We have recently shown that vascular endothelial growth factor-A (VEGFA), a major regulator of tumor vascularization, is essential for the organization of tumor cells into capillary-like structure (CLS), which is a hallmark of tumor vasculogenic mimicry (VM). Herein we further dissect the involvement of VEGFA and its downstream transducers, VEGF receptor 1 (VEGFR1), VEGFR2, and protein kinase C (PKC) in melanoma VM. The knockdown of VEGFR1 in three melanoma cell lines completely disrupts Matrigel-induced CLS formation, whereas inhibition of VEGFR2 kinase with a specific inhibitor, protein tyrosine kinase inhibitor II (PTKi-II), does not affect the process, indicating that VEGFR2 signaling is not involved in VEGFA-mediated melanoma VM. Furthermore, among tested PKC isoforms, only PKCα and δ are expressed in the melanoma cells during CLS formation. Pretreatment with selective PKCα and δ inhibitors blocked CLS formation. However, inhibition of PKCα, but not PKCδ, completely destroyed the previously formed CLS. Moreover, knockdown of PKCα, but not PKCδ, using small interfering RNAs abrogated CLS formation, suggesting that PKCα is the major contributory factor in melanoma VM. In-vivo experiments indicate that disruption of PKCα signaling significantly reduces the signs of VM in allografted B16/F10 melanoma. These findings may contribute to the development of new therapeutic agents that target melanoma VM.
Diadenosine oligophosphates (Ap n A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5P P,5R R-P 1 ,P 3 -triphosphate to diadenosine 5P P,5R R-P 1 ,P 4 -tetraphosphate (Ap 3 A/Ap 4 A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap 3 A decreases significantly while that of Ap 4 A increases. Here, we have examined the effects of exogenously added Ap 3 A and Ap 4 A on apoptosis and morphological differentiation. Penetration of Ap n A into cells was achieved by cold shock. Ap 4 A at 10 W WM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap 4 A breakdown as hydrolysis-resistant analogues of Ap 4 A were inactive. On its own, Ap 3 A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap 4 A and Ap 3 A are physiological antagonists in determination of the cellular status: Ap 4 A induces apoptosis whereas Ap 3 A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.z 1999 Federation of European Biochemical Societies.
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