Sulforaphane-induced G2/M Phase Cell Cycle Arrest Involves Checkpoint Kinase 2-mediated Phosphorylation of Cell Division Cycle 25C

Abstract

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9-and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G 2 /M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3␤. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G 2 /M arrest. HCT116 human colon cancer-derived Chk2 ؊/؊ cells were significantly more resistant to G 2 /M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G 2 /M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.Epidemiological studies have revealed an inverse correlation between the dietary intake of cruciferous vegetables and the risk for certain types of cancers, including prostate cancer (1-5). Laboratory studies indicate that the anticancer effect of cruciferous vegetables is caused by isothiocyanates that exist as thioglucoside conjugates (glucosinolates) in a variety of edible plants including broccoli, cabbage, watercress, and so forth (6 -9). Cruciferous vegetable-derived organic isothiocyanates are generated by hydrolytic cleavage of corresponding glucosinolates through catalytic mediation of myrosinases, which are released when the plant cells are damaged because of cutting or chewing (6 -9). Sulforaphane (SFN) 1 is one such isothiocyanate analog that has received a great deal of attention not only because it is present in high concentrations in certain varieties of broccoli but also because of its potent anticancer activity (10 -15). For example, oral administration of SFN (1-isothiocyanato-4-(methylsulfinyl)butane; CH 3 -SO-(CH 2 ) 4 -NϭCϭS)) caused a statistically significant reduction in 9,10-dimethyl-1,2-benzanthr...

p14ARF Triggers G2 Arrest Through ERK-Mediated Cdc25C Phosphorylation, Ubiquitination and Proteasomal Degradation

“…Interestingly, it was recently proposed that Ser216 phosphorylation could control Cdc25C degradation. 22 To clarify the mechanisms by which p14 ARF affects Cdc25C expression, we incubated p14 ARF -inducible cells with the proteasome inhibitor MG132 and studied the expression of Cdc25C by immunoblotting. As shown in Figure 6A (compare lanes 2 and 4), treating cells with MG132 abolished the Cdc25C protein decay observed upon p14 ARF expression.…”

“…Downregulation of Cdc25C has been previously reported in response to UVB exposure, 38 γ radiations, 39 BRCA1 expression 34 or infection by adenovirus 40 and related either with a decrease of transcription 39 or a proteasomal degradation. 22,34,40 Interestingly, Singh and colleagues recently proposed that serine 216 could control Cdc25C stability. 22 Consistent with such idea, we demonstrate that phosphorylation at serine 216 by active ERK1/2 targets Cdc25C for ubiquitination and further proteasomal degradation in response to p14 ARF .…”

A novel indole ethyl isothiocyanate (7Me-IEITC) with anti-proliferative and pro-apoptotic effects on platinum-resistant human ovarian cancer cells

“…ITC such as phenethyl ITC (PEITC), benzyl ITC (BITC) and naphthyl ITC (NITC) have been reported to inhibit function of ABC efflux proteins, P-glycoproteins and other factors [3,4] implicated in emergence of MDR [5,6,7]. Further mechanisms of ITC activity in cancer prevention have been proposed, such as induction of G2/M cell-cycle arrest and apoptosis (8) or suppression of angiogenesis with a disruption of microtubulin polymerization and mitotic progression of endothelial cells [9,10].…”

Design, Synthesis, and Evaluation of ω‐(Isothiocyanato)alkylphosphinates and Phosphine Oxides as Antiproliferative Agents

“…Reaction progress was monitored by 31 PNMR, and the reaction was continued until compound 34 was no longer present in the mixture (for 35:1 5min;f or 36:7 0min, for 37:7 0min and for 38:5 0min). Thereafter,e xcess sodium was removed from the reaction mixture and 1,n-dibromoalkane (2 equiv) was added dropwise.…”

“…Synthesis of (w-isothiocyanatoalkyl)diphenylphosphine oxides [29][30][31][32][33] New electrophiles-N-Boc protected w-bromoalkylamines-easily availablef rom parenta mines such as N-Boc 2-bromoethylamine( 14), [49] N-Boc 3-bromopropylamine ( 15), [50] bis-N-Boc 4-bromobutylamine (16), [51] bis-N-Boc 5-bromopentylamine (17), [52] and bis-N-Boc 6-bromohexylamine( 18) [53] were applied in the alkylation of diphenylphosphine oxide.A ll reactions were performed in pressure vials at 100 8C under microwave-assisted phase-transfer catalysis reaction (PTC) using toluene and 56 %a queousp otassium hydroxide as solvents andt etra-n-butylammonium bromide (TBABr)a sc atalyst. The protected diphenyl(w-aminoalkyl)phosphine oxides 19-23 were isolated in yields of 57-86 %a fter flash chromatography (Scheme 2).…”