PST Analogue & Curcumin for Osteosarcoma Therapy Figure 9. JCTH-4 and CC do not dissipate MMP in HOb and NFF cells. Effect of JCTH-4 and CC on MMP in (A) HOb and (B) NFF cells after 72 hours of treatment was examined by TMRM staining. Cells were grown on coverslips, treated with the indicated concentrations of JCTH-4, CC, and solvent control (Me2SO), and stained with TMRM and Hoechst dye. Images were taken at 4006 magnification on a fluorescent microscope. Red fluorescent punctuate marks are indicative of mitochondria with intact MMP. Scale bar = 15 mm. All images are representative of 3 independent experiments. doi:10.1371/journal.pone.0028780.g009 condensation, apoptotic body formation, phosphatidylserine externalization, and MMP dissipation (Figure 4,6,8). Moreover, by the end of 7 days of treatment, no viable cells were present, indicating the absence of any resistance to this compound by OS cells (data not shown). OS cells treated with JCTH-4 alone were negative for PI staining, a plasma membrane impermeable DNA stain, suggesting these cells were not dying by necrosis, a form of cell death characterized by lysis of the plasma membrane (Figure 11A,B) [38]. However, some PI positive staining was present in the combination treatment with CC, which could be a result of the permeabilization of cell membranes of late apoptotic cells (Figure 11A,B) [39]. Considerable toxicity to non-malignant tissue has been associated with a majority of the chemotherapeu- tics used for OS [40–44]. However, HOb cells, a normal non- cancerous equivalent to the OS cells used in this study, as wells as NFF cells exhibited markedly decreased sensitivity and did not show evident signs of apoptotic induction with JCTH-4 treatment (Figure 2C,5,7,9); therefore, JCTH-4 is selective towards OS cells and may prove to be a safer alternative for OS treatment to the current chemotherapeutics in use. Such selectivity suggests a specific target in cancer cells. Previously, we have provided supporting evidence suggesting that PST targets cancer cell mitochondria [13,17,18]. Compli- menting this data, we report similar results with JCTH-4. In addition to the JCTH-4-induced collapse of MMP in OS cells, observations made after direct JCTH-4-insult to isolated OS mitochondria strongly suggest this specific apoptotic activity to be attributed to mitochondrial targeting; with regards to isolated mitochondria from OS cells, JCTH-4 caused an increase in ROS generation and the release of apoptogenic factors (Figure 10). Likewise, PST selectively dissipated MMP with whole cell treatment and caused ROS production in isolated cancer cell mitochondria and not in non-cancerous cell mitochondria [13,14]. The well known tumor suppressor p53 causes the expression of proapoptotic proteins that permeabilize the mitochondria in response to different forms of intracellular stress [45]. Our results proved JCTH-4-induced apoptosis to be p53 independent; Saos-2 cells, which do not express p53, and U-2 OS cells, with wild type p53, were equally sensitive to JCTH-4 insult (Figure 2A,B,4) [46,47]. This circumvention in p53 activity strengthens the argument for a mitochondrial target as all p53 cellular events upstream of mitochondrial permeabilization have been proven to be insignificant in JCTH-4-induced cytotoxicity. As well as taking part in apoptosis pathways involved with the mitochondria, caspases are also utilized in apoptosis pathways independent of the mitochondria [35]. Akin to PST, we have found caspases to be nonessential in JCTH-4-induced apoptosis (Figure 10E), and that AIF and EndoG, two apoptogenic factors involved in caspase- independent pathways of apoptosis, are released from the mitochondria by direct JCTH-4 insult (Figure 10C,D); thus, caspase signalling upstream of mitochondrial membrane permea- bilization and independent of mitochondria are not vital for the induction of apoptosis by JCTH-4, thereby further supporting the notion of a mitochondrial target by JCTH-4 [17,23,35,48,49]. It is this targeting of the mitochondria that may provide the means to cancer selectivity by JCTH-4. Cancer cells possess many distinct characteristics, such as marked differences in their mitochondria and energy metabolism, which may be exploited for cancer selective therapy [50,51]. Cancer cells possess a distinct metabolic phenotype, a phenom- PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28780