br Gottingen Germany br Nu Nu mice were intraperitoneally in
Nu/Nu mice were intraperitoneally injected with 1 106 SKOV3-Luc cells to form tumors. After 5 days, BMS-777607 (50 mg/ kg daily) or an equal volume of water (control) was administered orally to these tumor-bearing mice. The tumor growth was moni-tored in terms of luminescence activity and determined using a noninvasive IVIS system (Xenogen, Grantham, UK).To determine the tumor burden, chemiluminescent images of the tumor areas of each mouse were calculated using ImageJ software.
Inhibition of c-MET phosphorylation by BMS-777607 reduced the viability of ovarian cancer cells
Effects of BMS-777607 on the c-MET signaling transduction pathway and bio-behavior of SKOV3 cells
Induction of c-MET signaling transduction has been considered to be involved in cell proliferation, cell motility, and cell cycles. For evaluating the effects of BMS-777607 on the SKOV3 cells, we first analyzed the pattern of expression levels and phosphorylation status of c-MET downstream proteins after BMS-777607 treatment. The expression and phosphorylation of immediate c-MET-associated Gab1 and Gab2 proteins decreased (Fig. 2A). The phosphorylation of
Fig. 1. Relationship between constitutive phospho-c-MET expression and cell viability after treatment with BMS-777607. (A) Western blot analysis of total c-MET and phospho-c-MET expression in various human ovarian cancer cell lysates. (B) MTT assay of SKOV3, ES-2, and A2780 cells co-cultured in media containing various concentra-tions of BMS-777607 for 72 h. The results demonstrated that the SKOV3 cells with constitutively phosphorylated c-MET were more sensitive to BMS-777607 than the ES-2 cells with overexpressed c-MET and c-MET-negative A2780 cells. Error bars in the figure represents the standard error. *p < 0.05, ***p < 0.0001.
Effects of BMS-777607 on the AUY922 (NVP-AUY922) and mitosis of the SKOV3 cells
Because BMS-777607 inhibited the proliferation of the SKOV3 cells, the effects of this compound on the cell cycle and mitosis in SKOV3 cells were also investigated. Expression of cyclin family proteins was first detected by Western blot analysis after BMS-777607 treatment. The expression of both cyclin A2 and cyclin B1 decreased, where as the protein level of cyclin D1 increased (Fig. 3A). These results demonstrate that the cell cycle regulation of the BMS-777607-treated SKOV3 cells occurred at the G2/M transition.
Apart from c-MET, BMS-777607 is known to inhibit other ki-nases, particularly Aurora kinases. Thus, the effect of BMS-777607 on the expression and activity of Aurora kinases of SKOV3 cells
Fig. 2. Effects of BMS-777607 on c-MET-associated signaling pathways and behavior of the SKOV3 cells. (A) Western blot of c-MET signaling-associated proteins of the SKOV3 cells treated with BMS-777607. (B) Cell apoptosis assays. The percentage of apoptotic cells was determined through flow cytometry based on PI and annexin V staining. (C) Wound healing analysis. These results imply that inhibition of c-MET phosphorylation by BMS-777607 can increase apoptosis and inhibit the migration of SKOV3 cells. Bars in each chart in this figure represent the standard error. *p < 0.05; **p < 0.001; ***p < 0.0001.
was also investigated. The levels of Aurora A and Aurora B (pro-teins) decreased (Fig. 3A). This effect also caused a decrease in Histone H3 phosphorylation and led to the formation of highly polyploid cells (Fig. 3B and C) as well as a-tubulin spindle assem-bling disorder (Fig. 3D). All these results demonstrate that BMS-777607 causes cell cycle arrest and polyploidy through the inhibi-tion of Aurora A and B expression.
Antitumor effects of BMS-777607 on the SKOV3 cells in vivo
Because BMS-777607 inhibited the proliferation and division of the SKOV3 cells with constitutively phosphorylated c-MET, we investigated whether this compound could inhibit tumor growth in vivo. BMS-777607 (50 mg/kg) was orally administered to SKOV3 tumor-bearing mice daily. The tumors in the BMS-777607-treated
Fig. 3. Effect of BMS-777607 on mitosis in SKOV3 cells. (A) Western blot of cell cycle- and mitosis-associated proteins in SKOV3 cells treated with BMS-777607 for 48 h. (B) Immunofluorescent staining of a-tubulin in SKOV3 cells treated with BMS-777607. The colors red and blue in the figure represent tubulin and DNA, respectively. (C) Flow cytometric analysis of cell cycle with PI DNA staining in SKOV3 cells treated with BMS-777607. (D) Changes in the nuclear morphology of the SKOV3 cells treated with BMS-777607. These data demonstrate that BMS-777607 can affect the cell cycle, mitosis regulation, protein expression, and Histone H3 phosphorylation in SKOV3 cells. The SKOV3 cells treated with BMS-777607 also induced a-tubulin assembling disorder and polyploidy.