JS-K has potent anti-angiogenic activity in vitro and inhibits tumour angiogenesis in a multiple myeloma model in vivo

Abstract

Objectives: Glutathione S-transferases (GSTs) play an important role in multidrug resistance and are upregulated in multiple cancers. We have designed a prodrug class that releases nitric oxide on metabolism by GST. O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antineoplastic activity.

Methods: We studied the effect of JS-K on angiogenesis in human umbilical vein endothelial cells (HUVECs), OPM1 multiple myeloma cells, chick aortic rings and in mice.

Key findings: JS-K inhibited the proliferation of HUVECs with a 50% inhibitory concentration (IC50) of 0.432, 0.466 and 0.505 microm at 24, 48 and 72 h, respectively. In the cord formation assay, JS-K led to a decrease in the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 microm, respectively. JS-K inhibited cell migration at 5 h using VEGF as a chemoattractant. Migration inhibition occurred with an IC50 of 0.493 microm. In the chick aortic ring assay using VEGF or FGF-2 for vessel growth stimulation, 0.5 microm JS-K completely inhibited vessel growth. JS-K inhibited tumour angiogenesis in vivo in NIH III mice implanted subcutaneously with OPM1 multiple myeloma cells.

Conclusions: JS-K is a potent inhibitor of angiogenesis in vitro and tumour vessel growth in vivo. As such, it establishes a new class of antineoplastic agent that targets the malignant cells directly as well as their microenvironment.

Figure 1. JS-K inhibits HUVEC growth

HUVEC were cultured with the indicated concentrations of JS-K. At the indicated time points cell growth was scored as outlined in the Methods section. JS-K treatment led to a dose-dependent inhibition of HUVEC growth. The IC₅₀ at 24, 48, and 72 hours was 0.432, 0.466, and 0.505 µM, respectively. (Averages and SEM of 3 separate experiments).

Figure 2. JS-K inhibits cord formation

HUVECs were plated in Matrigel after treatment with the indicated concentrations of JS-K. Twenty four hours later cord formation was evaluated by scoring cord length and cord junctions. JS-K treatment led to a dose-dependent inhibition of cord formation. The IC₅₀s for cord junction and cord length were 0.637 and 0.696 µM, respectively. (Averages and SEM of 3 separate experiments. Asterisks indicate statistically significant differences between treatments and controls).

Figure 3. JS-K inhibits HUVEC migration

HUVEC migration at 5 hours with the indicated concentrations of JS-K was assayed using 10 ng/ml VEGF as a chemoattractant. JS-K treatment led to a dose-dependent inhibition of HUVEC migration. The IC₅₀ for migration inhibition was 0.496 µM. At the 5-hours time point there was no evidence of growth inhibition. (Averages and SEM of 3 different experiments. Asterisks indicate statistically significant differences between treatments and control).

Figure 4. JS-K inhibits vessel growth in the chick aortic ring assay

Chick aortic rings were cultured in vitro with different additives as detailed in the Methods section. After 2 days in culture, rings were evaluated for vessel growth. A- No additive control; B- FGF-b 50 ng/ml; C- FGF-b 50 ng/ml + JS-K 0.1 µM; D- FGF-b 50 ng/ml + JS-K 0.5 µM. Pictures are representative of 2 separate experiments. Similar results were observed when 50 ng/ml VEGF was used to stimulate angiogenesis (not shown).

Figure 5. JS-K inhibits tumor angiogenesis in an in vivo plasmacytoma model

NIH III mice were implanted with OPM1 MM cells and treated with vehicle control (A) or JS-K at a dose of 4 µmol/kg thrice weekly (B). Control and JS-K-treated mice were sacrificed starting 21 and 43 days after starting treatment, respectively. Tumor explants were stained with a CD34 antibody to mark vessels and slides were evaluated under 400X magnification. JS-K significantly inhibited tumor angiogenesis in vivo. Pictures are representative of slides from 8 control and 9 JS-K-treated animals, respectively.