Nnexin V/PI staining. NB4-R2 cells were treated with VX-
Nnexin V/PI staining. NB4-R2 cells were treated with VX-680 at different concentrations for 24 hr and 48 hr. (A) Apoptotic cells were measured by Annexin V/PI staining. (B) Data summarized three independent experiments, **p < 0.01, ***p < 0.001, compared to control.suppressed Akt-1 activation, reduced mitochondrial membrane potentials and induced NB4-R2 cells apoptosis by activation of caspase pathway.Discussion Aurora kinases are important for the accurate execution of mitotic events. Aur-A played a significant role inensuring the centrosome segregation and spindle assemble [20,21]. The expression of Aur-A were commonly increased in various malignant tumors [9,10]. Our recent work has showed that inhibition of Aur-A induced cell apoptotic death of laryngeal and oral squamous cell carcinoma as well as nasopharyngeal carcinoma [22-24]. In addition, Aur-A was overexpressed inXu PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 et al. Journal of Translational Medicine 2011, 9:74 http://www.translational-medicine.com/content/9/1/Page 8 ofA24h48h2 VX-680(nM)BApoptosis ( ) Apoptosis of percentage( )120 100 80 60 40 20 0 0 1 2 VX-680(nM) 5 10 *** 24h 48h *** *** ***Figure 6 Morphological changes in nucleus after induction of apoptosis by VX-680. (A) VX-680 treated or untreated cells were stained with Hoechst 33342, and observed by fluorescence microscopy (Avermectin B1a cost magnification, 400?. (B) Data summarized three independent experiments, ***p < 0.001, compared to control.bone marrow mononuclear cells (BMMCs) in a significant proportion of de novo AML patients [16]. Smallmolecule Aurora kinase inhibitor VX-680 had anti-leukemic effect for various leukemic cell types and was considered to be a potential targeting agent (Figure 1). However, the role of VX-680 in treating ATRA-resistant APL cells has not been evaluated. In this study, we showed that NB4-R2 cells were resistant to ATRA by detecting expression of CD11b (Figure 2). VX-reduced the autophosphorylation of Aur-A at the activation site, Thr288 (Figure 3A) and caused formation of monopolar structures in NB4-R2 cells (Figure 3B). In both dose- and time-dependent manners, VX-680 suppressed NB4-R2 cells growth (Figure 4A) and induced cells apoptosis (Figure 4B, 5, and 6). Moreover, we observed VX-680 induced mitochondrial depolarization by flow cytometry (Figure 7A) and importantly, caspase pathway was activated, which was associated with down-Xu et al. Journal of Translational Medicine 2011, 9:74 http://www.translational-medicine.com/content/9/1/Page 9 ofA0VX680(nM)2 5B24h0 1 2VX680(nM) 48h10 0 1 2 5aggregatesmonomerpAkt pGSK-3 Cleaved caspase3 Cleaved PARP GAPDHFigure 7 VX-680 induces mitochondrial depolarization and cellular caspase activation in NB4-R2 cells. (A) VX-680 treated NB4-R2 cells were stained with JC-1 probe and measured by flow cytometry. X- and Y-axes were indicative of monomer and aggregates, respectively. Data shown is a representative of three independent experiments. (B) NB4-R2 cells were collected, lysed and subjected to Western blot analysis with cleaved caspase-3, cleaved-PARP, pAkt-1 (Ser473), pGSK-3b (Ser9) specific antibodies. GAPDH was used as a loading control. Data shown is a representative of three independent experiments.regulation of Akt-1 phosphorylation at the activation site, Ser473 (Figure 7B). Our results suggest that VX680 is a potential novel agent for APL treatment, and Aurora kinase may serve as a promising therapeutic target for ATRA-resistant APL patients. APL is characterized by a balanced reciprocal transloc.
