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  • br Fig Apatinib enhanced the pro

    2020-08-03


    Fig. 3. Apatinib enhanced the pro-apoptotic effect of cisplatin on MDA-MB-231 cells. MDA-MB-231 Methylpiperidino pyrazole were treated with cisplatin (5 μM) or/and apatinib (10 μM) for 72 h. (A) Apoptotic cells were measured with Annexin V and PI double staining. (B) The apoptosis cell rates were calculated. (C) The expressions of Bax, Bcl-2 and active caspase 3 were analyzed by western blotting in MDA-MB-231 cells. (D) The relative expression of Bax was quantified. (E) The relative expression of Bcl-2 was quantified. (F) The relative expression of active caspase 3 was quantified. **P < 0.01, compared with control group; ##P < 0.01, compared with 5 μM cisplatin treatment group.
    Fig. 4. Apatinib enhanced the inhibitory effects of cisplatin on the migration and invasion of MDA-MB-231 cells. MDA-MB-231 cells were treated with cisplatin (5 μM) or/and apatinib (10 μM) for 72 h. (A) Transwell mi-gration assay was performed to determine the migratory capacity of MDA-MB-231 cells. (B) The migratory cells in each group were quan-tified. (C) Transwell invasion assay was per-formed to determine the invasive capacity of MDA-MB-231 cells. (D) The invasion cells in each group were quantified. *P < 0.05, **P < 0.01, compared with control group; ##P < 0.01, compared with 5 μM cisplatin treatment group.
    Fig. 5. Apatinib enhanced the anti-tumor effect of cisplatin on MDA-MB-231 cells via inhibiting VEGFR2-Akt-mTOR pathway.
    expressions of p-Akt and p-mTOR were significantly decreased by cis-platin, which were further reduced in the presence of apatinib. These data suggested that apatinib enhanced the anti-tumor effect of cisplatin on MDA-MB-231 cells via inhibiting VEGFR2-Akt-mTOR pathway.
    4. Discussion
    Many studies have suggested that migration, invasion and metas-tasis are considered as the primary causes of cancer-related mortality [18]. TNBC always exhibits a high malignancy, which immensely lead to patient morbidity and mortality [19]. Angiogenesis is important for progression of malignancies; vascular endothelial growth factor (VEGF) is a main mediator of blood vessel biology [20]. VEGF plays its pro-angiogenic effects through combining and activating VEGFR2 [21]. In this study, we found that the expression of VEGFR2 was higher in pa-tients with TNBC. According to the results of clinicopathological char-acteristics, the high VEGFR-2 expression in 60 cases of patients with TNBC was 71.67% (43/60). In addition, the study of the association between VEGFR-2 and the clinicopathological factors of TNBC showed that the expression of VEGFR-2 was not related to the age of the pa-tients. However, it is related to tumor size, nuclear grade, LN metas-tasis, TNM stage and Ki-67. These data showed that VEGFR-2 is a biomarker for TNBC.
    Previous studies indicated that apatinib could inhibit metastasis and tumorigenesis in patients with TNBC [3,22,23]. In this study, apatinib enhanced the pro-apoptotic, anti-migratory and anti-invasive effects of cisplatin on MDA-MB-231 cells. In addition, apatinib is an anti-angio-genic drug and a small-molecule inhibitor of VEGFR2, which could markedly inhibit VEGF signaling pathway [24,25]. Moreover, apatinib induced cell apoptosis and inhibited cell proliferation via blocking VEGF pathway [26,27]. Prior study also demonstrated that apatinib induce apoptosis in intrahepatic cholangiocarcinoma via inhibiting VEGF signaling [27]. Thus, we hypothesized that apatinib enhance the pro-apoptotic, anti-migratory and anti-invasive effects of cisplatin in TNBC through VEGFR2 signaling. Our results indicated that cisplatin has no effect on the expression of p-VEGFR2, while combination cis-platin with apatinib significantly decreased the expressions of p-VEGFR2. Therefore, we supposed that apatinib could enhance the anti-tumor effect of cisplatin via VEGFR2. Whether other factors are in-volved in this process needs to be clarified.
    Angiogenesis and PI3K/Akt/mTOR pathway are the major mole-cular targets for the treatment of breast cancer [28]. Hong et al illu-strated that VEGF activated VEGFR2 and initiated a PI3K-AKT-mTOR pathway, which exhibited an anti-apoptotic effect [27]. Inhibition of angiogenesis through the blocking of the VEGFR2-Akt-mTOR signaling pathway has exerted as a potential method in anti-tumor therapy [29,30]. In this study, we also found that cisplatin decreased the ex-pressions of p-Akt and p-mTOR, while the levels of these proteins were further reduced in the presence of apatinib. Therefore, the possible mechanism was that apatinib decreased the VEGF-mediated Akt/mTOR signaling activity. Thus, we infer that intracellular VEGFR2 inhibitors, apatinib, could enhance the anti-tumor effect of cisplatin via VEGFR2-Akt-mTOR signaling pathway.