PST Analogue & Curcumin for Osteosarcoma Therapy Figure 10. JCTH-4 directly causes mitochondrial ROS production and release of apoptogenic factors independent of caspases. (A) Saos-2 and (B) U-2 OS isolated mitochondria were treated directly with JCTH-4, CC, PQ, and solvent control (Me2SO), and incubated with Amplex Red and horseradish peroxidase for 2 hours. Subsequently, fluorescence readings were taken at Ex. 560 nm and Em. 590 nm and expressed as relative fluorescence units (RFU). Statistics were performed using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean 6 SD of quadruplicates of 1 independent experiment. *p,0.05, **p,0.01, ***p,0.001 versus solvent control (Me2SO); {p,0.01 versus 0.25 mM JCTH-4; @p,0.01 versus 0.5 mM JCTH-4; #p,0.01 versus 5 mM CC. Isolated mitochondria samples treated directly with JCTH-4, CC, and solvent control (Me2SO) for 2 hours were also centrifuged, producing mitochondrial pellets and post mitochondrial supernatants which were examined for retention and release of apoptogenic factors respectively via western blot analyses; (C) Retention of AIF and release of EndoG by U-2 OS cell mitochondria and (D) release of AIF by Saos-2 cell mitochondria was monitored. Mitochondrial pellets were probed for SDHA to serve as loading controls. Densitometric analyses were performed 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,0.01, **p,0.001 versus solvent control (Me2SO); {p,0.01 versus 0.25 mM JCTH-4; #p,0.01 versus 5 mM CC. (E) Saos-2 cells were treated with broad spectrum caspase inhibitor Z-VAD-FMK with and without JCTH-4 for 72 hours. WST-1 reagent was used to quantify cell viability. Absorbance was read at 450 nm and expressed as a percent of solvent control (Me2SO). Values are expressed as mean 6 SD from quadruplicates of 3 independent experiments. *p,0.001 versus solvent control (Me2SO); ns = not significant. doi:10.1371/journal.pone.0028780.g010 enon referred to as the Warburg effect, in which levels of aerobic glycolysis are elevated; thus, these cells have high glucose uptake and through the production of lactate, produce an acidic microenvironment [52–54]. Consequently, this glycolytic shift confers a proliferative advantage and an acquired resistance to apoptosis in cancer cells; intermediates of glycolysis provide a vast source of resources needed for nucleotide and lipid synthesis and alterations of the outer mitochondrial membrane have rendered it less susceptible to permeabilization [55–57]. Therefore, a promising strategy in cancer therapy would be to divert cancer cell metabolism away from glycolysis, which was demonstrated by other efforts via the promotion of oxidative phosphorylation [58]. Such a strategy may be employed by JCTH-4. A major factor promoting the resistance to apoptosis as a result of this metabolic shift is the glycolytic enzyme hexokinase; its association to the outer mitochondrial membrane has been shown to discourage mitochondrial membrane permeabilization [59,60]. Cancer cells have also been reported to highly express antiapoptotic members of the Bcl-2 family of proteins and peripheral-type benzodiazepine, all of which inhibit mitochondrial outer membrane permeabiliza- tion [61,62]. Thus, JCTH-4 may act by targeting these proteins. Traditional medicines have long used natural products in treating various disorders. Modern science is now rediscovering many of these products and their potential in rectifying numerous PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28780