We show that doxorubicin treatment of PC3 carcinoma cells halts protein translation and that this effect does not occur at initiation but elongation of translation and is mediated by phosphorylation of EF-2. The effect of doxorubicin on EF-2 phosphorylation, loss of cFLIPS and enhanced sensitivity to TRAIL was recapitulated by treatment with hydrogen peroxide. Cell cycle distribution in hydrogen peroxide treated cells also trended towards G2/M arrest. Free radical generation did not only result in reduced levels of cFLIPS but also in the loss of XIAP and survivin, which like cFLIPS have short half-lives and function as antagonists in the apoptotic pathway. Pro-apoptotic proteins including DR5, caspases-8, -9, and –3 as well as Bax were not affected by hydrogen peroxide treatment. Overall these data suggest that free radical generation, phosphorylation of EF-2 and subsequent inhibition of protein elongation results in a preferential loss of short half-life proteins with anti-apoptotic function leaving a cell vulnerable to apoptotic stimuli. Doxorubicin treatment accomplishes this sensitization by generating free radicals upon association with iron, since decreased expression of cFLIPS and XIAP was abrogated by pre-treatment with an iron chelator. Our data suggest that the phosphorylation status of EF-2 is exquisitely sensitive to oxidative stress and inactivation of EF-2 by phosphorylation quickly results in a lowering of defenses against apoptosis. This built-in sensitivity to abnormal free radical abundance may be an important self-diagnostic capability of cells that naturally results in a vulnerable physiological state. Our proposed model of how doxorubicin affects cancer cells is more comprehensive than has been elucidated previously (). The inclusion of the EF-2 arm of this pathway opens a new area of inquiry into the role of free radicals in controlling cancer growth.
A rigorous conversation about free radicals, antioxidants, and cancer therapies is well underway and timely. Currently, highly concentrated doxorubicin therapy is sometimes coupled with iron chelation therapy. The iron chelation prevents free radical formation as demonstrated in a variety of studies and confirmed here [3
]. The purpose of the dual therapy is to protect cardiac myocytes which are particularly sensitive to free radical damage, perhaps due to a reduced intrinsic ability to process free radicals as compared to other normal cells such as cardiac fibroblasts [35
]. The danger inherent in this approach is that the iron chelators may have the potential to be cancer-protective as well as cardiac-protective. Interestingly, our DFO dose curve and TRAIL sensitization studies ( and ) show that a relatively high concentration of DFO is required to protect PC3 cancer cells (at least 40 μM) while cardiac cells are protected by doses as low as 10 μM [36
]. These results suggest that there may be a therapeutic window for DFO, which would be important to quantify in order to give patients the maximally effective treatment. Dexrazoxane (DRZ), another iron chelating agent, has been evaluated for cardiac-protective effects when used in combination with ABVE (doxorubicin, bleomycin, vincristine and etoposide) therapy in children with Hodgkin’s disease [37
]. The authors found that secondary acute myeloid leukemia and myelodysplastic syndrome occurred more frequently among children in the DRZ arm of the study, which approached but did not reach statistical significance. Two patients in the DRZ group also developed secondary solid tumors earlier than expected. The authors concluded that “the incidence of and the time of onset of the specific leukemias and solid tumors noted after DRZ with doxorubicin and etoposide heightens our concern about using DRZ to reduce long-term cardiopulmonary toxicity in the context of ABVE-based therapy”. Our data suggest a mechanism for this observation that should be considered when designing further studies on amelioration of side effects associated with doxorubicin.
Debate about the role of free radicals in cancer development is ongoing. Our proposed model of doxorubicin’s toxic effect on cancer cells is counter-intuitive to the long-standing belief that free radicals typically cause cancer rather than facilitate eradication of cancer. However, a recent study in Cancer Cell
revealed that the ROS-forming compound beta-phenylethyl isothiocyanate, found in vegetables such as broccoli and cabbage, actually destroys cancer in a mouse model through a ROS-mediated mechanism [38
]. Another recent mouse model of cancer treatment, which unexpectedly hinged on ROS production is the use of manganese superoxide dismutase (MnSOD) in combination with TRAIL. The MnSOD produces hydrogen peroxide, which was reported to activate caspase-8 and downregulate Bcl-2 [39
]. Our data suggest that anti-apoptotic proteins in addition to Bcl-2 may also have been down-regulated by this approach and that caspase-8 activation may have been the result of reduced c-FLIP expression. Furthermore, a group which focused specifically on TRAIL activation of caspase-8 in prostate cancer cells recently published their finding that caspase activation could be blocked by the antioxidants Vitamin C or catalase [40
]. Our data suggest the antioxidants may be preventing EF-2 phosphorylation, thereby protecting the cell by preserving anti-apoptotic proteins such as cFLIPS
, XIAP, and survivin.
In addition to illuminating a therapeutic mechanism of doxorubicin, our data suggest that EF-2 phosphorylation plays a role in the side effects caused by the drug. For instance, anemia and nausea may be attributed in part to cell cycle arrest in cells, which normally proliferate (blood cells and gut epithelial cells, respectively). Several studies have shown that iron chelators and antioxidants may help to ameliorate such doxorubicin-induced side effects [41
]. Early work on EF-2 suggested that EF-2 phosphorylation may impact cell cycling by downregulating protein synthesis and thereby clearing the cell of short-half life proteins such as cyclins [17
]. The authors of this study state that, “Temporary inhibition of translation may trigger the transition of a cell from one physiologic state into another because of the disappearance of short-lived repressors.” We propose that this mechanism is common between cell cycle modulation and TRAIL sensitization. In this paper we connect phosphorylation of EF-2 by doxorubicin to TRAIL sensitization via changes in the profile of apoptosis-related proteins and also conclude that the phosphorylation of EF-2 by free radicals may contribute to the cell cycle arrest seen upon doxorubicin treatment.
Our model also clarifies some of our previous findings. For instance, we have recently shown that downregulation of cFLIPS
was sufficient to sensitize PC3 cells to TRAIL [13
]. Because these cells have such low levels of Bcl-2 and Mcl-1, cFLIPS
may be the critical determinant of its apoptotic phenotype. In other cell systems, the global downregulation of anti-apoptotic proteins may be required to sensitize cells to TRAIL. We also previously demonstrated that cFLIPS
was decreased sufficiently after four hours of doxorubicin treatment to allow TRAIL-induced apoptosis to occur [11
]. However, levels of cFLIPS
rebounded by 24 hours, suggesting the effect of doxorubicin on inhibition of protein elongation is transient and reversible. Since translational control is an excellent modifier of the protein profile of a cell, we see EF-2 as an interesting new target for cancer researchers.