Kim et al. BMC Cancer 2012, 12:147 http://www.biomedcentral.com/1471-2407/12/147 known to target DNA integrity or synthesis are often cor- related to the induction of pro-apoptotic signaling and the generation of oxidative stress [7,20]. In the present report, the mild cytotoxicity displayed by 5-FU as a single agent was ROS independent while TM/5-FU combina- tion treatment led to a significant ROS based reduction of ovarian cancer cell viability suggesting that expanded in vitro studies are needed to establish the parameters for the use of these two drugs in animal tumor models. Similarly, our study suggests that the combination treatment with 4-HPR and TM can be considered to treat ovarian or other cancers. 4-HPR is a synthetic reti- noid with antitumor activity and is known to cause cellu- lar metabolic perturbations and induction of apoptosis in ovarian and other cancer cells via ROS-dependent mechanisms involving ER stress and MAPK (e.g. JNK) activation [18,37]. In recent phase II trials, patients with recurrent ovarian and primary peritoneal carcinoma received 4-HPR. It was well tolerated and displayed clini- cal responses [17]. Based on our findings, ovarian tumors may be sensitized to the treatment with 4-HPR by combi- nation treatment with TM. Because both drugs were well tolerated by patients in previous studies, we suggest exploring the efficacy of TM/4-HPR in animal tumor models and ultimately in clinical applications. As shown in the present report, TM increases the effi- cacy of doxorubicin, MMC, 4-HPR and 5-FU in ovarian cancer cells. Accordingly, lower concentrations of these drugs are needed to cause significant cytotoxicity. A simi- lar effect during in vivo treatments could potentially reduce administered doses and toxic side effects of these or other anticancer drugs when combined with TM. The drug effects of doxorubicin, MMC, 4-HPR or 5-FU are potentiated when TM is co-applied to the cells unless an antioxidant is added; proving that increased ROS genera- tion is responsible for drug action when used in combina- tion. ROS are byproducts of normal cellular metabolism and tightly regulated in balance with cellular antioxidants. Cancer cells, through mitochondria dysfunction and increased metabolism, generate a relatively high level of ROS [38,39]. Further elevation of cellular ROS beyond a toxic threshold, as shown here after combinational treat- ment with TM, is an attractive strategy to selectively target tumor cells during chemotherapy [40,41]. These phenom- ena are not unique to TM combination treatment because ROS generation is a key mechanism of apoptosis for a variety of common chemotherapeutic drugs such as dau- norubicin, cyclophosphamide or cisplatin [42-44]. Poten- tially, combination treatment of TM with doxorubicin, MMC, 4-HPR, or 5-FU may exert additional synergistic effects when combined with other agents thought to mod- ulate the antioxidant functions of cancer cells such as 2-methoxyestradiol (SOD inhibitor) or drugs leading to glutathione depletion such as buthionine-sulfoximine. Page 8 of 10 This approach might, in particular, be applied to treat MDR tumors because, for example, SOD1 is a therapeutic target of TM [6] and inhibition of SOD1 was shown to restore the cisplatin-sensitivity in resistant ovarian cancer cells [45]. It has been reported that chemotherapy-induced generation of ROS is often correlated with activation of the JNK and p38 MAPK signaling pathways [46,47] which play a crucial role in the response of ovarian cancer cells to common anticancer agents such as cisplatin [48]. Even though a detailed analysis of the modulation of these sig- naling pathways is beyond the scope of the present report we conducted a limited study to the effect of one drug combination (TM/doxorubicin) on the activation of these apoptotic markers in ovarian cancer cells. Combination treatment led to a dramatic increase of JNK and p38 acti- vation which could be blocked by ROS scavenging and, thus, was directly correlated to the increase of ROS levels. Accordingly, apart from the destabilization of the cellular oxidative defense system, the subsequent potentiation of stress kinase signals also plays a vital role in the improved efficacy by TM combination therapy. Conclusions The present study reveals that TM increases the efficacy of various anticancer drugs in multi-drug resistant ovar- ian cancer cells in a ROS-dependent manner. In addi- tion, to the study of medical applications of TM, as discussed in the previous sections, we suggest to expand the analysis of biochemical and mechanistic effects of TM combination therapy in vitro and in animal tumor models. These may include the use of redox-modulating agents, cell lines derived from refractory ovarian or other cancers, and a broader panel of cancer drugs. Ulti- mately, the use of TM or TM-derivatives in combina- tion therapy may improve current cancer treatment options. Acknowledgements RGM is partially supported by NCI Grant #1 RO1 CA136491-01 and Grants from Swim Across America. RKS is partially supported by Grants from Swim Across America. Author details 1Molecular Therapeutics Laboratory, Program in Women’s Oncology, Departments of Obstetrics and Gynecology, Women and Infants Hospital, Alpert Medical School, Brown University, 101 Dudley Street, Providence, RI 02905, USA. 2Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA. 3Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, IL 62794, USA. Authors’ contributions Coordinated the study (KK, TL, RS, RM, LB). Carried out the experiments (KK, RS). Analyzed the data (KK, TL). Wrote the manuscript (KK, TL, RS, RM, LB). All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests.