A computational modelling framework to quantify the effects of passaging cell lines

AbstractIn vitro cell culture is routinely used to grow and supply a sufficiently large number of cells for various types of cell biology experiments. Previous experimental studies report that cell characteristics evolve as the passage number increases, and various cell lines can behave differently at high passage numbers. To provide insight into the putative mechanisms that might give rise to these differences, we perform in silico experiments using a random walk model to mimic the in vitro cell culture process. Our results show that it is possible for the average proliferation rate to either increase or decrease as the passaging process takes place, and this is due to a competition between the initial heterogeneity and the degree to which passaging damages the cells. We also simulate a suite of scratch assays with cells from near–homogeneous and heterogeneous cell lines, at both high and low passage numbers. Although it is common in the literature to report experimental results without disclosing the passage number, our results show that we obtain significantly different closure rates when performing in silico scratch assays using cells with different passage numbers. Therefore, we suggest that the passage number should always be reported to ensure that the experiment is as reproducible as possible. Furthermore, our modelling also suggests some avenues for further experimental examination that could be used to validate or refine our simulation results.

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In Silico and In Vitro Analysis of lncRNA XIST Reveals a Panel of Possible Lung Cancer Regulators and a Five-Gene Diagnostic Signature

Cancers ◽

10.3390/cancers12123499 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3499

Author(s):

Periklis Katopodis ◽

Qiduo Dong ◽

Heerni Halai ◽

Cristian I. Fratila ◽

Andreas Polychronis ◽

...

Keyword(s):

Lung Cancer ◽

Cell Lines ◽

In Silico ◽

Cell Biology ◽

Tumour Progression ◽

Cell Communication ◽

Nucleic Acid Metabolism ◽

Rna Seq ◽

Diagnostic Potential

Long non-coding RNAs (lncRNAs) perform a wide functional repertoire of roles in cell biology, ranging from RNA editing to gene regulation, as well as tumour genesis and tumour progression. The lncRNA X-inactive specific transcript (XIST) is involved in the aetiopathogenesis of non-small cell lung cancer (NSCLC). However, its role at the molecular level is not fully elucidated. The expression of XIST and co-regulated genes TSIX, hnRNPu, Bcl-2, and BRCA1 analyses in lung cancer (LC) and controls were performed in silico. Differentially expressed genes (DEGs) were determined using RNA-seq in H1975 and A549 NSCLC cell lines following siRNA for XIST. XIST exhibited sexual dimorphism, being up-regulated in females compared to males in both control and LC patient cohorts. RNA-seq revealed 944 and 751 DEGs for A549 and H1975 cell lines, respectively. These DEGs are involved in signal transduction, cell communication, energy pathways, and nucleic acid metabolism. XIST expression associated with TSIX, hnRNPu, Bcl-2, and BRCA1 provided a strong collective feature to discriminate between controls and LC, implying a diagnostic potential. There is a much more complex role for XIST in lung cancer. Further studies should concentrate on sex-specific changes and investigate the signalling pathways of the DEGs following silencing of this lncRNA.

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Dual SMO/BRAF Inhibition by Flavonolignans from Silybum marianum

Antioxidants ◽

10.3390/antiox9050384 ◽

2020 ◽

Vol 9 (5) ◽

pp. 384 ◽

Cited By ~ 1

Author(s):

Antonia Diukendjieva ◽

Maya M. Zaharieva ◽

Mattia Mori ◽

Petko Alov ◽

Ivanka Tsakovska ◽

...

Keyword(s):

Cell Lines ◽

In Silico ◽

Experimental Studies ◽

Braf V600e ◽

Silybum Marianum ◽

Anticancer Activities ◽

Protein Targets ◽

Braf Inhibition ◽

Approved Drugs

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

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Quantitative Structural Activity Relationship Studies for In Silico Prediction of In Vitro Cytotoxicity of NSAIDs in Colon Cancer Cell Lines

Letters in Drug Design & Discovery ◽

10.2174/157018012802652895 ◽

2012 ◽

Vol 9 (8) ◽

pp. 755-763 ◽

Cited By ~ 1

Author(s):

Honey Goel ◽

Suresh Thareja ◽

Priyanka Malla ◽

Manoj Kumar ◽

V. R Sinha

Keyword(s):

Colon Cancer ◽

Cell Lines ◽

In Silico ◽

In Vitro Cytotoxicity ◽

Colon Cancer Cell ◽

In Silico Prediction ◽

Colon Cancer Cell Lines ◽

Structural Activity Relationship ◽

Quantitative Structural Activity Relationship

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10-gingerol induces oxidative stress through HTR1A in cumulus cells: in-vitro and in-silico studies

Journal of Complementary and Integrative Medicine ◽

10.1515/jcim-2019-0042 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Kiptiyah Kiptiyah ◽

Widodo Widodo ◽

Gatot Ciptadi ◽

Aulanni’am Aulanni’Am ◽

Mohammad A. Widodo ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Culture ◽

Superoxide Dismutase ◽

In Silico ◽

Cumulus Cell ◽

Cumulus Cells ◽

Sod Activity ◽

In Silico Studies ◽

Incubation Periods

AbstractBackgroundWe investigated whether 10-gingerol is able to induce oxidative stress in cumulus cells.MethodsFor the in-vitro research, we used a cumulus cell culture in M199, containing 10-gingerol in various concentrations (0, 12, 16, and 20 µM), and detected oxidative stress through superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentrations, with incubation periods of 24, 48, 72, and 96 h. The obtained results were confirmed by in-silico studies.ResultsThe in-vitro data revealed that SOD activity and MDA concentration increased with increasing incubation periods: SOD activity at 0 µM (1.39 ± 0.24i), 12 µM (16.42 ± 0.35ab), 16 µM (17.28 ± 0.55ab), 20 µM (17.81 ± 0.12a), with a contribution of 71.1%. MDA concentration at 0 µM (17.82 ± 1.39 l), 12 µM (72.99 ± 0.31c), 16 µM (79.77 ± 4.19b), 20 µM (85.07 ± 2.57a), with a contribution of 73.1%. Based on this, the in-silico data uncovered that 10˗gingerol induces oxidative stress in cumulus cells by inhibiting HTR1A functions and inactivating GSK3B and AKT˗1.Conclusions10-gingerol induces oxidative stress in cumulus cells through enhancing SOD activity and MDA concentration by inhibiting HTR1A functions and inactivating GSK3B and AKT˗1.

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Histone/protein deacetylases and T-cell immune responses

Blood ◽

10.1182/blood-2011-10-292003 ◽

2012 ◽

Vol 119 (11) ◽

pp. 2443-2451 ◽

Cited By ~ 85

Author(s):

Tatiana Akimova ◽

Ulf H. Beier ◽

Yujie Liu ◽

Liqing Wang ◽

Wayne W. Hancock

Keyword(s):

T Cell ◽

Cell Biology ◽

T Regulatory Cells ◽

Hdac Inhibitors ◽

Experimental Studies ◽

Cell Subset ◽

Histone Protein ◽

Anti Inflammatory

Abstract Clinical and experimental studies show that inhibition of histone/protein deacetylases (HDAC) can have important anti-neoplastic effects through cytotoxic and proapoptotic mechanisms. There are also increasing data from nononcologic settings that HDAC inhibitors (HDACi) can exhibit useful anti-inflammatory effects in vitro and in vivo, unrelated to cytotoxicity or apoptosis. These effects can be cell-, tissue-, or context-dependent and can involve modulation of specific inflammatory signaling pathways as well as epigenetic mechanisms. We review recent advances in the understanding of how HDACi alter immune and inflammatory processes, with a particular focus on the effects of HDACi on T-cell biology, including the activation and functions of conventional T cells and the unique T-cell subset, composed of Foxp3+ T-regulatory cells. Although studies are still needed to tease out details of the various biologic roles of individual HDAC isoforms and their corresponding selective inhibitors, the anti-inflammatory effects of HDACi are already promising and may lead to new therapeutic avenues in transplantation and autoimmune diseases.

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An in vitro–in silico interface platform for spatiotemporal analysis of pattern formation in collective epithelial cells

Integrative Biology ◽

10.1039/c6ib00073h ◽

2016 ◽

Vol 8 (8) ◽

pp. 861-868 ◽

Cited By ~ 6

Author(s):

M. Hagiwara

Keyword(s):

Cell Culture ◽

Pattern Formation ◽

Epithelial Cells ◽

In Silico ◽

Reaction Diffusion ◽

Spatiotemporal Analysis ◽

Bronchial Epithelial ◽

Reaction Diffusion Model ◽

2D Pattern

The mechanisms of 2D pattern formation in bronchial epithelial cells were dynamically analyzed by controlled cell culture and a reaction-diffusion model.

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Quantitative analysis of tumour spheroid structure

eLife ◽

10.7554/elife.73020 ◽

2021 ◽

Vol 10 ◽

Author(s):

Alexander P Browning ◽

Jesse A Sharp ◽

Ryan J Murphy ◽

Gency Gunasingh ◽

Brodie Lawson ◽

...

Keyword(s):

Cell Lines ◽

Tumour Microenvironment ◽

Culture Conditions ◽

Experimental Models ◽

Seeding Density ◽

Modelling Framework ◽

Tumour Spheroid ◽

Tumour Spheroids ◽

Wide Range

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

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Which cytochrome P450 metabolizes phenazepam? Step by step in silico, in vitro, and in vivo studies

Drug Metabolism and Personalized Therapy ◽

10.1515/dmpt-2017-0036 ◽

2018 ◽

Vol 33 (2) ◽

pp. 65-73 ◽

Cited By ~ 6

Author(s):

Dmitriy V. Ivashchenko ◽

Anastasia V. Rudik ◽

Andrey A. Poloznikov ◽

Sergey V. Nikulin ◽

Valeriy V. Smirnov ◽

...

Keyword(s):

Cell Culture ◽

Cytochrome P450 ◽

In Silico ◽

In Vivo Studies ◽

Cyp3a Activity ◽

Cell Culture Study ◽

Study Results ◽

Three Stages

Abstract Background: Phenazepam (bromdihydrochlorphenylbenzodiazepine) is the original Russian benzodiazepine tranquilizer belonging to 1,4-benzodiazepines. There is still limited knowledge about phenazepam’s metabolic liver pathways and other pharmacokinetic features. Methods: To determine phenazepam’s metabolic pathways, the study was divided into three stages: in silico modeling, in vitro experiment (cell culture study), and in vivo confirmation. In silico modeling was performed on the specialized software PASS and GUSAR to evaluate phenazepam molecule affinity to different cytochromes. The in vitro study was performed using a hepatocytes’ cell culture, cultivated in a microbioreactor to produce cytochrome P450 isoenzymes. The culture medium contained specific cytochrome P450 isoforms inhibitors and substrates (for CYP2C9, CYP3A4, CYP2C19, and CYP2B6) to determine the cytochrome that was responsible for phenazepam’s metabolism. We also measured CYP3A activity using the 6-betahydroxycortisol/cortisol ratio in patients. Results: According to in silico and in vitro analysis results, the most probable metabolizer of phenazepam is CYP3A4. By the in vivo study results, CYP3A activity decreased sufficiently (from 3.8 [95% CI: 2.94–4.65] to 2.79 [95% CI: 2.02–3.55], p=0.017) between the start and finish of treatment in patients who were prescribed just phenazepam. Conclusions: Experimental in silico and in vivo studies confirmed that the original Russian benzodiazepine phenazepam was the substrate of CYP3A4 isoenzyme.

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Novel N-bridged pyrazole-1-carbothioamides with potential antiproliferative activity: design, synthesis, in vitro and in silico studies

Future Medicinal Chemistry ◽

10.4155/fmc-2021-0066 ◽

2021 ◽

Vol 13 (20) ◽

pp. 1743-1766

Author(s):

Islam H El Azab ◽

Essa M Saied ◽

Alaa A Osman ◽

Amir E Mehana ◽

Hosam A Saad ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Anticancer Agents ◽

Antiproliferative Activity ◽

In Silico ◽

Human Cancer ◽

Cancer Cell Lines ◽

Molecular Docking Study ◽

In Silico Studies

Thiazole-substituted pyrazole is an important structural feature of many bioactive compounds, including antiviral, antitubercular, analgesic and anticancer agents. Herein we describe an efficient and facile approach for the synthesis of two series of 36 novel N-bridged pyrazole-1-phenylthiazoles. The antiproliferative activity of a set of representative compounds was evaluated in vitro against different human cancer cell lines. Among the identified compounds, compound 18 showed potent anticancer activity against the examined cancer cell lines. The in silico molecular docking study revealed that compound 18 possesses high binding affinity toward both SK1 and CDK2. Overall, these results indicate that compound 18 is a promising lead anticancer compound which may be exploited for development of antiproliferative drugs.

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