Augmented inhibition of angiogenesis by combination of HER2 antibody chA21 and trastuzumab in human ovarian carcinoma xenograft

Abstract

Background: chA21 is a novel tumor-inhibitory antibody which recognized subdomain I of HER2 extracellular domain with an epitope distinct from other HER2 antibodies. Previously, we demonstrated that chA21 inhibits human ovarian carcinoma cell line SKOV-3 growth in vitro and in vivo study. In this study, we further investigated the anti-angiogenic efficacy combination of chA21 with trastuzumab in SKOV-3 xenograft model.

Methods: Nude mice were s.c. challenged with SKOV-3 cells and received treatment of chA21 alone, trastuzumab alone or both antibodies together twice a week for 21 days. Tumor volume and microvessel density (MVD) were evaluated. The effect of chA21 plus trastuzumab treament on vascular endothelial growth factor (VEGF) secretion, endothelial cells proliferation and migration, and the status of HER2 downstream pathway AKT/phosphorylated AKT (pAKT) were evaluated in vitro.

Results: In vivo study combination of chA21 with trastuzumab resulted in reduce tumor growth and angiogenesis than each monotherapy. In vitro study, the combination of chA21 with trastuzumab inhibits VEGF secretion, endothelial cells proliferation and migration. Furthermore, the combination treatment inhibits pAKT expression.

Conclusion: Our findings suggested that the combination of chA21 with trastuzumab can cause augmented inhibition of angiogenesis in SKOV-3 xenograft model. Inhibition of agniogenesis may through suppression of AKT pathway. The therapeutic benefits of combination chA21 with trastuzumab warrant further study in an attempt to make the translation into the clinic.

Figure 1

The tumor volume and the weight of SKOV-3 xenograft in the different treatment groups. (A) Either chA21 or trastuzumab treatment cause a marked growth inhibition in SKOV-3 xenograft compared with the control (P < 0.01), and the combination of chA21 with trastuzumab treatment induced a more efficient efficacy than the each antibody alone (P < 0.05). (B) When the experiment ended, all tumors were removed and weighted. Results are representative of the mean ± SD of 8 animals in each group. *, P < 0.01 compared with control. **, P < 0.01 compared with chA21 or trastuzumab alone.

Figure 2

Tumor microvessel density (MVD) and VEGF expression. (A) The VEGF and CD34 (marker for MVD) expression in SKOV-3 xenografts were detected by immunohistochemistry. (B) Tumor MVD and MOD of VEGF expression were calculated and the values were shown as percents of the control treatment. *, P < 0.01 compared with control. **, P < 0.01 compared with chA21 or trastuzumab alone.

Figure 3

Detection on secretion of VEGF from SKOV-3 cells. (A) After co-culture of SKOV-3 cells with chA21 (5 μg/ml), trastuzumab (5 μg/ml) or chA21(5 μg/ml) plus trastuzumab (5 μg/ml) for 12 h, secreted content of VEGF in the medium was detected by ELISA. *, P < 0.01 compared with control. **, P < 0.01 compared with chA21 or trastuzumab alone. (B) VEGF protein expression in the SKOV-3 cells was detected by western blot.

Figure 4

HUVECs proliferation and migration assay. (A) By the MTS assay, HUVECs number was measured by the MTS assay upon different treatment for 72 h. (B) The HUVECs migration was measured by Transwell assay in the co-cultured system (×400). (C) Data was shown as the mean of the number of migration HUVECs in five representative fields. *, P < 0.01 compared with control. **, P < 0.01 compared with chA21 or trastuzumab alone.

Figure 5

Protein expression of AKT and phopsho-AKT (pAKT) in xenograft tumors. (A) After SKOV-3 cells were treated with antibodies for 12 h, the cell lysates were analyzed. pAKT (Ser⁴⁷³) expression educed upon treament withchA21 or trastuzumab treatment and more dramatic reduction was observed with chA21 plus trastuzumab treatment. The levels of total AKT protein were not altered significantly upon various interventions. (B) The ratios of p-AKT/AKT were calculated and the values were shown as ratios compared with control, which was arbitrarily taken as 1.0. *, P < 0.01 compared with control. **, P < 0.01 compared with chA21 or trastuzumab alone.