Kim et al. BMC Cancer 2012, 12:147 http://www.biomedcentral.com/1471-2407/12/147 Page 6 of 10 elevation of ROS or MAPK activation. We investigated if TM potentially can sensitize cancer cells to a panel of drugs including MMC, 4-HPR, and 5-FU. SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells treated for another 24 h with MMC or another 48 h with 4-HPR or 5-FU (Figure 4a). Cells treated solely with MMC (2.5, 5, 10 μM), similarly to doxorubi- cin, did not show a clear dose response (Figure 1a) and reduced cell viability by only 27% as compared to untreated controls. 4-HPR exerted dose-dependent cyto- toxicity on SKOV-3 cells (viability of 83.1% at 5 μM; 50.4% at 10 μM). 5-FU displayed partial effects (viability of 78.3% at 1 mM). As seen for doxorubicin, treatment with TM potentiated the cytotoxic effect of all three drugs tested. Under these conditions the viability was reduced to 48.2% (TM + 10 μM MMC), 40.3% (TM + 5 μM 4-HPR) and 48.2% (TM + 1 mM 5-FU), respec- tively (Figure 4a). In summary, depending on the drug and concentration used, TM increased the cytotoxicity between 1.6 and 2.9 fold after collecting the effect of TM alone (Table 1). Our experiments suggest that TM treatment improves the response of ovarian cancer cells to doxorubicin, MMC, 4-HPR, and 5-FU treatment in vitro. To confirm that the potentiation of the cytotoxicity of these drugs by TM treatment is accompanied by Figure 4 TM increases ROS mediated cytotoxicity of various anticancer drugs in ovarian cancer cells. (a) SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated for another 24 h with MMC or for another 48 h with 4-HPR or 5-FU in the concentrations indicated. The cell viability was evaluated as described in Methods. Data are expressed as the mean of the triplicate determinations (X ± SD) compared to untreated cells [100%]. (b) SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated for another 24 h with doxorubicin (0, 5 μM), MMC (0, 5 μM), 4-HPR (0, 10 μM) or 5-FU (0, 1000 μM). Immunoblotting was carried out with primary antibodies against cleaved PARP, pro-survival marker XIAP or GAPDH as internal control for equal loading. (c) SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated for another 24 h with MMC (0, 5 μM) or for another 48 h with 4-HPR (0, 5 μM) or 5-FU (0, 1000 μM) in the presence or absence of radical scavenger ascorbic acid (750 μM).