The present study was undertaken to determine the molecular mechanism by which kaempferol induces cell death in human glioma cells. Kaempferol resulted in loss of cell viability and inhibition of proliferation in a dose- and time-dependent manner, which were largely attributed to cell death. Kaempferol caused an increase in reactive oxygen species (ROS) generation and the kaempferol-induced cell death was prevented by antioxidants, suggesting that ROS generation is involved in kaempferol-induced cell death. Kaempferol caused depolarization of mitochondrial membrane potential. Western blot analysis showed that kaempferol treatment caused a rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. The ERK inhibitor U0126 and the Akt inhibitor LY984002 increased the kaempferol-induced cell death and overexpression of MEK, the upstream kinase of ERK, and Akt prevented the cell death. The expression of anti-apoptotic proteins XIAP and survivin was down-regulated by kaempferol and its effect was prevented by overexpression of MEK and Akt. Kaempferol induced activation of caspase-3 and kaempferol-induced cell death was prevented by caspase inhibitors. Taken together, these findings suggest that kaempferol results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of XIAP and survivin regulating by ERK and Akt.

Anticancer activity of silibinin, a flavonoid, has been demonstrated in various cancer cell types. However, the underlying mechanism and in vivo efficacy in glioma were not elucidated. The present study was undertaken to determine the effect of silibinin on glioma cell proliferation in vitro and to examine whether silibinin inhibits tumor growth in vivo. Silibinin resulted in inhibition of proliferation in a dose- and time-dependent manner, which was largely attributed to cell death. Silibinin induced a transient increase in intracellular Ca2+ followed by an increase in reactive oxygen species (ROS) generation. The silibinin-induced cell death was prevented by EGTA, calpain inhibitor and antioxidants (N-acetylcysteine and Trolox). Western blot analysis showed that silibinin also induced ROS-dependent activation of extracellular signal-regulated kinase, p38 kinase, and c-Jun N-terminal kinase. Inhibitors of these kinases prevented the silibinin-induced cell death. Silibinin caused caspase activation and the silibinin-induced cell death was prevented by caspase inhibitors. Glioma cell migration was also decreased by silibinin treatment. Oral administration of silibinin in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed a decrease in Ki-67 positive cells, an increase in TUNEL-positive cells, and caspase activation. These results indicate that silibinin induces a caspase-dependent cell death via Ca2+/ROS/MAPK-mediated pathway in vitro and inhibits glioma growth in vivo. These data suggest that silibinin may serve as a potential therapeutic agent for malignant human gliomas.

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruit extracts on cell viability in vitro in human glioma cells and the anticancer efficacy in vivo. This study was undertaken to examine the effect of mulberry fruit (Moris fructus; MF) extracts on cell viability in vitro and anticancer efficacy in vivo. Cell viability and cell death were estimated by MTT assay and trypan blue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with DiOC(6)(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Cell migration was estimated using the scratched wound model. In vivo anticancer efficacy of MF extracts was evaluated using a subcutaneously injected mouse tumor model. Changes in proliferation and apoptosis were estimated by immunohistochemistric analysis. MF extracts resulted in apoptotic cell death in a dose- and time-dependent manner. MF extracts increased ROS generation, and the MF extract-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in the MF extract-induced cell death. Western blot analysis showed that treatment of MF extracts caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). MF extracts induced depolarization of mitochondrial membrane potential, and its effect was inhibited by the antioxidants NAC and catalase. MF extracts induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by MF extracts, and caspase inhibitors prevented the MF extract-induced cell death. Treatment of MF extracts inhibited cell migration. Oral MF extracts administration in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed a decrease in PCNA-positive cells, an increase in TUNEL-positive cells, and caspase activation. From these data, we concluded that MF extracts reduce glioma tumor growth through inhibition of cell proliferation resulting from induction of apoptosis. These findings suggest that MF extracts result in human glioma cell death in vitro through ROS-dependent mitochondrial pathway and glioma tumor growth in vivo via reduction of tumor cell proliferation and induction of apoptosis.

The present study was undertaken to determine the underlying mechanism of silibinin-induced cell death in human breast cancer cell lines MCF7 and MDA-MB-231. Silibinin-induced cell death was attenuated by antioxidants, N-acetylcysteine (NAC) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, suggesting that the effect of silibinin was dependent on generation of reactive oxygen species (ROS). Western blot analysis showed that silibinin induced downregulation of extracellular signal-regulated kinase (ERK) and Akt. When cells were transiently transfected with constitutively active (ca) mitogen-activated protein kinase (MEK), an upstream kinase of ERK and caAkt, they showed resistance to silibinin-induced cell death. Silibinin decreased the cleavage of Notch-1 mRNA and protein levels. Notch-1-overexpressed cells were resistant to the silibinin-induced cell death. Inhibition of Notch-1 signaling was dependent on ROS generation. Overexpression of Notch-1 prevented silibinin-induced inhibition of ERK and Akt phosphorylation. Silibinin-induced cell death was accompanied by increased cleavage of caspase-3 and was prevented by caspase-3 inhibitor in MDA-MB-231 cells but not in MCF7 cells. Silibinin induced translocation of apoptosisinducing factor (AIF), which was blocked by NAC, and transfection of caMEK and caAkt. Silibinin-induced cell death was prevented by silencing of AIF expression using small interfering AIF RNA in MCF7 cells but not in MDA-MB-231 cells. In conclusion, silibinin induces cell death through an AIF-dependent mechanism in MCF7 cells and a caspase-3-dependent mechanism in MDA-MB-231 cells, and ROS generation and Notch-1 signaling act upstream of the ERK and Akt pathway. These data suggest that silibinin may serve as a potential agent for induction of apoptosis in human breast cancer cells.