MODULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND INTERLEUKIN-8 BY CARBOXYAMIDOTRIAZOLE IN MELANOMA CELLS

Abstract

Angiogenesis, the development of new blood vessels, is required for proliferation and growth of tumors and has now become an important target for cancer chemotherapeutics. The synthetic compound carboxyamido-triazole (CAI) has been demonstrated to be an anti-angiogenic agent and is currently in Phase II trials at the National Cancer Institute in patients with refractory ovarian cancers. CAI inhibits calcium influx through nonvoltage and voltage-gated calcium channels and subsequent down regulation of calcium-dependent signal transduction cascades. The current study was undertaken to examine the effect of CAI on melanoma cells in vitro and in vivo and determine a possible mechanism by which CAI acts. In vivo studies demonstrate an 89% reduction in melanoma tumor volume and confirm its anti-angiogenic effect by showing a reduced number of endothelial cells in CAI-treated tumors versus control tumors. In vivo analysis demonstrated that CAI reduced secretion of the proangiogenic cytokines vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in A2058 human melanoma xenografts by 56% and 88%, respectively. In an effort to elucidate the possible mechanism for the role of CAI in reducing melanoma growth, in vitro investigation was employed. Two important conditions of the tumor environment were explored: acidosis and relative hypoxia. A marked reduction in secreted VEGF was measured under hypoxic-like conditions at either neutral or acidic pH following CAI treatment (P=0.0003 and P=0.0006, respectively). Protein expression was similarly affected; however, mRNA expression showed a greater reduction under normoxic-like conditions. Expression of hypoxia inducible factor-1 alpha (HIF-1α), a transcriptional regulator of VEGF, was reduced under hypoxic like conditions after CAI treatment, but no significant inhibition was observed during normoxic conditions. Secretion and expression of IL-8 demonstrated a contradictory result to the in vivo data but correlated with a statistically significant CAI dose-dependent decrease in culture pH. Examination of endothelial migration showed that the presence of CAI alone could inhibit both basal unstimulated or VEGF-stimulated migration, and this effect was further enhanced when cells were pre-exposed to CAI. These observations demonstrate a role for CAI in the disruption between the tumor and host microenvironment leading to reduced tumor production and secretion of proangiogenic cytokines and endothelial cell response to VEGF.