PST Analogue & Curcumin for Osteosarcoma Therapy Figure 11. JCTH-4 induces autophagy in OS cells alone and with CC. The presence of autophagic vacuoles in (A) Saos-2 after 96 hours of treatment and (B) U-2 OS cells after 72 hours of treatment with JCTH-4, CC, and solvent control (Me2SO) was determined by MDC staining. Bright blue punctate marks are indicative of autophagic vacuoles. Corresponding phase and PI micrographs are shown below the MDC images. Scale bar = 15 mm. All images are representative of 3 independent experiments. (C) Cell lysates of Saos-2 cells treated with JCTH-4, CC, TAM as positive control, and solvent control (Me2SO) for 72 hours were examined for the conversion of LC3-I to LC3-II by western blot analyses. b-actin was probed to serve as a loading control. Densitometric analyses were done using ImageJ software and statistics were calculated using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean 6 SD of triplicates of one independent experiment. *p,0.05, **p,0.01 versus solvent control (Me2SO); #p,0.01 versus 5 mM CC. doi:10.1371/journal.pone.0028780.g011 pathological conditions [17,63,64]. The natural compound CC, used to treat an array of ailments, has been recognized to possess potent activity against numerous cancers, including OS [4,65]. As determined by the WST-1 colorimetric assay, other reports have established IC50 values of 17.2 mM and 21.6 mM in Saos-2 and U- 2 OS cells respectively after 72 hours [4]. By similar means, we found 5 mM CC to have very little to no effect on these cell lines (Figure 3A,B). However, when used with JCTH-4, CC was found to potentiate the cytotoxic effects of JCTH-4 in both Saos-2 and U-2 OS cells with HOb and NFF cells being markedly less sensitive to this combinatorial treatment (Figure 3A–D). Like JCTH-4 treatment alone, this combinatorial treatment exerts its cytotoxicity against OS cells selectively through induction of apoptosis (Figure 4–7). Although whole cell treatment of OS cells with 5 mM CC did not appear to effect viability of mitochondria (Figure 8), direct insult to isolated OS mitochondria induced ROS production and release of AIF (Figure 10A,B,D); such discrepan- cies between whole cell and direct mitochondrial treatment may be attributed to low solubility or uptake of CC [66]. In accordance with our findings, although at higher doses, other studies report whole cell treatment with CC to induce ROS production and AIF release from mitochondria [67,68]. Interestingly, direct treatment of CC on OS mitochondria enhanced ROS production and AIF release (Figure 10A,B,D). CC is known to bind and target numerous proteins; however, these findings suggest CC to bind either one of these known targets and/or an unknown protein, both associated to the mitochondria [65,69]. As JCTH-4 is shown to elicit its cancer selective effects via mitochondrial targeting, such targeting by CC may be responsible for sensitizing OS cells to JCTH-4-induced apoptosis. Dual roles, both pro-death and pro-survival, have been implicated in autophagic induction elicited by chemotherapeutic insult [36]. JCTH-4 was found to induce autophagy at doses between 0.25 mM and 1 mM while 5 mM CC yielded no observable autophagic response in OS cells (Figure 11). The autophagic response produced by the combination treatment of 5 mM CC and 0.25 mM JCTH-4 was not significantly different from response made by the 0.25 mM JCTH-4 treatment alone; this may indicate the enhancement in activity of JCTH-4 by CC to be a result of targeting the apoptosis pathway rather than that of autophagy by CC. Previous work has shown CC alone to induce autophagy, although at much higher doses used in this study; PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e28780