth in a dose-dependent fashion. Furthermore, topotecan elicited potent antileukemic activity in three different severe combined immunodeficiency (SCID) mouse models of human poor prognosis ALL and markedly improved event-free survival of SCID mice challenged with otherwise fatal doses of human leukemia cells at systemic drug exposure levels that can be easily achieved in children with leukemia.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

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