PST Analogue & Curcumin for Osteosarcoma Therapy Figure 6. JCTH-4 alone and in combination with CC induces phosphatidylserine externalization in OS cells. Annexin V binding to externalized phosphatidylserine was monitored to confirm induction of apoptosis in (A) Saos-2 cells after 96 hours of treatment and (B) U-2 OS cells after 72 hours of treatment with the indicated concentrations of JCTH-4, CC, and solvent control (Me2SO). Cells were also stained with Hoechst dye. Images were taken at 4006 magnification on a fluorescent microscope. Green fluorescence is indicative of Annexin V binding to externalized phosphatidylserine of the plasma membrane. Scale bar = 15 mm. All images are representative of 3 independent experiments. doi:10.1371/journal.pone.0028780.g006 apoptogenic factor AIF (Figure 10C). JCTH-4 also caused the release of the apoptogenic factor EndoG in a dose dependent manner from these U-2 OS mitochondria, deduced by the analysis of the post mitochondrial supernatants (Figure 10C). Similarly, JCTH-4 and 5 mM CC alone caused the release of AIF from isolated mitochondria of Saos-2 cells; however, combinatorial treatment yielded the greatest release of AIF (Figure 10D). The execution of apoptosis by released apoptogenic factors from the mitochondria can be achieved with the activation of proteins known as caspases, a family of cysteine proteases, or directly by the apoptogenic factor [23,35]. To determine the dependence of caspases in JCTH-4-induced apoptosis, the broad spectrum caspase inhibitor Z-VAD-FMK was used at various doses in combination with JCTH-4. However, as determined through the WST-1 based colorimetric assay for cell viability, this inhibitor was not able to protect Saos-2 cells from JCTH-4 insult (Figure 10E). Thus, JCTH-4 exerts its cytotoxicity against OS cells in a manner independent of caspase activation. JCTH-4 selectively induces autophagy alone and with CC in OS cells Various studies have reported chemotherapeutics to trigger both pro-death and pro-survival autophagy [36]. Moreover, because JCTH-4 produced oxidative stress, a known autophagy inducer, we monitored OS cells for autophagic induction following treatment with JCTH-4 and CC. As illustrated MDC staining, OS cells treated with various doses of JCTH-4 produced blue punctate staining, indicative of autophagic vacuoles (Figure 11A,B). Such staining was also seen with the combinatorial treatment of 0.25 mM JCTH-4 and 5 mM CC, but not with 5 mM CC treatment alone (Figure 11A,B). Corresponding phase and PI images show cell shrinkage and blebbing with JCTH-4 treatment alone and in combination, but not with CC alone, while only combination treatment gave some positive PI staining (Figure 11A,B). During autophagy, LC3 situated in the cytosol, referred to as LC3-I, is converted to LC3-II, a lipidated form of LC3 that is recruited to autophagosomal membranes [37]. To confirm the induction of autophagy in OS cells, western blot analyses were carried out on cell lysates of Saos-2 cells treated with the indicated concentrations of JCTH-4 and CC for 72 hours to monitor the conversion of LC3-I to LC3-II. TAM, a known inducer of autophagy, was used as a positive control. This conversion was enhanced with JCTH-4 at various concentrations (Figure 11C). With the treatment of 5 mM CC alone, LC3-II conversion decreased, and when used with 0.25 mM JCTH-4, the effect was not significantly different from 0.25 mM JCTH-4 alone (Figure 11C). In HOb and NFF cells, autophagic induction was only seen in the TAM positive control groups but not in cells treated with JCTH-4 and CC alone and in combination (Figure 12A,B). Therefore, JCTH-4 selectively induces autophagy in OS cells. Discussion In this study, we demonstrate JCTH-4 to be a highly potent agent against OS cells. More specifically, JCTH-4 exerts its cytotoxicity against OS by way of apoptotic induction; OS cells treated with JCTH-4 exhibited cell shrinkage, blebbing, nuclear PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28780