Tumorigenesis of HCT116 isogenic variants in nude mice.

<p>Indicated HCT116 cells (5×10⁶ cells) were transplanted into nude mice. Size of tumors was measured every other day. Five nude mice per cell type were used, and experiments were repeated at least three times. Statistical analysis was determined by the ANOVA analysis.</p

Phosphorylation of mTOR and Akt is elevated in chemoresistant tumors.

<p>Levels of indicated proteins and their phosphorylation were studied in MK-8745-resistant HCT116 Puma(-), p53(-), Chk2(-), Bax(-), p21(-) cells, and VX680-resistant HCT116 p21(-) cells. As references (indicated with *), HCT116 cells were transiently treated for 24 h with VX680 or MK8734, and HCT116 Puma(-), p53(-), Chk2(-), Bax(-) and p21(-) cells were transiently treated with VX680 for 24 h. Those cells were also immunoblotted.</p

Integrity of p53 Associated Pathways Determines Induction of Apoptosis of Tumor Cells Resistant to Aurora-A Kinase Inhibitors

<div><p>We have previously shown that mammary tumorigenesis in MMTV-Aurora-A mice is further enhanced when p53 is inactivated, demonstrating that integrity of p53 pathway determines phenotypes induced by this oncogenic kinase. In this study, we investigated the roles of genes involved in p53 pathway (p53, Puma, p21, Bax, and Chk2) in response to Aurora-A inhibitors, VX680 and MK-8745, and explored whether chemoresistant tumor cells would further undergo apoptosis with other therapeutic agents. Isogenic HCT116 cell lines were treated with VX680 or MK-8745. Cell cycle analysis, apoptosis, and tumorigenesity were studied. Chemoresistant cells were recovered from xenograft, and further induction of apoptosis was studied. Induction of apoptosis and aneuploidy with VX680 is much stronger than MK-8745. Xenograft assay indicates that tumor growth of HCT116 and HCT116 p53(-) cells are strongly inhibited by VX680, while that of other cell types are similarly inhibited by two compounds. Among the established cell lines recovered from xenografts, MK-8745-resistant clones contain elevated phosphorylation of mTOR and Akt. When further treated with inhibitors of both mTOR and Akt, those cells undergo apoptosis. These results indicate that p53-associated pathway plays a crucial role in regulating growth inhibition of tumor cells when treated with Aurora-A inhibitors. Combined treatment with Akt/mTOR inhibitors can further induce apoptosis of Aurora-A tumors.</p> </div

The effects of VX680 or MK-8745 on cell cycle.

<p>HCT116 and their isogenic variants, HCT116 p53(-), HCT116 Puma(-), HCT116 Bax(-) HCT116 p21(-) and HCT116 Chk2(-), were treated with VX680 (800 nM) or MK8734 (800 nM) for 48 h. Experiments were repeated in at least five independent experiments. Statistical analysis was determined by the Student's <i>t</i>-test (p<0.05).</p

Tumor growth of HCT116 variants resistant to either VX680 or MK-8745.

<p>Parental and VX680/MK-8745-resistant HCT116 Puma(-) cells, parental and MK-8745-resistant HCT116 Bax(-) cells, parental and MK-8745-resistant HCT116 p53(-) cells, parental and MK-8745-resistant HCT116 p21(-) cells, parental and MK-8745-resistant HCT116 Chk2(-) cells were transplanted into nude mice. Size of developed tumors was monitored every other day. Five mice per cell type were used, and experiments were repeated at least two times. Statistical analysis was determined by the 2 way ANOVA analysis.</p

Anti-proliferative effects of VX680 or MK-8745 in vivo.

<p>Each of the indicated HCT116 variants was transplanted into both flanks of a mouse. When tumor size reached at 200–400 mm3, VX680 or MK-8745 (800 nM) was injected into one of the developed tumors per mouse. Tumor size was measured every day. Total five mice were used for these studies in two independent experiments. Statistical analysis was determined by the 2 way ANOVA analysis.</p