A Tropical Lichen, Dirinaria consimilis Selectively Induces Apoptosis in MCF-7 Cells through the Regulation of p53 and Caspase-Cascade Pathway

2020 ◽  
Vol 20 (10) ◽  
pp. 1173-1187
Author(s):  
Anil K. Shendge ◽  
Sourav Panja ◽  
Tapasree Basu ◽  
Nripendranath Mandal
Keyword(s):  
Western Blotting ◽  
Bioactive Compounds ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Phytochemical Analysis ◽  
Crude Mortality Rate ◽  
Crude Mortality ◽  
Caspase Cascade ◽  
Mcf 7

Background: Breast cancer is the most leading cause of death, with 49.9% of crude incidence rate and 12.9% of crude mortality rate. Natural resources have been extensively used throughout history for better and safer treatment against various diseases. Objectives: The present study was aimed to investigate the antioxidant and anticancer potential of a tropical lichen Dirinaria consimilis (DCME) and its phytochemical analysis. Methods: The DCME was preliminarily evaluated for ROS, and RNS scavenging potential. Furthermore, DCME was evaluated for in vitro anticancer activity through cell proliferation assay, cell cycle analysis, annexin V/PI staining, morphological analysis, and western blotting study. Finally, the HPLC and LC-MS analyses were done to identify probable bioactive compounds. Results: The in vitro antioxidant studies showed promising ROS, and RNS scavenging potential of DCME. Moreover, the in vitro antiproliferative study bared the cytotoxic nature of DCME towards MCF-7 (IC50 - 98.58 ± 6.82μg/mL) and non-toxic towards WI-38 (IC50 - 685.85 ± 19.51μg/mL). Furthermore, the flow-cytometric analysis revealed the increase in sub G1 population as well as early apoptotic populations dose-dependently. The results from confocal microscopy showed the DNA fragmentation in MCF-7 upon DCME treatment. Finally, the western blotting study revealed the induction of tumor suppressor protein, p53, which results in increasing the Bax/Bcl-2 ratio and activation of caspase-cascade pathways. Conclusion: The activation of caspase-3, -8, -9 and PARP degradation led us to conclude that DCME induces apoptosis in MCF-7 through both intrinsic and extrinsic mechanisms. The LC-MS analysis showed the presence of various bioactive compounds.

Design, Synthesis and In Vitro Anti-Cancer Evaluation of Novel Derivatives of 2-(2-Methyl-1,5-diaryl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-3- yl)acetamide

2020 ◽  
Vol 16 (3) ◽  
pp. 340-349
Author(s):  
Ebrahim S. Moghadam ◽  
Farhad Saravani ◽  
Ernest Hamel ◽  
Zahra Shahsavari ◽  
Mohsen Alipour ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Peripheral Neuropathy ◽  
Cell Line ◽  
Normal Cell ◽  
Caspase 3 ◽  
Annexin V ◽  
Normal Cell Line ◽  
Anti Cancer ◽  
Mcf 7

Objective: Several anti-tubulin agents were introduced for the cancer treatment so far. Despite successes in the treatment of cancer, these agents cause toxic side effects, including peripheral neuropathy. Comparing anti-tubulin agents, indibulin seemed to cause minimal peripheral neuropathy, but its poor aqueous solubility and other potential clinical problems have led to its remaining in a preclinical stage. Methods: Herein, indibulin analogues were synthesized and evaluated for their in vitro anti-cancer activity using MTT assay (on the MCF-7, T47-D, MDA-MB231 and NIH-3T3 cell lines), annexin V/PI staining assay, cell cycle analysis, anti-tubulin assay and caspase 3/7 activation assay. Results: One of the compounds, 4a, showed good anti-proliferative activity against MCF-7 cells (IC50: 7.5 μM) and low toxicity on a normal cell line (IC50 > 100 μM). All of the tested compounds showed lower cytotoxicity on normal cell line in comparison to reference compound, indibulin. In the annexin V/PI staining assay, induction of apoptosis in the MCF-7 cell line was observed. Cell cycle analysis illustrated an increasing proportion of cells in the sub-G-1 phase, consistent with an increasing proportion of apoptotic cells. No increase in G2/M cells was observed, consistent with the absence of anti-tubulin activity. A caspase 3/7 assay protocol showed that apoptosis induction by more potent compounds was due to activation of caspase 3. Conclusion: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of current scaffold would be beneficial.

Derivatives of Deoxypodophyllotoxin Induce Apoptosis Through Bcl-2/Bax Proteins Expression

2020 ◽  
Vol 20 ◽  
Author(s):  
Ya-Nan Li ◽  
Ni Ning ◽  
Lei Song ◽  
Yun Geng ◽  
Jun-Ting Fan ◽  
...  
Keyword(s):  
Annexin V ◽  
Mtt Method ◽  
Anthriscus Sylvestris ◽  
Anti Tumor Activity ◽  
Derivatives Of ◽  
Toxicity In Vitro ◽  
Ht 29 ◽  
Mcf 7

Background: Deoxypodophyllotoxin, isolated from theTraditional Chinese Medicine Anthriscus sylvestris, is well-known because of its significant antitumor activity with strong toxicity in vitro and in vivo. Objective: In this article, we synthesized a series of deoxypodophyllotoxin derivatives, and evaluated their antitumor effectiveness.Methods:The anti tumor activity of deoxypodophyllotoxin derivatives was investigated by the MTT method. Apoptosis percentage was measured by flow cytometer analysis using Annexin-V-FITC. Results: The derivatives revealed obvious cytotoxicity in the MTT assay by decreasing the number of late cancer cells. The decrease of Bcl-2/Bax could be observed in MCF-7, HepG2, HT-29 andMG-63 using Annexin V-FITC. The ratio of Bcl-2/Bax in the administration group was decreased, which was determined by the ELISA kit. Conclusion: The derivatives of deoxypodophyllotoxin could induce apoptosis in tumor cell lines by influencing Bcl-2/Bax.

P13.08 Novel Thioridazine derivates: Antiproliferative and apoptosis-inducing activity on glioblastoma cells in vitro

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii34-ii34
Author(s):  
S G Schwab ◽  
K Sarnow ◽  
E Alme ◽  
R Goldbrunner ◽  
H Bjørsvik ◽  
...  
Keyword(s):  
Imaging System ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Therapeutic Effects ◽  
Cell Viability Assay ◽  
Selective Cytotoxicity ◽  
Viability Assay

Abstract BACKGROUND Although withdrawn from the market due to cardiotoxicity, we have shown that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of the presented project was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. MATERIAL AND METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay as well as a real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p<0,0001, U251 p<0,0001, LN18 p=0,0004) as well as on glioma stem cells (GSC) (P3 p<0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in the GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We have developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

FLOW CYTOMETRIC ANALYSIS OF APOPTOTIC LYMPHOCYTES WITH ANNEXIN V AND DiOC6 AFTER IN VITRO IRRADIATION

1996 ◽  
Vol 24 (4) ◽  
pp. 607S-607S
Author(s):  
K. Hertveldt ◽  
J. Philippé ◽  
H. Thierens ◽  
A. Vral ◽  
L. De Ridder
Keyword(s):  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Flow Cytometric

Impaired Ca++ mobilization in rituximab-resistant cells (RRCL) is associated with changes in the structure of CD20 antigen, down-regulation of Bax/Bak pro-apoptotic proteins and up-regulation of the endoplasmic reticulum (ER) Ca++ pump protein SERCA-3

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2516-2516 ◽  
Author(s):  
F. J. Hernandez-Ilizaliturri ◽  
H. Kaur ◽  
A. Bhinder ◽  
S. Olejniczak ◽  
J. Knight ◽  
...  
Keyword(s):  
Western Blotting ◽  
Flow Cytometric Analysis ◽  
Acetoxymethyl Ester ◽  
Down Regulation ◽  
Raji Cells ◽  
External Domain ◽  
Cd20 Expression ◽  
Membrane Reorganization ◽  
Apoptotic Proteins

2516 Ca++ mobilization leading to apoptosis has been observed following rituximab biding to CD20 in lymphoma cells. Extending rituximab (R) exposure (e.g. as via R maintenance regimens) could potentially accelerate the emergence of resistance to rituximab. To define the molecular basis for rituximab resistance we developed several RRCL and demonstrated changes in CD20 expression/structure and membrane reorganization following rituximab therapy. Specifically, changes in the N-terminal and the C-terminal region of the internal domain of CD20 were observed in RRCL as determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. In our current work we evaluated the effects that CD20 structure/expression changes have upon Ca++ mobilization. Extracellular and intracellular Ca++ mobilization was measured by flow cytometric analysis using FLUO-3 AM (acetoxymethyl ester). Optimization of the Ca++ indicator and calibration curves were performed for each cell line. Raji and RRCL were labeled under optimal conditions with FLUO-3 AM/Pluronic Acid F-127. Subsequently, cells were then re-suspended in HANKS media with or without Ca++ and exposed to rituximab or isotype (10μg/ml) ± human serum (25%). Surface CD20 expression was similar between Raji cells and RRCL. Expression of pro- or anti-apoptotic proteins and Ca++ regulatory proteins SERCA3, SERCA2 and Calreticulin was also determined by western blotting. In vitro exposure of Raji cells to rituximab + HS resulted in Ca++ mobilization even in Ca++ depleted media. Notably, Ca++ mobilization was impaired in RRCL when compared to Raji parental cells. In addition, down-regulation of Bax/Bak and up-regulation of SERCA3 was demonstrated in RRCL. Our data suggest that the acquirement of rituximab resistance is associated with changes in the intracellular domain of CD20 and in Ca++ regulator/pro-apoptotic proteins (SERCA3 and Bax/Bak) resulting in a decrease in the intracellular mobilization of Ca++. No significant financial relationships to disclose.

Combination therapy in childhood leukaemia: in vitro studies of thiopurines and inhibitors of purine metabolism on apoptosis

2003 ◽  
Vol 40 (1) ◽  
pp. 70-74 ◽  
Author(s):  
Ronney A. De Abreu ◽  
Robert C. Trueworthy ◽  
André B.P. van Kuilenburg ◽  
Trude M. Vogels-Mentink ◽  
Lambert H.J. Lambooy ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Childhood Leukaemia ◽  
Therapeutic Success ◽  
Drug Concentrations ◽  
Sequential Administration ◽  
Induction Of Apoptosis ◽  
Chemotherapy Agents

Background: Methotrexate (MTX) followed by 6-mercaptopurine (6MP) is one of the best known combinations for the treatment of childhood acute lymphoblastic leukaemia. Tiazofurin (TF) and 6-thioguanine (TG) are also used as chemotherapy agents in the treatment of malignancies. We have examined the induction of apoptosis by combinations of these drugs to gain more insights into their efficacy in the treatment of malignancies. Methods: The induction of apoptosis was examined in Molt-4, a human malignant acute lymphoblastic T-cell line. The cells were exposed to increasing drug concentrations at various exposure times. Annexin V/FITC and propidium iodide (PI) were used as markers for apoptosis and cell death. Annexin V/FITC positive and PI positive cells were detected by flow-cytometric analysis. Results: Sequential 24-h exposure with MTX (0·005-0·02 µmol) followed by 6MP (1-10 µmol) and 24-h exposure with TF (5·20 µmol) followed by TG (0·5-2 µmol) showed a more than additive induction of apoptosis compared with single-drug exposure. Simultaneous administration of the drugs does not show an additive effect on apoptosis. Conclusions: The results of this study indicate that sequential administration of MTX before 6MP and of TF before TG may be essential for therapeutic success in the treatment of leukaemia.

scholarly journals Corrigendum to: Screening of Ipomoea tuba Leaf Extract for Identification of Bioactive Compounds and Evaluation of Its in vitro Antiproliferative Activity Against MCF-7 and HeLa Cells (Published: Food Technol. Biotechnol. 58 (1) 71-75 (2020) https://doi.org/10.17113/ftb.58.01.20.6351)

2020 ◽  
Vol 58 (3) ◽  
pp. 356-356
Author(s):  
Thirupati Chinna Venkateswarulu ◽  
Gaddam Eswaraiah ◽  
Srirrama Krupanidhi ◽  
Karlapudi Abraham Peele ◽  
Indira Mikkili ◽  
...  
Keyword(s):  
Cell Lines ◽  
Bioactive Compounds ◽  
Hela Cells ◽  
Antiproliferative Activity ◽  
Leaf Extract ◽  
Mcf 7

The authors requested the replacement of Fig. 1. Morphology of MCF-7 cells after the treatment with the extract of Ipomoea tuba leaf: a) untreated MCF-7 cell lines, and b-g) treated with different concentrations (5, 10, 25, 50, 75 and 100 μg/mL respectively) of the leaf extract The change includes the replacemend of images 1c-1g.

scholarly journals Pathogenesis of Yersinia pestis Infection in BALB/c Mice: Effects on Host Macrophages and Neutrophils

2005 ◽  
Vol 73 (11) ◽  
pp. 7142-7150 ◽  
Author(s):  
Roman A. Lukaszewski ◽  
Dermot J. Kenny ◽  
Rosa Taylor ◽  
D. G. Cerys Rees ◽  
M. Gill Hartley ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Fluorescent Protein ◽  
Virulent Strain ◽  
Late Phase ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Intracellular Bacteria ◽  
Necrosis Factor Alpha ◽  
Green Fluorescent

ABSTRACT The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 103 to 104 CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.

scholarly journals Effect of Sterols Isolated fromMyrtillocactus geometrizanson Growth Inhibition of Colon and Breast Cancer Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Mario Augusto Bolaños-Carrillo ◽  
Jose Luis Ventura-Gallegos ◽  
Arturo David Saldivar-Jiménez ◽  
Alejandro Zentella-Dehesa ◽  
Mariano Martínez-Vázquez
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cancer Cells ◽  
Biological Activities ◽  
Annexin V ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Cycle Dependent ◽  
Mcf 7

Objective. To explore the effect of peniocerol and macdougallin on HCT-15 and MCF-7 cells proliferation, cell cycle, apoptosis, and PARP cleavage.Methods. HCT-15 and MCF-7 cells were treated with various concentrations of peniocerol and macdougallin (10–80 μM) during 24 or 48 h. Crystal Violet Assay was used to evaluate the inhibition effect. Cell cycle regulation was examined by a propidium iodide method. Cell apoptosis was detected through both Annexin–V FLUOS/PI double-labeled cytometry assays and Western blot was applied to assess PARP cleavage.Results. Peniocerol and macdougallin induced growth inhibition and apoptosisin vitroin a time- and dose-dependent manner. Moreover, peniocerol and macdougallin induced arrest of cell cycle-dependent manner and increased the proportion of cells in G0/G1phase. PARP cleavage in HCT-15 and MCF-7 cells was induced by treatment with peniocerol and macdougallin after 36 hours.Conclusions. Our results showed that the mechanism of cytotoxicity displayed by peniocerol and macdougallin is related to cell cycle arrest and apoptosis in both cell lines. This is a significant observation because it helps to understand the way some oxysterols isolated fromMyrtillocactus geometrizansdevelop their biological activities against cancer cells.

scholarly journals Influence of opioid peptides on human neutrophil apoptosis and activationin vitro

2002 ◽  
Vol 11 (4) ◽  
pp. 245-250 ◽  
Author(s):  
Zofia Sulowska ◽  
Ewa Majewska ◽  
Katarzyna Krawczyk ◽  
Magdalena Klink ◽  
Henryk Tchórzewski
Keyword(s):  
Opioid Peptides ◽  
Oxidative Metabolism ◽  
Acute Stress ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Human Neutrophils ◽  
Apoptotic Cells ◽  
Mtt Reduction ◽  
Neutrophil Survival

Background: It has been shown that cells of the immune system release opioid peptides and possess receptors for them. The concentrations of opioid peptides in the peripheral circulation rapidly increase during inflammation and acute stress response.Aims: The effect of opioid peptides Met-enkephalin (M-ENK) and beta-endorphin (β-END) on the oxidative metabolism of normal human neutrophils and their death by apoptosisin vitrowas investigated.Methods: Isolated from peripheral blood, neutrophils were incubated in the presence or absence of 10-6to 10-10M of M-ENK and β-END for 12 and 18 h. Apoptosis of neutrophils was determinedin vitroby flow cytometric analysis of cellular DNA content and Annexin V-FITC protein binding to the cell surface. The MTT-reduction assay was employed to estimate the oxidative metabolism of neutrophils.Results: Treatment with M-ENK caused a significant increase in apoptotic cells after 18 h of culture:∗0M (control) versus 10-10M,p≤0.02;∗∗10−10M versus 10-10M,p≤0.02. Treatment with β-END caused a significant increase in apoptotic cells after 12 h of culture: 0 M versus 10-8M,p≤0.03;∗∗0MM versus 10-10M,p≤0.04. We found the significant increase in MTT reduction by neutrophils in the presence of M-ENK and β-END both before and after the culture. However, the ability of neutrophils to reduce the MTT salt to formazan decreased significantly after the culture.Conclusions: We observed that thein vitroeffect of opioid peptides on the neutrophil survival and their functional state was time and dose dependent. The presence of antioxidants in the culture medium modifies neutrophil survival.

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