Semaphorin 3A Attenuates Melanoma Progression Figure 5. Sema 3A overexpressed clone exhibits increased drug sensitivity in B16F10 cells. (A) Control or clone 2 cells were treated with melanoma specific drug, Dacarbazine (DTIC) (0–400 mM) for 24 h and cells survival was analyzed by MTT assay. #p = 0.393, *p,0.001, **p = 0.002 vs. respective treatment group within B16F10 and clone 2 cells. (B) Similarly, both cells were treated with curcumin (0–50 mM) for 12 h and cell viability was checked by MTT assay. The data are represented in the form of bar graph and the mean value of triplicate experiments is indicated. #p = 0.074, *p,0.001, **p = 0.044 vs. respective treatment group within B16F10 and clone 2 cells. (C) Both cells were treated with indicated concentrations of curcumin, fixed and stained with PI (red) and photographed under fluorescence microscope at 606 magnifications. The apoptotic nuclei are indicated by arrows. (D) Cells were treated with two doses of curcumin for 12 h. Fragmentation of genomic DNA was extracted and resolved on 2% agarose gel. Apoptotic DNA fragmentation was visualized by ethidium bromide staining. (E) Curcumin induced apoptosis in control and clone 2 cells were also analyzed by Western blot using anti-PARP antibody. Cells were treated with 0–50 mM curcumin for 12 h and then analyzed by Western blot. a-Tubulin was used as loading control. The experiments showed here are representative of triplicate independent experiments with similar results. doi:10.1371/journal.pone.0033633.g005 demonstrating the paracrine effect of secreted Sema 3A in regression of melanoma growth. The tumor sections were analyzed histopathologically using H&E staining and the photographs were taken at 106 and 606 magnifications (Fig. 6B). The data showed that tumors generated by control B16F10 cells exhibit higher infiltration, poorly differentiated structure, enhanced nuclear polymorphism and increased number of tumor giant cells as compared to the tumors generated by clone 2 or CM of clone 2 (Fig. 6B). The data demonstrated that Sema 3A significantly attenuates in vivo melanoma growth. The tumor sections were also analyzed immunohistochemically using anti-vWF antibody. The results showed that there is a significant enhancement of tumor angiogenesis in tumor sections generated by control B16F10 cells as compared to clone 2 (Fig. 6B), indicating that overexpression of Sema 3A attenuates melanoma growth and angiogenesis in allograft tumor models via an autocrine or paracrine mechanism. To further examine the potential role of Sema 3A in tissue specific metastasis in melanoma models, the allograft melanoma tumors as shown in Fig. 6A, were ventrally dissected and metastatic lesions such as liver, intestine and kidney were separated and analyzed by histopathologically (606) (Fig. 7A, panels II–IV). The data indicated that tumors generated by control cells augment metastasis in liver, intestine and kidney whereas Sema 3A clone 2 or CM of clone 2 dramatically suppressed these metastasis (Fig. 7A, panels I–IV) demonstrating that Sema 3A inhibits melanoma growth, angiogenesis and metastasis in subcutaneous C57BL/6 mice models. PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33633