Inhibition of angiogenesis induces chromaffin differentiation and apoptosis in neuroblastoma - PubMed (original) (raw)

Inhibition of angiogenesis induces chromaffin differentiation and apoptosis in neuroblastoma

E Wassberg et al. Am J Pathol. 1999 Feb.

Abstract

Inhibition of angiogenesis has been shown to reduce tumor growth, metastasis, and tumor microvascular density in experimental models. To these effects we would now like to add induction of differentiation, based on biological analysis of xenografted human neuroblastoma (SH-SY5Y, WAG rnu/rnu) treated with the angiogenesis inhibitor TNP-470. Treatment with TNP-470 (10 mg/kg s.c., n = 15) reduced the tumor growth by 66% and stereological vascular parameters (Lv, Vv, Sv) by 36-45%. The tumor cell apoptotic fraction increased more than threefold, resulting in a decrease in viable tumor cells by 33%. In contrast, the mean vascular diameter (29 microm) and the mean tumor cell proliferative index (49%) were unaffected. TNP-470-treated tumors exhibited striking chromaffin differentiation of neuroblastoma cells, observed as increased expression of insulin-like growth factor II gene (+88%), tyrosine hydroxylase (+96%), chromogranin A, and cellular processes. Statistical analysis revealed an inverse correlation between differentiation and angiogenesis. It is suggested that by inhibiting angiogenesis, TNP-470 induces metabolic stress, resulting in chromaffin differentiation and apoptosis in neuroblastoma. Such agonal differentiation may be the link between angiostatic therapy and tumor cell apoptosis.

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Figures

Figure 1.

Microvascular corrosion cast at scanning electron microscopy. Human neuroblastoma xenotransplanted to nude rat. A: control; B: treated with TNP-470. Top: overview; bottom: close-up of the tumor capsule. The tumor microcirculation consists of newly formed sinusoidal tumor vessels, 10–80 μm in diameter, exhibiting extensive anastomoses. Only the number of vessels is affected by treatment with TNP-470, not the angioarchitecture. There are a few true arteriovenous capillaries, 4–10 μm in diameter. Scale bars: overview, 1 mm; close-up, 500 μm.

Figure 2.

Markers of proliferation, apoptosis, and chromaffin differentiation in neuroblastoma tumors treated with TNP-470, perivascular cuffs. A: Ki 67; proliferative cells in the inner and middle layers. B: TUNEL; apoptotic cells mainly located in the outer layers. C: Insulin-like growth factor II gene (IGF2) expression, dark field view. Note the distinct ring-like expression peripheral to the central vessel. D: IGF2 expression, light field view. E: TH; positive cells located in the middle and outer layers, essentially the same distribution as IGF2. F: TH; Cellular processes (arrows) in close-up. G: Chromogranin A. H: Hematoxylin and eosin. Scale bars: F, 20 μm; all others, 100 μm.

Figure 3.

Plasma concentration of chromogranin A (CgA) versus tumor volume. CgA levels were directly proportional to tumor volume and intact CgA was liberated to the rat circulation from viable tumor cells, as we have reported earlier. Treatment with TNP-470 (in a separate correlation study) did not affect this correlation, despite a 33% reduction of viable neuroblastoma cells. Our interpretation of this phenomenon is that TNP-470 causes increased CgA secretion as a sign of chromaffin differentiation. CgA was measured with a specific competitive radioimmunoassay. The heparin plasma was kept frozen until measurements. •, Treatment with 10 mg/kg body weight of TNP-470 s.c. every other day for 12 to 24 days (n = 7); ○, no treatment (n = 20, control group).

Figure 4.

Agonal differentiation and apoptosis. Based on the cellular dynamics and differentiation observed, we suggest that TNP-470 induces metabolic stress (eg, hypoxia, reduction of endothelium-derived growth factors), leading to agonal chromaffin differentiation and apoptosis of neuroblastomal cells. Abbreviations: Ki 67, proliferating cells; TUNEL, apoptotic cells; IGF2, insulin-like growth factor II gene; TH, tyrosine hydroxylase; CgA, chromogranin A.

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Inhibition of angiogenesis induces chromaffin differentiation and apoptosis in neuroblastoma - PubMed (original) (raw)