Kim et al. BMC Cancer 2012, 12:147 http://www.biomedcentral.com/1471-2407/12/147 Page 5 of 10 Figure 2 Generation of intracellular ROS in ovarian cancer cells after individual or combinational treatment with TM and doxorubicin. (a) SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with doxorubicin (0, 10 μM) for another 5.5 h. Generation of intracellular ROS was measured by flow cytometry (see Methods). Data are presented as relative fluorescence intensity (DCF). (b) SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with doxorubicin (0, 5 μM) for another 24 h in the presence or absence of radical scavenger ascorbic acid (750 μM) before the viability of cells was measured. Data are expressed as the mean of the triplicate determinations (X ± SD) compared to untreated cells [100%]. To clarify whether an enhanced ROS production caused by the drugs alone or in combination potentiated the MAPK activations described above, we employed antioxidant ascorbic acid (750 μM) alongside to TM (30 μM) and doxorubicin (5 μM) treatment. As shown in Figure 3b, the potentiation of JNK and p38 activation upon TM/doxorubicin combination treatment was clearly attenuated by ascorbic acid, resulting in decreased kinase activation levels similar to those found in the cells treated with doxorubicin alone. Furthermore, the strong inactivation of PARP (an indicator for apoptotic pro- cesses) observed upon TM/doxorubicin combination treatment was blocked by ascorbic acid co-treatment and, thus, ROS dependent (Figure 3b). In summary, increased ROS generation caused by TM/doxorubicin combination potentiated cytotoxic effects (viability assay; Figure 2b) and pro-apoptotic signaling. TM induces sensitivity of ovarian cancer cells to various anticancer drugs via promotion of apoptotic signaling and ROS generation A multitude of commonly used anticancer drugs display mechanistic features similar to those ascribed to doxor- ubicin when used to treat cells in vitro, such as the Figure 3 Activation of pro-apoptotic JNK and p38 MAPK after TM/doxorubicin treatment with or without inhibition of ROS. (a) SKOV-3 cells (left panel) were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with doxorubicin (0, 5 μM) for 1, 4, 7 or 24 h as indicated. The same procedure was employed to treat A2780 cells (right panel) with different concentrations of TM (0, 7.5 μM) and doxorubicin (0, 2.5 μM). Immunoblotting was carried out with primary antibodies against native and activated/phosphorylated JNK or p38 MAPK. As an internal standard for equal loading blots were probed with an anti-GAPDH antibody. (b) SKOV-3 cells were treated as described in (a) in the presence or absence of radical scavenger ascorbic acid (750 μM). Immunoblotting was carried out with primary antibodies against JNK, p-JNK, p38, p-p38, cleaved PARP or GAPDH.