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. 2017 Nov;14(5):4263-4271.
doi: 10.3892/etm.2017.5028. Epub 2017 Aug 24.

Vascular endothelial growth receptor 1 acts as a stress-associated protein in the therapeutic response to thalidomide

Affiliations

Vascular endothelial growth receptor 1 acts as a stress-associated protein in the therapeutic response to thalidomide

Qin Liu et al. Exp Ther Med. 2017 Nov.

Abstract

Thalidomide (THD) exhibits antitumor effects in several types of cancer. However, the failure of THD to inhibit tumor growth has also been observed in a number of murine models in vivo. The mechanism involved in the therapeutic failure of THD remains unclear. The present study demonstrated that, accompanied by growth-arresting and apoptosis-inducing effects (P<0.05), THD upregulated vascular endothelial growth factor receptor 1 (VEGFR1) expression levels in CT26 murine colorectal carcinoma cell lines. This in vitro phenomenon was also observed in various other cell lines, including human umbilical vein endothelial cells, SW480, SW620 and HCT116. Reactive oxygen species (ROS) levels were increased compared with those in the untreated control when cells were exposed to THD (P<0.05). Furthermore, results suggested that ROS suppression may have provoked the induction of VEGFR1 expression to some extent. In addition, the results revealed that THD failed to inhibit CT26 tumor growth in vivo and the expression of VEGFR1 protein was elevated by THD treatment compared with the control group in the murine colorectal tumor model (P<0.05). The results of further experiments suggested that VEGFR1 was elevated in response to various stress-associated situations, including chemotherapy, radiotherapy and thermotherapy, which indicate that it may act as a stress-associated protein. The present findings provide a foundation for the future study of VEGFR1-targeted therapy to enhance the efficacy of current therapies.

Keywords: CT26; human umbilical vein endothelial cells; reactive oxygen species; stress response; thalidomide; vascular endothelial growth factor receptor 1.

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Figures

Figure 1.
Figure 1.
In vitro effects of THD on the growth of CT26 tumor cells and HUVECs. (A) CT26 cells and HUVECs were incubated with increasing concentrations of THD for 0, 24, 48 and 72 h, and the number of surviving cells was counted. (B and C) The percentages of apoptotic and necrotic CT26 cells and HUVECs were analyzed using an Annexin V/propidium iodide assay. Data are presented as the mean ± standard deviation. **P<0.01 and ***P<0.001 vs. Con. THD, thalidomide; HUVECs, human umbilical vein endothelial cells; Con, control.
Figure 2.
Figure 2.
Effect of THD on cell cycle and apoptosis-associated proteins. (A) CT26 tumor cells and HUVECs were incubated with increasing concentrations of THD for 24 h. Cell cycle analysis was performed using flow cytometry. (B and C) Expression of the CDK6, cyclin D1, C-MYC, cleaved-caspase-3, BCL-2 and BAX proteins in the CT26 cells and HUVECs was determined by western blot analysis. Data are presented as the mean ± standard deviation. THD, thalidomide; Con, control; HUVECs, human umbilical vein endothelial cells; CDK6, cyclin dependent kinase 6; BCL-2, B-cell lymphoma-2; BAX, BCL-2-associated X protein.
Figure 3.
Figure 3.
Effect of THD on VEGFR1 protein expression levels in vitro. (A) HUVECs and (B) CT26, (C) HCT116, SW480 and SW620 cells were treated with various concentrations of THD for 24 h. Protein expression levels of VEGFR1 in the various cell types were determined using western blot analysis. THD, thalidomide; Con, control; VEGFR1, vascular endothelial growth factor receptor 1; HUVECs, human umbilical vein endothelial cells.
Figure 4.
Figure 4.
Effect of THD-induced VEGFR1 upregulation on ROS. (A and B) CT26 tumor cells and HUVECs were incubated with indicated concentrations of THD for 6 h. Cells were stained with DCFH-DA and ROS levels were evaluated using flow cytometry. (C) HUVECs and CT26 tumor cells were treated with various concentrations of NAC and then exposed to 100 µM THD for 24 h. Expression of VEGFR1 in the CT26 cells and HUVECs was determined by western blotting. Data are presented as the mean ± standard deviation. *P<0.05 and ***P<0.001 vs. Con. THD, thalidomide; VEGFR1, vascular endothelial growth factor receptor 1; ROS, reactive oxygen species; Con, control; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; MFI, mean fluorescence index; HUVECs, human umbilical vein endothelial cells; NAC, N-acetyl-L-cysteine.
Figure 5.
Figure 5.
Effect of THD on VEGFR1 protein expression levels in vivo. (A) CT26 cells were implanted into the abdominal cavity of BALB/c mice. Tumor-bearing mice received THD therapy or saline. Body weight, ascites volume and tumor weight were assessed. (B) Representative immunohistochemical images of VEGFR1 protein expression in the control group and THD-treated group. Brown staining indicates that tissues are positive for VEGFR1. Data are presented as the mean ± standard deviation. THD, thalidomide; Con, control; VEGFR1, vascular endothelial growth factor receptor 1.
Figure 6.
Figure 6.
VEGFR1 acts as a stress-inducible molecule. (A) HUVECs and CT26 tumor cells were incubated with indicated concentrations of cisplatin, doxorubicin, paclitaxel and 5-FU, respectively. (B and C) HUVECs and CT26 tumor cells were irradiated with 2 Gy X-rays or heated at 47°C for 3 min. Protein expression levels of VEGFR1 in the CT26 and HUVECs cells were determined using western blot analysis. THD, thalidomide; Con, control; VEGFR1, vascular endothelial growth factor receptor; 5-FU, 5-fluorouracil; HUVECs, human umbilical vein endothelial cells.

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