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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

Time dependent relationship between changes in energy dissipation factor and vinculin immunofluorescence staining 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 the following inhibitors: 100 nM PD158780 (row A), 1 µM cytochalasin D (row B), 10 µM L779450 (row C), 10 µM LY294002 (row D), and 5 µM U73122 (row E). (AA), (BA), (CA), (DA), and (EA) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure of focal adhesions (mean ± SEM; n = 10). A strong correlation is shown between the normalized ΔD-responseand the normalized RFU of focal adhesions. To ensure that the values of RFU and ΔD could be compared, each of the values was normalized, i.e., was divided by the range covered. For each quantity, the range was taken as the highest value (at 0 min) minus the lowest value (at 60 min). All correlations are highly statistically significant (p < 0.005). In each of rows (A) to (E), column (B) shows the fluorescence images of focal adhesions in a monolayer of cells prior to inhibition, labeled as uninhibited. Columns (C) to (G) show the fluorescence images of focal adhesions in a monolayer of cells after being exposed first to the inhibitor for 40 min, then to 10 nM EGF for: (C) 0 min, (D) 30 min, (E) 60 min, and (F) 100 min, and (G) 150 min.
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biosensors-02-00448-f003: Time dependent relationship between changes in energy dissipation factor and vinculin immunofluorescence staining 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 the following inhibitors: 100 nM PD158780 (row A), 1 µM cytochalasin D (row B), 10 µM L779450 (row C), 10 µM LY294002 (row D), and 5 µM U73122 (row E). (AA), (BA), (CA), (DA), and (EA) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure of focal adhesions (mean ± SEM; n = 10). A strong correlation is shown between the normalized ΔD-responseand the normalized RFU of focal adhesions. To ensure that the values of RFU and ΔD could be compared, each of the values was normalized, i.e., was divided by the range covered. For each quantity, the range was taken as the highest value (at 0 min) minus the lowest value (at 60 min). All correlations are highly statistically significant (p < 0.005). In each of rows (A) to (E), column (B) shows the fluorescence images of focal adhesions in a monolayer of cells prior to inhibition, labeled as uninhibited. Columns (C) to (G) show the fluorescence images of focal adhesions in a monolayer of cells after being exposed first to the inhibitor for 40 min, then to 10 nM EGF for: (C) 0 min, (D) 30 min, (E) 60 min, and (F) 100 min, and (G) 150 min.

Mentions: Next we investigated the time-dependent effect of each inhibitor by focusing on changes in areal density of focal adhesions (Figure 3). Figure 3(AC–AG) show fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells that had been pretreated with 100 nM PD158780 and then exposed to 10 nM EGF for various times (0, 30, 60, 100, and 150 min). Because of the high fluorescence background, some of the images in Figure 3 look brighter than others, even though the intensities of immunostained vinculin in those images may not be necessarily higher than others. The appearance of dark holes enclosed within bright boundaries was probably due to immunostained vinculin at sites of cell-cell contact. Prior to exposure to EGF (0 min), numerous prominent focal adhesions are present as short bright streaks of vinculin in both the central regions and the peripheries of the cells that had been pretreated with 100 nM PD158780 (Figure 3(AC)). Upon exposure to 10 nM EGF for 30 min, the cells exhibit fewer, smaller, and less intense spots of stained vinculin (Figure 3(AD)), indicating a lowered level of focal adhesions. A 60-min exposure to EGF further diminished the spots of stained vinculin in size and number, as shown in Figure 3(AE). However, a longer exposure to EGF, e.g., 100 min, did not cause any further diminution of stained vinculin compared with the 60 min exposure. In fact, they show a slight increase in both size and number, which is indicative of an increase in level of focal adhesions (Figure 3(AF)). After a 150 min exposure to EGF, stained vinculin spots (Figure 3(AG)) become even more noticeable compared with the 100-min exposure. Overall, when exposed to EGF, a monolayer of mutant MCF10A cells underwent time-dependent restructuring of focal adhesions, which corresponds to de-adhesion, transition (around 60 min), and re-adhesion. This pattern is consistent with the one revealed with the QCM-D measurements in Figure 1(A). The cells in the presence of other inhibitors in this study also showed similar patterns to that of PD158780 (Figure 3).


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)

Time dependent relationship between changes in energy dissipation factor and vinculin immunofluorescence staining 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 the following inhibitors: 100 nM PD158780 (row A), 1 µM cytochalasin D (row B), 10 µM L779450 (row C), 10 µM LY294002 (row D), and 5 µM U73122 (row E). (AA), (BA), (CA), (DA), and (EA) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure of focal adhesions (mean ± SEM; n = 10). A strong correlation is shown between the normalized ΔD-responseand the normalized RFU of focal adhesions. To ensure that the values of RFU and ΔD could be compared, each of the values was normalized, i.e., was divided by the range covered. For each quantity, the range was taken as the highest value (at 0 min) minus the lowest value (at 60 min). All correlations are highly statistically significant (p < 0.005). In each of rows (A) to (E), column (B) shows the fluorescence images of focal adhesions in a monolayer of cells prior to inhibition, labeled as uninhibited. Columns (C) to (G) show the fluorescence images of focal adhesions in a monolayer of cells after being exposed first to the inhibitor for 40 min, then to 10 nM EGF for: (C) 0 min, (D) 30 min, (E) 60 min, and (F) 100 min, and (G) 150 min.
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biosensors-02-00448-f003: Time dependent relationship between changes in energy dissipation factor and vinculin immunofluorescence staining 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 the following inhibitors: 100 nM PD158780 (row A), 1 µM cytochalasin D (row B), 10 µM L779450 (row C), 10 µM LY294002 (row D), and 5 µM U73122 (row E). (AA), (BA), (CA), (DA), and (EA) Quantitation of the areal densities of stained vinculin in relative fluorescence units (RFU) as a measure of focal adhesions (mean ± SEM; n = 10). A strong correlation is shown between the normalized ΔD-responseand the normalized RFU of focal adhesions. To ensure that the values of RFU and ΔD could be compared, each of the values was normalized, i.e., was divided by the range covered. For each quantity, the range was taken as the highest value (at 0 min) minus the lowest value (at 60 min). All correlations are highly statistically significant (p < 0.005). In each of rows (A) to (E), column (B) shows the fluorescence images of focal adhesions in a monolayer of cells prior to inhibition, labeled as uninhibited. Columns (C) to (G) show the fluorescence images of focal adhesions in a monolayer of cells after being exposed first to the inhibitor for 40 min, then to 10 nM EGF for: (C) 0 min, (D) 30 min, (E) 60 min, and (F) 100 min, and (G) 150 min.
Mentions: Next we investigated the time-dependent effect of each inhibitor by focusing on changes in areal density of focal adhesions (Figure 3). Figure 3(AC–AG) show fluorescence images of immunostained vinculin within focal adhesions of mutant MCF10A cells that had been pretreated with 100 nM PD158780 and then exposed to 10 nM EGF for various times (0, 30, 60, 100, and 150 min). Because of the high fluorescence background, some of the images in Figure 3 look brighter than others, even though the intensities of immunostained vinculin in those images may not be necessarily higher than others. The appearance of dark holes enclosed within bright boundaries was probably due to immunostained vinculin at sites of cell-cell contact. Prior to exposure to EGF (0 min), numerous prominent focal adhesions are present as short bright streaks of vinculin in both the central regions and the peripheries of the cells that had been pretreated with 100 nM PD158780 (Figure 3(AC)). Upon exposure to 10 nM EGF for 30 min, the cells exhibit fewer, smaller, and less intense spots of stained vinculin (Figure 3(AD)), indicating a lowered level of focal adhesions. A 60-min exposure to EGF further diminished the spots of stained vinculin in size and number, as shown in Figure 3(AE). However, a longer exposure to EGF, e.g., 100 min, did not cause any further diminution of stained vinculin compared with the 60 min exposure. In fact, they show a slight increase in both size and number, which is indicative of an increase in level of focal adhesions (Figure 3(AF)). After a 150 min exposure to EGF, stained vinculin spots (Figure 3(AG)) become even more noticeable compared with the 100-min exposure. Overall, when exposed to EGF, a monolayer of mutant MCF10A cells underwent time-dependent restructuring of focal adhesions, which corresponds to de-adhesion, transition (around 60 min), and re-adhesion. This pattern is consistent with the one revealed with the QCM-D measurements in Figure 1(A). The cells in the presence of other inhibitors in this study also showed similar patterns to that of PD158780 (Figure 3).

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