Sub-Lethal Irradiation of Human Colorectal Tumor Cells Imparts Enhanced and Sustained Susceptibility to Multiple Death Receptor Signaling Pathways

Background: Death receptors (DR) of the TNF family function as anti-tumor immune effector molecules. Tumor cells, however, often exhibit DR-signaling resistance. Previous studies indicate that radiation can modify gene expression within tumor cells and increase tumor cell sensitivity to immune attack. The aim of this study is to investigate the synergistic effect of sub-lethal doses of ionizing radiation in sensitizing colorectal carcinoma cells to death receptor-mediated apoptosis. Methodology/Principal Findings: The ability of radiation to modulate the expression of multiple death receptors (Fas/ CD95, TRAILR1/DR4, TRAILR2/DR5, TNF-R1 and LTbR) was examined in colorectal tumor cells. The functional significance of sub-lethal doses of radiation in enhancing tumor cell susceptibility to DR-induced apoptosis was determined by in vitro functional sensitivity assays. The longevity of these changes and the underlying molecular mechanism of irradiation in sensitizing diverse colorectal carcinoma cells to death receptor-mediated apoptosis were also examined. We found that radiation increased surface expression of Fas, DR4 and DR5 but not LTbR or TNF-R1 in these cells. Increased expression of DRs was observed 2 days post-irradiation and remained elevated 7-days post irradiation. Sub-lethal tumor cell irradiation alone exhibited minimal cell death, but effectively sensitized three of three colorectal carcinoma cells to both TRAIL and Fasinduced apoptosis, but not LTbR-induced death. Furthermore, radiation-enhanced Fas and TRAIL-induced cell death lasted as long as 5-days post-irradiation. Specific analysis of intracellular sensitizers to apoptosis indicated that while radiation di

Functional enhancement of Fas receptor pathway in sub-lethally irradiated colorectal tumor cells.

<p>Human tumor cells were mock-irradiated (0 Gy) or irradiated with 2.5, 5 or 10 Gy and re -cultured for 72 h (3-days). Cells were harvested and incubated with the indicated concentrations of Fas crosslinking antibody CH11 or IgM isotype control antibody for 3 h. A. Non-functional Fas receptor signaling in SW620 cells 72 h post-IR. B. Functional Fas receptor signaling in WiDr cells 72 h post-IR. C. Functional Fas receptor signaling in HCT116 cells 72 h post-IR. Experiment was repeated 3 times with similar results. D. Surface Fas levels, as determined by flow cytometry of stained cells 72 h post-IR.</p

Radiation-induced increase in DR4 expression is sustained for up to a week.

<p>HCT116 cells were irradiated with 0, 5 or 10 Gy irradiation. Cells were re-cultured, harvested and subsequently stained for surface DR4 after 2 days (A–C), 4 days (D–F), and 7 days (G–I). Each experiment was repeated twice with similar results. Cells stained with a PE-labeled isotype control antibody were below 5% (not shown).</p

Tumor cells continue to proliferate following sub-lethal doses of radiation.

<p>Proliferation was measured 72 h after receiving 0, 2.5, 5, or 10 Gy of radiation by measuring BRDU incorporation. Cells receiving 25 or 30 Gy of irradiation were used as a positive control for inhibition of proliferation. A. Proliferation of SW620 cells. B. Proliferation of WiDr cells. C. Proliferation of HCT116 cells. Samples were done in triplicate at each dose. Experiment was repeated 3 times with similar results.</p

Cell death through the LTβR is not enhanced by sub-lethal irradiation of colorectal tumor cells.

<p>Cells were incubated on plates coated with 10 µg/ml or 1 µg/ml of anti- LTβR mAb (31G4D8) for 48–72 h. Cell viability was determined by 7-AAD uptake comparing cells treated with 31G4D8 versus cells cultured in medium alone and isotype control mAb. A. As a positive control HT-29 cells were incubated with 31G4D8 in conjunction with 50 units/ml IFN-γ (+IFN- γ), or without (-IFN- γ) as a negative control. B–D. Human tumor cells were mock-irradiated (0 Gy) or irradiated with 5 or 10 Gy and re-cultured for 72 h. SW620 (B), Widr (C) and HCT116 (D) cells were then harvested and incubated on plates coated with 10 µg/ml or 1 µg/ml of 31G4D8 for 48–72 h. Experiments were repeated 3 times with similar results.</p

Functional enhancement of TRAIL receptor pathway in sub-lethally irradiated colorectal tumor cells is sustained.

<p>Human tumor cells were mock-irradiated (0 Gy) or irradiated with 2.5, 5 or 10 Gy and re-cultured for the indicated times. Cells were harvested and incubated with the indicated concentrations of recombinant TRAIL protein or media for 3 h. Functional TRAIL receptor signaling in (A) SW620, (B) WiDr and (C) HCT116 cells 72 h post-IR (3-days). SW620 and WiDr cells were incubated with 100 ng/mL of recombinant protein and HCT116 cells were incubated with 25 ng/mL of protein. Experiments were repeated 3 times with similar results. Functional TRAIL receptor signaling in (D) SW620, (E) WiDr, and (F) HCT116 cells 120 h (5-days) post-IR. Experiments were repeated 2 times with similar results.</p

Radiation can modulate surface expression of some but not all TNF family death receptors in colorectal tumor cell lines.

<p>SW620, WiDr and HCT116 cells received 0 (black bar), 2.5 (dark grey bar), 5 (light gray bar) and 10 Gy (white bar) of radiation. Cells were re-cultured for 72 h and then analyzed by flow cytometry for surface (A) LTβR, (B) TNF-R1, (C) DR4, and (D) DR5 surface expression (inset; MFI of DR5 expression is shown for SW620 and HCT116 cells). Percent of cells expressing each death receptor is graphed. Cells stained with a fluorescently-labeled isotype control antibodies were negative (not shown). Staining was repeated 3 times with similar results.</p

Tumor cells remain viable after 2.5, 5 and 10 Gy of radiation.

<p>A. SW620 (squares), WiDr (circles) and HCT116 (triangles) cells were irradiated with 2.5, 5 or 10 Gy of ionizing radiation. Irradiation and non-irradiated cells (0 Gy) were cultured for 72 h. Adherent cells were subsequently harvested and cell death was analyzed by 7AAD staining and flow cytometric analysis. Experiment was repeated 3 times with similar results.</p

Enhanced sensitivity to receptor-mediated cell death by sub-lethal irradiation is apoptotic.

<p>Kinetics of dead and apoptotic cells were analyzed by annexin V–PE/7AAD staining of cells and flow cytometry. A–F. WiDr tumor cells were mock-irradiated (0 Gy) or irradiated with 5 or 10 Gy and re -cultured for 72 h. Cells were harvested and incubated with the indicated concentrations of Fas crosslinking antibody CH11 (A–C) or IgM isotype control antibody (D–F) for 5 h. Percent of apoptotic cells are represented in the upper left quadrant as Annexin-V⁺ and 7AAD⁻. B. Induction of apoptosis (Annexin-V⁺ and 7AAD⁻) following Fas receptor signaling in SW620 cells 120 h post-IR. Cells were treated with 1 µg/ml of CH11 mAb for 5 h. C. Induction of apoptosis (Annexin-V⁺ and 7AAD⁻) following TRAIL receptor signaling in HCT116 cells 72 h post-IR. Cells were incubated with 100 ng/ml of recombinant TRAIL protein for 5 h. Experiment was repeated 3 times with similar results.</p

Functional enhancement of Fas receptor pathway in sub-lethally irradiated colorectal tumor cells is sustained.

<p>Human tumor cells were mock-irradiated (0 Gy) or irradiated with 2.5, 5 or 10 Gy and re -cultured for 120 h (5-days). Cells were harvested and incubated with the indicated concentrations of Fas crosslinking antibody CH11 or IgM isotype control antibody for 3 h. A. Functional Fas receptor signaling in SW620 cells 120 h post-IR. B. Functional Fas receptor signaling in WiDr cells 120 h post-IR. C. Functional Fas receptor in HCT116 cells 120 h post-IR. Experiment was repeated 3 times with same results.</p