Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies

Pauwels, Bea; Korst, A.E.C.; Pooter, Christel M J De; Pattyn, G; Lambrechts, Hilde; Baay, Marc; Lardon, Filip; Vermorken, Jan B.

doi:10.1007/s00280-002-0557-9

Cited by 120 publications

(56 citation statements)

References 11 publications

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“…After an overnight recovery period, cells were treated either with 0–10 nM cetuximab for 168 h or with 0–20 μM erlotinib for 72 h. Cells were incubated under normal or reduced oxygen conditions for 24 or 72 h immediately after addition of the drug. After incubation, cell survival was evaluated by the sulphorhodamine B assay, as previously described [23]. Optical density (OD) was determined at 540 nM with the iMark microplate reader (Bio-Rad, Temse, Belgium).…”

Section: Methodsmentioning

confidence: 99%

The hypoxic tumor microenvironment and drug resistance against EGFR inhibitors: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines

et al. 2015

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BackgroundIncreased expression of the epidermal growth factor receptor (EGFR) is observed in more than 90% of all head and neck squamous cell carcinomas (HNSCC). Therefore, EGFR has emerged as a promising therapeutic target. Nevertheless, drug resistance remains a major challenge and an important potential mechanism of drug resistance involves the hypoxic tumor microenvironment. Therefore, we investigated the cytotoxic effect of the EGFR-targeting agents cetuximab and erlotinib under normoxia versus hypoxia.FindingsThree cetuximab-sensitive HNSCC cell lines (SC263, LICR-HN2 and LICR-HN5) were treated with either cetuximab or erlotinib. Cells were incubated under normal or reduced oxygen conditions (<0.1% O2) for 24 or 72 h immediately after drug addition. Cell survival was assessed with the sulforhodamine B assay. Cetuximab and erlotinib established a dose-dependent growth inhibition under both normal and prolonged reduced oxygen conditions in all three HNSCC cell lines. However, a significantly increased sensitivity to cetuximab was observed in SC263 cells exposed to hypoxia for 72 h (p = 0.05), with IC50 values of 2.38 ± 0.59 nM, 0.64 ± 0.38 nM, and 0.10 ± 0.05 nM under normoxia, hypoxia for 24 h and hypoxia for 72 h, respectively. LICR-HN5 cells showed an increased sensitivity towards erlotinib when cells were incubated under hypoxia for 24 h (p = 0.05).ConclusionsOur results suggest that both EGFR-inhibitors cetuximab and erlotinib maintain their growth inhibitory effect under hypoxia. These results suggest that resistance to anti-EGFR therapy in HNSCC is probably not the result of hypoxic regions within the tumor and other mechanisms are involved.

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Section: Methodsmentioning

confidence: 99%

The hypoxic tumor microenvironment and drug resistance against EGFR inhibitors: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines

et al. 2015

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“…It is a robust and reproducible technique but is low throughput and impractical for drug screening. Various methods have been used to screen for radiation sensitizers, such as cell proliferation colorimetric assay 13 , colorimetric sulforhodamine B assay 14 , or γH2AX foci formation assay 15 , but such approaches do not appropriately identify compounds that inhibit low cell density clonogenic survival and therefore may not appropriate for radiation screening of compounds 16 . We sought to develop a method that would facilitate drug screen with radiation, capitalizing on the power of the traditional clonogenic survival assay in a higher throughput, less cumbersome format.…”

Section: Introductionmentioning

confidence: 99%

A High Content Clonogenic Survival Drug Screen Identifies MEK Inhibitors as Potent Radiation Sensitizers for KRAS Mutant Non–Small-Cell Lung Cancer

Lin¹,

Zhang²,

Giri

et al. 2014

Journal of Thoracic Oncology

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Introduction Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method which we call the High Content Clonogenic Survival Assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers. Methods Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000® (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds). Results Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild type lung cancer cells. Conclusions Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.

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“…In contrast, the SRB assay measures the total protein content and is independent of metabolic conditions of the cell line. This assay does not need specific optimization for each individual cell line [20]. Therefore, slightly lower cell inactivation that was detected by SRB could be explained by possible presence of the proteins coming from the dead cells.…”

Section: Discussionmentioning

confidence: 99%

Radiosensitivity of human ovarian carcinoma and melanoma cells to γ-rays and protons

Keta

Todorović

Popović

et al. 2014

aoms

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IntroductionProton radiation offers physical advantages over conventional radiation. Radiosensitivity of human 59M ovarian cancer and HTB140 melanoma cells was investigated after exposure to γ-rays and protons.Material and methodsIrradiations were performed in the middle of a 62 MeV therapeutic proton spread out Bragg peak with doses ranging from 2 to 16 Gy. The mean energy of protons was 34.88 ±2.15 MeV, corresponding to the linear energy transfer of 4.7 ±0.2 keV/µm. Irradiations with γ-rays were performed using the same doses. Viability, proliferation and survival were assessed 7 days after both types of irradiation while analyses of cell cycle and apoptosis were performed 48 h after irradiation.ResultsResults showed that γ-rays and protons reduced the number of viable cells for both cell lines, with stronger inactivation achieved after irradiation with protons. Surviving fractions for 59M were 0.91 ±0.01 for γ-rays and 0.81 ±0.01 for protons, while those for HTB140 cells were 0.93 ±0.01 for γ-rays and 0.86 ±0.01 for protons. Relative biological effectiveness of protons, being 2.47 ±0.22 for 59M and 2.08 ±0.36 for HTB140, indicated that protons provoked better cell elimination than γ-rays. After proton irradiation proliferation capacity of the two cell lines was slightly higher as compared to γ-rays. Proliferation was higher for 59M than for HTB140 cells after both types of irradiation. Induction of apoptosis and G2 arrest detected after proton irradiation were more prominent in 59M cells.ConclusionsThe obtained results suggest that protons exert better antitumour effects on ovarian carcinoma and melanoma cells than γ-rays. The dissimilar response of these cells to radiation is related to their different features.

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Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies

Cited by 120 publications

References 11 publications

The hypoxic tumor microenvironment and drug resistance against EGFR inhibitors: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines

The hypoxic tumor microenvironment and drug resistance against EGFR inhibitors: preclinical study in cetuximab-sensitive head and neck squamous cell carcinoma cell lines

A High Content Clonogenic Survival Drug Screen Identifies MEK Inhibitors as Potent Radiation Sensitizers for KRAS Mutant Non–Small-Cell Lung Cancer

Radiosensitivity of human ovarian carcinoma and melanoma cells to γ-rays and protons

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