The present report reveals that combination treatment of ovarian cancer cells in vitro
with TM improved the efficacy of a panel of anticancer drugs, namely doxorubicin, MMC, 4-HPR and 5-FU. We recently reported that TM sensitized drug-resistant endometrial cancer cell lines to doxorubicin [7
] similar to ovarian cancer cells. Doxorubicin 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 cellular 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
]. It has been proposed that the pharmacological suppression of cellular antioxidants or elevation of ROS levels can sensitize tumor cells to doxorubicin [28
] 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 concentration-dependent toxic effects [30
]. Interestingly, independent 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 toxicity during chemotherapy. Similar to doxorubicin, MMC contains a quinone moiety and leads to radical formation in vitro
]. 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 classifying 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 successfully been used alongside other drugs such as irinotecan (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
], and based on our findings we suggest to establish trials to treat resistant and/or refractory ovarian cancer by combination 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 previously that 5-FU can improve the efficacy of platinum based drugs in combination for the treatment of ovarian cancer [15
]. 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 known to target DNA integrity or synthesis are often correlated to the induction of pro-apoptotic signaling and the generation of oxidative stress [7
]. In the present report, the mild cytotoxicity displayed by 5-FU as a single agent was ROS independent while TM/5-FU combination 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 retinoid with antitumor activity and is known to cause cellular 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
]. In recent phase II trials, patients with recurrent ovarian and primary peritoneal carcinoma received 4-HPR. It was well tolerated and displayed clinical responses [17
]. Based on our findings, ovarian tumors may be sensitized to the treatment with 4-HPR by combination 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 efficacy 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 similar 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 generation is responsible for drug action when used in combination. 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
]. Further elevation of cellular ROS beyond a toxic threshold, as shown here after combinational treatment with TM, is an attractive strategy to selectively target tumor cells during chemotherapy [40
]. These phenomena 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 daunorubicin, cyclophosphamide or cisplatin [42
]. Potentially, combination treatment of TM with doxorubicin, MMC, 4-HPR, or 5-FU may exert additional synergistic effects when combined with other agents thought to modulate the antioxidant functions of cancer cells such as 2-methoxyestradiol (SOD inhibitor) or drugs leading to glutathione depletion such as buthionine-sulfoximine. 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
] 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 signaling 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 activation 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.