The aforementioned studies showed that GGTI-298 inhibits VSMC growth and induces apoptosis. Because nitric oxide (NO) is known to inhibit VSMC proliferation, we investigated the effects of GGTI-298 on NO formation as a possible mechanism of action of GGTI-298 growth-inhibiting effects. To this end, we treated VSMC with GGTI-298 prior to treatment with IL-ip, a known inducer of NOS-2. We first analyzed the effects of GGTI-298 on IL-ip-induced nitrites as a measure ofmedium-released nitric oxide. Figure 12 shows that in the absence ofinhibitors, IL-ip (0-10 ng/mL) induced a concentration-dependent, but modest, increase in the medium levels of nitrite. Pretreatment with GGTI-298 (10 pM) caused a dramatic increase in IL-ip-induced nitrite formation (from 4 to 52 pM). The medium of cells treated with 1 ng/mL IL-ip alone accumulated nitrite levels of 4 pM, whereas the medium of cells treated with GGTI-298 prior to IL-ip accumulated levels of nitrites that were more than 10-fold higher (42 pM) (Fig. 12). In the absence of IL-1 p, GGTI-298 increased basal levels of nitrites to levels comparable to those obtained by stimulation of cells with 1 ng/mL IL-ip alone (67). In contrast to the effects of GGTI-298, treatment of cells with FTI-277 inhibited IL-ip-stimulated nitrite production (Fig. 12).
The dramatic increase in the level of nitrite brought about by GGTI-298 could be owing to direct activation ofNOS-2 enzymatic activity or superinduction ofNOS-2 protein. To determine the effects ofGGTI-298 on NOS-2 protein levels, VSMC were treated with GGTI-298 for 24 h and then stimulated with IL-ip for a further 24 h. Figure 13 shows
Fig. 12. Effect of FTI-277 and GGTI-298 on IL-1ß-stimulated nitrite formation. Cells were pre-treated for 24 hwithFTI-277 (10 pM)orGGTI-298 (10 pM)orwereleftuntreated. Fresh medium with orwithout FTI-277or GGTI-298 andcontainingIL-1 ß at0, 1, 3, or 10 ng/mLwasthenadded. After a further 24 h, the medium was harvested and assayed for nitrites. The curves show the response of IL-1 ß of untreated cells (♦) or cells treated with FTI-277 (A) or GGTI-298 (□).
Fig. 13. Effect of GGTI-298 on IL-ip stimulated expression of NOS-2 protein levels in VSMC. Cells were treated with and without GGTI-298 in the manner described in the legend to Fig. 12. Following 24 h of IL-ip stimulation, cells were harvested, and NOS-2 protein levels were determined by Western analysis.
that in the absence ofGGTI-298 (control), significant induction ofNOS-2 protein levels in VSMC occurred only at 10 ng/mL IL-1p, whereas in the presence of inhibitor, NOS-2 was induced at concentrations as low as 1 ng/mL. In VSMC treated with 10 ng/mL IL-ip, GGTI-298 enhanced the ability of IL-ip to induce NOS-2 protein by fivefold.
Was this article helpful?