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Evaluating Inhibition of the Epidermal Growth Factor (EGF)-Induced Response of Mutant MCF10A Cells with an Acoustic Sensor.

Garcia MP, Shahid A, Chen JY, Xi J - Biosensors (Basel) (2012)

Bottom Line: Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions.Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response.Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA. mpg36@drexel.edu.

ABSTRACT
Many cancer treatments rely on inhibition of epidermal growth factor (EGF)-induced cellular responses. Evaluating drug effects on such responses becomes critical to the development of new cancer therapeutics. In this report, we have employed a label-free acoustic sensor, the quartz crystal microbalance with dissipation monitoring (QCM-D), to track the EGF-induced response of mutant MCF10A cells under various inhibitory conditions. We have identified a complex cell de-adhesion process, which can be distinctly altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The dose dependencies of the inhibitors provide IC50 values which are in strong agreement with the values reported in the literature, demonstrating the sensitivity and reliability of the QCM-D as a screening tool. Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions. Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response. Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.

No MeSH data available.


Related in: MedlinePlus

Real-time quartz crystal microbalance with dissipation monitoring (QCM-D) measurements (at the order of overtone n = 3) of the ΔD-responses of mutant MCF-10A cells to 10 nM epidermal growth factor (EGF) at 37 °C. The corresponding sequential EGF-induced de-adhesion processes were indicated. (A) The ΔD-responsesof the cells were suppressed by PD158780, a known inhibitor of EGFR tyrosine kinase, at various doses (0, 10, 20, 50, 100, 200, and 400 nM). (B) The ΔD-responsesof the cells were suppressed by cytochalasin D, a known inhibitor of actin polymerization, at various doses (0, 0.1, 0.3, 0.6, 1, 2, and 4 µM). (C) The ΔD-responsesof the cells were suppressed by L779450, a known inhibitor of Raf kinase in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, at various doses (0, 0.1, 0.5, 5, 8, and 10 µM). (D) The ΔD-responsesof the cells were suppressed by LY294002, a known inhibitor of PI3K in the PI3K pathway, at various doses (0, 0.5, 1, 3, 5, 8, and 10 µM). (E) The ΔD-responsesof the cells were increased by U73122, a known inhibitor of phospholipase C (PLC)γ in the PLC pathway, at various doses (0, 0.5, 1, 5, 8, and 10 µM).
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biosensors-02-00448-f001: Real-time quartz crystal microbalance with dissipation monitoring (QCM-D) measurements (at the order of overtone n = 3) of the ΔD-responses of mutant MCF-10A cells to 10 nM epidermal growth factor (EGF) at 37 °C. The corresponding sequential EGF-induced de-adhesion processes were indicated. (A) The ΔD-responsesof the cells were suppressed by PD158780, a known inhibitor of EGFR tyrosine kinase, at various doses (0, 10, 20, 50, 100, 200, and 400 nM). (B) The ΔD-responsesof the cells were suppressed by cytochalasin D, a known inhibitor of actin polymerization, at various doses (0, 0.1, 0.3, 0.6, 1, 2, and 4 µM). (C) The ΔD-responsesof the cells were suppressed by L779450, a known inhibitor of Raf kinase in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, at various doses (0, 0.1, 0.5, 5, 8, and 10 µM). (D) The ΔD-responsesof the cells were suppressed by LY294002, a known inhibitor of PI3K in the PI3K pathway, at various doses (0, 0.5, 1, 3, 5, 8, and 10 µM). (E) The ΔD-responsesof the cells were increased by U73122, a known inhibitor of phospholipase C (PLC)γ in the PLC pathway, at various doses (0, 0.5, 1, 5, 8, and 10 µM).

Mentions: Figure 1(A) shows the results of a typical dose-response inhibition of the EGF-induced cellular response measured with the QCM-D. In this experiment, seven different doses (0, 10, 20, 50, 100, 200, and 400 nM) of PD158780, a potent inhibitor of EGFR tyrosine kinase [51] were introduced into confluent monolayers of mutant MCF-10A cells on QCM-D sensor crystals. After incubation at 37 °C for 40 min, each assay solution was replaced with a solution of 10 nM EGF. The ΔD-responses at the order of overtone, n = 3, were recorded at 37 °C for 3 h. Although the QCM-D is able to provide simultaneous measurements of both ΔD- and Δf-responses, we focused on the ΔD-responses because it is a far more sensitive measure of the EGF-induced cellular response than the Δf-response [39].


Evaluating Inhibition of the Epidermal Growth Factor (EGF)-Induced Response of Mutant MCF10A Cells with an Acoustic Sensor.

Garcia MP, Shahid A, Chen JY, Xi J - Biosensors (Basel) (2012)

Real-time quartz crystal microbalance with dissipation monitoring (QCM-D) measurements (at the order of overtone n = 3) of the ΔD-responses of mutant MCF-10A cells to 10 nM epidermal growth factor (EGF) at 37 °C. The corresponding sequential EGF-induced de-adhesion processes were indicated. (A) The ΔD-responsesof the cells were suppressed by PD158780, a known inhibitor of EGFR tyrosine kinase, at various doses (0, 10, 20, 50, 100, 200, and 400 nM). (B) The ΔD-responsesof the cells were suppressed by cytochalasin D, a known inhibitor of actin polymerization, at various doses (0, 0.1, 0.3, 0.6, 1, 2, and 4 µM). (C) The ΔD-responsesof the cells were suppressed by L779450, a known inhibitor of Raf kinase in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, at various doses (0, 0.1, 0.5, 5, 8, and 10 µM). (D) The ΔD-responsesof the cells were suppressed by LY294002, a known inhibitor of PI3K in the PI3K pathway, at various doses (0, 0.5, 1, 3, 5, 8, and 10 µM). (E) The ΔD-responsesof the cells were increased by U73122, a known inhibitor of phospholipase C (PLC)γ in the PLC pathway, at various doses (0, 0.5, 1, 5, 8, and 10 µM).
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Related In: Results  -  Collection

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biosensors-02-00448-f001: Real-time quartz crystal microbalance with dissipation monitoring (QCM-D) measurements (at the order of overtone n = 3) of the ΔD-responses of mutant MCF-10A cells to 10 nM epidermal growth factor (EGF) at 37 °C. The corresponding sequential EGF-induced de-adhesion processes were indicated. (A) The ΔD-responsesof the cells were suppressed by PD158780, a known inhibitor of EGFR tyrosine kinase, at various doses (0, 10, 20, 50, 100, 200, and 400 nM). (B) The ΔD-responsesof the cells were suppressed by cytochalasin D, a known inhibitor of actin polymerization, at various doses (0, 0.1, 0.3, 0.6, 1, 2, and 4 µM). (C) The ΔD-responsesof the cells were suppressed by L779450, a known inhibitor of Raf kinase in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, at various doses (0, 0.1, 0.5, 5, 8, and 10 µM). (D) The ΔD-responsesof the cells were suppressed by LY294002, a known inhibitor of PI3K in the PI3K pathway, at various doses (0, 0.5, 1, 3, 5, 8, and 10 µM). (E) The ΔD-responsesof the cells were increased by U73122, a known inhibitor of phospholipase C (PLC)γ in the PLC pathway, at various doses (0, 0.5, 1, 5, 8, and 10 µM).
Mentions: Figure 1(A) shows the results of a typical dose-response inhibition of the EGF-induced cellular response measured with the QCM-D. In this experiment, seven different doses (0, 10, 20, 50, 100, 200, and 400 nM) of PD158780, a potent inhibitor of EGFR tyrosine kinase [51] were introduced into confluent monolayers of mutant MCF-10A cells on QCM-D sensor crystals. After incubation at 37 °C for 40 min, each assay solution was replaced with a solution of 10 nM EGF. The ΔD-responses at the order of overtone, n = 3, were recorded at 37 °C for 3 h. Although the QCM-D is able to provide simultaneous measurements of both ΔD- and Δf-responses, we focused on the ΔD-responses because it is a far more sensitive measure of the EGF-induced cellular response than the Δf-response [39].

Bottom Line: Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions.Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response.Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA. mpg36@drexel.edu.

ABSTRACT
Many cancer treatments rely on inhibition of epidermal growth factor (EGF)-induced cellular responses. Evaluating drug effects on such responses becomes critical to the development of new cancer therapeutics. In this report, we have employed a label-free acoustic sensor, the quartz crystal microbalance with dissipation monitoring (QCM-D), to track the EGF-induced response of mutant MCF10A cells under various inhibitory conditions. We have identified a complex cell de-adhesion process, which can be distinctly altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The dose dependencies of the inhibitors provide IC50 values which are in strong agreement with the values reported in the literature, demonstrating the sensitivity and reliability of the QCM-D as a screening tool. Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions. Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response. Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.

No MeSH data available.


Related in: MedlinePlus