Involvement of caspase-2 activation in aurora kinase inhibitor-induced cell death in axin-expressing L929 cells.

Axin is a multifunctional protein that participates in many cellular events including Wnt signaling and cell fate determination. Aurora kinase inhibitor (AKI)-induced cell death and cell membrane rupture is facilitated in L929 cells expressing axin (L-axin cells) through the activation of poly ADP-ribose polymerase (PARP). We observed that caspase-2 activity is required for AKI-induced cell death. Inhibition of caspase-2 activity suppressed AKI-induced PARP activation and mitochondrial dysfunction, resulting in a decrease in AKI-induced cell death. When an axin mutant deleted for the glycogen synthase kinase 3ß (GSK3ß)-binding domain was expressed in L929 cells (L-ΔGSK cells), AKI-induced caspase-2 activation and cell death decreased. AKI treatment reduced the expression of a 32-kDa caspase-2 splicing variant (caspase-2S) in most L-axin cells, but not in L-ΔGSK cells. These results suggest that AKI-induced caspase-2 activation in L-axin cells might be due to a decrease in the expression of caspase-2S, which inhibits caspase-2 activity. In addition, AKI treatment failed to activate caspase-8 and treatment with necrostatin inhibited AKI-induced cell death in L-axin cells, suggesting that the absence of caspase-8 activation might favor necrotic cell death. Axin expression may facilitate AKI-induced caspase-2 activation followed by activation of PARP and initiation of the necrotic cell death pathway.

Axin1 expression facilitates cell death induced by aurora kinase inhibition through PARP activation.

Axin, a negative regulator of Wnt signaling, participates in apoptosis, and Axin1 localizes to centrosomes and mitotic spindles, which requires Aurora kinase activity. In this study, Aurora inhibition of Axin1-expressing cells (L-Axin) produced polyploid cells, which died within 48 h posttreatment, whereas Axin2-expressing cells (L-Axin2) survived the same period. These cell death events showed apoptotic signs, such as chromatin condensation and increased sub-G1 populations, as well as cell membrane rupture. Further analysis showed that Aurora kinase inhibitor (AKI) treatment of L-Axin cells induced poly(ADP-ribose) polymerase (PARP) activation, which increased the poly(ADP-ribosyl)ation of cellular proteins and reduced cellular ATP content. PARP inhibition reduced a proportion of dead cells, suggesting PARP involvement in AKI-induced cell death. Also, AKI treatment of L-Axin cells induced mitochondrial apoptosis-inducing factor (AIF) release, but not mitochondrial cytochrome c release or caspase-3 activation. Knockdown of AIF attenuated AKI-induced cell death in L-Axin cells. Thus, our results suggest that Axin1 expression renders L929 cells sensitive to Aurora inhibition-induced cell death in a PARP- and AIF-dependent manner.

Axin localizes to mitotic spindles and centrosomes in mitotic cells.

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3beta) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3beta in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.

Reduction of the CD16(-)CD56bright NK cell subset precedes NK cell dysfunction in prostate cancer.

BACKGROUND: Natural cytotoxicity, mediated by natural killer (NK) cells plays an important role in the inhibition and elimination of malignant tumor cells. To investigate the immunoregulatory role of NK cells and their potential as diagnostic markers, NK cell activity (NKA) was analyzed in prostate cancer (PCa) patients with particular focus on NK cell subset distribution. METHODS: Prospective data of NKA and NK cell subset distribution patterns were measured from 51 patients initially diagnosed with PCa and 54 healthy controls. NKA was represented by IFN-γ levels after stimulation of the peripheral blood with Promoca®. To determine the distribution of NK cell subsets, PBMCs were stained with fluorochrome-conjugated monoclonal antibodies. Then, CD16(+)CD56(dim) and CD16(-)CD56(bright) cells gated on CD56(+)CD3(-) cells were analyzed using a flow-cytometer. RESULTS: NKA and the proportion of CD56(bright) cells were significantly lower in PCa patients compared to controls (430.9 pg/ml vs. 975.2 pg/ml and 2.3% vs. 3.8%, respectively; p<0.001). Both tended to gradually decrease according to cancer stage progression (p for trend = 0.001). A significantly higher CD56(dim)-to-CD56(bright) cell ratio was observed in PCa patients (41.8 vs. 30.3; p<0.001) along with a gradual increase according to cancer stage progression (p for trend = 0.001), implying a significant reduction of CD56(bright) cells in relation to the alteration of CD56(dim) cells. The sensitivity and the specificity of NKA regarding PCa detection were 72% and 74%, respectively (best cut-off value at 530.9 pg/ml, AUC = 0.786). CONCLUSIONS: Reduction of CD56(bright) cells may precede NK cell dysfunction, leading to impaired cytotoxicity against PCa cells. These observations may explain one of the mechanisms behind NK cell dysfunction observed in PCa microenvironment and lend support to the development of future cancer immunotherapeutic strategies.

Multinuclear giant cell formation is enhanced by down-regulation of Wnt signaling in gastric cancer cell line, AGS.

AGS cells, which were derived from malignant gastric adenocarcinoma tissue, lack E-cadherin-mediated cell adhesion but have a high level of nuclear beta-catenin, which suggests altered Wnt signal. In addition, approximately 5% of AGS cells form multinuclear giant cells in the routine culture conditions, while taxol treatment causes most AGS cells to become giant cells. The observation of reduced nuclear beta-catenin levels in giant cells induced by taxol treatment prompted us to investigate the relationship between Wnt signaling and giant cell formation. After overnight serum starvation, the shape of AGS cells became flattened, and this morphological change was accompanied by decrease in Myc expression and an increase in the giant cell population. Lithium chloride treatment, which inhibits GSK3beta activity, reversed these serum starvation effects, which suggests an inverse relationship between Wnt signaling and giant cell formation. Furthermore, the down-regulation of Wnt signaling caused by the over-expression of ICAT, E-cadherin, and Axin enhanced giant cell formation. Therefore, down-regulation of Wnt signaling may be related to giant cell formation, which is considered to be a survival mechanism against induced cell death.