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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

Fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells in response to 10 nM EGF. Examples of focal adhesions are indicated with arrows. Scale bar: 10 µm. The cells had been pretreated with an inhibitor described in (B–G). (A) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure for the level of focal adhesions (mean ± SEM; n = 10) in (B–G). (B) The control without the presence of an inhibitor. (C) 100 nMPD158780. (D) 1 µM cytochalasin D. (E) 10 µM L77945. (F) 10 µM LY294002. (G) 5 µM U73122. The difference in fluorescence intensities between the control and each inhibited sample is significant as indicated by P < 0.001 for all five inhibited samples.
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biosensors-02-00448-f002: Fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells in response to 10 nM EGF. Examples of focal adhesions are indicated with arrows. Scale bar: 10 µm. The cells had been pretreated with an inhibitor described in (B–G). (A) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure for the level of focal adhesions (mean ± SEM; n = 10) in (B–G). (B) The control without the presence of an inhibitor. (C) 100 nMPD158780. (D) 1 µM cytochalasin D. (E) 10 µM L77945. (F) 10 µM LY294002. (G) 5 µM U73122. The difference in fluorescence intensities between the control and each inhibited sample is significant as indicated by P < 0.001 for all five inhibited samples.

Mentions: First we examined the inhibitory effect of each inhibitor on the EGF-induced changes in level of focal adhesions. Figure 2(B–G) show fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells. Each image was taken from the cell sample that had been pretreated with a specific inhibitor (or the assay buffer for the control) for 40 min and then exposed to 10 nM EGF for 60 min. The overall comparison based on the quantitation of fluorescence intensities of stained vinculin is shown in Figure 2(A). With the presence of the inhibitors including PD158780 (100 nM, Figure 2(C)), cytochalasin D (1 µM, Figure 2(D)), L779450 (10 µM, Figure 2(E)), and LY294002 (10 µM, Figure 2(F)), the cells exhibit slightly higher fluorescence intensities of vinculin compared to the control which does not have any inhibitors (Figure 2(B)). These results indicate that these inhibitors enhanced the cell adhesion by suppressing the EGF-induced de-adhesion, which is consistent with the observed reduction of the amplitudes of the ΔD-responses in Figure 1(A–D). For the sample that had been pretreated with 5 µM U73122 (Figure 2(G)), the cells exhibit a slightly lower fluorescence intensity compared to the control (Figure 2(A)), which is consistent with the increasing amplitudes of the ΔD-responses in the presence of U73122 (Figure 1(E)), a sign of an enhanced cell de-adhesion.


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)

Fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells in response to 10 nM EGF. Examples of focal adhesions are indicated with arrows. Scale bar: 10 µm. The cells had been pretreated with an inhibitor described in (B–G). (A) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure for the level of focal adhesions (mean ± SEM; n = 10) in (B–G). (B) The control without the presence of an inhibitor. (C) 100 nMPD158780. (D) 1 µM cytochalasin D. (E) 10 µM L77945. (F) 10 µM LY294002. (G) 5 µM U73122. The difference in fluorescence intensities between the control and each inhibited sample is significant as indicated by P < 0.001 for all five inhibited samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263556&req=5

biosensors-02-00448-f002: Fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells in response to 10 nM EGF. Examples of focal adhesions are indicated with arrows. Scale bar: 10 µm. The cells had been pretreated with an inhibitor described in (B–G). (A) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure for the level of focal adhesions (mean ± SEM; n = 10) in (B–G). (B) The control without the presence of an inhibitor. (C) 100 nMPD158780. (D) 1 µM cytochalasin D. (E) 10 µM L77945. (F) 10 µM LY294002. (G) 5 µM U73122. The difference in fluorescence intensities between the control and each inhibited sample is significant as indicated by P < 0.001 for all five inhibited samples.
Mentions: First we examined the inhibitory effect of each inhibitor on the EGF-induced changes in level of focal adhesions. Figure 2(B–G) show fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells. Each image was taken from the cell sample that had been pretreated with a specific inhibitor (or the assay buffer for the control) for 40 min and then exposed to 10 nM EGF for 60 min. The overall comparison based on the quantitation of fluorescence intensities of stained vinculin is shown in Figure 2(A). With the presence of the inhibitors including PD158780 (100 nM, Figure 2(C)), cytochalasin D (1 µM, Figure 2(D)), L779450 (10 µM, Figure 2(E)), and LY294002 (10 µM, Figure 2(F)), the cells exhibit slightly higher fluorescence intensities of vinculin compared to the control which does not have any inhibitors (Figure 2(B)). These results indicate that these inhibitors enhanced the cell adhesion by suppressing the EGF-induced de-adhesion, which is consistent with the observed reduction of the amplitudes of the ΔD-responses in Figure 1(A–D). For the sample that had been pretreated with 5 µM U73122 (Figure 2(G)), the cells exhibit a slightly lower fluorescence intensity compared to the control (Figure 2(A)), which is consistent with the increasing amplitudes of the ΔD-responses in the presence of U73122 (Figure 1(E)), a sign of an enhanced cell de-adhesion.

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