Multiplexed and reproducible high content screening of live and fixed cells using the Dye Drop method

High throughput measurement of cell perturbation, by libraries of small molecules or gene knockouts, is a key step in functional genomics and pre-clinical drug development. However, it is difficult to perform viable, single-cell assays in 384-well plates, limiting many studies to simple well-average measurements (e.g. CellTiter-Glo). Here we describe a public domain "Dye Drop" method in which sequential density displacement is used to perform multi-step assays for cell viability and EdU incorporation followed by immunofluorescence imaging. The method is rapid, reproducible, can be readily customized, and is compatible with either manual or automated laboratory equipment. We demonstrate Dye Drop in the collection of dose-response data for 67 drugs in 58 breast cancer cell lines and separate cytostatic and cytotoxic responses, thereby providing new insight into the effects of specific drugs on cell cycle progression and cell viability. Dye Drop substantially improves the tradeoff between data content and cost, enabling collection of large information-rich datasets.

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Differential Regulation of Cell Cycle Progression in Human Breast Cancer Cell Lines by the Estrogen Receptor

10.21236/ada394036 ◽

2000 ◽

Author(s):

James Direnzo ◽

Myles Brown

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Human Breast Cancer ◽

Cell Cycle Progression ◽

Differential Regulation ◽

Human Breast Cancer Cell ◽

Breast Cancer Cell Lines ◽

Human Breast ◽

Cycle Progression ◽

Regulation Of Cell Cycle

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Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

Pharmaceuticals ◽

10.3390/ph14070682 ◽

2021 ◽

Vol 14 (7) ◽

pp. 682

Author(s):

Jianling Bi ◽

Garima Dixit ◽

Yuping Zhang ◽

Eric J. Devor ◽

Haley A. Losh ◽

...

Keyword(s):

Cell Cycle ◽

Endometrial Cancer ◽

Tyrosine Kinase ◽

Cell Viability ◽

Cell Lines ◽

Cell Cycle Progression ◽

Kinase Inhibitor ◽

Cell Cycle Checkpoint ◽

Mitotic Catastrophe ◽

M Cell

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

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The Olive Leaves Extract Has Anti-Tumor Effects against Neuroblastoma through Inhibition of Cell Proliferation and Induction of Apoptosis

Nutrients ◽

10.3390/nu13072178 ◽

2021 ◽

Vol 13 (7) ◽

pp. 2178

Author(s):

Fabio Morandi ◽

Veronica Bensa ◽

Enzo Calarco ◽

Fabio Pastorino ◽

Patrizia Perri ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Viability ◽

Cell Cycle Progression ◽

Treatment Strategies ◽

Annexin V ◽

Dependent Manner ◽

Induction Of Apoptosis ◽

Dose Dependent

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.

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Cytotoxic activity of crude saponins from Gaultheria trichophylla against human breast cancer cells MCF-7 and MDA-MB-468

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v10i2.22992 ◽

2015 ◽

Vol 10 (2) ◽

pp. 443

Author(s):

Fiaz Alam ◽

Qazi Najam us Saqib ◽

Abdul Waheed

Keyword(s):

Breast Cancer ◽

Cell Viability ◽

Crude Extract ◽

Human Breast Cancer ◽

Neutral Red ◽

Breast Cancer Cell Lines ◽

Dependent Manner ◽

Human Breast ◽

Uptake Assay ◽

Mcf 7

This study was conducted to evaluate Gaultheria trichophylla crude extract and respective saponins fraction against human breast cancer cell lines. In MTT assay, cell viability was inhibited by G. trichophylla crude extract (500 µg/mL) and saponins (200 µg/mL) in a dose dependent manner with maximum inhibition of (82% and 85%) and (71% and 42%) against MCF-7 and MDA MB-468, respectively. In neutral red uptake assay, the cell viability was inhibited by crude extract and saponins (100 µg/mL) in a similar manner with maximum inhibitions of (96% and 93%) and (87% and 61%) against MCF-7 and MDA MB-468, respectively, with respect to 91% and 93% inhibition by actinomycin-D (4 µM). The DAPI (4',6-diamidino-2-phenylindole) (10 µg/mL) staining of MCF-7 cells treated with crude saponins showed shrunken nuclei with apparent nuclear fragmentation indicating apoptosis and in contrast, MDA MB-468 showed necrosis mode of cell death. The study exhibited that the G. trichophylla provides new evidences to further explore this plant for the novel targets in anticancer drug development.

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