To show the reactivity of our FMD agents with

To show the reactivity of our FMD agents with antioxidants in human normal and cancer cells, we measured the intracellular reduced glutathione (GSH) levels of both human normal cells (GM05757) and human cervical cancer cells (ME-180) in control (with no FMDs) and treated by various concentrations of an exemplary FMD compound (FMD-2Br-DAB) using a GSH detection kit (Abcam), following the manufacturer\'s protocol. Briefly, 2×105 GM05757/ME-180 cells were plated into T25 flasks in 8ml of MEM/McCoy\'s 5A with 10% FBS and were cultured overnight at 37°C before the addition of the test FMD compound (0, 50, and 100μM). Cells were incubated with the compound for 24h, and were harvested and washed with PBS. For all samples, the cells were counted and lysed by 5% metaphosphoric alk pathway at the concentration 8×105 cells/100μl. The cell lysates were centrifuged at 12,000rpm for 5min and supernatants were collected and diluted using the assay buffer by 10 times for our experiments. The GSH levels were detected with a 96-well fluorometric microplate reader for an excitation wavelength at 485nm and emission wavelength at 538nm, which were close to the ex/em=490nm/520nm suggested by the manufacturer. To derive the mouse xenograft models of ME-180 cervical cancer, A549 lung cancer, MDA-MB-231 breast cancer and NIH:OVCAR-3 ovarian cancer for in vivo growth delay studies involving a FMD compound, 6–8week old female SCID mice were injected s.c. in the flank with 1.5×106 ME-180 cells or 5×106 A549/MDA-MB-231/NIH:OVCAR-3 cells. The treatments were started when xenografts reached a volume of 100–150mm3. For each treatment, mice were allocated in complete randomness into two groups with 5 mice per group: (1) control (no FMD); (2) FMD-2Br-DAB at 7mg/kg or FMD-2I-DAB at 5mg/kg was administrated for totally 5/10 IP injections at each other day. Mice were weighed, and flank tumors were measured every 2–5days with calipers, and tumor volumes were calculated. Growth delay was calculated as the time difference (in days) between treatment and control groups to grow to 500mm3. For acute toxicity assay, liver and kidney toxicity biomarkers were measured through proteins, enzymes, metabolites, and electrolytes. Blood samples were collected by a terminal cardiac puncture of the mice at the end of the treatment with and 7mg/kg FMD-2Br-DAB or 5mg/kg FMD-2I-DAB daily for 10days. Analyses of blood serum samples were provided by the Animal Health Laboratory of the University of Guelph. Moreover, mice were observed for any physical toxicity. These physical indicators included: body weight changes, dull sunken eyes, rapid/shallow breathing, hunched back, and lethargy. Wax-embedded tumor tissue samples from SCID mice in the control group and treatment groups (FMD-2Br-DAB at 7mg/kg×5; FMD-2I-DAB at 5mg/kg×5) were obtained at 24h post-treatment and sectioned at 4μm onto polarized slides for colorimetric TUNEL assay. To visualize and quantify apoptotic cells in tumor tissue, TUNEL assay was performed using the DeadEnd™ Colorimetric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer\'s protocol. Apoptotic cells were visualized by staining them in brown color. Nuclei were counterstained using Harris-modified hematoxylin, and slides were mounted. Percentage of apoptotic cells is expressed as a percentage of total cells in the measured regime. Data are presented as mean value±SEM, and statistically analyzed with two-tailed and paired t-tests. A P value<0.05 was considered statistically significant (P*<0.05; **P<0.01; ***P<0.001).
Results
Discussion Our studies of FMD in cancer have led to the discoveries of a reductive damaging mechanism in DNA (Wang et al., 2009; Nguyen et al., 2011) and living cells (Lu et al., 2013) and of the molecular mechanisms of existing anti-cancer agents (Lu, 2007; Lu et al., 2007; Wang et al., 2006; Wang and Lu, 2007, 2010). These have offered unique opportunities to develop new effective drugs for targeted chemotherapy of cancer. Our recent results on cell experiments showed that an exogenous antioxidant induced significant DNA damage and apoptosis (cell death) in human normal cells through a reductive mechanism (Lu et al., 2013). Antioxidants may have a promotion effect in causing cancer (The ATBC, 1994; Albanes et al., 1996; Omenn et al., 1996; DeNicola et al., 2011; Lu et al., 2013), and reduce the effect of an exogenous reducing agent in killing tumor cells (Lu et al., 2013). This is probably because weakly-bound electrons in antioxidants can cause serious reductive DNA damage, which if not repaired properly, can lead to apoptosis, genetic mutations and likely diseases notably cancer. Thus, it is reasonable to speculate that abnormal (cancer) cells may have a more reduced intracellular/intranuclear environment and may therefore have some resistance to an exogenous reducing agent (antioxidant). Our finding of a reductive DNA-damaging mechanism might be an important step for effective prevention and therapy of cancer.