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Consequently, we reinvestigated the metabolic response of LM8-lacZ cells to honokiol with the WST-1 assay and shortened the treatment from 72 hours to 4 hours. It is worth noting that these short-time incubations with honokiol inhibited the metabolic activity of LM8-lacZ cells almost as effectively as the 72-hour treatment. This was reflected by an IC50 value for honokiol of 15 ��g/mL, which was only 2 times higher than that observed for the 72-hour treatment (Fig. 4A, Table 1) and, again, was representative of the other cell types that initially were investigated with the 72-hour protocol. It has been demonstrated that cyclosporin A protects against honokiol-induced cell death in esophageal carcinoma, breast cancer, and acute myeloid leukemia cells.25, 26 Likewise, http://www.selleckchem.com/products/PTC124.html we also observed this effect with LM8-lacZ cells. The inhibition of metabolic activity by honokiol could be reversed in part by preincubation of the cells with 10 ��M cyclosporin A for 2 hours (Fig. 4A). Microscopic inspection of the cells as early as 1 hour after the beginning of honokiol treatment revealed extensive cytoplasmic vacuolation (Fig. 4Bii), which also has been reported in human leukemia cells.26, 27 It is noteworthy that the vacuolation provoked by 15 ��g/mL honokiol could not be prevented by a 2-hour pretreatment of LM8-lacZ cells with 10 ��M cyclosporin A (Fig. 4Biv). http://www.selleck.cn/products/lapatinib.html Cyclosporine A alone had no effect on cell morphology http://www.selleckchem.com/products/Avasimibe(CI-1011).html (Fig. 4Biii). All observations taken together suggested that rapid inhibition of metabolic activity and early vacuolation in honokiol-treated LM8-lacZ cells were not interdependent. To further exclude the possibility that the unexpected observation of honokiol-evoked vacuolation was related to apoptosis or necrosis, we induced apoptosis in LM8-LacZ cells with staurosporine and induced necrosis with H2O2 to compare morphologic and nuclear changes in these cells with the changes in cells that were treated with honokiol. Incubation of LM8-LacZ cells with 1 ��M staurosporine for 6 hours induced apoptosis, as indicated by cell rounding, the formation of apoptotic bodies, chromatin condensation observed by 4��,6-diamidino-2-phenylindole (DAPI) staining, and karyopyknosis revealed by a 24% reduction of the nuclear area compared with control cells (P 95% of the cells, indicating loss of membrane integrity and necrosis (results not shown). Apoptotic bodies were not observed. The percentage of propidium iodide-stained control cells and staurosporine-treated cells was