concentrations for 96 and 72 hours respectively. As depicted by Hoechst staining, JCTH-4 produced brightly stained, condensed nuclei, and apoptotic bodies while corresponding phase micro- graphs revealed cell condensation and blebbing in a dose dependent manner; such features are indicative of apoptosis (Figure 4A,B). This morphology was not present in OS cells treated with 5 mM CC alone (Figure 4A,B); however, the frequency of these morphological changes induced by JCTH-4 was enhanced when used with 5 mM CC (Figure 4A,B). All of the aforementioned treatments did not produce apoptotic morphology in both HOb and NFF cells (Figure 5A,B). Selective induction of apoptosis by JCTH-4 alone and in combination with CC in OS cells was confirmed by Annexin V binding to externalized phosphatidylserine, a marker for apoptosis, indicated by green fluorescence (Figure 6–7) [30]. JCTH-4 alone and in combination with CC targets OS cell mitochondria In preceding studies, PST has been found to exert its effects by way of mitochondrial targeting; thus, mechanistic studies were performed to verify such targeting by JCTH-4 in OS cells [13– 15,16,17]. Saos-2 and U-2 cells were treated for 96 and 72 hours respectively with JCTH-4 and CC at the indicated concentrations and stained with Hoechst dye and TMRM, an indicator of intact MMP and impermeabilized mitochondrial membrane, depicted by red fluorescence. Greatest dissipation of MMP was seen in OS cells treated with 1 mM JCTH-4 (Figure 8A,B). Dissipation of MMP was also seen with lower doses of JCTH-4; this dissipation by JCTH-4 was enhanced with the addition of 5 mM CC, which PST Analogue & Curcumin for Osteosarcoma Therapy had no observable effect on MMP its own (Figure 8A,B). Additionally, none of these treatments yielded MMP dissipation in HOb and NFF cells (Figure 9A,B). Oxidative stress produced in the mitochondria has been associated the dysfunction of this organelle; in particular, mitochondrial permeabilization and apoptogenic factor release have both been linked to increases in ROS production [31–34]. After apoptogenic factors are released into the cytosol from the mitochondria, some can execute apoptosis by translocating to the nucleus or participating in cytosolic downstream pathways [23]. To evaluate the ability of JCTH-4 to induce the generation of ROS directly in mitochondria of OS cells, mitochondria were isolated from Saos-2 and U-2 OS cells, treated directly with various doses of JCTH-4 and CC for 2 hours, and analyzed for ROS production with Amplex Red. JCTH-4 alone at several concentrations caused an increase in ROS production in OS cells which was further enhanced by the addition of 5 mM CC (Figure 10A,B). 5 mM CC alone also increased ROS generation in isolated OS cell mitochondria (Figure 10A,B) PQ, known to cause ROS production in mitochondria, was used as a positive control [34]. Furthermore, isolated mitochondria of OS cells treated with JCTH-4 alone or with CC for 2 hours were monitored for the release of apoptogenic factors. Post treatment, mitochondrial samples were resuspended and centrifuged; resul- tant post mitochondrial supernatants and mitochondrial pellets were monitored by western blot analyses for the release and retention of apoptogenic factors respectively. By analyzing the resultant mitochondrial pellet samples of U-2 OS cells, isolated U- 2 OS cell mitochondria treated with JCTH-4 retained less of the Figure 5. JCTH-4 and CC do not yield apoptotic morphology in HOb and NFF cells. Nuclear and cellular morphology of (A) HOb and (B) NFF 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.g005 PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28780