Kim et al. BMC Cancer 2012, 12:147 http://www.biomedcentral.com/1471-2407/12/147 the mechanism of action by TM remain largely unknown. TM in tumor animal studies activated pro-apoptotic MAPK signaling, down regulated survival proteins, such as XIAP, and reduced cancer cell motility and invasive- ness [10]. TM, in phase I trials in patients with a variety of metastatic cancers including breast, colon, lung, and prostate and in phase II trials in patients with advanced renal cancer and mesothelioma, was well tolerated and showed stabilization of the disease in a proportion of patients [11-13]. We recently reported that TM can sensitize drug-resis- tant endometrial cancer cell lines to doxorubicin [7]. The present study explores the ability of TM to potentiate the effect of doxorubicin and several other anticancer drugs including mitomycin C (MMC), fenretinide (4-HPR) and 5-fluorouracil (5-FU) in ovarian cancer cells. While these four drugs differ with respect to their application to treat ovarian cancer doxorubicin is used in recurrent disease, MMC showed activity in phase II trials, and 5-FU and 4- HPR showed positive responses in combination with plati- num based drugs or as a single agent in phase II trials respectively [14-17]. These drugs were chosen for this study since they share one common target, the cellular oxidative defense system. Treatment with any of these drugs can lead to the elevation of oxidative stress promot- ing cell death in cancer cells [7,18-21]. Their efficacy may be improved if the oxidative balance in cancer cells is dis- rupted by agents that target cellular antioxidants such as TM. In the present study we describe the effect of TM combination treatment with doxorubicin, 4-HPR, 5-FU, and MMC on various cellular apoptotic markers and determine the correlation between ROS activity and cell death in ovarian cancer cells. Methods Cell lines, cell culture, and material SKOV-3 (human ovarian adenocarcinoma) cells were pur- chased from ATCC (Manassas, VA) and A2780 (human ovarian adenocarcinoma) cells were supplied by Dr. Alex- ander Brodsky (Brown University, Providence, RI). Both cell lines were grown in Dulbecco’s modified eagle’s med- ium (DMEM) supplemented with 10% fetal calf serum or bovine calf serum and 1% penicillin/streptomycin; referred to as a complete medium. Cells were cultured at 37°C with 5% CO2 in a humidified incubator. Reagents were purchased as follows: Sulforhodamine B (SRB), doxorubi- cin, ammonium tetrathiomolybdate, phenylmethylsulfonyl fluoride, ascorbic acid, propidium iodide, rhodamine 123, 4-HPR and 5-FU (Sigma Aldrich, Madison, WI); MMC (Tocris Bioscience, Ellisville, MO); carboxy-H2DCFDA (Invitrogen, Carlsbad, CA); trichloroacetic acid and acetic acid (Fisher Scientific, Pittsburgh, PA); XIAP, JNK, p-JNK, p38, p-p38, cleaved PARP, cleaved caspase-3 and -7 anti- Page 2 of 10 bodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). GAPDH antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SRB cell viability assay Cells were plated into a 96-well microtiter plate in com- plete medium and treated with TM (0, 30 μM) for 24 h, after which the cells were treated with either doxorubi- cin, MMC, 4-HPR, 5-FU of different concentrations and durations as indicated. For treatment with doxorubicin or MMC, 1 × 104 cells were seeded per well, for 4-HPR and 5-FU 7.5 × 103 cells were seeded per well. Cell viabi- lity, as represented by the amount of cell protein-bound SRB, was measured as previously described [22]. Analysis of mitochondrial membrane potential (ΔΨm) SKOV-3 cells (1 × 105) were seeded into 6-well plates and treated with TM (0, 30 μM) after which the cells were treated with doxorubicin (0, 5 μM) for another 24 h. The cells were incubated with rhodamine 123 (13 μM) for 30 min at 37°C prior to completion of the drug treat- ment. Rhodamine 123 is a cationic dye which localizes in the mitochondria of viable cells. The cells were harvested, resuspended in medium containing propidium iodide (7.5 μM) and analyzed by flow cytometry. Data was acquired on a BD FACSort flow cytometer using Cell- Quest software (BD Immunocytometry-Systems, San Jose, CA) and analyzed (ModFit LT software, Verity Soft- ware House, Inc., Topsham, ME). Ten thousand cells were analyzed for each sample. Western blot Analysis SKOV-3 or A2780 cells were treated with TM and anticancer drugs (doxorubicin, MMC, 4-HPR or 5-FU) alone and in combination under the conditions indi- cated. Cells were lysed, protein concentration of the lysates quantified, proteins separated by NuPAGE sys- tem (Invitrogen, Carlsbad, CA), and immunoblotted as described previously [7]. Detection of intracellular ROS SKOV-3 cells (3 × 105/well) were seeded into 6-well plates and treated with TM (0, 30 μM) for 24 h, after which the cells were additionally treated with doxorubi- cin (0, 10 μM) for another 5.5 h and the assay carried out as described previously [23]. After treatment, the cells were incubated with carboxy-H2DCFDA (25 μM) for 30 min at 37°C with 5% CO2. Carboxy-H2DCFDA is the acetylated form of a reduced fluorescein derivative that is cell-permeable and becomes fluorescent in the presence of cellular oxidants. Cells were harvested and resus- pended in phosphate buffered saline buffer before being analyzed by flow cytometry.