Mechanism Of Action Studies

Much effort has been directed toward elucidating the mechanism by which TNP-470 affects endothelial cell proliferation. Several laboratories have investigated how and when within the cell cycle TNP might block growth factor-induced proliferation. Time-course studies suggest that TNP affects events that occur late in G1, but prior to the transition to S-phase (5,34,34). The actual degree to which specific CDKs are inhibited is dependent on the source of endothelial cells, and the timing of exposure to TNP relative to that of mitogen. The effects of TNP within G1 of HUVECs include inhibition or attenuation of several cyclins, attenuation of bFGF-induced activation of cyclin-depen-dent kinases cdc2 and cdk2, and inhibition of phosphorylation of the retinoblastoma (RB) protein (35).

Maier et al. (36) report that TNP inhibits the activity of urokinase-type plasminogen activator (uPA) in endothelial cells. This same inhibition is observed when an antibody to endoglin (a component of the TGF-p receptor system) is used; there is no additive effect when the antibody is used in conjunction with TNP. The authors suggest that TNP and endoglin may have similar targets, or affect a similar pathway. It is difficult to draw conclusions from this report, because it is unclear whether cytostatic or cytotoxic concentrations of drug were used.

It has also been demonstrated that the addition of TNP to bovine aortic endothelial cells (BAE) leads to an increase in E-selectin mRNA and protein. That this increase was more pronounced in subconfluent than in confluent cells may suggest a mechanism by which TNP can inhibit tumor growth in vivo with no obvious effect on the normally quiescent endo-thelium (37). It is counterintuitive that TNP would enhance the expression of E-selectin, which is expressed by dividing endothelial cells. This is further complicated by the fact that the concentration of TNP used in these experiments was in the cytotoxic range; it would be interesting to repeat these experiments using cytostatic concentrations of the drug.

There are several reports that indicate that TNP can also affect nonendothelial cells. As mentioned in Section 2., TNP enhances the proliferation of normal human, but not tumor-derived, B-lymphocytes; in vitro, this phenomenon requires the presence of normal T-cells (9). Also in vitro, TNP stimulates the proliferation of murine B/T-lympho-cytes in the presence of phytohemagglutinin (13). Follow-up in vivo experiments showed an increase in the size of axillary or mesenteric nodes in a murine model; following treatment with TNP; this increase was not apparent in T-cell-deficient mice (9). The mechanism by which TNP modulates the immune system warrants further investigation.

TNP inhibits PDGF- or IGF-induced proliferation of bovine aortic vascular smooth-muscle cells; this inhibition is associated with decreased cdk2 activity and mRNA levels induced by both mitogens, and with a partial inhibition of cdk4 mRNA induced by PDGF. At concentrations up to 100 ng/mL (cytotoxic), TNP could only partially inhibit the proliferation induced by fetal calf serum (FCS) (38). This suggests that the effectiveness of TNP regarding cell proliferation depends not only on the cell type, but on the mechanism of proliferation induction.

There are also reports of TNP directly affecting tumor cells. A decreased secretion of bFGF has been reported for squamous cell carcinoma (39) and uterine endometrial carcinoma cells (40). Kondo (41) has reported that TNP affects the cell cycle of colon 26 cells; however, the concentration of study drug used in these experiments was in the cytotoxic range.

It has recently been shown that a biotinylated derivative of fumagillol is able to bind to methioninaminopeptidase-2 (MetAP-2), a highly conserved, cobalt-dependent metalloproteinase expressed by endothelial and nonendothelial cells (42). This same report demonstrated that fumagillin selectively inhibits the growth of yeast that are dependent on MetAP-2 for proliferation. MetAP-2, also known as p67, a eukaryotic initiation factor 2 (eIF2)-associated protein, is able to interfere with the phosphorylation of eIF2 (43,44), and is involved on posttranslational processing (45,46). It may be that fumagillin-TNP is able to interfere with endothelial cell proliferation by affecting the activity or stability of proteins specifically involved in endothelial cell translation and/ or cell-cycle signaling.

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