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  • br Results br The expression of VEGFR was upregulated

    2020-08-12


    3. Results
    3.1. The expression of VEGFR2 was upregulated in TNBC
    To explore the expression of VEGFR2 in TNBC, 60 cases of TNBC samples and adjacent samples were analyzed using IHC. As shown in Fig. 1A, the level of VEGFR2 was significantly increased in tumor tis-sues compared with that in adjacent tissues. In addition, according to the proportion of positive Oxidopamine and staining intensity, the TNBC samples were divided into high or low VEGFR2 expression group (Table 1). Moreover, VEGFR2 expression correlated with clinic-pathological parameters including tumor size, nuclear grade, LN metastasis, TNM stage and Ki-67 (Table 1). These data indicated that the upregulation of VEGFR2 was observed in TNBC, which was associated with poor prognosis of patients.
    3.2. Apatinib enhanced the anti-proliferation effect of cisplatin on MDA-MB-231 cells
    CCK-8 assay was used to assess the anti-proliferation effect of cis-platin or/and apatinib on MDA-MB-231 cells. Both cisplatin (range from 5 to 50 μM) and apatinib (range from 5 to 50 μM) inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner, re-spectively (Fig. 1B and 1C). In addition, apatinib (5, 10, 20 or 50 μM) markedly enhanced the anti-proliferative effect of cisplatin on MDA-MB-231 cells (Fig. 1D). Since, combination of cisplatin (5 μM) with apatinib (10 μM) treatment induced 50% inhibition of cell growth (Fig. 1D), cisplatin (5 μM) and apatinib (10 μM) were utilized in the following experiments. Furthermore, the immunofluorescence assay indicated cisplatin significantly inhibited the proliferation of MDA-MB-
    231 cells. The anti-proliferation effect of cisplatin was notably in-creased in the presence of apatinib, compared with cisplatin alone treatment (Fig. 2A and B). These results suggested that apatinib could enhance the anti-proliferation effect of cisplatin on MDA-MB-231 cells. 
    Table 1
    The association between the expression of VEGFR-2 and the clinicopathological characteristics in patients with TNBC.
    Clinicopathologic factors VEGFR-2 low VEGFR-2 high P
    Age
    Tumor size
    Nuclear grade
    LN metastasis
    TNM stage
    3.3. Apatinib enhanced the pro-apoptotic effect of cisplatin on MDA-MB-
    In order to investigate the effects of cisplatin or/and apatinib on the apoptosis of MDA-MB-231 cells, flow cytometry was used. As indicated in Fig. 3A and B, cisplatin-induced cell apoptosis was markedly en-hanced by the treatment of apatinib, compared with cisplatin alone treatment. In addition, western blotting was used to evaluate the ex-pressions of apoptosis-related proteins Bax, Bcl-2 and active caspase-3.
    Fig. 2. Apatinib enhanced the anti-proliferation effect of cisplatin on MDA-MB-231 cells. MDA-MB-231 cells were treated with cisplatin (5 μM) or/and apatinib (10 μM) for 72 h. (A) The proliferation of cells were detected with Ki67 staining. (B) Relative fluorescence expression levels were quantified by Ki67 and DAPI staining. **P < 0.01, compared with control group; ##P < 0.01, compared with 5 μM cisplatin treatment group.
    The results showed cisplatin significantly increased the expressions of Bax and active caspase 3 in cells, while decreased the expression of Bcl-
    2. As expected, the levels of Bax and active caspase 3 were further in-creased, and the expression of Bcl-2 was further decreased in the pre-sence of apatinib (Fig. 3C–F). These data indicated that apatinib en-hanced the pro-apoptotic effect of cisplatin on MDA-MB-231 cells.
    3.4. Apatinib enhanced the inhibitory effects of cisplatin on the migration and invasion of MDA-MB-231 cells
    Previous study demonstrated that TNBC is a severe aggressive tumor [2]. Therefore, we decided to investigate whether apatinib could en-hance the anti-migratory and anti-invasive effects of cisplatin in MDA-MB-231 cells. As illustrated in Fig. 4A–D, cisplatin significantly in-hibited cell migration and invasion. As expected, the anti-migratory and
    anti-invasive effects of cisplatin was markedly enhanced by the treat-ment of apatinib, compared with cisplatin alone treatment. These data illustrated that apatinib enhanced the inhibitory effects of cisplatin on the migration and invasion of MDA-MB-231 cells.
    3.5. Apatinib enhanced the anti-tumor effect of cisplatin on MDA-MB-231 cells via inhibiting VEGFR2-Akt-mTOR pathway
    The data reported above indicated that the expression of VEGFR2 were upregulated in patients with TNBC. Therefore, we further in-vestigated whether combination of cisplatin with apatinib could affect the fate of TNBC cells via regulating VEGFR2-Akt-mTOR pathway. The data showed combination of cisplatin with apatinib decreased the ex-pressions of p-VEGFR2, compared with the cisplatin alone treatment group (Fig. 5A and B). In addition, as indicated in Fig. 5A, C and D, the