Thrombospondin 1, a mediator of the antiangiogenic effects of low-dose metronomic chemotherapy

Abstract

Chemotherapeutic drugs chronically administered to tumor-bearing mice, using a frequent schedule at doses substantially lower than the maximum tolerated dose (MTD) (i.e., metronomic dosing), can cause sustained and potent antiangiogenic effects by targeting the endothelial cells of newly growing tumor blood vessels. These effects appear to occur in the absence of an increase in the severity of side effects caused by destruction of other cell types normally sensitive to MTD chemotherapy, suggesting a marked and selective sensitivity of activated endothelial cells, the basis of which is unknown. Here we report that protracted exposure of endothelial cells in vitro to low concentrations of several different anticancer agents, including microtubule inhibitors and an alkylating agent, caused marked induction of gene and protein expression of TSP-1, a potent and endothelial-specific inhibitor of angiogenesis. Increases in circulating TSP-1 were also detected in the plasma of human tumor-bearing severe combined immunodeficient mice treated with metronomic low-dose cyclophosphamide. Most importantly, the antiangiogenic and antitumor effects of low-dose continuous cyclophosphamide were lost in TSP-1-null C57BL/6 mice, whereas, in contrast, these effects were retained by using a MTD schedule of the same drug. Taken together, the results implicate TSP-1 as a secondary mediator of the antiangiogenic effects of at least some low-dose metronomic chemotherapy regimens.

Fig. 1.

(A) Northern blot analysis with its densitometric quantification (Upper Right) and Western blot analysis (Lower Right) of human dermal microvascular endothelial cell samples after 6 days of low-dose treatment with different cytotoxic drugs. β-Actin and CRM-1 were used as loading controls. (B) Human TSP-1 competitive enzyme immunoassay of vascular endothelial cell-conditioned media after 6 days of treatment using low concentrations of different cytotoxic drugs. The results are expressed as the percentage increase of secreted TSP-1 versus control samples and are the mean of two independent experiments with at least two replicates per sample. (C) Effect of protracted (144 h) low-dose cytotoxic drug treatments, in the presence or absence of neutralizing monoclonal antibody A4.1 directed against human TSP-1, on in vitro microvascular endothelial cell proliferation. Shown are mean values ± SEM.

Fig. 2.

Inhibition of angiogenesis in vivo by daily low-dose (≈25 mg/kg p.o.) and MTD CTX (150 mg/kg i.v.) in wild-type and TSP-1-null C57BL/6 mice. Angiogenesis was induced in s.c. implanted matrigel (matr.) plugs by bFGF. After 10 days of treatment, mice were injected i.v. with FITC-dextran (4). Macroscopic appearance of representative sample of matrigel alone (negative control) (A), matrigel plus bFGF (positive control) (B), matrigel plus bFGF plus low-dose CTX (C), and matrigel plus bFGF plus MTD CTX (D). Quantification of intravascular FITC content showed a significant inhibition of neovascularization in wild-type mice treated with a low-dose CTX regimen (E) but a complete lack of antiangiogenic activity of the same regimen (CTX 25) in TSP-1-null mice, whereas MTD CTX (CTX 150) retained activity in these mice (F). Shown are mean values ± SD (*, P < 0.05).

Fig. 3.

Effects of MTD CTX (150 mg/kg i.v.), which was then switched to low-dose CTX (25 mg/kg p.o.) or normal saline on LL/2 tumor in syngeneic C57BL/6 mice. The two regimens were compared in wild-type (A) and TSP-1-null (B) mice. Results showed a complete loss of antitumor activity after a continuous low-dose CTX in TSP-1-null C57BL/6 mice (B) compared with wild-type controls (A), whereas the MTD regimen suppressed tumor growth to an equivalent extent in both types of mice.

Fig. 4.

(A) Effect of two different metronomic chemotherapy regimens (continuous administration of CTX 20 mg/kg p.o. or VBL 0.3 mg/kg administered three times a week) and their combination at the same doses (CTX + VBL) or consecutively and alternatively (CTX/VBL) in human PC-3 tumor xenografts. At day 20, treatment was started (arrow), and at days 40 and 41, mice were bled from the retroorbital sinus, and tumor volumes were measured, respectively (gray rectangle). (B) The TSP-1-competitive enzyme immunoassay was performed on those blood samples, and the results are given as the ratio of TSP-1 plasma concentration to tumor volume. The ratio significantly increased in all treated groups and strongly correlated with the response to the low-dose metronomic chemotherapy regimens. Shown are mean values ± SD (*, P < 0.05; **, P < 0.005).