The EZMTT cell proliferation assay provides precise measurement for drug combinations and better correlation between in vitro and in vivo efficacy

10568 Background: Adrenomedullin (AM) is a 52 amino acid peptide that has an important role on tumor cell proliferation and neoangiogenesis through its receptors CRLR/RAMP2 and CRLR/RAMP3. AM gene expression is stimulated by Hypoxia Inductible Factor-1 (HIF- 1) and 60–80% of the human conventional renal carcinomas (cRCC) display mutations in the tumor suppressor protein von Hippel-Lindau, which leads to constitutively elevated HIF-1. Methods: Tumors and non-malignant kidney tissues were obtained from patients who underwent unilateral nephrectomy. We studied VEGFA, AM and its receptors expression by quantitative RT-PCR in 62 frozen renal tumors including: 40 cRCC; 5 cRCC metastasis; 5 chromophobe carcinomas; 5 Papillary carcinomas; 2 oncocytomas; 2 collecting duct carcinomas; 2 normal adjacent renal tissue; 1 unclassified renal tumor. AM and AM-receptors expression was studied in 42 paraffin-embedded renal tumors by immunohistochemistry using rabbit anti-AM, anti-CRLR, anti-RAMP2 and anti-RAMP3 polyclonal antibodies. Effects of these anti AM or anti AM- receptors antibodies on cell proliferation were examined in vitro on BIZ cell line (cRCC cells). Results: VEGFA, AM and AM-receptors genes were overexpressed in cRCC compared to other renal tumors (Except AM gene in chromophobe carcinoma). Their expressions were independent of classical prognostic factors (such as Fuhrman Grade and pT status). In cRCC, there was a strong positive correlation between VEGF-A gene expression and AM (r=0.7; p=0.01) and AM-receptors genes expressions (RAMP2 r=0.61; p= 0.01 and RAMP3 r=0.58; p= 0.01). Immunohistochemistry confirmed a tumor expression of CRLR, RAMP2 and AM in more than 80% cRCC. RAMP3 was expressed by inflammatory cells but not by tumoral cells. The cell proliferation assay showed a significant inhibition of BIZ cell proliferation by AM antibody or AM-receptors antibodies. Conclusion: AM and its receptor CRLR/RAMP2 are overexpressed in cRCC. In vitro cell proliferation assay results support that AM inhibition may suppress cRCC growth, independently of potential antiangiogenic effects. Further studies on animal models are needed but AM pathway may be a potential therapeutic target in cRCC. No significant financial relationships to disclose.

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Use of a Tetrazolium-based Cell Proliferation Assay to Measure Effects of In Vitro Conditions on Perkinsus marinus (Apicomplexa) Proliferation

Journal of Eukaryotic Microbiology ◽

10.1111/j.1550-7408.1995.tb01598.x ◽

1995 ◽

Vol 42 (4) ◽

pp. 379-388 ◽

Cited By ~ 42

Author(s):

CHRISTOPHER F. DUNGAN ◽

ROSALEE M. MAMILTON

Keyword(s):

Cell Proliferation ◽

Perkinsus Marinus ◽

Proliferation Assay ◽

Cell Proliferation Assay

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Targeting the androgen receptor as a novel therapeutic strategy for high-grade gliomas by suppressing glioma cancer stem cells.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e13504 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e13504-e13504 ◽

Cited By ~ 1

Author(s):

Nan Zhao ◽

Shaheen Ahmed ◽

Fei Wang ◽

DiMaio J Dominick ◽

Chi Lin ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Proliferation ◽

Androgen Receptor ◽

Cancer Stem Cells ◽

Cell Lines ◽

Proliferation Assay ◽

Cell Proliferation Assay ◽

Expression Levels

e13504 Background: Glioblastoma (GBM) is the most aggressive and most common type of primary brain malignancies with median overall survival being only 20.9 months. The incidence of GBM is 50% greater in men than in women, and GBM transplanted into animals grow at a slower rate in females compared with males. Gender difference in GBM indicates that sex hormones such as androgen receptor (AR) may be involved in the tumorigenesis of GBM. A newer generation of AR antagonist, Enzalutamide, is available for prostate cancer treatment in clinic and can pass the blood-brain barrier, thus a good candidate for GBM treatment. Methods: Cell proliferation assay, cell cycle analysis, and cell apoptosis assay were performed on different GBM cell lines after Enzalutamide treatment. After treating GBM cells with or without Enzalutamide in mono-layer cell culturing or tumor spheres, cancer stem cell sub-population (CD133+ cells) in different groups was compared using flow cytometry. After enriching GBM cancer stem cells by sorting CD133+ U87MG cells out, cell proliferation assay was performed on CD133+ U87MG cells. Western blotting was performed comparing marker gene expression levels including CD133 and c-Myc with total protein isolated from GBM cells treated with Enzalutamide at different time points. A syngeneic orthotopic GBM mouse model was used for in vivo study. The size of tumors in the brain was monitored weekly with and without Enzalutamide treatment by in vivo imaging system for the luciferase activities. Results: Enzalutamide inhibited the proliferation of GBM cells both in vitro and in vivo. Enzalutamide induced apoptosis of GBM cells as well as arrested the cell cycle at G2/M phase in a cell cycle that has a potential of radio-sensitizing effect. Enzalutamide decreased the cancer stem cells population both in cultured mono-layer cells and in tumor spheres. Enzalutamide inhibited the proliferation of CD133+ U87MG cells after four days’ treatment. c-Myc is a proto-oncogene and required for maintenance of GBM cancer stem cells. Both CD133 and c-Myc expression levels decreased in GBM cell lines after Enzalutamide treatment in a time-dependent manner. Conclusions: Enzalutamide targets cancer stem cells and inhibits the proliferation of GBM.

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In Vitro Cell Proliferation Assay Method Using Rat Gastric Cultured Cells and Effect of Anti-ulcer Drugs on the Proliferation of Cultured Cells

YAKUGAKU ZASSHI ◽

10.1248/yakushi1947.114.5_316 ◽

1994 ◽

Vol 114 (5) ◽

pp. 316-324 ◽

Cited By ~ 1

Author(s):

Keiji IMAI ◽

Reiko UKAI ◽

Kazuhiro ISHIKAWA ◽

Hiromoto AOKI ◽

Tetsuo KATO ◽

...

Keyword(s):

Cell Proliferation ◽

Cultured Cells ◽

Assay Method ◽

Proliferation Assay ◽

Cell Proliferation Assay

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Designing a Long Acting Erythropoietin by Fusing Three Carboxyl-Terminal Peptides of Human Chorionic GonadotropinβSubunit to theN-Terminal andC-Terminal Coding Sequence

International Journal of Cell Biology ◽

10.1155/2011/275063 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 16

Author(s):

Fuad Fares ◽

Avri Havron ◽

Eyal Fima

Keyword(s):

Cell Proliferation ◽

Clinical Trials ◽

Cho Cells ◽

Therapeutic Efficacy ◽

Proliferation Assay ◽

Coding Sequence ◽

Carboxyl Terminal ◽

Long Acting

A new analog of EPO was designed by fusing one and two CTPs to theN-terminal andC-terminal ends of EPO (EPO-(CTP)3), respectively. This analog was expressed and secreted efficiently in CHO cells. Thein vitrotest shows that the activity of EPO-(CTP)3in TFI-1 cell proliferation assay is similar to that of EPO-WT and commercial rHEPO. However,in vivostudies indicated that treatment once a week with EPO-(CTP)3(15 μg/kg) dramatically increased (~8 folds) haematocrit as it was compared to rHuEPO. Moreover, it was found that EPO-(CTP)3is more effective than rHuEPO and Aranesp in increasing reticulocyte number in mice blood. The detected circulatory half-lives of rHuEPO, Aranesp, and EPO-(CTP)3following IV injection of 20 IU were 4.4, 10.8, and 13.1 h, respectively. These data established the rational for using this chimera as a long-acting EPO analog in clinics. The therapeutic efficacy of EPO-CTP analog needs to be established in higher animals and in human clinical trials.

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