Kim et al. BMC Cancer 2012, 12:147 http://www.biomedcentral.com/1471-2407/12/147 Table 1 Effect of drug combinations on SKOV-3 cell viability Cell Viability DOX MMC 4-HPR 5-FU [% of Untreated Control] [10 μM] [10 μM] [5 μM] [1 mM] No TM With TM, 30 μM Cytotoxicity Increase 70.7% 20.9% 2.5 fold 73.0% 48.2% 1.6 fold 83.1% 40.3% 2.9 fold 78.3% 48.2% 1.9 fold suppressed anti-apoptotic events we analyzed the inacti- vation/cleavage of PARP, a DNA repair factor, and expression of the X-linked inhibitor of apoptosis (XIAP), by immunoblotting. SKOV-3 cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with doxorubicin (0, 5 μM), MMC (0, 5 μM), 4-HPR (0, 10 μM) or 5-FU (0, 1000 μM) for another 24 h. As shown, PARP inactivation/cleavage over background levels (untreated controls) was observed for individual treatment with MMC, 4-HPR, and 5-FU (Figure 4b) as shown for doxorubicin (Figure 1d) but not for TM treatment. Combination treatment of SKOV-3 cells with TM potentiated the effects of all drugs tested on PARP. PARP was partially inactivated/cleaved after TM/5-FU treatment and drastically inactivated after TM/MMC, TM/4-HPR (Figure 4b) and TM/doxorubicin (Figure 1d) treatment. XIAP expression was reduced after TM/5-FU and barely detectable after TM/doxorubicin, TM/MMC and TM/4-HPR combination treatment (Figure 4b). In summary, TM sensitized ovarian cancer cells to treat- ment with these four anticancer drugs and potentiated cell death via induction of apoptosis. We showed that the generation of ROS by TM/doxoru- bicin combination treatment is a key mechanism of cyto- toxic action (Figure 2) in ovarian cancer cells. To assess if ascorbic acid can similarly quench the effects of 5-FU, MMC and 4-HPR when combined with TM, we per- formed viability assays using SKOV-3 cells. Cells were treated with TM (0, 30 μM) for 24 h, after which the cells were treated with MMC (0, 5 μM) for another 24 h or 4- HPR (0, 5 μM) or 5-FU (0, 1000 μM) for another 48 h. As shown in Figure 4c scavenging of ROS by ascorbic acid did not significantly reduce the partial cytotoxic effects of any single drug. In contrast, upon TM/MMC combination treatment, ascorbic acid restored cell viability effectively from 51.0 to 78.1%. Upon TM/4-HPR treatment, ascorbic acid restored cell viability dramatically from 37.6 to 94.1%. Upon TM/5-FU treatment, ascorbic acid restored the via- bility effectively from 22.7 to 51.3% (Figure 4c). Discussion The present report reveals that combination treatment of ovarian cancer cells in vitro with TM improved the effi- cacy of a panel of anticancer drugs, namely doxorubicin, MMC, 4-HPR and 5-FU. We recently reported that TM Page 7 of 10 sensitized drug-resistant endometrial cancer cell lines to doxorubicin [7] similar to ovarian cancer cells. Doxorubi- cin is among the most widely used agents to treat tumors, including lymphomas, leukemias, lung, breast, and ovarian cancers [16]. Doxorubicin is classified as a topoisomerase II inhibitor but its cytotoxic mechanisms remain unclear. The interaction of doxorubicin with cel- lular iron and its quinone moiety with oxo-reductive enzymes leads to the formation of radicals and excessive generation of ROS promoting cancer cell death [7,21,26,27]. It has been proposed that the pharmacologi- cal suppression of cellular antioxidants or elevation of ROS levels can sensitize tumor cells to doxorubicin [28,29] as shown here by combination treatment with TM. Such a treatment modality would provide a benefit to cancer patients because the use of doxorubicin and other anticancer agents is often challenged by concentra- tion-dependent toxic effects [30]. Interestingly, indepen- dent of improved anti-tumor efficacy, TM treatment was shown in mice to protect against doxorubicin-induced cardiac toxicity [31]. Cardiotoxicity is the major limiting factor in the clinical use of doxorubicin implicating that combination therapy with TM may also provide clinical benefits secondary to the mitigation of doxorubicin toxi- city during chemotherapy. Similar to doxorubicin, MMC contains a quinone moiety and leads to radical formation in vitro [19]. In addition, as for doxorubicin, the efficacy of MMC in ovarian cancer cells was improved and was linked to an increased ROS activity when the cells were treated in combination with TM as shown in the present report. MMC was originally isolated as an antibiotic from Streptomyces and is a potent DNA cross-linker classify- ing it as a potential agent to treat a broad range of solid tumors [32]. Phase II trials of low-dose MMC in patients with refractory ovarian cancer revealed higher survival rates and no significant toxicity [14]. MMC has also suc- cessfully been used alongside other drugs such as irinote- can (topoisomerase inhibitor) to treat refractory ovarian cancer [33]. Because TM was well tolerated in patients with Wilson disease and in phase I and II trials to treat patients with a variety of different tumors [11-13,34], and based on our findings we suggest to establish trials to treat resistant and/or refractory ovarian cancer by combi- nation therapy with TM and doxorubicin or alternatively with MMC. TM in ovarian cancer cells also potentiated the effect of the pyrimidine analog 5-FU which acts as a thymidylate synthase inhibitor, thereby strongly affecting rapidly dividing cancerous cells [35]. It has been shown pre- viously that 5-FU can improve the efficacy of platinum based drugs in combination for the treatment of ovarian cancer [15,36]. 5-FU in these studies was well tolerated and can be used at relatively high concentrations without toxic side effects. The effects of anticancer drugs that are