Figure 1.
LLL12 inhibits angiogenesis in vitro.
HUVECs were grown under serum-deficient conditions and stimulated with VEGF (10 ng/ml) in the absence or presence of LLL12 (100 nM). A. Proliferation/viability was determined after 2 days by Calcein AB staining. Migration was determined using the crystal violet assay as described in Materials and Methods as well as scratch assay/wound-healing. Invasion was determined using Matrigel coated membranes (photomicrographs show representative fields) and is quantified (bottom right panel). Each data set represents the mean ± SE for at least 3 independent experiments. B. To study the effect of LLL12 (100 nM) on the cell migration in HUVEC cells, wound-healing assays were carried out by allowing the cells to move to the scar region for 24 hours using VEGF (10 ng/ml) as a positive control in the presence and absence of LLL12 (original magnification ×40).
Figure 2.
Inhibition of angiogenesis by LLL12 correlates with inhibition of STAT3 phosphorylation.
A) Suppression of STAT3 phosphorylation by LLL12 in HUVEC cells after VEGF (10 ng/ml) stimulation. HUVEC cells were grown and stimulated with VEGF (10 ng/ml) for 10 mins and treated with LLL12 for 18–20 hrs. Total cellular protein was extracted and both total STAT3 and pSTAT3(Tyr705) expression was determined. GAPDH was used as a loading control. B) LLL12 inhibits HUVEC tube formation. HUVECs were grown in M200, and incubated with PBS or VEGF (10 ng/ml) for 18–20 hr in the absence or presence of LLL12 (100 nM). Tube formation was quantified as described in Materials and Methods.
Figure 3.
The STAT3 inhibitor, LLL12, induces cytoskeletal changes in cultured HUVEC cells.
HUVEC cells cultured in 4-well chamber slides were treated with PBS, VEGF (10 ng/mL) alone, VEGF with DMSO or LLL12 (100 nM) for 18 hrs. The cultures were then probed using anti-β-tubulin primary antibodies (green), and F-actin was stained using phalloidin (red). White arrows highlight F-actin localization at the leading edge, while white arrowheads indicate the curling of microtubules at the cell periphery. 200× magnification. Slice depth = 1 µm. Scale bar = 20 µm. Inset 400× magnification.
Figure 4.
LLL12 inhibits angiogenesis in mice.
Matrigel plugs containing PBS (Control), or VEGF (100 ng/ml) were implanted subcutaneously in mice that were or were not treated with LLL12 (5 mg/kg daily). The Matrigel plugs were excised on day 7, fixed with formalin and 5-µm sections were stained for CD34 staining. The numbers of CD34 positive vessels per high power field (HPF, magnification, 200×) were counted for each experimental condition. Results are mean (n = 4) ± SE. *P<0.05; versus VEGF alone.
Figure 5.
LLL12 inhibits tumor growth in vivo by inhibition of STAT3. A, LLL12 inhibits tumor growth in osteosarcoma xenograft mice.
OS-1 tumors were transplanted into 6-week-old CB17SC scid−/− female mice. After tumors grew to ∼130 mm3, mice were randomized to receive no treatment (control), DMSO or LLL12 (5 mg/kg/d) for a planned six weeks. LLL12 inhibited tumor growth as measured by tumor volume. Representative tumors at the termination of each group are shown. B. Western blot showing STAT3, and p-STAT3 level in six independent tumors from each treatment group. LLL12 completely blocks pSTAT3 levels with compassion to control and DMSO control group.
Figure 6.
LLL12 inhibits angiogenesis in OS-1 xenografts.
Analysis of vascularity and tumor cell proliferation: Tumors were harvested at completion of the study and examined by hematoxylin and eosin staining. Vascularity of osteosarcoma tumor xenografts in mice was evaluated by CD34 related antigen staining (brown) for endothelial cells, and Ki 67 for proliferation. Apoptotic cells were identified by TUNEL staining. There is a significant effect of LLL12 as compared to vehicle control on tumor vasculature as well as cell proliferation (quantified by staining with anti–Ki-67) in osteosarcoma xenografts, but no significant change in apoptosis in drug treated tumors. All values are expressed as mean plus or minus SEM. *P<.01 by Student t test.
Figure 7.
LLL12 downregulates several angiogenic factors.
LLL12 induced changes in angiogenic factors in OS-1 xenografts were determined using a Proteome profiler antibody array as described in Materials and Methods. The effect of LLL12 treatment is quantified in the histogram, untreated tumors (−), tumors from mice receiving LLL12 (+), 5 mg/kg).
Table 1.
Sensitivity of sarcoma cell lines to LLL12.