Dynamic Assessment of Apoptosis for In Vitro Design of Bortezomib Combination Therapies for Lymphoid Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2494-2494
Author(s):  
Lauren C. Wallis ◽  
Matthew J. Streetly ◽  
Rebecca Auer ◽  
John Gribben ◽  
Dean Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Growth Characteristics ◽  
Cytotoxic Agents ◽  
Alamar Blue ◽  
Apoptosis Induction ◽  
Flow Cytometric ◽  
Induction Of Apoptosis

Abstract Conventional techniques for assessing drug response and apoptosis induction rely on static assessment of cellular changes at predetermined time points (e.g. detection of exposed membrane phospholipids by Annexin V). The Kinetics of Optical Response assay (KOR) is a new technique that detects induction of apoptosis dynamically. It employs a spectrophotometric methodology to detect changes in optical density associated with membrane blebbing related to growth and death, allowing detection of apoptosis in real time. The KOR assay has already predicted the response to cytotoxic agents of AML cell lines and primary samples. This study uses the KOR assay in lymphoid malignancy and shows sensitivity to apoptosis induction by conventional and novel agents including bortezomib. The lymphoma cell line DOHH2 (t(14;18)), U266 (myeloma), K562 (CML) and primary CLL cells were used in this study with HL60 (AML) as a control. Cells were seeded in 96 well plates and treated with a variety of drugs alone or in combination (cytarabine, fludarabine, doxorubicin, daunorubicin, etoposide, melphalan, bortezomib) at multiple concentrations. Measurements were made at 5 min. intervals for up to 48 hrs and analysed using KORSoft™ software to generate apoptotic response curves. To validate this approach conventional techniques were used for comparison (Alamar Blue for cytotoxicity and flow cytometric analysis of cell cycle and apoptosis using propidium iodide and Annexin V staining respectively). The KOR assay can show changes in growth characteristics, induction of apoptosis and necrosis in response to drugs permitting a continuous analysis for maximum sensitivity (Smax). DOHH2 was found to be dose responsive to four of the drugs used, with the Smax for 10μM daunorubicin at 6 hours (48%), 1μM doxorubicin at 8 hours (38%), 100μM etoposide at 8 hours (52%), and minimally to 100μM cytarabine at 16 hours (21%). There was no effect from fludarabine. The addition of bortezomib increased Smax to 89% with etoposide and to a lesser degree with the other cytotoxic drugs. U266 showed a similar spectrum of results with greatest Smax with 100μM melphalan at 9 hours (57%) enhanced to 78% with the addition of bortezomib. There was minimal response to cytarabine and fludarabine. Parallel flow cytometric analysis using Annexin V and PI showed similar results to those from the KOR assay confirming the assessment of apoptosis to be valid. Cell cycle analysis showed an increased sub-G1 peak in keeping with apoptosis at times of Smax assessed by the KOR assay. The Alamar Blue cytotoxicity assay showed a dose dependent decrease in cell proliferation in response to increasing drug dose again paralleling other apoptosis measurements implying an apoptotic effect due to drug action and correlate well with those from the KOR assay. Primary CLL samples following CD19 selection were cultured with and without IL4 and exposed to the KOR assay with cytotoxics and bortezomib. Culture with IL4 alone gave good growth characteristics and revealed the combination of etoposide and bortezomib to provide the best induction of apoptosis (Smax 82%) compared to etoposide (26%) or bortezomib (32%) alone. The KOR assay is a microtitre approach to the assessment in real time of apoptosis. This study suggests the combination of bortezomib and etoposide is effective for lymphoma. Such approaches can accelerate the development of effective clinical trials.

The Inhibitory Receptor FcgRIIB Is Overexpressed, and Its Ligation by Anti- FcgRIIB Antibodies Suppresses IgM Production and Induces Apoptosis in Waldenstrom's Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3736-3736 ◽  
Author(s):  
Guang Yang ◽  
Ping Gong ◽  
Lian Xu ◽  
Zachary Hunter ◽  
Jenny Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Real Time Pcr ◽  
Flow Cytometric Analysis ◽  
Cross Linking ◽  
Inhibitory Receptor ◽  
Downstream Signaling ◽  
Flow Cytometric ◽  
Induction Of Apoptosis ◽  
Waldenström's Macroglobulinemia

Abstract Abstract 3736 Poster Board III-672 Introduction Waldenstrom's macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by overproduction of a monoclonal IgM paraprotein which can produce morbidity including hyperviscosity, as well as autoimmune related neuropathy, hemolysis, and thrombocytopenia. Therefore approaches aimed at both suppressing IgM production, as well as selectively inducing apoptosis of WM cells represent an ideal treatment strategy for WM. FcgRIIB is an inhibitory receptor that is expressed on B-cells, and whose expression we recently identified as highly over-expressed in WM. Importantly, FcgRIIB possesses an immunoreceptor tyrosine-based inhibitory motif (ITIM), and which becomes phosphorylated at Tyr 292 upon activation, and is then followed by inhibition of BCR signaling and induction of apoptosis. We therefore validated the expression of this receptor in WM, and examined the impact of its ligation on tumor cell killing, IgM secretion and downstream signaling events in WM cells. Patients and Methods Bone marrow lymphoplasmacytic cells (LPC) from 12 WM patients which were sorted for CD19+ and CD138+, and BCWM.1 WM cells were subjected to real-time PCR and flow cytometric analysis. Cells were then subjected to co-culture with anti- FcgRIIB (AT10, 7.3) or control antibodies for 24-48 hours, and their effects on survival, IgM production and downstream signaling were assessed. Results Real-time PCR and flow-cytometric analysis demonstrated strong expression of FcgRIIB in WM patient bone marrow CD19+ and CD138+ cells, thus confirming our recent microarray results. Importantly, the expression of FcgRIIB in WM LPC correlated with the memory B-cell marker CD27. Anti-FcgRIIB antibody treatment dramatically reduced constitutive, and/or IL-6 induced IgM production in CD19+ and CD138+ sorted primary WM LPC, as well as CD32hiCD138hi BCWM.1 cells. This effect was observed in some experiments at an early time point that had not effected survival. Among primary CD138+ WM LPC and CD32hiCD138hi expressing BCWM.1 cells, treatment with anti-FcgRIIB antibodies for 48 hours led to increased apoptosis in 10 of 12 patients, as assessed by Annexin V and PI staining which occurred despite blockade with a pan-caspase inhibitor, and was even more pronounced when anti-FcgRIIB antibodies were cross-linked. Western blot analysis revealed that treatment of CD32hiCD138hi expressing BCWM.1 cells with cross-linked anti- FcgRIIB antibodies led to phosphorylation of Tyr 292 of the FcgRIIB ITIM which was not observed in the absence of cross-linking. Binding of FcgRIIB by both the AT10 and 7.3 antibodies resulted in dephosphorylation of Akt, which was further reduced in the presence of cross-linking. Coincident with the above, the pro-apoptotic molecule JNK also underwent phosphorylation in the presence of anti-FcgRIIB binding. Conclusion Taken together, these studies validate our previous microarray data by showing that the inhibitory receptor FcgRIIB is strongly expressed on LPC from WM patients, and ligation thereof leads to suppression of IgM production and induction of apoptosis thereby identifying FcgRIIB as a novel therapeutic target in WM. Disclosures: No relevant conflicts of interest to declare.

Novel 1,3,4-Triaryl Pyrazoles: Synthesis, QSAR Studies and Cytotoxicity against Breast Cancer

2019 ◽  
Vol 19 (7) ◽  
pp. 948-959 ◽  
Author(s):  
Magda M.F. Ismail ◽  
Amel M. Farrag ◽  
Marwa F. Harras
Keyword(s):  
Cell Cycle ◽  
Cytotoxic Activity ◽  
Flow Cytometric Analysis ◽  
Structural Features ◽  
Pharmacophore Modeling ◽  
Cytotoxic Agents ◽  
Pyrazole Derivatives ◽  
Mcf7 Cells ◽  
Qsar Studies ◽  
Induction Of Apoptosis

Background:The existence of drug-resistance and lack of selectivity encourages scientists to search for novel and more selective cytotoxic agents.Objective:In this work, novel 1,3,4-triarylpyrazole derivatives were synthesized to study their cytotoxicity on MCF7 (human breast Cell Line). In addition, QSAR studies were performed to show the relation between the cytotoxic activity and the structural features of our new synthesized pyrazole derivatives.Methods:Pyrazole-4-carbaldehyde derivative 3 was utilized as a starting material for the preparation of the new pyarazole derivatives. These target compounds were screened for their cytotoxic activity against MCF-7 followed by study cell cycle of the most active compounds. Finally, pharmacophore modeling and QSAR Studies was carried out.Results:Among these compounds; 5d and 8b showed the highest anti-proliferative activity (IC50 = 4.9 and 2.11 µM, respectively). Flow cytometric analysis showed that, compounds 5d and 8b arrested the cell cycle in addition to induction of apoptosis in MCF7 cells. Moreover, their stimulation effect on caspases 3/7 was examined to explore their mechanism of induction of apoptosis and the results showed that their proapoptotic activity could be due to the activation of caspases 3/7.Conclusion:Pyrazole derivatives 5d and 8b displayed potent bioactivities, indicating that these compounds could be considered as a new lead for more investigation in the future

scholarly journals Naringenin inhibits human breast cancer cells (MDA-MB-231) by inducing programmed cell death, caspase stimulation, G2/M phase cell cycle arrest and suppresses cancer metastasis

2021 ◽  
Vol 67 (2) ◽  
pp. 8-13
Author(s):  
Zhaozhen Qi ◽  
Shuangxi Kong ◽  
Shunyu Zhao ◽  
Qiu Tang
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Apoptotic Cell ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Cycle Arrest ◽  
Flow Cytometric ◽  
M Phase

The current study was designed to unveil the anticancer effects of naringenin against breast cancer MDA-MB-231 cells. Cytotoxic effects were estimated via MTT viability assay. Clonogenic assay was performed to assess clonogenic potential of MDA-MB-231 cells. Apoptosis was examined via AO/EB staining, quantified via annexin V/PI staining and western blotting was performed to monitor apoptosis allied protein expressions. Cell cycle was analyzed through flow cytometric analysis. Transwell chambers assay was executed for determination of cell migration and cell invasion tendency of MDA-MB-231 breast cancer cells. Results indicated significant anticancer potential of naringenin drug against MDA-MB-231 cells. On evaluation of cell proliferation rate of breast cancer cells by MTT assay, it was observed that naringenin inhibited proliferation rate in dose as well as time dependent manner. AO/EB staining assay revealed potential morphological changes indicating apoptotic cell death. Annexin V/PI staining assay revealed increased apoptotic cell percentage with increased drug doses. The apoptosis inducing potential of naringenin drug was observed to be mediated via caspase activation. Flow cytometric analysis predicted cell cycle arrest at G2/M phase of cell cycle. Further cell migration as well as cell invasion tendency of MDA-MB-231 cells was reduced to minimum upon application of naringenin drug.

Triggering of CD44 by Ultra-Low-Molecular-Weight Hyaluronan Induces Cell Death by Autophagy Via Endoplasmic Reticulum Stress in Acute Lymphoblastic Leukemia with MLL Gene Rearrangements

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1361-1361 ◽  
Author(s):  
Shin Kasai ◽  
Yoshiyuki Furuichi ◽  
Norie Ando ◽  
Keiko Kagami ◽  
Kumiko Goi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Cell Death ◽  
Er Stress ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Low Molecular Weight ◽  
Gene Rearrangements ◽  
Flow Cytometric ◽  
Induction Of Apoptosis

Abstract Abstract 1361 Acute lymphoblastic leukemia (ALL) with MLL gene rearrangements (MLL+ALL) is chemoresistant and its prognosis is still dismal. MLL+ALL shows a unique gene profile clearly distinguishable from other types of ALL and AML, and CD44 is known as one of specific molecules expressed very highly in MLL+ALL. CD44, a transmembrane glycoprotein recently attracting an attention as a cancer stem cell marker for several types of cancers, is expressed on a variety of cells, and has the specific binding sites for hyaluronic acid (HA, formally hyaluronan). High-molecular-weight (HMW)-HA which exists as extracellular matrix in tissues is incorporated into cells in a CD44-dependent manner, and then is secreted from cells as low-molecular-weight (LMW) to ultra-low-molecular-weight (ULMW)-HA in normal and pathological conditions. The purpose of this study is to investigate the biological events elicited after ligand stimulation of CD44 by using distinct molecular sizes of HA (2.5mg/ml) in MLL+ALL. When cultured in the presence of ULMW-HA (6.4kDa), but not of LMW-HA (31kDa) or HMW-HA (980kDa), thymidine uptakes of cell lines expressing CD44 at very high levels (KOPB-26 and YACL-95) were gradually inhibited in the course of culture from day3 through day5 (greater than 90% decrease at day5), although those expressing CD44 at very low levels (KOCL58, KOCL69) were largely unaffected. Cell cycle analysis of KOPB-26 and YACL-95 using propidium iodide (PI) staining after ULMW-HA stimulation revealed no increase in G0/G1 and G2/M phases and subdiploid population, suggesting that a marked inhibition of thymidine uptakes is neither due to cell cycle arrest nor induction of apoptosis. Dye exclusion test showed a marked decrease in viability at days 3–5 after ULMW-HA stimulation, and flow cytometric analysis showed a gradual increase in the low FSC/high SSC population doubly labeled by Annexin V and PI. However, cytospine smears revealed a massive cell death lacking features characteristic of apoptosis. In addition, pan-caspase inhibitor Z-VAD did not block cell death and cleaved caspase 3 was not detected on flow cytometric analysis after ULMW-HA stimulation, resulting in the conclusion that ULMW-HA-induced cell death is not elicited by induction of apoptosis. Although levels of high-mobility group protein B1 (HMGB1) usually used as a necrosis marker was progressively elevated in culture media after ULMW-HA stimulation, the necrosis inhibitor Necrostatin-1 did not block the ULMW-HA-induced cell death. Finally, electromicroscopic examination was performed and revealed a marked extension of endoplasmic reticulum (ER) and an increase in autophagsomes, and the autophagy inhibitor 3-methyladenine specifically suppressed cell death, indicating that ULMW-HA strongly induces ER stress leading to cell death by autophagy. Changes in ER stress and autophagy-associated molecules after ULMW-HA stimulation were also demonstrated. In conclusion, triggering of CD44 by ULMW-HA stimulation strongly elicits cell death by autophagy via ER stress in chemoresistant MLL+ALL. CD44-targeted therapy by ULMW-HA possibly becomes a useful strategy not only for MLL+ALL but also for CD44-high hematological and non-hematological tumors particularly for those in which the cancer stem cell population is highly positive for CD44. Disclosures: No relevant conflicts of interest to declare.

TGFβ1 Induces MLL-Rearranged Acute Lymphoblastic Leukemia into Chemoresistant Quiescence in a Coordinated Manner with FLT3 Ligand

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3972-3972
Author(s):  
Minori Tamai ◽  
Yoshiyuki Furuichi ◽  
Shin Kasai ◽  
Norie Ando ◽  
Kumiko Goi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Marrow Stromal Cells ◽  
Mrna Expression Level ◽  
Flt3 Ligand ◽  
Flow Cytometric

Abstract Background Acute lymphoblastic leukemias with rearrangements of mixed-lineage leukemia gene (MLL+ALL) frequently develop in infants and have a dismal prognosis. The refractory nature of this leukemia is known to be associated with persistence of high levels of minimal residual disease (MRD). We previously reported that FLT3 ligand (FL) stimulation of MLL+ALL cells, which express FLT3 at high levels, induced quiescence resistant to anti-leukemic agents, to the process of which up-regulation of p27, one of cyclin-dependent kinase inhibitors (CDKIs), was closely attributable. Because a vast amount of FL is secreted from bone marrow stromal cells, it was postulated that this FLT3/FL interaction should contribute, at least in part, to the high levels of MRD in MLL+ALL (Furuichi et. al. Cancer Res 2007). Of interest, in B-cell precursor ALLs other than MLL+ALL, FL stimulated their proliferation. Therefore, we performed the microarray analysis using the MLL+ALL cell line and the ETV6-RUNX1-positive cell line for comparing changes in the gene expression profiles at 24h after FL stimulation, and picked up the transforming growth factor β1 (TGFβ1) as a gene whose mRNA level was conversely regulated between two cell lines. TGFβ1 is a growth inhibitory cytokine for some cancer cells and known to have a potential to induce hematopoietic stem cell quiescence. In the present study, we examined the effects of TGFβ1 and/or FL on MLL+ALL cells particularly in terms of induction of quiescence and chemosensitivity. Materials and Methods The MLL+ALL cell line KOCL58 (MLL-AF4+) was used throughout the study to examine TGFβ1 mRNA, cell cycle progression, induction of apoptosis, and changes in chemosensitivity in the presence of TGFβ1 and/or FL. Results 1. We first examined changes in TGFβ1 mRNA after FL (20ng/ml) stimulation by real time RT-PCR in KOCL58 cells, and confirmed that TGFβ1 mRNA expression level was significantly increased (1.5 fold at 24h). Of note, when the cells were stimulated with FL and TGFβ1 (10ng/ml), TGFβ1 mRNA expression level was further increased (3.5 fold) between 7 and 24h. 2. We next evaluated the effect of TGFβ1 on proliferation. When KOCL58 cells were incubated with a low concentration of TGFβ1 (0.33ng/ml) in the presence or absence of FL (20ng/m), flow cytometric analysis using the BrdU/PI double staining showed that the G0/G1 population (35.2%; before stimulation) was increased in the presence of TGFβ1 (48.4%) or FL (53.0%), and further in the presence of TGFβ1+FL (75.1%). We also examined expression levels of cell cycle regulating molecules, and found that p27 was significantly increased in the presence of FL and further in the presence of TGFβ1+FL. This p27 up-regulation was due to post-transcriptional mechanism, because p27 mRNA remained unchanged but the half-life of p27 protein was elongated in the presence of FL and/or TGFβ1. 3. To elucidate whether TGFβ1 affects the sensitivity of MLL+ALL cells to a cell cycle dependent anti-leukemic agent AraC, we pre-incubated KOCL58 cells in the presence of TGFβ1 and/or FL for 72h and then exposed to AraC (500nM) for 24h. Flow cytometric analysis using FITC-conjugated Annexin V demonstrated that the Annexin V-negative viable population (%) was decreased by AraC exposure (25.6±1.4%), but it was significantly restored by pre-incubation with FL (39.4±1.7%, P<0.0005) and further by pre-incubation with TGFβ1+FL (49.3±5.9%, P<0.003), although not significantly different by pre-incubation with TGFβ1 alone. Flow cytometric analysis using anti-active caspase 3 antibody also demonstrated that the positive population was increased to 50.8% by AraC exposure, but it was decreased by pre-incubation with either TGFβ1 (27.5%) or FL (24.8%), and further by pre-incubation with TGFβ1+FL (10.2%) (Figure). 4.We performed chmosensitivity experiments to AraC (800nM) using KOCL58 cells adhering to bone marrow stromal cells, which secrete FL and TGFβ1, in the presence of FLT3 inhibitor (CEP701, 5nM) and/or TGFβ-receptor 1 inhibitor (HTS, 5mM), and found that KOCL58 cells adhering to stromal cells showed a marked resistance to AraC (60% increase in Annexin V-negative population), but this restoring effect was partially (~20%) alleviated by each of inhibitors. [Discussion] TGFβ1 should be involved in the development of MRD in MLL+ALL in synergy with FL, and a combined use of inhibitors against FLT3 and TGFβ1 might be effective for eradicating MRD in MLL+ALL patients. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Synthesis of New Dihydroquinopimaric Acid Analogs with Nitrile Groups as Apoptosis-Inducing Anticancer Agents

2019 ◽  
Vol 19 (9) ◽  
pp. 1172-1183 ◽  
Author(s):  
Elena V. Tretyakova ◽  
Elena V. Salimova ◽  
Lyudmila V. Parfenova ◽  
Milyausha M. Yunusbaeva ◽  
Lilya U. Dzhemileva ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Antitumor Activity ◽  
Tumor Cell ◽  
Anticancer Agents ◽  
Flow Cytometric Analysis ◽  
Acid Methyl Ester ◽  
Annexin V ◽  
Cytotoxic Agents ◽  
Dependent Manner ◽  
Triethylbenzylammonium Chloride

Background: Cyan-containing compounds are of great interest as potential anticancer agents. Terpenoids can severe as a natural matrix for the development of promising derivatives with antitumor activity. Methods: The 2-cyanoethoxy methyl dihydroquinopimarate derivatives (5-9) were synthesized by the reaction of the intermediates (1-4) with acrylonitrile in the presence of alkali (30% KOH solution) using triethylbenzylammonium chloride. The cytotoxicity evaluation was carried out according to the National Cancer Institute (NCI) Protocol, while apoptosis was studied by flow cytometric analysis of Annexin V and 7-aminoactinomycin D staining and cell cycle was analyzed using the method of propidium iodide staining. Results: Synthesis of new dihydroquinopimaric acid derivatives with nitrile groups was carried out. The obtained cyanoethyl derivatives were converted into tetrazole, amine, oxadiazole and amidoxime analogs. The primary screening for antitumor activity showed the highest cytotoxic potency of the cyanoethyl-substituted compounds. The introduction of cyanoethyl groups at C-1, C-4 and C-1, C-4, C-20 positions of dihydroquinopimaric acid methyl ester provided antiproliferative effect towards the Jurkat, K562, U937, and HeLa tumor cell cultures (CC50=0.045-0.154µM). These nitrile derivatives are effective inducers of tumor cell apoptosis affecting the S and G2 phases of the cell cycle in a dose-dependent manner. Conclusion: The cyanoethyl analogs of dihydroquinopimaric acid reported herein are apoptosis inducers and cytotoxic agents. These findings will be useful for the further design of more potent cytotoxic agents based on natural terpenes.

Design, Synthesis, Anti-Proliferative Evaluation and Cell Cycle Analysis of Hybrid 2-Quinolones

2019 ◽  
Vol 19 (9) ◽  
pp. 1132-1140
Author(s):  
Heba A.E. Mohamed ◽  
Hossa F. Al-Shareef
Keyword(s):  
Cell Cycle ◽  
Anticancer Agents ◽  
Biological Activities ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Cytotoxic Activities ◽  
Significant Group ◽  
Design Synthesis ◽  
Standard Drug ◽  
Wide Range

Background: Quinolones are a significant group of nitrogen heterocyclic compounds that exist in therapeutic agents, alkaloids, and synthetic small molecules that have important biological activities. A wide range of quinolones have been used as antituberculosis, antibacterial, anti-malarial, antifungal, anticonvulsant, anticancer agents and urease inhibitors. Methods: Ethyl 3,3-disubstituted-2-cyano propionates containing hybride quinolones derivatives were synthesized by the reaction of 1-amino-7-hydroxy-4-methylquinolin-2(1H)-one and its dibromo derivative with α, β-unsaturated carbonyl in ethanol. Results: A novel series of hybrid 2-quinolone derivatives was designed and synthesized. The compounds structures were confirmed using different spectroscopic methods and elemental analysis. The cytotoxic activities of all the compounds were assessed against HepG2 cell line in comparison with doxorubicin as a standard drug. Conclusion: Most compounds revealed superior anti-proliferative activity than the standard. Compound 4b, is the most active compound (IC50 = 0.39mM) compared with doxorubicin (IC50 = 9.23mM). DNA flow cytometric analysis of compound 4b showed cell cycle arrest at G2/M phase with a concomitant increase of cells in apoptotic phase. Dual annexin-V/ propidium iodide staining assay of compound 4b revealed that the selected candidate increased the apoptosis of HepG-2 cells more than control.

Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 210-215 ◽  
Author(s):  
Mona Diab-Assaf ◽  
Josiane Semaan ◽  
Marwan El-Sabban ◽  
Soad K. Al Jaouni ◽  
Rania Azar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Dependent Manner ◽  
Apoptosis Induction ◽  
Inhibition Of Proliferation ◽  
Human T Cell

Introduction: Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. Objective: The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. Materials and Methods: Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. Results: At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. Conclusion: Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

Elucidation of the Chemopreventive Role of Stigmasterol Against Jab1 in Gall Bladder Carcinoma

2019 ◽  
Vol 19 (6) ◽  
pp. 826-837 ◽  
Author(s):  
Pratibha Pandey ◽  
Preeti Bajpai ◽  
Mohammad H. Siddiqui ◽  
Uzma Sayyed ◽  
Rohit Tiwari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bladder Cancer ◽  
Gall Bladder ◽  
Cancer Cells ◽  
Caspase 3 ◽  
Annexin V ◽  
Gall Bladder Cancer ◽  
Apoptosis Induction ◽  
Bladder Cancer Cells ◽  
Hoechst Staining

Background:Plant sterols have proven a potent anti-proliferative and apoptosis inducing agent against several carcinomas including breast and prostate cancers. Jab1 has been reported to be involved in the progression of numerous carcinomas. However, antiproliferative effects of sterols against Jab1 in gall bladder cancer have not been explored yet.Objective:In the current study, we elucidated the mechanism of action of stigmasterol regarding apoptosis induction mediated via downregulation of Jab1 protein in human gall bladder cancer cells.Methods:In our study, we performed MTT and Trypan blue assay to assess the effect of stigmasterol on cell proliferation. In addition, RT-PCR and western blotting were performed to identify the effect of stigmasterol on Jab1 and p27 expression in human gall bladder cancer cells. We further performed cell cycle, Caspase-3, Hoechst and FITC-Annexin V analysis, to confirm the apoptosis induction in stigmasterol treated human gall bladder cancer cells.Results:Our results clearly indicated that stigmasterol has up-regulated the p27 expression and down-regulated Jab1 gene. These modulations of genes might occur via mitochondrial apoptosis signaling pathway. Caspase-3 gets activated with the apoptotic induction. Increase in apoptotic cells and DNA were confirmed through annexin V staining, Hoechst staining, and cell cycle analysis.Conclusion:Thus, these results strongly suggest that stigmasterol has the potential to be considered as an anticancerous therapeutic agent against Jab1 in gall bladder cancer.

scholarly journals The Olive Leaves Extract Has Anti-Tumor Effects against Neuroblastoma through Inhibition of Cell Proliferation and Induction of Apoptosis

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2178
Author(s):  
Fabio Morandi ◽  
Veronica Bensa ◽  
Enzo Calarco ◽  
Fabio Pastorino ◽  
Patrizia Perri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Cycle Progression ◽  
Treatment Strategies ◽  
Annexin V ◽  
Dependent Manner ◽  
Induction Of Apoptosis ◽  
Dose Dependent

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.

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