Semaphorin 3A Attenuates Melanoma Progression Figure 4. Sema 3A augments p53 phosphorylation and attenuates tumor-endothelial interaction via neuropilin 1 (NRP1) mediated paracrine mechanism. (A) Tumor-endothelial interaction was performed in modified Boyden chamber or matrigel coated invasion chamber. Endothelial cells (HUVEC) were placed on upper chamber whereas tumor (control B16F10 or clone 2) cells were used in lower chamber. In separate experiments, HUVEC were treated with anti-NRP1 blocking antibody and used on the upper chamber. After 18 h, migrated or invaded cells were stained with Giemsa, photographed (Fig. 4A, panel 1), counted in three high power field (C/HPF) and represented in the form of bar graph (panel II, *p = 0.005 vs. control, **p,0.04 vs. control, #p = 0.048 vs. control). The experiments were performed in triplicate. (B) Overexpression of Sema 3A inhibits cell viability of B16F10 cells. Equal number of control B16F10 or clone 2 cells (16104) were grown in 96 well tissue culture plates and cell viability was performed by MTT assay. The data are represented in the form of bar graph (*p = 0.011) and the mean value of triplicate experiments is indicated. (C) Sema 3A augments Ser-15 phosphorylation of p53. The phosphorylation at Ser-15 of p53 was analyzed by immunofluorescence using specific antibody followed by Cy3 (red) labeled secondary antibody. Photographs were taken under confocal microscope at 606 magnification (Fig. 4C, panel I). The typical photographs have shown here represents three independent experiments exhibiting similar results. Similarly, SK-Mel-28 cells were treated with Sema 3A (100 ng/ml) and the level of phospho-p53 at Ser-15 was analyzed by confocal microscopy (Fig. 4C, panel II). doi:10.1371/journal.pone.0033633.g004 overexpressing melanoma cells as compared to control B16F10 cells (Fig. 5B). To further elucidate the effect of curcumin on cell survival in presence of Sema 3A, both control and clone 2 cells were incubated with two doses of curcumin, fixed and nuclei were stained with propidium iodide (PI) and visualized under fluorescence microscope. The data showed that curcumin even in lower doses in clone 2 cells is able to induce apoptotic morphology as compared to parental cells (Fig. 5C). To further validate the effect of curcumin on cell death, DNA fragmentation assay was performed. The data showed that clone 2 cells along with low doses of curcumin significantly induce apoptosis as characterized by marked DNA fragmentation (Fig. 5D). The curcumin-induced apoptosis was also analyzed by Western blot using anti-PARP antibody (Fig. 5E). The results also indicated that curcumin even in low dose is able to induce PARP cleavage in clone 2 cells. Sema 3A suppresses in vivo melanoma growth and angiogenesis in C57BL/6 allograft melanoma model Our in vitro experimental results prompted us to investigate the role of Sema 3A on in vivo melanoma progression. Accordingly, control B16F10 and clone 2 cells were injected subcutaneously to C57BL/6 mice (n = 6/group). In separate experiments, condi- tioned media (CM) collected from clone 2 cells were injected intratumorally, twice a week to the tumors generated by injecting B16F10 cells. After 4 weeks, mice were sacrificed and photo- graphed, tumors were removed and weighed and represented in the form of bar graph (Fig. 6A and C). Tumor volumes were measured, analyzed and represented graphically (Fig. 6D). The data showed that overexpression of Sema 3A significantly suppressed in vivo tumor load in C57BL/6 mice. Moreover, intratumoral injection of CM from clone 2 attenuates tumor growth of control B16F10 cells in these mice (Fig. 6A and C) PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33633