Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model

Abstract

Introduction: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF).

Materials and methods: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy.

Results: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8.

Conclusion: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.

Figure 1

Analysis of cellular proliferation. (A) In SKOV3ip1 tumors, paclitaxel significantly decreased the number of proliferative (PCNA-positive) cells, and this effect was enhanced in response to combination treatment with paclitaxel and rhLK8. (B) In HeyA8 tumors, treatment with paclitaxel alone or rhLK8 alone did not change the cellular proliferative index (PCNA-positive cells), but combination treatment with paclitaxel and rhLK8 significantly decreased the number of PCNA-positive cells.

Figure 2

MVD of tumors by immunohistochemical staining of CD31. (A) In SKOV3ip1 tumors, rhLK8 significantly reduced the number of CD31-positive cells, and this effect was enhanced by combination treatment with rhLK8 and paclitaxel. (B) In HeyA8 tumors, treatment with paclitaxel alone significantly decreased MVD as determined by the number of CD31-positive cells, and this effect was intensified by rhLK8 treatment and most significantly by the combination of paclitaxel and rhLK8.

Figure 3

Apoptosis of tumor cells and tumor-associated endothelial cells. Tumor specimens from (A) SKOV3ip1 or (B) HeyA8 tumors were subjected to immunofluorescence double labeling, and the co-localization of signals for CD31 and TUNEL was determined. Treatment with paclitaxel alone significantly induced apoptotic (TUNEL-positive, green) tumor cells and tumor-associated endothelial cells (TUNEL-positive and CD31-positive, yellow), and this effect was intensified by treatment with rhLK8 alone. Combination treatment (paclitaxel plus rhLK8) had the most significant effect on the induction of apoptosis.

Figure W1

Effect of rhLK8 on VEGF production by human ovarian cancer cells.

Figure W2

Effects of rhLK8 and/or paclitaxel treatment on the Bcl-2 and Bax expression.

Figure W3

Effects of rhLK8 and/or paclitaxel treatment on the expression of VEGF in the tumor tissues.