scholarly journals Identification of Antitumor Constituents in Toad Venom by Spectrum-Effect Relationship Analysis and Investigation on Its Pharmacologic Mechanism

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4269
Author(s):  
Ji-Heng Wu ◽  
Yue-Ting Cao ◽  
Hong-Ye Pan ◽  
Long-Hu Wang
Keyword(s):  
Flow Cytometry ◽  
Susceptibility Gene ◽  
In Vitro Assays ◽  
Breast Cancer Susceptibility Gene ◽  
Effect Relationship ◽  
Relationship Analysis ◽  
Effect Model ◽  
Toad Venom ◽  
Spectrum Effect

(1) Background: Toad venom (Bufonis Venenum, known as ‘Chansu’ in Chinese), the secretion of the ear-side gland and skin gland of Bufo gargarizans cantor or Duttaphrynus melanostictus Schneider, has been utilized to treat several diseases in China for thousands of years. However, due to the chemical variability of the components, systematic chemical composition and the key pharmacophores in toad venom have not yet fully understood. Besides, it contains a variety of effective compounds with different physiological activity and chemotypes, mainly including alkaloids, bufogenins, bufotoxins, and so on. The recent pharmacological researches have demonstrated that several bufogenins have remarkable pharmacological effects, such as anti-inflammatory, analgesic effects, and anti-tumor effects. Aim of the study: To identify the bioactive compounds and pharmacophores originating from toad venom based on analyzing spectrum-effect relationship by chemometrics and to explore the anti-cancer mechanism primarily. (2) Materials and methods: Fingerprint of the 21 batches of samples was established using HPLC (High Performance Liquid Chromatography). The anti-tumor activity of extracts were determined by in-vitro assays. Chemometric analysis was used to establish the spectrum-effect model and screen for active ingredients. Pharmacodynamic tests for the screened active compound monomers were conducted with in-vitro assays. Further anti-tumor mechanisms were investigated using western blot and flow cytometry. (3) Results: The established spectrum-effect model has satisfactory fitting effect and predicting accuracy. The inhibitory effect of major screened compounds on lung carcinoma cells A549 were validated in vitro, demonstrating that arenobufagin, telocinobufogenin, and cinobufotalin had significant anti-tumor effects. Through further investigation of the mechanism by western blotting and flow cytometry, we elucidated that arenobufagin induces apoptosis in A549 cells with the enhanced expression of cleaved PARP (poly (ADP-ribose) polymerase). These results may provide valuable information for further structural modification of bufadienolides to treat lung cancer and a method for discovery of anti-tumor active compounds. Conclusions: Our research offers a more scientific method for screening the principal ingredients dominating the pharmacodynamic function. These screened compounds (arenobufagin, etc.) were proven to induce apoptosis by overactivation of the PARP-pathway, which may be utilized to make BRCA (breast cancer susceptibility gene) mutant cancer cells more vulnerable to DNA damaging agents and kill them.

scholarly journals Multivariate Statistical Analysis Uncovers Spectrum–Effect Relationship between HPLC Fingerprints and Antioxidant Activity of Saffron

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Ya You ◽  
Zijin Xu ◽  
Qingrou Zhong ◽  
Lin Zhu ◽  
Susu Lin ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Quality Control ◽  
Evaluation Method ◽  
Control Method ◽  
Partial Least Square ◽  
Least Square ◽  
Crocus Sativus ◽  
Effect Relationship ◽  
Spectrum Effect

Crocus sativus L. is commonly used as functional food and medicinal herb in traditional Chinese medicine. In this study, the spectrum–effect relationship was established between HPLC fingerprints and in vitro antioxidant activity of saffron to improve the quality evaluation method of saffron. The fingerprints of 21 batches of saffron collected from different regions were assessed, and the data were further analyzed by chemometric methods, including similarity analysis, hierarchical clustering analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. The spectrum–effect relationship between fingerprints and antioxidant effect of saffron was analyzed by grey relational analysis and partial least square methods to figure out the antioxidant component of saffron. Thirteen common peaks of 21 batches of saffron were included in the analysis, and peak 3 (picrocrocin), peak 7 (crocin I), and peak 10 (crocin II) were identified as the main active components responsible for antioxidant efficacy. Besides, a multi-index quality control method was developed for simultaneous determination of these three antioxidant components in saffron. Taken together, this study provided new strategies for the quality control and the development of new bioactive products of saffron in the future.

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

2019 ◽  
Vol 44 (6) ◽  
pp. 810-821
Author(s):  
Edibe Avci ◽  
Yeliz Z. Akkaya-Ulum ◽  
Digdem Yoyen-Ermis ◽  
Gunes Esendagli ◽  
Banu Balci-Peynircioglu
Keyword(s):  
Flow Cytometry ◽  
Cell Migration ◽  
Neutrophil Migration ◽  
Cost Effective ◽  
In Vitro Assays ◽  
Human Neutrophils ◽  
Neutrophil Granulocytes ◽  
Inflammatory Conditions ◽  
Migration Analysis

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

scholarly journals Host Cell Reactivation and Transcriptional Activation of Carboplatin-Modified BRCA1

2014 ◽  
Vol 8 ◽  
pp. BCBCR.S14224 ◽  
Author(s):  
Adisorn Ratanaphan ◽  
Bhutorn Canyuk
Keyword(s):  
Host Cell ◽  
Transcriptional Activation ◽  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Brca1 Gene ◽  
Breast Cancer Susceptibility ◽  
Breast Cancer Susceptibility Gene ◽  
Cell Reactivation ◽  
Host Cell Reactivation

The breast cancer susceptibility gene 1 ( BRCA1) has been shown to maintain genomic stability through multiple functions in the regulation of DNA damage repair and transcription. Its translated BRCT (BRCA1 C-terminal domain) acts as a strong transcriptional activator. BRCA1 damaged by carboplatin treatment may lead to a loss of such functions. To address the possibility of the BRCA1 gene as a therapeutic target for carboplatin, we investigated the functional consequences of the 3′-terminal region of human BRCA1 following in vitro platination with carboplatin. A reduction in cellular BRCA1 repair of carboplatin-treated plasmid DNA, using a host cell reactivation assay, was dependent on the platination levels on the reporter gene. The transcriptional transactivation activity of the drug-modified BRCA1, assessed using a one-hybrid GAL4 transcriptional assay, was inversely proportional to the carboplatin doses. The data emphasized the potential of the BRCA1 gene to be a target for carboplatin treatment.

scholarly journals Screening and Characterizing Tyrosinase Inhibitors from Salvia miltiorrhiza and Carthamus tinctorius by Spectrum-Effect Relationship Analysis and Molecular Docking

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Ya-Li Wang ◽  
Guang Hu ◽  
Qian Zhang ◽  
Yu-Xiu Yang ◽  
Qiao-Qiao Li ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Amino Acid ◽  
Salvia Miltiorrhiza ◽  
Carthamus Tinctorius ◽  
Amino Acid Residues ◽  
Analysis Method ◽  
Effect Relationship ◽  
Relationship Analysis ◽  
Ms Analysis ◽  
Spectrum Effect

Tyrosinase (TYR) is a rate-limiting enzyme in the synthesis of melanin, while direct TYR inhibitors are a class of important clinical antimelanoma drugs. This study established a spectrum-effect relationship analysis method and high-performance liquid chromatography-mass spectrometry (LC-MS) analysis method to screen and identify the active ingredients that inhibited TYR in Salvia miltiorrhiza–Carthamus tinctorius (Danshen–Honghua, DH) herbal pair. Seventeen potential active compounds (peaks) in the extract of DH herbal pair were predicted, and thirteen of them were tentatively identified by LC-MS analysis. Furthermore, TYR inhibitory activities of five pure compounds obtained from the DH herbal pair were validated in the test in which kojic acid served as a positive control drug. Among them, three compounds including protocatechuic aldehyde, hydroxysafflor yellow A, and tanshinone IIA were verified to have high TYR inhibitory activity (IC50 value of 455, 498, and 1214 μM, resp.) and bind to the same amino acid residues in TYR catalytic pocket according to the results of the molecular docking test. However, the other two compounds lithospermic acid and salvianolic acid A had a weak effect on TYR, as they do not combine with the active amino acid residues or act on the active center of TYR. Therefore, the developed methods (spectrum-effect relationship analysis and molecular docking) could be used to effectively screen TYR inhibitors in complex mixtures such as natural products.

DNA damage hypersensitivity in cells lacking BRCA2: a review of in vitro and in vivo data

2005 ◽  
Vol 33 (4) ◽  
pp. 715-717 ◽  
Author(s):  
T. Hay ◽  
A.R. Clarke
Keyword(s):  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Short Review ◽  
Breast Cancer Susceptibility ◽  
Breast Cancer Susceptibility Gene ◽  
Functional Protein ◽  
Increased Sensitivity ◽  
Cancer Susceptibility Gene

Since the discovery of the tumour suppressor BRCA2 (encoded by breast-cancer susceptibility gene 2), cells lacking the fully functional protein have consistently been found to show increased sensitivity to a variety of DNA-damaging agents, particularly those that cross-link DNA. In this short review, we will bring together these findings and discuss them in the light of our recent in vivo data in the mouse small intestine, which suggests that deletion of cells lacking Brca2 is necessary to avoid the development of potentially tumorigenic clones in this tissue, a system that may be less effective in the mammary glands of humans with germline mutations in BRCA2.

Characterization of the interaction between the human DNA topoisomerase IIβ-binding protein 1 (TopBP1) and the cell division cycle 45 (Cdc45) protein

2007 ◽  
Vol 409 (1) ◽  
pp. 169-177 ◽  
Author(s):  
Uta Schmidt ◽  
Yvonne Wollmann ◽  
Claudia Franke ◽  
Frank Grosse ◽  
Hans-Peter Saluz ◽  
...  
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Glutathione Transferase ◽  
Cancer Susceptibility ◽  
Binding Protein ◽  
Susceptibility Gene ◽  
Cell Division Cycle ◽  
Breast Cancer Susceptibility Gene

TopBP1 (topoisomerase IIβ-binding protein 1) is a BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminal]-domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and DNA damage signalling. Experiments with fission yeast and Xenopus revealed that the TopBP1 homologues of these organisms are required for chromatin loading of the replication protein Cdc45 (cell division cycle 45). To improve our understanding of the physiological functions of human TopBP1, we investigated the interplay between human TopBP1 and Cdc45 proteins in synchronized HeLa-S3 cells. Using GST (glutathione transferase) pull-down and co-immunoprecipitation techniques, we showed a direct interaction between TopBP1 and Cdc45 in vitro and in vivo. The use of deletion mutants in GST pull-down assays identified the first and second as well as the sixth BRCT domains of TopBP1 to be responsible for the functional interaction with Cdc45. Moreover, the interaction between Cdc45 and the first and second BRCT domains of TopBP1 inhibited their transcriptional activation both in yeast and mammalian one-hybrid systems. Both proteins interacted exclusively at the G1/S boundary of cell cycle; only weak interaction could be found at the G2/M boundary. The overexpression of the sixth BRCT domain led to diminished loading of Cdc45 on to chromatin. These results suggest that human TopBP1 is involved in the formation of the initiation complex of replication in human cells and is required for the recruitment of Cdc45 to origins of DNA replication.

scholarly journals Design and in Vitro Evaluation of a CAR-T Prototype (ARI-0003) Targeting CD123 for Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4799-4799
Author(s):  
Alex Bataller Torralba ◽  
Néstor Tirado ◽  
Diego Sánchez-Martínez ◽  
Talía Velasco-Hernández ◽  
Aina Oliver-Caldés ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Target Cell ◽  
In Vitro Assays ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Abstract Introduction The outcome of patients with Acute Myeloid Leukemia (AML) is still dismal, especially in patients with a relapsed or refractory (R/R) disease. In these patients, innovative treatment strategies must be considered in order to improve survival. Chimeric Antigen Receptor T-cell (CART) immunotherapy has demonstrated its efficacy and safety in diverse B-cell neoplasms and therefore is being investigated in other hematological malignancies. In AML, CART development is challenging due to the absence of a universal target antigen across AML subtypes, as well as on-target/off-tumor toxicity in healthy tissues. Among all explored AML antigens, CD123 seems to be a safe antigen to target, given its expresion in a high proportion of bulk and leukemic AML stem cells and with a higher level compared to normal hematopoietic progenitors cells. Herein we detail the generation and the in vitro assays of a CD123 CAR-T model (ARI-0003) for AML. Methods We developed an anti-CD123 antibody-secreting hybridoma and thereafter identified the variable domains of heavy and light chains of the immunoglobulin to create the scFv domain. The CAR (ARI-0003) genetic sequence was designed and cloned into a pCCL plasmid, and together with a VSV-G, RRE and REV plasmids we transfected HEK 293T cells to obtain 3 rd generation lentivirus (Figure 1A). T-lymphocytes were obtained from healthy volunteers and were transduced with lentiviral vectors to generate CAR-T cells. CAR expression in infected T-cells was monitored with flow cytometry using anti-F(ab) 2 antibodies. To test in vitro activity against AML, non-transduced T-lymphocytes (NT) and CAR-T cells were incubated with AML cell lines and AML primary samples from diverse genetic subtypes, and cytotoxicity was evaluated by flow cytometry at 24 and 48 hours using CD123 and CD33 antibodies. Results At the time of co-incubation of target cells and lymphocytes, the mean scFv expression of ARI-0003 in CAR-T cells was 37% (range, 20-57). Cytotoxicity assays with AML cell lines (THP-1, Kasumi1) showed higher target cell mortality with CAR-T cells, compared to NT cells (mean percentage of alive cells at 48h, obtained with an effector-target cell ratio of 1:1, of 0 and 1% [CAR] vs 45 and 57 [NT] in THP-1 and Kasumi1, respectively; Figure 1B-C). This differential cytotoxic activity was maintained using distinct effector ratio, being statistically significant in higher ratios (namely, 1:1, 1:2 and 1:4). Moreover, cytotoxicity induced by CAR-T cells was higher at 48 hours compared to 24 hours. To demonstrate antigen specificity of ARI-0003 CAR-T cells, CD123 negative cell lines (Raji, 697) were used, and target cell mortality was not statistically significant between CAR-T and NT in CD123-negative cell lines. Finally, ARI-0003 CAR-T efficacy was tested against 8 primary AML samples with a distinct genetic risk, including 5 unfavorable cases according to the European LeukemiaNet classification and 3 AML samples refractory to multiple high-intensity regimens (Figure 1F). Interestingly, a significant cytotoxic effect was observed against all 8 samples after incubation with ARI-0003 CAR-T cells at higher E:T ratio (p < 0.05 for ratios 1:1, 1:2 and 1:4; Figure D-E). Conclusions In vitro assays showed that immunotherapy with CAR-T cells ARI0003 against CD123 could be an effective approach for R/R AML. In vivo experiments are needed in order to confirm these results before translating this therapy to clinical phases. Moreover, on-target/off-tumor toxicity induced by ARI-0003 CAR-T needs to be explored, especially myelotoxicity due to target antigen expression in hematopoietic precursor cells. Figure legend A) Structure of the ARI0003 CAR. B) Cytotoxicity of CAR-T cells against cell lines (normalized to background toxicity observed with NT cells). C) Flow cytometry of THP1 cells against NT and CAR-T cells. D) Cytotoxicity of CAR-T cells against AML primary samples (normalized to NT percentage). E) Absolut number of remaining AML cells after 48h of coincubation with CAR or NT cells. F) Characteristics of AML primary samples used for in vitro assays. Figure 1 Figure 1. Disclosures Diaz-Beyá: Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Esteve: Abbvie: Consultancy; Astellas: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Jazz: Consultancy; Novartis: Research Funding.

scholarly journals Dynamic Change of Secondary Metabolites and spectrum-effect relationship of Malus halliana Koehne flowers during blooming

2018 ◽  
Vol 16 (1) ◽  
pp. 362-370 ◽  
Author(s):  
Lili Cui ◽  
Nan He ◽  
Xiaofeng Zhang ◽  
Shiming Li ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
High Performance ◽  
Dynamic Change ◽  
Inhibitory Effect ◽  
Partial Least Square ◽  
Least Square ◽  
Effect Relationship ◽  
Relationship Of ◽  
Spectrum Effect

AbstractMalus halliana Koehne flowers have been used as a Chinese traditional medicine to treat metrorrhagia. In this study, the dynamic changes in its secondary metabolites and spectrum-effect relationship of inhibition on α-glucosidase during blooming were investigated. The changes in the contents of three flavonoids (phloretin-4’-O-glycosidase, afzeloside, and 3-hydroxyphloridzin) were determined by high performance liquid chromatography (HPLC) and changes in inhibitory effect on α-glucosidase were evaluated in vitro. Then, spectrum-effect relationship was evaluated by partial least square method. The results indicated that the contents of three flavonoids and inhibition of α-glucosidase activity in vitro showed a fluctuating downward trend, thereinto, the maximum contents of phloretin-4’-O-glycosidase, afzeloside, and 3-hydroxyphloridzin reached 157.43±0.36, 17.27±0.06 and 22.67±0.35 (mg/g), respectively. In spectrum-effect relationship assay, matched 40 mutual peaks, thereinto, P2, P5, P6-P12, P14 (3-hydroxyphloridzin), P16-P19, P20 (phloretin-4’-O-glycosidase), P24, P26, P29, P31, P33, P34, P36, P39 and P40 were positively correlated to inhibitory effect on α-glucosidase in vitro. P1, P3, P4, P13, P15, P21-P23, P27, P28, P30 (afzeloside), P32, P35, P37 and P38 were negatively related to inhibitory effect on α-glucosidase in vitro.

scholarly journals In vitro assays for cell death determination

2008 ◽  
Vol 16 (3-4) ◽  
pp. 49-54 ◽  
Author(s):  
Vladimir Jurisic ◽  
Vladimir Bumbasirevic
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
In Vitro Assays ◽  
Activity Determination ◽  
Advantages And Disadvantages ◽  
Enzymes Activity ◽  
Death Determination

In this paper, we focused on commonly used in vitro assays for estimation of cell death: morphological analyses of cell death, cytotoxic assays based on enzymes activity determination, flow cytometry, and western blot techniques. We discussed advantages and disadvantages of several assays used in the modern research for estimation of cell death.

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