availability for preclinical and clinical work. We have recently synthesized and screened 7-deoxypancratistatin derivatives and have identified a C-1 acetoxymethyl analogue, JC-TH-acetate-4 (JCTH-4), with comparable efficacy and specificity to PST against several cancer cell lines [19]. Evasion of apoptosis, or type I programmed cell death, as a result of abnormalities in pathways leading to apoptosis plays a major role in the development of cancer; therefore, much effort has been made to manipulate and restore apoptosis as a way to treat cancer [20,21]. Apoptosis can be activated in response to a death ligand binding to its corresponding death receptor or in response an internal stress stimulus such as DNA damage [22]. In response to such internal stress, various proapoptotic proteins are upregulated and translocated to the mitochondria where they induce mitochondrial membrane permeabilization, collapse of mitochondrial membrane potential (MMP), and release of apoptogenic factors which subsequently execute apoptosis directly or indirectly [23]. Execution of this pathway yields nuclear and cellular condensation, membrane blebbing, and formation of apoptotic bodies which are phagocytosed by phagocytes or neighbouring cells [24]. As well as apoptosis, much focus has been put on the implications of other cell death pathways in cancer therapy [25,26]. Autophagy has been recognized as a cellular pro-survival response to various forms of sublethal cellular stress such as DNA damage, deficiencies in growth factors and nutrients, protein aggregates, reactive oxygen species, hypoxia, pathogens, and defective organelles [25]. Once this pathway is activated, cytoplasmic material is sequestered by double-membraned vesicles known as autophagosomes. These autophagosomes fuse with lysosomes to form autolysosomes which degrade the contained cytoplasmic contents [26]. Extensive activation of autophagy however, can result in a pro-death response leading to autophagic cell death, classified as type II programmed cell death [27]. In this report, we demonstrate JCTH-4 to be a potent inducer of apoptosis and autophagy in OS cells (Saos-2, U-2 OS) via mitochondrial targeting. Furthermore, CC was able to potentiate the cytotoxic effects of JCTH-4 in Saos-2 and U-2 OS cells. The normal human fetal fibroblast (NFF) and normal human osteoblast (HOb) cell lines utilized in this study were drastically less sensitive to JCTH-4 alone and in combination with CC, presenting a potential therapeutic window for this aggressive malignancy. Materials and Methods Cell Culture An OS cell line, Saos-2 (American Type Culture Collection, Cat. No. HTB-85, Manassas, VA, USA), was grown in McCoy’s 5A Medium Modified (Sigma-Aldrich Canada, Mississauga, ON, Canada) supplemented with 15% (v/v) fetal bovine serum (FBS) standard (Thermo Scientific, Waltham, MA, USA) and 10 mg/ mL gentamicin (Gibco BRL, VWR, Mississauga, ON, Canada). An additional OS cell line, U-2 OS (American Type Culture Collection, Cat. No. HTB-96, Manassas, VA, USA), was grown in McCoy’s 5A Medium Modified (Sigma-Aldrich Canada, Mis- sissauga, ON, Canada) supplemented with 10% (v/v) FBS standard (Thermo Scientific, Waltham, MA, USA) and 10 mg/ mL gentamicin (Gibco BRL, VWR, Mississauga, ON, Canada). Apparently NFF cells (Coriell Institute for Medical Research, Cat. No. AG04431B, Camden, NJ, USA) were cultured in Dulbecco’s Modified Eagle’s Medium, High Glucose (Thermo Scientific, Waltham, MA, USA) supplemented with 15% (v/v) FBS standard (Thermo Scientific, Waltham, MA, USA) and 10 mg/mL gentamicin (Gibco BRL, VWR, Mississauga, ON, Canada). PST Analogue & Curcumin for Osteosarcoma Therapy HOb cells (Cell Applications, Inc., Cat. No. 406-05a, San Diego, CA, USA) were cultured in Osteoblast Growth Medium (Cell Applications, Inc., Cat. No. 417-500, San Diego, CA, USA). All cells were grown at 37uC and 5% CO2. Cell Treatment After culturing cells to 60–70% confluence, they were treated with CC (Sigma-Aldrich Canada, Cat. No. C7727, Mississauga, ON, Canada), tamoxifen (TAM) citrate salt (Sigma-Aldrich, Cat. No. T9262, Mississauga, ON, Canada), the broad spectrum caspase inhibitor Z-VAD-FMK (EMD Chemicals, Gibbstown, NJ, USA), and JCTH-4 (((1S, 2S, 3R, 4S, 4aR, 11bR)-2, 3, 4- Trihydroxy-6-oxo-1, 2, 3, 4, 4a, 5, 6, 11b-octahydro-[1,3]diox- olo[4,5-j]phenanthridin-1-yl)methyl Acetate) at the indicated doses and durations. As per a previously published protocol, JCTH-4 was produced by chemoenzymatic synthesis from bromobenzene [19]. All stock solutions of drugs were made with dimethylsulfoxide (Me2SO). Nuclear Staining Post treatment and incubation with the aforementioned drugs, Hoechst 33342 dye (Molecular Probes, Eugene, OR, USA) was used to stain the nuclei. Cells were incubated with 10 mM Hoechst 33342 dye for 5 minutes and images were taken at 4006 magnification on a Leica DM IRB inverted fluorescence microscope (Wetzlar, Germany). Annexin V Binding Assay The Annexin V binding assay was performed to verify the induction of apoptosis. Following drug treatment, cells were washed two times using phosphate buffer saline (PBS), resus- pended in Annexin V binding buffer (10 mM HEPES, 10 mM NaOH, 140 mM NaCl, 1 mM CaCl2, pH 7.6), and incubated with Annexin V AlexaFluor-488 (1:50) (Sigma-Aldrich Canada, Mississauga, ON, Canada) for 15 minutes. Micrographs were taken at 4006 magnification on a Leica DM IRB inverted fluorescence microscope (Wetzlar, Germany). WST-1 Assay for Cell Viability The WST-1 based colorimetric assay was performed according to the manufacturer’s protocol (Roche Applied Science, India- napolis, IN, USA) to quantify cell viability as a function of active cell metabolism. 96-well clear bottom tissue culture plates were seeded with approximately 6.06103 Saos-2 cells/well, 7.56103 U-2 OS cells/well, 5.06103 NFF cells/well, or 4.06103 HOb cells/well and treated with JCTH-4 and CC at the indicated concentrations and for the indicated durations. Following treatment, the WST-1 reagent, which is processed to formazan by cellular enzymes, was added to each well and incubated for 4 hours at 37uC. Absorbance readings were taken at 450 nm on a Wallac Victor3TM 1420 Multilabel Counter (PerkinElmer, Woodbridge, ON, Canada) to quantify the formazan product and were expressed as percentages of the solvent control groups (Me2SO). Tetramethylrhodamine Methyl Ester (TMRM) Staining TMRM (Gibco BRL, VWR, Mississauga, ON, Canada) was used as an indicator for MMP. Cells were grown on coverslips and treated with the indicated concentrations of JCTH-4 and CC for the indicated durations. Subsequent to drug treatment, cells were incubated with 200 nM TMRM for 45 minutes at 37uC. Micrographs were taken at 4006 magnification on a Leica DM IRB inverted fluorescence microscope (Wetzlar, Germany). PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28780