Antiangiogenic and anticolorectal cancer effects of metronomic irinotecan chemotherapy alone and in combination with semaxinib

Abstract

Metronomic chemotherapy refers to the administration of chemotherapy at low, nontoxic doses on a frequent schedule with no prolonged breaks. The aim of the study is to rationally develop a CPT-11 metronomic regimen in preclinical settings of colon cancer. In vitro cell proliferation, apoptosis and thrombospondin-1/vascular endothelial growth factor (TSP-1/VEGF) expression analyses were performed on endothelial (HUVEC, HMVEC-d) and colorectal cancer (HT-29, SW620) cells exposed for 144 h to metronomic concentrations of SN-38, the active metabolite of CPT-11. HT-29 human colorectal cancer xenograft model was used, and tumour growth, microvessel density and VEGF/TSP-1 quantification was performed in tumours. In vitro and in vivo combination studies with the tyrosine inhibitor semaxinib were also performed. SN-38 preferentially inhibited endothelial cell proliferation alone and interacted synergistically with semaxinib; it induced apoptosis and increased the expression and secretion of TSP-1. Metronomic CPT-11 alone and combined with semaxinib significantly inhibits tumour growth in the absence of toxicity, which was accompanied by decreases in microvessel density and increases in TSP-1 gene expression in tumour tissues. In vitro results show the antiangiogenic properties of low-concentration SN-38, suggesting a key role of TSP-1 in this effect. In vivo, the CPT-11 metronomic schedule is effective against tumour and microvessel growth without toxic effect on mice.

Figure 1

Effect of low-dose SN-38, the active metabolite of CPT-11, on in vitro cell proliferation (A and B). The antiproliferative effects of the drug were studied using prolonged continuous exposures (144 h) on HUVEC, HMVEC-d, HT-29 and SW620 cell lines (A) and shorter ones (72 h) on HMVEC-d cells (B). Symbols and bars, mean values±s.e., respectively. ^*P<0.05 vs vehicle-treated controls. IC₅₀, the concentration of drug that reduced cell proliferation by 50%. Pro-apoptotic effects of (C) SN-38 on proliferating endothelial cells treated at their experimental IC₅₀s (lower concentrations) for 144 h and (D) SN-38 on proliferating colorectal cancer cells treated at their experimental IC₅₀s (higher concentrations) for 144 h. Columns and bars, mean values ±s.e., respectively. ^*P<0.05 vs vehicle-treated controls.

Figure 2

Combination index–fraction affected plot of semaxinib and SN-38 simultaneous 144-h combination in HMVEC-d cells (A). Isobologram analysis of HMVEC-d cell growth inhibition by simultaneous combination of semaxinib and SN-38 (B). The IC₅₀ values of each drug are plotted on the axes; the solid line represents the additive effect, whereas the point representing the concentrations of semaxinib and SN-38 resulting in 50% growth inhibition of the combination is reported on the left of the connecting line, indicating synergism.

Figure 3

Thrombospondin-1 (TSP-1) gene expression and secretion in HMVEC-d (A), HUVEC (B), HT-29 (C) and SW620 (D) cells exposed to SN-38 at a concentration corresponding to the experimental IC₅₀ of cell proliferation and at a lower and inactive concentration or with vehicle alone for 144 h. Vascular endothelial growth factor (VEGF) expression and secretion in HMVEC-d (E), HUVEC (F), HT-29 (G) and SW620 (H) cells exposed to the above-mentioned SN-38 concentrations. Columns and bars, mean values ±s.e., respectively. ^*P<0.05 vs vehicle-treated controls. Thrombospondin-1 and VEGF concentrations in conditioned media were measured with EIA and ELISA kits, respectively, and they were normalised to total protein concentration.

Figure 4

Thrombospondin-1 (TSP-1) and VEGF secretion in HMVEC-d (A) cells exposed to SN-38 at a concentration corresponding to the experimental IC₅₀ of cell proliferation or with vehicle alone for 72 h. Thrombospondin-1 and VEGF concentrations in conditioned media were normalised to total protein concentration. Modulation of Akt (pThr³⁰⁸) phosphorylation by SN-38 in HMVEC-d cells after 72 h of treatment (B). Columns and bars, mean values ±s.e., respectively. ^*P<0.05 vs vehicle-treated controls.

Figure 5

(A) Antitumour effect of (i) metronomic CPT-11 4 mg kg⁻¹ i.p. daily, (ii) CPT-11 100 mg kg⁻¹ i.p. every week (MTD schedule) and (iii) an initial single dose of CPT-11 100 mg kg⁻¹ i.p. followed by metronomic CPT-11 4 mg kg⁻¹ i.p. daily on HT-29 tumours xenotransplanted in CD nu/nu mice. ^*P<0.05 with respect to controls. Symbols and bars, mean±s.e. (B) Body weight of HT-29 tumour-bearing control mice and mice treated with metronomic CPT-11, MTD CPT-11 and the single high dose followed by the metronomic schedule. MTD CPT-11 caused a severe loss of weight that required veterinary assistance with an immediate fluid therapy (↑; 0.9% saline, 40–80 ml kg⁻¹ s.c. every 24 h) to save the animals. The group of animal at MTD CPT-11 needed a continued supportive fluid therapy for the duration of the treatment, whereas mice treated with a single initial dose of 100 mg kg⁻¹ and metronomic maintenance did not need any additional fluid therapy until the end of the study. Symbols and bars, mean±s.e.

Figure 6

Representative images of immunohistochemistry of mouse CD31 and human VEGF in HT-29 xenografts in vehicle-treated mice (control group) (A and B, respectively), in CPT-11 100 mg kg⁻¹ i.p. every week (MTD schedule) group of mice (C and D, respectively), in metronomic CPT-11 4 mg kg⁻¹ i.p. daily group (E and F, respectively) and in the group treated with an initial single dose of CPT-11 100 mg kg⁻¹ i.p. followed by metronomic CPT-11 4 mg kg⁻¹ i.p. daily (G and H, respectively). Arrowheads, positively stained cells. Magnification, × 200.

Figure 7

(A) Quantification of microvessel density and VEGF positivity in HT-29 tumour xenografts administered with MTD and metronomic CPT-11 schedules. (B) Thrombospondin-1 (TSP-1) and VEGF gene expression in explanted tumour samples from mice treated with MTD and metronomic CPT-11 schedules. Columns and bars, mean values ±s.e., respectively. ^*P<0.05 vs vehicle-treated controls.

Figure 8

In vivo effects of the simultaneous combination of metronomic CPT-11 schedules and semaxinib on HT-29 tumours xenotransplanted in CD nu/nu mice expressed as % T/C value. Columns, % T/C values. ^*P<0.05, initial single dose of CPT-11 100 mg kg⁻¹ followed by metronomic CPT-11 4 mg kg⁻¹ daily combined with semaxinib 10 mg kg⁻¹ twice weekly vs initial single dose of CPT-11 100 mg kg⁻¹ followed by metronomic CPT-11 4 mg kg⁻¹ daily.