β-Lapachone Significantly Increases the Effect of Ionizing Radiation to Cause Mitochondrial Apoptosis via JNK Activation in Cancer Cells
Figure 7
β-lap in combination with IR causes Bax cleavage and translocation of cleaved Bax to mitochondria.
(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. Cell lysates were subjected to Western blot analysis. 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. Mitochondrial fractions of NQO1+-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (C) NQO1+-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 12 h. Whole cell lysates and mitochondrial fractions of NQO1+-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (D) NQO1+-MDA-MB-231 cells were treated with combination of IR and β-lap for 24 h in the presence or absence of MG132. The data are representative a typical experiment conducted three times. (E) NQO1+-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. After 12 h, the concentration of retained DiOC6(3) in cells was measured by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (F) NQO1+-MDA-MB-231 cells were treated with combination of IR and β-lap for 12 h in the presence or absence of NAC, PD98059, Sal or SP600125. Mitochondrial fractions were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (G) NQO1+-MDA-MB-231 cells were treated with combination of IR and β-lap for 12 h in the presence or absence of NAC, PD98059, Sal or SP600125. After 12 h, the concentration of retained DiOC6(3) in cells was measured by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (H) Schematic model of combined treatment (IR+β-lap)-induced apoptotic cell death. Combined treatment with IR and β-lap increases mitochondrial apoptotic cell death in an NQO1 dependent manner. As described in detail in the text, positive feedback regulation between ERK and ROS induced by combined treatment plays a critical role in the induction of ER stress. This enhanced ER stress is required for JNK activation, which leads to subsequent mitochondrial apoptotic cell death. Moreover, JNK activation induces cleavage of Bax and mitochondrial translocation of cleaved Bax, which causes loss of mitochondrial transmembrane potential and consequent release of AIF.