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. 2013 Jan 18:15:015008.
doi: 10.1088/1367-2630/15/1/015008.

Fluid Shear Stress Sensitizes Cancer Cells to Receptor-Mediated Apoptosis via Trimeric Death Receptors

Michael J Mitchell  1 , Michael R King  1
Affiliations

Fluid Shear Stress Sensitizes Cancer Cells to Receptor-Mediated Apoptosis via Trimeric Death Receptors

Michael J Mitchell et al. New J Phys. .

Abstract

Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor (TNF) apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer (NK) cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to receptor-mediated apoptosis has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlated with the application of fluid shear stress, and TRAIL-induced apoptosis increased in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis was not affected by the application fluid shear stress. Interestingly, fluid shear stress did not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments revealed that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear force can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

Keywords: TRAIL; cancer; caspase; death receptors; mechanotransduction; shear stress.

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Figures

Figure 1
Figure 1
Fluid shear stress sensitizes cancer cells to TRAIL. COLO 205 cancer cells exposed to static conditions (A) and 2.0 dyn/cm2 of fluid shear stress (B) for 120 min at 37°C, respectively. COLO 205 cells treated with 0.1 µg/mL TRAIL and then exposed to static conditions (C) and 2.0 dyn/cm2 of fluid shear stress (D) for 120 min at 37°C. Percent viable (E) and apoptotic (F) COLO 205 cells after treatment with 0.1 µg/mL TRAIL followed by exposure to static conditions and 2.0 dyn/cm2 of fluid shear stress (n = 3). Percent viable (G) and apoptotic (H) PC-3 cells after treatment with 0.1 µg/mL TRAIL followed by exposure to static conditions and 2.0 dyn/cm2 of fluid shear stress (n = 3). Lower lefthand and righthand quadrants of each flow cytometry plot represent viable and early apoptotic cells, respectively. Upper lefthand and righthand quadrants of each flow cytometry plot represent necrotic and late apoptotic cells, respectively. PI: propidium iodide. FITC: Fluorescein isothiocyanate. Error bars represent 95% confidence intervals. *P < 0.05. **P < 0.01. NS: non-significant.
Figure 2
Figure 2
Brightfield microscopy images of untreated COLO 205 cells exposed to static conditions (A) and 2.0 dyn/cm2 of fluid shear stress (B) for 120 min at 37°C. COLO 205 cells treated with 0.1 µg/mL TRAIL and then exposed to static conditions (C) and 2.0 dyn/cm2 of fluid shear stress (D) for 120 min at 37°C. Untreated PC-3 cells exposed to static conditions (E) and 2.0 dyn/cm2 of fluid shear stress (F) for 120 min at 37°C. PC-3 cells treated with 0.1 µg/mL TRAIL and then exposed to static conditions (G) and 2.0 dyn/cm2 of fluid shear stress (H) for 120 min at 37°C. Scale bars = 30 µm.
Figure 3
Figure 3
Percent necrotic COLO 205 cells (A) after treatment with 0.1 µg/mL TRAIL followed by exposure to static conditions and 2.0 dyn/cm2 of fluid shear stress (n = 3) 120 min at 37°C. Percent necrotic PC-3 cells (B) after treatment with 0.1 µg/mL TRAIL followed by exposure to static conditions and 2.0 dyn/cm2 of fluid shear stress (n = 3) 120 min at 37°C. Error bars represent 95% confidence intervals. NS: non-significant.
Figure 4
Figure 4
Increasing fluid shear stress sensitizes cancer cells to TRAIL. Percent viable (A) and apoptotic (B) COLO 205 cells (n = 3). Shear stress magnitude was varied in separate experiments from 0.05 – 2.0 dyn/cm2 for 120 min at 37°C. COLO 205 cells were treated with 0.1 µg/mL TRAIL prior to the onset of fluid shear stress. Error bars represent 95% confidence intervals. *P < 0.05 for all measurements.
Figure 5
Figure 5
Shear-induced sensitization to TRAIL increases with increasing fluid shear stress duration. Percent viable (A) and apoptotic (B) COLO 205 cells (n = 3). Time dependence of shear-induced sensitization was determined by increasing the fluid shear stress exposure time from 10 – 120 min at a uniform shear stress of 2.0 dyn/cm2 at 37°C. COLO 205 cells were treated with 0.1 µg/mL TRAIL prior to the onset of fluid shear stress. Error bars represent 95% confidence intervals. *P < 0.05 for all measurements.
Figure 6
Figure 6
Cancer cells develop sensitization to TRAIL-mediated apoptosis with increasing shear stress magnitude (A) and exposure time (B) (n = 3). Resistance is plotted as a function of the log10 of shear stress (dyn/cm2) or log10 of time (min). Error bars represent 95% confidence intervals.
Figure 7
Figure 7
Fluid shear stress does not sensitize cancer cells to doxorubicin. COLO 205 cells exposed to static conditions (A) and 2.0 dyn/cm2 of fluid shear stress (B) for 120 min at 37°C. COLO 205 cells treated with 20 µM doxorubicin and then exposed to static conditions (C) and 2.0 dyn/cm2 of fluid shear stress (D) for 120 min at 37°C. Percent apoptotic (E) COLO 205 cells after treatment with 20 µM doxorubicin followed by exposure to static conditions and 2.0 dyn/cm2 of fluid shear stress (n = 3). Comparison of cancer cell shear-induced sensitization to TRAIL and doxorubicin with increasing shear stress magnitude (F) and exposure time (G) (n = 3). Resistance is plotted as a function of the log10 of shear stress (dyn/cm2) or log10 of time (min). Error bars represent 95% confidence intervals. Gated region of flow cytometry histograms represent apoptotic COLO 205 cells. Gates determined by labeling viable COLO 205 control samples with Annexin-V APC staining. APC: allophycocyanin. NS: non-significant.
Figure 8
Figure 8
Fluid shear stress sensitization to TRAIL-mediated apoptosis is caspase-dependent. COLO 205 cells (A,B) treated with 0.1 µg/mL TRAIL (C,D), negative control inhibitor Z-FA-FMK followed by 0.1 µg/mL TRAIL (E,F), and pan caspase inhibitor Z-VAD-FMK followed by 0.1 µg/mL TRAIL (G,H), exposed to static conditions and 2.0 dyn/cm2 of fluid shear stress for 120 min at 37°C, respectively. Percent apoptotic COLO 205 cells (n = 3) after exposure to various treatments (I). Gated region of flow cytometry histograms represent apoptotic COLO 205 cells. Gates determined by labeling viable COLO 205 control samples with Annexin-V FITC staining. FITC: fluorescein isothiocyanate. NS: non-significant.
Figure 9
Figure 9
Fluid shear stress does not alter death receptor surface expression. COLO 205 cells exposed to static conditions and 2.0 dyn/cm2 of fluid shear stress for 120 min at 37°C were labeled with anti-DR4 (A) and anti-DR5 (B) antibodies, respectively. (C) QSC receptor quantification of DR4 and DR5 on the surface of COLO 205 cells exposed to static conditions and fluid shear stress (n = 3). NS: non-significant.
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