PST Analogue & Curcumin for Osteosarcoma Therapy Figure 12. JCTH-4 and CC do not induce autophagy in Hob and NFF cells. MDC staining was used to detect the presence of autophagic vacuoles in (A) HOb and (B) NFF cells after 72 hours of treatment with JCTH-4, CC, and solvent control (Me2SO) at the indicated concentrations. 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. doi:10.1371/journal.pone.0028780.g012 inconsistencies between these findings and that of this report again may be attributed to the low solubility and uptake of CC [66,70]. JCTH-4 treatment yielded autophagic induction which was accompanied by significant cytotoxicity and cell death, as depicted in the corresponding phase images and previous figures (Figure 2A,B, 3A,B, 4, 6, 11A,B); this may indicate JCTH-induced autophagy to be a detrimental response in OS cells. Such induction was not evident in HOb and NFF cells (Figure 12A,B); therefore, autophagic induction by JCTH-4 is selective towards OS cells. As established in this report, JCTH-4 causes mitochon- drial ROS production and mitochondrial dysfunction. Oxidative stress is a known inducer of autophagy and thus, JCTH-4 may trigger autophagy as a default mechanism [25]. Regardless, because JCTH-4 permeabilizes OS cell mitochondria, these cells ultimately die from apoptosis. Recapitulating the findings of this report, JCTH-4, was found to effectively induce apoptosis and autophagy in OS cells with evidence suggesting this to be a result of mitochondria targeting. Additionally, the natural compound CC was able to potentiate these cytotoxic effects induced by JCTH-4 against OS cells. Importantly, JCTH-4 treatment alone and in combination proved to be selective towards OS cells as non-cancerous cells were markedly less sensitive and did show evident signs of apoptotic and autophagic induction. Thus, we present a promising novel strategy, combining the novel synthetic com- pound JCTH-4 and the natural compound CC for the treatment of OS. Acknowledgments Thank you to Cynthia Tran for her initial work on this project and Manika Gupta for the critical review of this manuscript. Author Contributions Conceived and designed the experiments: DM SP. Performed the experiments: DM PT KM JC TH. Analyzed the data: DM SP. Wrote the paper: DM PT KM SP. Synthesized and provided JCTH-4 used in this study: JC TH. PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e28780