Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent endogenous activator of the cell death pathway and functions by activating the cell surface death receptors 4 and 5 (DR4 and DR5). TRAIL is nontoxic in vivo and preferentially kills neoplastically transformed cells over normal cells by an undefined mechanism. Radiotherapy is a common treatment for breast cancer as well as many other cancers. Here we demonstrate that ionizing radiation can sensitize breast carcinoma cells to TRAIL-induced apoptosis. This synergistic effect is p53-dependent and may be the result of radiation-induced up-regulation of the TRAIL-receptor DR5. Importantly, TRAIL and ionizing radiation have a synergistic effect in the regression of established breast cancer xenografts. Changes in tumor cellularity and extracellular space were monitored in vivo by diffusion-weighted magnetic resonance imaging (diffusion MRI), a noninvasive technique to produce quantitative images of the apparent mobility of water within a tissue. Increased water mobility was observed in combined TRAIL- and radiation-treated tumors but not in tumors treated with TRAIL or radiation alone. Histological analysis confirmed the loss of cellularity and increased numbers of apoptotic cells in TRAIL- and radiation-treated tumors. Taken together, our results provide support for combining radiation with TRAIL to improve tumor eradication and suggest that efficacy of apoptosis-inducing cancer therapies may be monitored noninvasively, using diffusion MRI.

Figure 1

Polyhistidine-tagged recombinant TRAIL is nontoxic to mice. (A) TRAIL was run on a native SDS/PAGE gel (Left) or under reduced, denaturing conditions (Right). Monomeric TRAIL runs at ≈27 kDa whereas multimeric TRAIL runs at ≈120 kDa. (B) His-tagged TRAIL is nontoxic to NIH III nude mice. Mice were injected intravenously with saline, 50 μg of TRAIL, or 10 μg of Jo2 mAb (anti-CD95, PharMingen) once. Alternatively, mice were treated with daily injections of 5, 50, or 100 μg of TRAIL for 5 consecutive days (Chronic TRAIL). Mice were weighed 2–3 times per week with no significant decreases in control or TRAIL-treated mice (data not shown). Mice treated with Jo2 mAb died within 3 hr of injection whereas control or TRAIL-treated mice survived past 45 days. Ratios represent the number of mice that survived past 45 days over the total number treated. (C) Liver pathology of TRAIL-treated mice. Livers were isolated and sectioned in control mice and TRAIL treated mice 45 days after treatment whereas anti-CD95 livers were isolated 2 hr after injection of Jo2 mAb. Tissue sections were hematoxylin- and eosin-stained. TRAIL treatment chronically (for 45 days) or acutely (for 2 hr, data not shown) did not alter liver histology as compared with the control. However, activation of the CD95 pathway induced massive hepatocyte cell death (as evidenced by the numerous apoptotic figures), hemorrhage, and other alterations in morphology.

Figure 2

TRAIL and radiation have a combined apoptotic effect in p53 wild-type breast carcinoma cells. (A) MCF7 cells (p53 wild-type) were treated with 8 Gy radiotherapy (RT) or TRAIL (1,000 ng/ml) alone or in combination. Apoptosis was assessed 42 hr posttreatment by morphology (DNA staining) or sub-G₁ DNA content (flow cytometry). UnRx, untreated control. (B) SUM44 cells (p53 wild-type) were treated with TRAIL and selected doses of RT alone or in combination for 42 hr, and cell death was assessed by MTS assay. Cell death was determined relative to the percent viable cells in the untreated control. (C) SUM 149 cells (p53 mutant) were treated with 8 Gy RT and selected doses of TRAIL alone or in combination, and cell death was assessed by MTS assay 42 hr posttreatment. (D) SUM 190 (p53 mutant) and SUM225 (p53 mutant) cells were treated with 8 Gy RT and TRAIL (1,000 ng/ml) alone or in combination, and cell death was assessed by MTS assay 42 hr posttreatment. The data shown (mean ± SD) is from at least three independent experiments.

Figure 3

The synergistic interaction of TRAIL and radiation is p53-dependent and likely is mediated by up-regulation of the DR5 protein (TRAIL receptor). (A) Lysates were generated from parental MCF7 cells (p53 wild-type) treated with 8 Gy RT (radiotherapy) and harvested 0, 18, 36, and 72 hr later. After SDS/PAGE, samples were immunoblotted by using a polyclonal antibody directed toward DR4 and DR5. (B) MCF7 vector control cells (p53 wild-type) and MCF7 p53DN (dominant negative) expressing cells were treated with 5 Gy RT (radiotherapy) and TRAIL (1,000 ng/ml) alone or in combination and were harvested 24 hr later. Apoptosis was assessed by morphology (DNA staining). The data shown (mean ± SD) is from at least three independent experiments. (C) MCF7 vector control cells and MCF7 p53DN cells were treated with 5 Gy RT, and, 24 hr posttreatment, lysates were subjected to immunoblotting with anti-DR4 and anti-DR5 antibodies.

Figure 4

TRAIL and radiotherapy, in combination, induce regression and slow the growth of breast cancer xenografts. NIH III nude mice harboring MCF7 xenograft tumors were treated with saline, TRAIL (5 mg/kg for 7 days), RT (radiotherapy, three 5-Gy fractions), or a combination of TRAIL and RT. Mean tumor volumes were calculated 15 days after the start of treatment and are expressed as a percent change in tumor volume (A). Similarly, mean tumor volumes were assessed 2–3 times a week for over 4 weeks and are expressed in relation to the starting tumor volume (B). Six to ten xenografts were used in each experiment, and the data shown is derived from the mean volume ± SEM.

Figure 5

Noninvasive imaging of TRAIL- and radiation-induced apoptosis activity as monitored by diffusion MRI. (A) Serial histograms of ADC values in control and treated tumors. Mice were imaged at day 0 and 7 days after start of various treatments. The bar graphs displayed are the mean ADC value across pixels within region of interests defined on a representative animal from each treatment or control group. Region-of-interest areas were drawn over three consecutive MR slices near the rim of the respective images for each tumor studied. Graphed are the mean ± SD of ADC values for the regions of interest within a given tumor. (B) Isotropic ADC images of a representative tumor before and after TRAIL and radiation treatment. Pixel intensity is directly proportional to the measured ADC. There is a noticeable increase in water mobility after radiation- and TRAIL-combined treatment. Such an increase was not observed in the control or when the treatments were used individually (data not shown). (C) Imaged tumors were then assessed for apoptosis. Tumors were sectioned and subsequently stained with hematoxylin and eosin (H&E) or TUNEL. TUNEL-positive cells stain green. Nuclei are counterstained red with propidium iodide.