Induction of endothelial cell apoptosis by the antivascular agent 5,6-Dimethylxanthenone-4-acetic acid

Abstract

5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg x kg(-1)). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed. Apoptosis of central vessels in splenic white pulp was also detected in tumour-bearing mice but not in mice without tumours. Apoptosis was not observed in liver tissue. No apoptosis was observed with the inactive analogue 8-methylxanthenone-4-acetic acid. Positive TUNEL staining of tumour vascular endothelium was evident in one patient in a Phase I clinical trial, from a breast tumour biopsy taken 3 and 24 h after infusion of 5,6-Dimethylxanthenone-4-acetic acid (3.1 mg x m(-2)). Tumour necrosis and the production of tumour tumour necrosis factor were not observed. No apoptotic staining was seen in tumour biopsies taken from two other patients (doses of 3.7 and 4.9 mg x m(-2)). We conclude that 5,6-Dimethylxanthenone-4-acetic acid can induce vascular endothelial cell apoptosis in some murine and human tumours. The action is rapid and appears to be independent of tumour necrosis factor induction.

Figure 1

Induction of endothelial cell apoptosis by DMXAA. (A–E) Representative cryosections of Colon 38 tumours (100× magnification) were immunostained for apoptosis using TUNEL (black) and endothelial cells with antibodies to CD-31 (red): (A) untreated, (B) 30 min after DMXAA (25 mg kg⁻¹), (C) 1 h after DMXAA, (D) 3 h after DMXAA, (E) 3 h after 8-MeXAA (220 mg kg⁻¹). (F–H) Representative cryosections (100× magnification) immunostained for apoptosis using TUNEL (black): (F) liver tissue from Colon 38 tumour-bearing mice 3 h after DMXAA (25 mg kg⁻¹), (G) spleen tissue from Colon 38-bearing mice 3 h after DMXAA (25 mg kg⁻¹), and (H) spleen tissue from normal mice 3 h after DMXAA (25 mg kg⁻¹). (I–L) Representative cryosections (100× magnification) from a human breast carcinoma immunostained for apoptosis using TUNEL (black): (I) before treatment, (J) 3 h after DMXAA infusion (3.1 mg m⁻²), (K) 24 h after DMXAA. (L) Section at higher magnification (500×) of two vessels 3 h after DMXAA infusion.

Figure 2

Northern blots for mRNA expression. (A) mRNA for IP-10, TNF, IFN-α, IFN-γ in HECPP cells detected 2 h following in vitro treatment with DMXAA (400 μg ml⁻¹). Loading of each lane shown by GADPH mRNA levels. Untreated controls (lane C). (B) mRNA for IP-10, TNF, IFN-α, IFN-γ in murine splenocytes detected 2 h following in vivo treatment with DMXAA (25 mg kg⁻¹).

Figure 3

Induction of apoptosis by DMXAA in cultured HECPP endothelial cells. (A) Percentage of HECPP cells stained using TUNEL assay for apoptosis after various incubation times with DMXAA (400 μg ml⁻¹). (B) Percentage TUNEL-staining HECPP cells 24 h after exposure to different DMXAA concentrations. Mean±s.e.m. of at least three determinations, obtained by counting 500 cells.