A novel epigenetic drug conjugating flavonoid and HDAC inhibitor confers to suppression of acute myeloid leukemogenesis

Epigenetic dysregulation has long been identified as a key driver of leukemogenesis in acute myeloid leukemia (AML). However, epigenetic drugs such as histone deacetylase inhibitors (HDACi) targeting epigenetic alterations in AML have obtained only limited clinical efficiency without clear mechanism. Fortunately, we screened out a novel epigenetic agent named Apigenin-Vorinostat-Conjugate (AVC), which provides us a possibility to handle the heterogenous malignancy. Its inhibition on HDACs was presented by HDACs expression, enzyme activity, and histone acetylation level. Its efficacy against AML was detected by cell viability assay and tumor progression of AML mouse model. Apoptosis is the major way causing cell death. We found AVC efficiently suppresses leukemogenesis whereas sparing the normal human cells. Kasumi-1 cells are at least twenty-fold higher sensitive to AVC (IC50=0.024μM) than vorinostat (IC50=0.513μM) and Ara-C (IC50=0.4366μM). Furthermore, it can efficiently regress the tumorigenesis in AML mouse model while keeping the pivotal organs safe, demonstrating a feasibility and favorable safety profile in treatment of AML. Collectively, these pre-clinical data suggest a promising potential utilizing flavonoid-HDACi-conjugate as a next-generation epigenetic drug for clinical therapy against AML.

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Single-agent and combination activity of suberoylanilide hydroxamic acid (SAHA, vorinostat) in small cell lung cancer (SCLC) cell lines

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e14586 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e14586-e14586

Author(s):

C. Cubitt ◽

S. Zhang ◽

A. Chiappori

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Histone Deacetylase Inhibitors ◽

Single Agent ◽

Cell Viability Assay ◽

Viability Assay ◽

Potential Synergy ◽

Ic50 Values ◽

Blue Cell ◽

Drug Doses

e14586 Background: SCLC represents a major therapeutic challenge. Histone deacetylase inhibitors (HDAC-I) are a new class of drugs. Exposure to HDAC-Is results in hyperacetylation of core histone proteins, with subsequent chromatin decondensation, and increased topoisomerase inhibitor (TI) DNA binding, with potentiation of DNA damage and apoptosis. This synergy is only observed when the HDAC-I precedes the TI. We investigated the activity of SAHA, an HDAC-I with broad activity in different cancer cell lines, and the potential synergy between SAHA and TIs, in SCLC cell lines. Methods: TIs were obtained from chemical supply companies and SAHA from Merck. Four different cell lines (DMS-114, NCI-H69, NCI-H82, and NCI-H526) were grown and cryopreserved in the recommended media. Drug activity was determined by a high-throughput CellTiter-Blue cell viability assay. A luciferase based assay (Caspase-Glo 3/7) was used to confirm apoptosis as the cause of cell viability reductions. The Chou and Talalay method was used to optimize the drug doses to use in a combination, and to determine the influence of drug sequencing on any additive or synergistic anti- tumor effect. Results: The 72 hours inhibitory concentration 50 (IC50) values corresponding to each drug and cell line is reported in the Table . Using the CellTiter-Blue cell viability assay, the combination index (CI) for SAHA with each of 2 TIs (topotecan and etoposide) was calculated concurrently and sequentially. The strongest synergism was always detected when SAHA and the TI were combined sequentially (SAHA first). This observation was reproduced when the CI was calculated using the Caspase-Glo 3/7 luciferase based assay. Conclusions: The anti-tumor activity of SAHA in SCLC cell lines is comparable to that of common TIs. The synergism observed between SAHA and TIs is sequence specific and highest when drugs are used sequentially (SAHA first). Clinical confirmation of this synergism is warranted in patients with SCLC. [Table: see text] No significant financial relationships to disclose.

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LINC01255 combined with BMI1 to regulate Human Mesenchymal Stromal Senescence and Acute myeloid leukemia cell proliferation through repress the transcription of MCP-1

10.21203/rs.3.rs-25878/v1 ◽

2020 ◽

Author(s):

Qinghua Liu ◽

Jing Dong ◽

Jie Li ◽

Yanchao Duan ◽

Keqiang Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cell ◽

Acute Myeloid Leukemia Cell ◽

Cell Viability Assay ◽

Viability Assay ◽

Acute Myeloid ◽

Myeloid Leukemia Cell

Abstract Background: Long non-coding RNAs (lncRNAs) govern fundamental biochemical and cellular biology processes, for example, participate in chromatin remodeling, imprinting, splicing, transcriptional regulation and translation. Dysregulation of lncRNA expression is act as a feature of various diseases and cancers, including hematopoietic malignancies. However, the clinical relevance of myelodysplastic syndrome (MDS) and acute myeloid leukemia preceded by MDS (MDS-AML) requires further research. Recently, lncRNAs have been demonstrated plays an important role in hematopoiesis, thus, to further finding more functional lncRNA seemed particularly important.Methods: Western blotting, real-time PCR, RNA-pulldown, RIP, Chromatin immunoprecipitation (ChIP), cellular compartments extraction assays, SA-β-gal staining, lentivirus transfection, cell viability assay and cell proliferation assays were used to examine the relationship between lncRNA LINC01255 and its regulation of p53-p21 pathway in human mesenchymal stromal and acute myeloid leukemia cells.Results: LncRNA LINC01255 is highly expressed in bone marrow cells of AML patients, CD34+ cells of MDS-AML patients and AML cell lines and the higher expression of LINC01255 is assocoated with poor survival rate of AML patients. LINC01255 can interact with BMI1 and repress the transcription of MCP-1 to active p53-p21 pathway, thus inhibiting the senescence of human mesenchymal stromal and proliferation of acute myeloid leukemia cell.Conclusions: We discovered a novel functional lncRNA LINC01255, which can regulate the senescence of human mesenchymal stromal and the proliferation of acute myeloid leukemia cell through inhibiting the transcription of MCP-1.

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Carbon Nanotubes Interference with Luminescence-Based Assays

Materials ◽

10.3390/ma13194270 ◽

2020 ◽

Vol 13 (19) ◽

pp. 4270

Author(s):

Tomasz Szymański ◽

Marcelina Kempa ◽

Michael Giersig ◽

Jakub Dalibor Rybka

Keyword(s):

Carbon Nanotubes ◽

Cell Viability ◽

Multiwalled Carbon Nanotubes ◽

Articular Chondrocytes ◽

Multiwalled Carbon ◽

Cell Viability Assay ◽

Conflicting Information ◽

Viability Assay ◽

Normal Human

Carbon nanotubes (CNTs) are one of the most promising nanomaterials synthesized to date. Thanks to their unique mechanical, electronic, and optical properties, they have found a wide application in electronics in the production of biosensors and nanocomposites. The functionalization of multiwalled carbon nanotubes (MWCNTs) is aimed at making them biocompatible by adding hydrophilic groups on their surface, increasing their solubility and thus rendering them applicable in the regenerative medicine. So far, there is conflicting information about carbon nanotubes in biological systems. This paper investigates the effect of functionalized, oxidized, multiwalled carbon nanotubes (MWCNT-Ox) on the cytotoxicity of normal human articular chondrocytes (NHAC-kn cell line). Since absorbance-based and fluorescence-based assays were shown to interfere with carbon nanotubes, luminescence-based tests were carried out, as they work on a different method of detection and provide advantages over the mentioned ones. Cell viability and reactive oxygen species (ROS) tests were carried out. The cell viability assay showed that with the increasing MWCNTs concentration, the number of viable chondrocytes was significantly decreasing. Exposure to MWCNT-Ox indicated oxidative stress in the lowest investigated concentration with a decreased amount of ROS with higher concentrations. However, control experiments with adenosine triphosphate (ATP) and H2O2—molecules that are detected by the assays—showed that carbon nanotubes interfere directly with measurement, thus rendering the results unreliable. To understand the exact interference mechanisms, further studies must be taken. In conclusion, this study shows that luminescence-based tests yield erroneous results, confirming that in vitro experiments in the literature concerning carbon nanotubes should be analyzed with caution.

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Proliferative Effect of Tilapia Fish (Oreochromis niloticus) Lectin on BALB/c Mice Splenocytes

Protein and Peptide Letters ◽

10.2174/0929866526666190911144057 ◽

2019 ◽

Vol 26 (12) ◽

pp. 887-892

Author(s):

Cynarha Daysy Cardoso da Silva ◽

Cristiane Moutinho Lagos de Melo ◽

Elba Verônica Matoso Maciel Carvalho ◽

Mércia Andréa Lino da Silva ◽

Rosiely Félix Bezerra ◽

...

Keyword(s):

Cell Proliferation ◽

Oreochromis Niloticus ◽

Cytokine Production ◽

Thymidine Incorporation ◽

Con A ◽

Cell Viability Assay ◽

Proliferative Effect ◽

Viability Assay ◽

Apoptosis And Necrosis

Background: Lectins have been studied in recent years due to their immunomodulatory activities. Objective: We purified a lectin named OniL from tilapia fish (Oreochromis niloticus) and here we analyzed the cell proliferation and cytokine production in Balb/c mice splenocytes. Methods: Cells were stimulated in vitro in 24, 48, 72 hours and 6 days with different concentrations of OniL and Con A. Evaluation of cell proliferation was performed through [3H]-thymidine incorporation, cytokines were investigated using ELISA assay and cell viability assay was performed by investigation of damage through signals of apoptosis and necrosis. Results: OniL did not promote significant cell death, induced high mitogenic activity in relation to control and Con A and stimulated the cells to release high IL-2 and IL-6 cytokines. Conclusion: These findings suggest that, like Con A, OniL lectin can be used as a mitogenic agent in immunostimulatory assays.

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Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays

Current Drug Discovery Technologies ◽

10.2174/1570163815666180925095433 ◽

2020 ◽

Vol 17 (1) ◽

pp. 2-22 ◽

Cited By ~ 3

Author(s):

Abdel-Baset Halim

Keyword(s):

Cell Viability ◽

Data Interpretation ◽

Positive Control ◽

Live Cells ◽

Proof Of Concept ◽

Cell Viability Assay ◽

Viability Assay ◽

Current Cell ◽

Dying Cells ◽

Differential Permeability

:Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated.:A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

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SPARC regulates ferroptosis induced by sorafenib in human hepatocellular carcinoma

Cancer Biomarkers ◽

10.3233/cbm-200101 ◽

2021 ◽

pp. 1-9

Author(s):

Hong-Wei Hua ◽

Hao-Sheng Jiang ◽

Ling Jia ◽

Yi-Ping Jia ◽

Yu-Lan Yao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanism ◽

Cancer Progression ◽

Hepg2 Cells ◽

Cytotoxic Effect ◽

Cell Viability Assay ◽

Viability Assay ◽

Ldh Release ◽

Related Proteins ◽

Hcc Cells

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) is implicated in cancer progression, but its role and associated molecular mechanism in the sorafenib sensitivity of hepatocellular carcinoma cells (HCC) remains elusive. METHODS: Human HCC cell lines Hep3B and HepG2 were treated with sorafenib alone or combined with activator or inhibitor of ferroptosis. Cell viability assay, reactive oxygen species (ROS) assay, lactate dehydrogenase (LDH) assay and western blot were used to study the regulatory mechanism of SPARC on HCC cells. RESULTS: Overexpression of SPARC enhanced the cytotoxic effect of sorafenib in Hep3B and HepG2 cells compared with parental cells. Depletion of SPARC decreased the cytotoxic effect of sorafenib in Hep3B and HepG2 cells compared with parental cells. Moreover, overexpression of SPARC significantly induced LDH release, whereas depletion of SPARC suppressed the release of LDH in Hep3B and HepG2 cells. Inhibition of ferroptosis exerted a clear inhibitory role against LDH release, whereas activation of ferroptosis promoted the release of LDH in HCC cells, as accompanied with deregulated expression of ferroptosis-related proteins. Furthermore, overexpression of SPARC induced oxidative stress, whereas depletion of SPARC suppressed the production of ROS. Deferoxamine (DFX)-induced inhibition of ferroptosis suppressed the production of ROS, while activation of ferroptosis promoted the contents of ROS in HCC cells exposed to sorafenib. CONCLUSION: Our findings give a better understanding of ferroptosis and its molecular mechanism in HCC cells that is regulated by SPARC in response to sorafenib.

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Epigenetic drug combination induces remission in mouse xenograft models of pediatric acute myeloid leukemia

Leukemia Research ◽

10.1016/j.leukres.2017.05.004 ◽

2017 ◽

Vol 58 ◽

pp. 91-97 ◽

Cited By ~ 5

Author(s):

Anilkumar Gopalakrishnapillai ◽

E. Anders Kolb ◽

Suzanne M. McCahan ◽

Sonali P. Barwe

Keyword(s):

Acute Myeloid Leukemia ◽

Drug Combination ◽

Myeloid Leukemia ◽

Pediatric Acute Myeloid Leukemia ◽

Mouse Xenograft ◽

Epigenetic Drug ◽

Xenograft Models ◽

Acute Myeloid

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Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy

International Journal of Molecular Sciences ◽

10.3390/ijms22137063 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7063

Author(s):

Sharon Mordechay ◽

Shaun Smullen ◽

Paul Evans ◽

Olga Genin ◽

Mark Pines ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Motor Coordination ◽

Mdx Mice ◽

Cell Viability Assay ◽

Muscle Fibrosis ◽

Antifibrotic Therapy ◽

Racemic Form ◽

Viability Assay

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(−)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form’s effect. No effect was observed for (−)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (−)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (−)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

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BDMC protects AD in vitro via AMPK and SIRT1

Translational Neuroscience ◽

10.1515/tnsci-2020-0140 ◽

2020 ◽

Vol 11 (1) ◽

pp. 319-327

Author(s):

Chenlin Xu ◽

Zijian Xiao ◽

Heng Wu ◽

Guijuan Zhou ◽

Duanqun He ◽

...

Keyword(s):

Neurodegenerative Disorder ◽

Neuroprotective Effect ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Activity Measurement ◽

Therapeutic Approaches ◽

Cell Viability Assay ◽

Viability Assay ◽

Compound C

AbstractBackgroundAlzheimer’s disease (AD) is a common neurodegenerative disorder without any satisfactory therapeutic approaches. AD is mainly characterized by the deposition of β-amyloid protein (Aβ) and extensive neuronal cell death. Curcumin, with anti-oxidative stress (OS) and cell apoptosis properties, plays essential roles in AD. However, whether bisdemethoxycurcumin (BDMC), a derivative of curcumin, can exert a neuroprotective effect in AD remains to be elucidated.MethodsIn this study, SK-N-SH cells were used to establish an in vitro model to investigate the effects of BDMC on the Aβ1–42-induced neurotoxicity. SK-N-SH cells were pretreated with BDMC and with or without compound C and EX527 for 30 min after co-incubation with rotenone for 24 h. Subsequently, western blotting, cell viability assay and SOD and GSH activity measurement were performed.ResultsBDMC increased the cell survival, anti-OS ability, AMPK phosphorylation levels and SIRT1 in SK-N-SH cells treated with Aβ1–42. However, after treatment with compound C, an AMPK inhibitor, and EX527, an SIRT1inhibitor, the neuroprotective roles of BDMC on SK-N-SH cells treated with Aβ1–42 were inhibited.ConclusionThese results suggest that BDMC exerts a neuroprotective role on SK-N-SH cells in vitro via AMPK/SIRT1 signaling, laying the foundation for the application of BDMC in the treatment of neurodegenerative diseases related to AMPK/SIRT1 signaling.

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