Kim et al. BMC Cancer 2012, 12:147 http://www.biomedcentral.com/1471-2407/12/147 Results Tetrathiomolybdate increases the sensitivity of ovarian cancer cell lines to doxorubicin To evaluate whether TM can sensitize ovarian cancer cells toward doxorubicin treatment, the SRB assay was carried out. Platinum resistant SKOV-3 ovarian cancer cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with doxorubicin (0, 2.5, 5, 10 μM) for another 24 h. Cells treated solely with doxorubicin (follow- ing vehicle pre-treatment for 24 h) at all concentrations tested reduced cell proliferation by 29% as compared to untreated controls (Figure 1a). Cells treated solely with TM (30 μM) reduced cell viability by 5%. Pre-treatment with TM followed by treatment with doxorubicin revealed significant sensitization with viability inhibited by 42.1% (2.5 μM doxorubicin), 62.0% (5 μM) and 79.1% (10 μM). To verify TM-mediated sensitization of ovarian cancer cells to doxorubicin, platinum sensitive A2780 cells were treated as described above with the exception that TM was applied at the concentration of 7.5 μM. (Figure 1b). Treatment with TM alone resulted in 14% reduction of viability while doxorubicin alone exerted effects in a dose- dependent manner (41.7% viability reduction at 2.5 μM; 50% at 5 μM; 58.7% at 10 μM doxorubicin). The TM/dox- orubicin combination revealed significant sensitization of A2780 cells with viability inhibited by 74.8% (2.5 μM dox- orubicin), 87.5% (5 μM) or 97.1% (10 μM). Taken together, our results suggest that TM treatment sensitizes ovarian cancer cells to doxorubicin treatment in vitro. Doxorubicin-mediated apoptosis in ovarian cancer cells is enhanced by pre-treatment with TM We carried out flow cytometry of drug treated cells (TM, doxorubicin, TM/doxorubicin combination) to measure a potential loss of mitochondrial membrane potential (ΔΨm), an irreversible event during the process of apopto- sis [24,25]. SKOV-3 cells were double-stained to detect cells with active mitochondria ΔΨm, and to detect non- viable cells. As shown in Figure 1c, the cells treated with TM alone (30 μM, 48 h) only displayed minimal change in ΔΨm and remained mostly viable (Q4; 94.7%). Similarly, the majority of cells treated with doxorubicin alone remained mostly viable (Q4; 78.0%) with 8.1% of cells (Q1 + Q2) being dead and an additional 13.9% displaying a dis- rupted ΔΨm (Q3). In contrast, treatment with TM (30 μM, 24 h), followed by additional treatment with doxorubicin (5 μM, 24 h) increased the population of dead cells to 15.2% (Q1 + Q2) and also increased the population of cells with a disrupted ΔΨm to 26.8% (Q3). To define the cellular response of SKOV-3 cells upon TM and doxorubicin treatment we analyzed the activa- tion of caspases characteristic for induction of apopto- sis as well as the inactivation of PARP, a DNA repair factor, by immunoblotting. SKOV-3 cells were treated Page 3 of 10 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 (Figure 1d, left panel). PARP inactiva- tion/cleavage and caspase-7 activation were observed when SKOV-3 cells were treated with doxorubicin alone for 24 h. TM/doxorubicin combination treat- ment, however, led to a strong detection of cleaved caspase-3 and to an increased level of activated cas- pase-7 and inactivated PARP when compared to doxor- ubicin treatment alone (Figure 2d, left panel). The same procedure was employed to treat A2780 cells (Figure 2d, right panel) with different concentrations of TM (7.5 μM) and doxorubicin (2.5 μM). Similarly to SKOV-3 cells, the most prominent cleavage of these apoptotic markers was found when A2780 cells were treated with TM/doxorubicin combination. Collec- tively, these results verify that TM sensitizes ovarian cancer cells to doxorubicin therapy and potentiates cell death via induction of apoptosis. An increased generation of intracellular ROS upon TM/ doxorubicin combination treatment causes ovarian cancer cell death One potential strategy suggested to treat cancer is to gen- erate an excess amount of ROS in tumor tissue to induce cell death. We determined if SKOV-3 cell treatment with either TM or doxorubicin alone or in combination led to excess generation of ROS via flow cytometry. As shown in Figure 2a, ROS generation was slightly elevated over baseline after treatment with TM alone (shift in relative fluorescence intensity). Doxorubicin alone also caused a peak shift, which was further increased when the cells were first treated with TM followed by doxorubicin treatment. To confirm that the generation of ROS by TM/doxoru- bicin combination treatment is the predominant mechan- ism of cytotoxic action we performed viability assays with SKOV-3 cells in the absence or presence of antioxidant ascorbic acid. As shown in Figure 2b the growth inhibitory effect of TM was not notably affected by ascorbic acid treatment (90.2% viability with ascorbic acid; 88.1% with- out). Similarly, scavenging of ROS did not reduce the cyto- toxic action (70.4% viability with ascorbic acid; 68.9% without) by doxorubicin treatment. In contrast, in TM/ doxorubicin combination treatment, ascorbic acid partially restored the viability (69.9% viability with ascorbic acid; 42.6% without) (Figure 2b). The potentiation of JNK and p38 activation by TM/ doxorubicin combination treatment is ROS-dependant JNK and p38 are key regulators of stress-mediated apop- tosis and activation of these MAPK takes a center stage in cellular response to treatment with TM/doxorubicin as shown recently for endometrial cancer cells in vitro