PST Analogue & Curcumin for Osteosarcoma Therapy Figure 3. CC potentiates the cytotoxicity of JCTH-4 selectively in OS cells. Effect of JCTH-4 & CC in combination on cellular viability of OS cells was determined by the WST-1 based colorimetric assay. (A) Saos-2 (96 hours), (B) U-2 OS (72 hours), (C) HOb (72 hours), and (D) NFF (72 hours) cells were treated with JCTH-4 and CC and the WST-1 reagent was used to quantify cellular viability. Absorbance was read at 450 nm and expressed as a percent of the solvent control (Me2SO). Values are expressed as mean 6 SD from quadruplicates of 3 independent experiments. *p,0.05, **p,0.01, versus solvent control (Me2SO); {p,0.001 versus 0.25 mM JCTH-4; {{p,0.01 versus 0.5 mM JCTH-4; #p,0.001 versus 5 mM CC; @p,0.001 versus 0.25 mM JCTH-4+5 mM CC treatment with Saos-2 cells (Figure 3A); &p,0.01 versus 0.5 mM JCTH-4+5 mM CC treatment with U-2 OS cells (Figure 3B). doi:10.1371/journal.pone.0028780.g003 CC potentiates the cytotoxicity of JCTH-4 selectively in OS cells The natural compound CC has been shown to possess anti- cancer properties as well as the ability to enhance the efficacy of various chemotherapeutics [28,29]. Thus, we evaluated the combinatorial effects of CC and JCTH-4 in OS cells. CC alone at 5 mM had no significant effect on Saos-2 (96 hours) and U-2 OS cells (72 hours); however, when used in combination, CC was able to selectively potentiate the effect of JCTH-4 in these OS cell lines as HOb and NFF cells were markedly less sensitive to this combinatorial insult (Figure 3A–D). JCTH-4 alone and in combination with CC induces apoptosis selectively in OS cells Nuclear and cellular morphology was monitored in Saos-2 and U-2 OS cells treated with JCTH-4 and CC at the indicated Figure 4. JCTH-4 alone and in combination with CC yields apoptotic morphology in OS cells. Nuclear and cellular morphology of (A) Saos- 2 cells after 96 hours of treatment and (B) U-2 OS cells after 72 hours of treatment. Cells were treated with JCTH-4, CC, and solvent control (Me2SO). Post treatment, the cells were stained with Hoechst 33342 dye. Corresponding phase micrographs are shown below the Hoechst micrographs. Apoptotic morphology is evident in cells with bright and condensed nuclei accompanied by apoptotic bodies, as well as cell shrinkage and blebbing. Images were taken at 4006 magnification on a fluorescent microscope. Scale bar = 15 mm. All images are representative of 3 independent experiments. doi:10.1371/journal.pone.0028780.g004 PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28780