scholarly journals Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis

2021 ◽  
Vol 9 (12) ◽  
pp. 2516
Author(s):  
Javiera Ortiz-Severín ◽  
Julia I. Tandberg ◽  
Hanne C. Winther-Larsen ◽  
Francisco P. Chávez ◽  
Verónica Cambiazo
Keyword(s):  
Cell Culture ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Cultures ◽  
High Throughput ◽  
Primary Cell ◽  
Infection Cycle ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Piscirickettsia Salmonis

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.

Characteristics of the combination paclitaxel plus doxorubicin in breast cancer cell lines analyzed with the ATP-Cell Viability Assay

1993 ◽  
Vol 28 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Ossi R. Koechli ◽  
Bernd-Uwe Sevin ◽  
James P. Perras ◽  
Ting Chao Chou ◽  
Roberto Angioli ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Viability ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cancer Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Cell Viability Assay ◽  
Viability Assay

scholarly journals Comparison of Cantharidin Toxicity in Breast Cancer Cells to Two Common Chemotherapeutics

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Katie M. Kern ◽  
Jennifer R. Schroeder
Keyword(s):  
Breast Cancer ◽  
Cell Viability ◽  
Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Cell Viability Assay ◽  
Chemotherapy Drugs ◽  
Viability Assay ◽  
Systemic Side Effects ◽  
High Level ◽  
Cancerous Cells

As part of a larger study synthesizing a more directed form of chemotherapy, we have begun to assess the efficacy of different potential toxins that could be delivered locally rather than systemically. In doing so, we hope to reduce the systemic side effects commonly observed, while maintaining a high level of toxicity and eliminating the need for metabolic alterations. In a search for this more efficient method for killing cancerous cells, we have begun studying cantharidin, a toxin used in traditional Chinese medicine, as a potential chemotherapeutic. Using an MTT cell viability assay, the toxicity of cantharidin was compared to both cyclophosphamide and paclitaxel in three different breast cancer cell lines: MCF-7, MDA-MB-231, and SK-BR-3. Increasing the concentration of chemotherapy drugs did decrease cell viability in all cell lines when cantharidin and cyclophosphamide were applied; however differences for paclitaxel were cell-specific. Additionally, cantharidin exhibited the highest decrease in cell viability regardless of cell type, indicating it may be a much more potent and less specific chemotherapeutic. These results will help us move forward in developing a potentially more potent treatment for breast cancer that might eliminate the need for subtype-specific treatments.

Comparative Chemosensitivity Profiles in Three Human Breast Cancer Cell Lines with the ATP-Cell Viability Assay

Oncology ◽  
1994 ◽  
Vol 51 (6) ◽  
pp. 552-558 ◽  
Author(s):  
Ossi R. Koechli ◽  
Bernd-Uwe Sevin ◽  
James P. Perras ◽  
Roberto Angioli ◽  
Michael Untch ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Viability ◽  
Cell Lines ◽  
Breast Cancer Cell ◽  
Human Breast Cancer ◽  
Human Breast Cancer Cell ◽  
Breast Cancer Cell Lines ◽  
Human Breast ◽  
Cell Viability Assay ◽  
Viability Assay

ABT199 and ONC201 in Diffuse Large B Cell Lymphoma Cell Lines

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5129-5129
Author(s):  
Nadia Khan ◽  
Lanlan Zhou ◽  
Jawad Babar ◽  
Joshua Allen ◽  
Richard I. Fisher ◽  
...  
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Large B Cell Lymphoma ◽  
Equity Ownership ◽  
Large B Cell

Abstract Background: Diffuse Large B cell lymphoma, the most commonly diagnosed type of non-Hodgkin lymphoma is curable in many cases, despite this, up to 30% of patients will relapse after initial therapy, necessitating salvage chemotherapy and transplantation if feasible. There is a pressing need for novel treatment strategies in highly chemo refractory cases. ONC201 is a small molecule that induces p53-independent cell death in tumor cells while sparing normal cells through a number of putative mechanisms, including inactivation of the pro-survival kinases Akt and ERK. ONC201 is currently entering Phase I/II clinical trials as a monoagent in adult advanced cancers. ONC201 promotes up-regulation of TRAIL gene transcription by inactivating AKT and ERK kinases which leads to translocation of transcription factor Foxo3a into the nucleus. It appears to act on a p53 independent pathway. ABT-199 is a selective, potent and orally bioavailable small molecule that selectively inhibits BCL-2 and triggers apoptosis. The Bcl-2 family of proteins is key regulator of the apoptotic process, comprising proapoptotic and prosurvival proteins. BH3-only proteins (such as Bim) bind to pro-survival proteins and cause increased permeability of mitochondrial membrane, release of cytochrome c and activation of caspases through release of Bax and others. Methods: Cell lines Pfeiffer and Toledo were purchased from ATCC and one patient DLBCL cell specimen from the ascitic fluid was cultured for use in experiments. ABT-199 was purchased from MedKoo Biosciences and ONC201 was provided from Oncoceutics. Cytotoxicity was evaluated by using the CellTiter-Glo Luminescent Cell Viability Assay as per the manufacturerÕs instructions. Cell viability was measured over time in response to treatment with ONC201(1-64μM) and ABT-199(128 nM-4μM). Western Blotting was performed on treated cells with well-established methodologies, with antibodies to c-Myc, Bcl-2, pAKT, pERK. Results: Immunohistochemical Staining Pattern of Cell Lines Table 1. Patient Toledo Pfeiffer Bcl-2 +++ ++ + Bcl-6 + ++ ++ c-Myc +++ ++ + p-ERK + + ++ p-AKT + ++ + Bax ++ NA NA Bim ++ NA NA Mean IC50 Calculated for Cell Lines Table 2. Cell Line Therapeutic Agent ABT199 ONC201 Patient Sample 8 μM 5 nM Toledo 9 μM 28 nM Pfeiffer 6 μM 2 μM SHAPE Conclusion/Discussion: The patient cell line, an ascitic fluid sample of DLBCL was sensitive to both ONC201 and ABT-199 and manifested bright Bcl-2 expression, the target of ABT-199. In this series there was a higher sensitivity to ABT199 in DLBCL cells with higher Bcl-2 expression. ONC201 down regulated pAKT expression, as seen in Western Blots in treated cells, consistent with prior investigation with the agent. We further found that ONC201 synergizes to potentiate cytotoxicity with ABT199, as demonstrated in the cell viability assay for Toledo cell lines (at the 24 hour time point), which were the least sensitive to ONC201 (highest IC50) when given as a single agent. Yet, when combined with increasing doses of ABT199, there was synergistic lymphoma cell kill with a fixed dose of ONC201. Together these results suggest that ONC201 has potential as an antitumor agent in NHL as monoagent and in combination with ABT-199, which may be further explored in phase Ib/II trials. Further analysis in larger patient sample series may elucidate the biomarkers that predict for greater therapeutic sensitivity to these highly potent lymphoma agents. Figure 1. Figure 1. Disclosures Allen: Oncoceutics, Inc: Employment, Equity Ownership. Eldeiry:Oncoceutics, Inc: Equity Ownership.

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

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.

Synthesis and Antitumor Activity of Some N2-(Thien-3-yl)amidrazones

2014 ◽  
Vol 69 (7) ◽  
pp. 811-816 ◽  
Author(s):  
Mohammed M. Abadleh ◽  
Mustafa M. El-Abadelah ◽  
Salim S. Sabri ◽  
Hanan H. Mohammed ◽  
Malek A. Zihlif ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Antitumor Activity ◽  
Cell Viability ◽  
Cell Lines ◽  
K562 Cell ◽  
Cyclic Amine ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
A Cell ◽  
Mcf 7

6aA set of new N2-(thien-3-yl)amidrazones (-h) incorporating N-piperazines and related congeners has been synthesized by reacting the hydrazonoyl chloride 4(derived from 3-aminothiophene- 2-carboxylate) with the appropriate sec-cyclic amine. The antitumor activity of these compounds was evaluated on breast cancer (MCF-7) and leukemic (K562) cell lines by a cell viability assay utilizing the tetrazolium dye (MTT). The amidrazone 6d encompassing the N-piperazine moiety, was the most active against MCF-7 and K562 with IC50 of 7.28 and 9:91 μM, respectively.

Sensitivity of Oncogenic KRAS-Expressing Cells to CDK9 Inhibition

2021 ◽  
pp. 247255522110088
Author(s):  
Lick Pui Lai ◽  
Viviane Brel ◽  
Kanika Sharma ◽  
Julia Frappier ◽  
Nadia Le-Henanf ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Specific Activity ◽  
Mammalian Target Of Rapamycin ◽  
Mouse Embryonic Fibroblast ◽  
Wild Type ◽  
Compound Library ◽  
Cell Viability Assay ◽  
Viability Assay

Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAFV600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.

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