Combined blockade of HER2 and VEGF exerts greater growth inhibition of HER2-overexpressing gastric cancer xenografts than individual blockade

Rohit Singh, Woo Jin Kim, Pyeung-Hyeun Kim, Hyo Jeong Hong, Rohit Singh, Woo Jin Kim, Pyeung-Hyeun Kim, Hyo Jeong Hong

Abstract

Gastric cancer overexpressing the human epidermal growth factor 2 (HER2) protein has a poor outcome, although a combination of chemotherapy and the anti-HER2 antibody trastuzumab has been approved for the treatment of advanced gastric cancer. Vascular endothelial growth factor (VEGF) expression in gastric cancer is correlated with recurrence and poor prognosis; however, the anti-VEGF antibody bevacizumab has shown limited efficacy against gastric cancer in clinical trials. In this study, we evaluated the antitumor effects of trastuzumab; VEGF-Trap binding to VEGF-A, VEGF-B and placental growth factor (PlGF); and a combination of trastuzumab and VEGF-Trap in a gastric cancer xenograft model. Although trastuzumab and VEGF-Trap each moderately inhibited tumor growth, the combination of these agents exerted greater inhibition compared with either agent alone. Immunohistochemical analyses indicated that the reduction in tumor growth was associated with decreased proliferation and increased apoptosis of tumor cells and decreased tumor vascular density. The combined treatment resulted in fewer proliferating tumor cells, more apoptotic cells and reduced tumor vascular density compared with treatment with trastuzumab or VEGF-Trap alone, indicating that trastuzumab and VEGF-Trap had additive inhibitory effects on the tumor growth and angiogenesis of the gastric cancer xenografts. These data suggest that trastuzumab in combination with VEGF-Trap may represent an effective approach to treating HER2-overexpressing gastric cancer.

Figures

Figure 1
Figure 1
Human epidermal growth factor 2 (HER2) and vascular endothelial growth factor (VEGF) expression levels in gastric cancer cells. NCI-N87Luc+ cells express HER2 (a) and VEGF (b), as detected by flow cytometry. (c) Detection of the expression levels of VEGF and VEGF-related receptors (VEGFR) by reverse transcriptase-polymerase chain reaction (RT-PCR). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; NRP-1, neuropilin-1.
Figure 2
Figure 2
Analysis of bromodeoxyuridine (BrdU) incorporation and total DNA content. (a and b) Epidermal growth factor (EGF)-induced cell cycle progression in NCI-N87Luc+ cells. Cells were serum-starved overnight and treated with vascular endothelial growth factor (VEGF) or EGF for 24 h. The cells were then pulsed with BrdU for 6 h, fixed and stained with a allophycocyanin (APC)-coupled anti-BrdU antibody and 7-aminoactinomycin D (7-AAD). (c and d) Cells (70–80% confluence) were treated with immunoglobulin G (IgG), trastuzumab or VEGF-Trap for 48 h and pulsed with BrdU for 6 h. The cells were then fixed and stained with an APC-coupled anti-BrdU antibody and 7-AAD. The stained cells were analyzed by a FACSAria flow cytometer (n=3; *P<0.05 versus control).
Figure 3
Figure 3
Antitumor and antiangiogenic activity of trastuzumab (Tr) in vitro. (a) In vitro proliferation (WST-1 assay) of NCI-N87Luc+ cells. (b) Inhibition of Akt and Erk 1/2 phosphorylation by trastuzumab. (c) Measurement of vascular endothelial growth factor (VEGF) mRNA in response to trastuzumab treatment in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR). (d) In vitro trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) against human epidermal growth factor 2 (HER2)-positive gastric cancer cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 4
Figure 4
The combined inhibition of human epidermal growth factor 2 (HER2) and vascular endothelial growth factor (VEGF) reduces tumor growth more efficiently than single-agent inhibition. (a) Mice bearing NCI-N87Luc+ tumors were divided into four groups and treated with an isotype-control antibody, VEGF-Trap (VT), trastuzumab (Tr) or a combination of VEGF-Trap and trastuzumab (VT+Tr) for 28 days. (b) Changes in body weight. No significant change in body weight was observed among the treatment groups. (c) Dissected tumor weight after 28 days (n=7; *P<0.05 versus isotype control; #P<0.05 versus VEGF-Trap; $P<0.05 versus trastuzumab). IgG, immunoglobulin G.
Figure 5
Figure 5
Viable and necrotic areas of tumors at the end of treatment. (a) Viable (green) and necrotic (black) areas, as differentiated by YO-PRO-1 staining and quantified by the ImageJ software program. The scale bar represents 1 mm. Viable (b) and necrotic tumor areas (c) after 28 days of treatment. (d) Viable and necrotic tumor areas are presented as a ratio (n=7; *P<0.05 versus isotype control; #P<0.05 versus vascular endothelial growth factor (VEGF)-Trap (VT); $P<0.05 versus trastuzumab (Tr)). IgG, immunoglobulin G.
Figure 6
Figure 6
The effect of treatment on tumor cell proliferation and apoptosis. Immunohistochemical staining of phospho (p)-histone-H3 (a and c) and activated caspase-3 (b and d) after 28 days of treatment. The scale bar represents 100 μm. The inset high-magnification ( × 60 objective lens) image shows a YO-PRO-1 (green)-stained nucleus and phospho-histone-H3 or activated caspase-3 (red) (n=7; *P<0.05 versus isotype control; #P<0.05 versus vascular endothelial growth factor (VEGF)-Trap (VT); $P<0.05 versus trastuzumab (Tr)).
Figure 7
Figure 7
The effect of treatment on vascular density. Two-dimensional images constructed from Z-stack images showing CD31-positive blood vessels in the peritumoural (a and c) and intratumoral (a and d) regions. The black dotted line indicates the boundary of the tumors. The scale bar represents 100 μm (n=7; *P<0.05 versus isotype control; #P<0.05 versus vascular endothelial growth factor (VEGF)-Trap (VT); $P<0.05 versus trastuzumab (Tr)). IgG, immunoglobulin G.

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