Inhibition of renal cell carcinoma angiogenesis and growth by antisense oligonucleotides targeting vascular endothelial growth factor

Abstract

Angiogenesis is critical for growth and metastatic spread of solid tumours. It is tightly controlled by specific regulatory factors. Vascular endothelial growth factor has been implicated as the key factor in tumour angiogenesis. In the present studies we evaluated the effects of blocking vascular endothelial growth factor production by antisense phosphorothioate oligodeoxynucleotides on the growth and angiogenic activity of a pre-clinical model of renal cell carcinoma (Caki-1). In vitro studies showed that treating Caki-1 cells with antisense phosphorothioate oligodeoxynucleotides directed against vascular endothelial growth factor mRNA led to a reduction in expressed vascular endothelial growth factor levels sufficient to impair the proliferation and migration of co-cultured endothelial cells. The observed effects were antisense sequence specific, dose dependent, and could be achieved at a low, non-toxic concentration of phosphorothioate oligodeoxynucleotides. When vascular endothelial growth factor antisense treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential, the number of vessels initiated were approximately half that induced by untreated Caki-1 cells. To test the anti-tumour efficacy of vascular endothelial growth factor antisense, phosphorothioate oligodeoxynucleotides were administrated to nude mice bearing macroscopic Caki-1 xenografts. The results showed that the systemic administration of two doses of vascular endothelial growth factor antisense phosphorothioate oligodeoxynucleotides given 1 and 4 days after the tumours reached a size of approximately 200 mm(3) significantly increased the time for tumours to grow to 1000 mm(3).

Figure 1

VEGF levels in culture medium of Caki-1 tumour cells treated with different antisense PS-ODNs. The cells were either untreated (Untreated) or treated with delivery vehicle only (DOTAP), a 1 μM dose of control PS-ODNs sequences (Scramble, Sense and Inverted) or a 1 μM dose of antisense PS-ODNs targeted to a specific sequence of VEGF mRNA (V515). Each bar represents the mean±s.e. of three independent experiments. The star indicates a significant difference from the untreated or control treated groups (P<0.05, student's t-test). The 100% VEGF expression level of the untreated group corresponds to ∼850 pg ml⁻¹ 10⁶ cells⁻¹.

Figure 2

VEGF levels in the culture medium of Caki-1 tumour cells treated with different doses (0.5, 1, 2, 5 μM) of VEGF antisense PS-ODNs (V515). The 0 dose group was treated with scramble control PS-ODNs (5 μM). Each square represents the mean±s.e. of three independent experiments. The stars indicate significant differences from 0 dose (P<0.05, student's t-test).

Figure 3

Message RNA levels in Caki-1 cells which were either untreated or treated with a 1 μM dose of scramble control sequence or VEGF antisense PD-ODNs (V515). (A) Representative relative RT–PCR results, each group was performed in duplicate; 18s indicates the RT–PCR amplification band of 18s ribosomal RNA. (B) Relative VEGF mRNA levels of Caki-1 cells treated with different PS-ODNs. The values were obtained by dividing the densitometer reading of the VEGF band by that of the 18s band. Each bar is the mean±s.e. of three experiments. The star indicates a significant difference (P<0.05, student's t-test).

Figure 4

Effect of co-culturing Caki-1 tumour cells on the growth of endothelial cells (MHE and HMVEC-L). Caki-1 cells were untreated or pre-treated with DOTAP liposome vehicle, 1 μM scramble control PS-ODNs or 1 μM V515 antisense PS-ODNs. Each bar represents the mean±s.e. of three independent experiments. Stars indicate significant difference (P<0.05, student's t-test) from the untreated group.

Figure 5

Impact of co-culturing Caki-1 tumour cells on ability of MHE and HMVEC-L cells to migrate. Caki-1 cells were untreated or pre-treated with DOTAP liposome vehicle, a 1 μM dose of scramble control or a 1 μM dose of V515 antisense PS-ODNs. Each bar represents the mean±s.e. of three experiments. The stars indicate a significant difference (P<0.05, student's t-test) from the untreated groups. The horizontal bars show the levels of spontaneous migration of endothelial cells cultured alone; the bar widths correspond to the mean values±1 s.e.

Figure 6

Number of blood vessels induced 3 days after injecting 5×10⁴ Caki-1 cells intradermally in nude mice. Caki-1 cells were either untreated or pre-treated with a 1 μM dose of PS-ODNs for 3 h. The Scramble group refers to cells pre-treated with scramble control PS-ODNs, whereas the V515 group represents Caki-1 cells pre-treated with VEGF antisense PS-ODNs (V515). Each datum point represents one injection site, the bars show the median of 16 sites in each group. The V515 treated group was significantly different from the untreated or scramble control groups (P<0.05, Wilcoxon rank sum test).

Figure 7

Uptake and distribution of FITC-labelled PS-ODNs in Caki-1 xenografts determined 3 h after an i.v. injection of a 20 mg kg⁻¹ dose. PS-ODNs were prepared with DOTAP : DOPE. Sections were 20 μm.

Figure 8

VEGF protein levels in Caki-1 tumours at different time points after treatment of 10 mg kg⁻¹ VEGF antisense PD-ODNs (V515). (A) Representative VEGF Western blot results, showing two tumour samples of each group; (B) Relative VEGF protein levels of Caki-1 tumours treated VEGF antisense PD-ODNs (V515). Each bar is the mean±s.e. of six tumours. The star indicates a significant difference (P<0.05, student's t-test).

Figure 9

(A) Growth of median RCC Caki-1 tumours in nude mice treated systemically with antisense PS-ODNs against VEGF. Mice were untreated, treated with 10 mg kg⁻¹ scramble PS-ODNs, 5 mg kg⁻¹ VEGF antisense V515, or 10 mg kg⁻¹ VEGF antisense V515 at the time indicated by arrows. Each group consisted of 10 animals. (B) Tumour response of Caki-1 xenografts treated systemically with antisense PS-ODNs targeted to VEGF mRNA. Scramble control (Scramble) or VEGF antisense (V515) PS-ODNs were administrated with cationic liposome (DOTAP : DOPE) via the tail vein 1 and 4 days after the tumours reached a size of ∼200 mm³. Liposome administration alone had no effect on Caki-1 tumour growth (data not shown). Each circle represents a single tumour; the bars show the response of the median tumour in each group of 10 mice. The stars indicate significant differences (P<0.05, Wilcoxon rank sum test) from the untreated and scramble control groups.