Initial and post-treatment MR images of the radiation therapy group.

<p>The tumors increase in size and show extensive central necrosis with low signal intensity and peripheral enhancement.</p

Patterns of the time-intensity curves demonstrating the perfusion state of a tumor on dynamic contrast-enhanced MR images.

<p>(A) Pattern I: Rapid wash-in followed by the washout phase. (B) Pattern II: Rapid wash-in followed by a plateau after the peak enhancement. (C) Pattern III: Delayed wash-in and prolonged enhancement.</p

Initial and post-treatment MR images of the control group.

<p>The sizes of the tumors increase and show low signal intensity changes in the central portion of the tumors that indicate necrosis.</p

Initial and post-treatment MR images of the combination therapy (AART) group.

<p>The tumors in the AART group minimally increase and enhance without remarkable necrosis.</p

Tumor growth curves obtained by measuring the volumes of the tumors in the study groups.

<p>The volume ratio of the tumor was obtained from the initial volume (V₀) at the onset of the treatment (Day 0) to the following day. On the tumor growth curve, the mean volume ratio in each group on the follow-up dates was plotted. In the AART group, the tumor growth rate is much more suppressed than those in the other groups. RT, radiation therapy; AAT, antiangiogenic agent therapy; and AART, AAT plus RT.</p

β-lap in combination with IR induces apoptotic cell death via nuclear translocation of AIF.

<p>(A) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. The data represent a typical experiment conducted three times with similar results. (B) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 12 h. Cytosolic fractions from NQO1⁺-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. The data represent a typical experiment conducted three times with similar results. (C) NQO1⁺-MDA-MB-231 cells were treated with combination of IR and β-lap for 12 h in the presence or absence of z-VAD-fmk. The data represent a typical experiment conducted three times with similar results. (D) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of z-VAD-fmk (30 µM). After 24 h, the percentage of the cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (E) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h. Nuclear fractions from NQO1⁺-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (F) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of siRNA targeting AIF. After 24 h, the percentage of the cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (G) NQO1⁺-MDA-MB-231 cells were treated with combination of IR and β-lap for 24 h in the presence or absence of NAC, PD98059, Sal or SP600125. Nuclear fractions were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times.</p

β-lap in combination with IR induces positive feedback regulation between ERK and ROS.

<p>(A) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of NAC. The data represent a typical experiment conducted three times with similar results. (B) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 3 h in the presence or absence of siRNA targeting ERK1/2 or JNK2. After 3 h, the cells were incubated with 10 µM H2DCF-DA for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of PD98059 or SP600125. The data represent a typical experiment conducted three times with similar results.</p

β-lap in combination with IR rapidly activates ERK and JNK.

<p>(A) NQO1⁻ or NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. The data represent a typical experiment conducted three times with similar results. (B) and (C) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of PD98059 (30 µM), SP600125 (30 µM), SB203580 (30 µM), or siRNA targeting ERK1/2 or JNK2. The percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs.</p

β-lap induces radiosensitization in an NQO1-dependent manner.

<p>(A) NQO1⁻ or NQO1⁺-MDA-MB-231 cells were treated with various concentrations of β-lap. Cells were allowed to grow for 10 to 14 days and were stained with 0.5% crystal violet and scored for colony formation. Results from three independent experiments are expressed as means ± SEM. *Significant difference between NQO1⁻- and NQO1⁺-MDA-MB-231 cells after β-lap treatment at p<0.05. (B) Cells were treated with 2 µM β-lap and then exposed to increasing doses of IR 30 min after treatment with β-lap. After 14 days, cells were scored for colony formation. Results from three independent experiments are expressed as means ± SEMs. (C) Cells were treated with IR alone, β-lap alone or the combination of IR and β-lap for the indicated times. The percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs.</p

β-lap in combination with IR enhances ROS generation and leads to apoptotic cell death.

<p>(A) and (B) Cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. After 3 h, the cells were incubated with 10 µM H2DCF-DA and 4 µM DHE, respectively, for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1⁺-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of NAC (10 mM). After 24 h, the percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (D) NQO1⁺-MDA-MB-231 cells were treated with combination of IR (2 Gy) and β-lap (2 µM) in the presence or absence of NAC (10 mM). Cells were allowed to grow for 10 to 14 days and were stained with 0.5% crystal violet and scored for colony formation. Results from three independent experiments are expressed as means ± SEM. *Significant difference between cells in the presence or absence of NAC after combined treatment with IR and β-lap, at p<0.05.</p