Isoobtusilactone A Induces Cell Cycle Arrest and Apoptosis through Reactive Oxygen Species/Apoptosis Signal-Regulating Kinase 1 Signaling Pathway in Human Breast Cancer Cells

Abstract

This study is the first to investigate the anticancer effect of isoobtusilactone A (IOA) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. IOA exhibited effective cell growth inhibition by inducing cancer cells to undergo G 2 -M phase arrest and apoptosis. Further investigation revealed that IOA's inhibition of cell growth was also evident in a nude mice model. Cell cycle blockade was associated with increased levels of p21 and reduced amounts of cyclin B1, cyclin A, cdc2, and cdc25C. IOA also enhanced the levels of inactivated phosphorylated cdc2 and cdc25C. IOA triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/ Bcl-2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and caspase-9 activation. We also found that the generation of reactive oxygen species (ROS) is a critical mediator in IOA-induced cell growth inhibition. Enhancement of ROS by IOA activated apoptosis signalregulating kinase 1 (ASK1) resulted in the increased activation of c-Jun NH 2 -terminal kinase and p38. Antioxidants EUK8 and N-acetyl cystenine significantly decreased apoptosis by inhibiting the ASK1 dephosphorylation at Ser 967 and subsequently increased the interaction of ASK1 with thioredoxin or 14-3-3 proteins. Moreover, blocking ASK1 by small interfering RNA inhibition completely suppressed IOA-induced apoptosis. Taken together, these results imply a critical role for ROS and ASK1 in IOA's anticancer activity. [Cancer Res 2007;67(15):7406-20]

Dehydrocostuslactone disrupts signal transducers and activators of transcription 3 through up-regulation of suppressor of cytokine signaling in breast cancer cells

“…During the G 2 phase, cyclin B/cdc2 complex remains inactive by phosphorylation of cdc2 at Tyr15. Cdc25C, a phosphatase, is necessary for the G 2 -M transition that can remove the phosphate group of cdc2 at Tyr15 and then restore the function of cyclin B/cdc2 complex, resulting in M-phase entrance (18,23,25). By means of immunoblot data, we found that DHE causes cyclin D1 and cyclin D2 degradation.…”

“…Cell Proliferation and Clonogenic Assays Cells were plated in 96-well culture plates (1 × 10 4 per well) and treated with vehicle alone (0.1% DMSO) and various concentrations of DHE for 48 h. Inhibition of cell proliferation was measured by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay as described previously (18). To determine long-term effects, cells (1,000 per well) were treated with DHE at various concentrations for 3 h. After being rinsed with fresh medium, cells were allowed to form colonies for 14 d and then were stained with crystal violet (0.4 g/L; Sigma).…”

“…After extraction with phenol-chloroform (1:1), the DNA was separated in 2% agarose gel and visualized by UV after staining with ethidium bromide. Apoptotic cells were quantitatively assessed by the terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) method, which examines DNA strand breaks during apoptosis by using BD ApoAlert DNA Fragmentation Assay kit as described previously (18).…”

Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1

“…The release of serum-starved HEK 293 cells into S phase or cell cycle arrest at the G 2 /M phase in MCF-7 (human breast adenocarcinoma) cells enhanced ASK1 activity without affecting ASK1 protein expression level. 21,34 In order to investigate how ASK1 is regulated during mitosis, we used nocodazole, an antineoplastic agent that interferes with microtubule polymerization, to synchronize cells in prometaphase of mitosis. Both MCF-7 and HEK 293 cells were successfully synchronized in mitosis after 16 h of nocodazole treatment ( Figure 1a).…”

“…Although ASK1 phosphorylation is known to be involved in the regulation of apoptosis, only a few reports show that ASK1 phosphorylation or activity is dependent on the cell cycle. 21,34 In this study, we examined the functional relationship between Cdc25C and ASK1 and identified a novel function of Cdc25C phosphatase that can dephosphorylate and inhibit ASK1 in interphase but not in mitosis. Furthermore, we demonstrated that Cdc25C phosphorylation status plays a critical role in the interaction with and the activity of ASK1.…”