• 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br Fig Whole tumour lysates were prepared and protein levels


    Fig. 6. Whole tumour lysates were prepared and protein levels of the EMT markers E-cadherin, N-cadherin, Vimentin, and Snail were analysed by western blotting (**P b 0.01; ***P b 0.001).
    3.3. Apatinib suppresses the epithelial-mesenchymal transition (EMT) in ovarian cancer cells
    The EMT is closely related to tumour metastasis. Western blotting was performed to measure the levels of EMT-associated markers in SKOV3 and HO8910 Nutlin-3 after treatment with apatinib and investigate whether apatinib suppresses the EMT in ovarian cancer cells. As shown in Fig. 3, the level of the epithelial marker E-cadherin was in-creased, while the levels of the mesenchymal markers Vimentin and N-cadherin were decreased by the apatinib treatment. Moreover, the 
    levels of the transcription factors Snail and Zo-1 were also decreased by the apatinib treatment. Based on these results, apatinib inhibits the EMT in ovarian cancer cells.
    3.4. Apatinib inhibits the JAK/STAT3, PI3K/Akt and Notch signalling path-ways in human EOC cells
    The JAK/STAT3, PI3K/Akt, and Notch signalling pathways are associ-ated with the EMT. Therefore, western blotting was performed to mea-sure the levels of markers of these three signalling pathways, including
    JAK, phosphorylated (p)-JAK, Stat3, p-Stat3, PI3K, Akt, p-Akt, Notch1, Hes1 and Hey1, and to reveal the underlying molecular mechanisms as-sociated with apatinib-induced EMT inhibition in ovarian cancer cells. Apatinib decreased the levels of p-JAK, p-Stat3, p-Akt, PI3K, Notch1, Hey1 and Hes1. JAK, Stat3 and Akt levels were unchanged between the control group and the apatinib-treated group (Fig. 4). Therefore, apatinib inhibits the EMT in human EOC cells via the JAK/STAT3, PI3K/ Akt, and Notch signalling pathways.
    3.5. Apatinib inhibits tumour growth in vivo
    After characterizing the effects of apatinib on ovarian cancer cells in vitro, we studied its effect on tumour growth in vivo using a mouse xenograft model. SKOV3 cells were introduced into immunocompro-mised nude mice to evaluate the efficacy of apatinib in inhibiting tu-mour growth. As shown in Fig. 5A and B, the oral administration Nutlin-3 of apatinib significantly inhibited SKOV3 tumour growth compared to the control treatment (P b 0.05). Tumour volumes were measured and plotted after each treatment to evaluate the effect of apatinib on mouse xenografts of ovarian cancer cells. After 40 days, the tumour vol-ume of the 50 mg/kg apatinib-treated group (644 ± 28.6 mm3) was sig-nificantly smaller than the NC group (1139 ± 115 mm3) (P b 0.01). In this xenograft model of ovarian cancer, apatinib significantly delayed tumour growth in mice. No significant differences in the body weights of the mice were observed between the groups before or after treatment (data not shown).
    Tumours were harvested, homogenized, and the levels of down-stream protein targets of the drug were analysed using western blotting to further examine target modulation in vivo. As shown in Fig. 6, the level of the epithelial marker E-cadherin was increased, while the levels of the mesenchymal markers Vimentin and N-cadherin were decreased by the apatinib treatment. Moreover, the level of the transcription factor Snail was also decreased by the apatinib treatment. Thus, apatinib in-hibits the EMT in mice with ovarian cancer.
    4. Discussion
    As Judah Folkman first proposed in 1971, tumour growth depends on angiogenesis [11]. Inhibitors of angiogenesis have become an impor-tant therapeutic strategy in the treatment of various tumours. Tumour angiogenesis plays an important role in the occurrence, development, and metastasis of ovarian cancer. Apatinib is a small-molecule VEGFR TKI that tightly binds and inhibits VEGFR-2 [5]. It is now recognized as a first generation oral anti-angiogenesis drug in China, where it is also a potential new third-line option for treating refractory gastric cancer [12]. Currently, the definitive efficacy of apatinib cannot be estimated due to an insufficient number of patients recruited for clinical trials. However, in many pretreated patients, the survival rates, including overall survival and progression-free survival, have been improved [13]. Apatinib inhibits the migration and proliferation of endothelial cells stimulated by VEGF. Thus, it is considered a promising VEGFR-2 in-hibitor that blocks tumour-induced angiogenesis [5,14]. The present study was performed to explore the anti-tumour activity of apatinib in ovarian cancer both in vitro and in vivo. We aimed to provide evidence supporting the use of apatinib as a treatment for ovarian cancer in clin-ical practice.
    Recent studies have reported the direct anticancer activity of apatinib in various cancer cell lines [15,16]. The proliferation of colon cancer cells was inhibited upon treatment with different concentrations of apatinib (20 and 40 μM) [17]. According to the results of the MTT pro-liferation assay, significantly lower viability of melanoma MUM-2B cells is observed in apatinib-treated groups [18]. In another study, when HCT116 and SW480 cells were treated with 20 μM apatinib, the apopto-sis percentages were 3.7% and 5.8%, respectively. As the drug concentra-tion increased to 40 μΜ, the apoptosis percentages increased to 11.9% and 13.5%. Moreover, the cell cycle was also altered [17]. However, in