This study is the first to identify Dexra as a drug-based protection mechanism to prevent anthracycline toxicity in the ovary and to implicate Dexra as a promising candidate for continued in vivo experiments to prevent DXR-induced ovarian insult. In the mouse model granulosa cell line, a wide range of Dexra doses eliminated acute DNA damage and H2AFX activation in response to DXR. Similar reductions in DNA damage were obtained using Dexra to protect mouse primary granulosa cells and in vitro mouse ovary cultures from DXR insult. The putative protective agent also significantly increased cell viability following DXR treatment.
This study is also the first to identify an ovarian protective agent that prevents both the primary DNA insult and ensuing toxicity, rather than inhibiting only apoptosis that occurs subsequent to the initial insult. In the ovary, an organ dedicated to supplying, protecting, and maturing oocytes for reproduction, genetic fidelity is critical. Oocytes have extraordinary DNA repair machinery, and simply blocking apoptosis in response to radiation or cisplatin is sufficient to preserve follicles and viable oocytes [44
]. Oocytes exposed in vitro, however, cannot repair DNA damage after premature removal from DXR treatment [46
], so preventing the initial insult is critical to maintaining their genetic fidelity. Over the time course of our 3-h in vitro ovary treatment, oocytes did not exhibit DXR-induced DNA damage (). This is consistent with a TOP2-mediated damage model because a vast majority of oocytes in the ovary are meiotically quiescent. Whether oocytes would exhibit DXR-induced DNA damage, however, if followed over a longer period and as they mature remains to be seen. Future studies will determine whether Dexra protects oocyte DNA integrity in vivo in the face of DXR insult and whether the protection can be maintained to preserve development and maturation prior to ovulation and meiotic competence through to fertilization.
Maintaining the integrity of the other cell types comprising the ovary is also of paramount importance in preserving oocyte health. Granulosa cells sustain the oocyte; they are the primary site of DXR-induced apoptosis [6
] and therefore must also be protected to preserve follicular and oocyte integrity. Furthermore, granulosa cells produce estrogen that promotes endometrial proliferation for embryo implantation and have a role in protecting from osteoporosis and urogenital atrophy [47
]. Likewise, stroma/theca cells are integral to preserving ovarian structure and function, playing a key role in steroidogenesis and hormone synthesis. Data presented here show that Dexra prevented DXR insult in primary cultured murine granulosa cells and granulosa and stroma/theca cells from in vitro ovarian culture; future studies will determine whether Dexra also protects each ovarian cell type from DXR insult in vivo, thereby preserving follicular health and oocyte viability. The lack of Dexra protection of the oocytes in this study was due to the inherent lack of DXR toxicity on the oocyte in our model of ex vivo ovarian culture. While others have shown denuded oocytes cultured in vitro with DXR exhibit DNA damage [46
], we did not observe induced DNA damage in our ex vivo ovarian culture, suggesting the surrounding follicular cells and stromal tissue may play a role in protecting the oocyte from DXR toxicity. The potential to protect the ovary as a whole from chemotherapy, regardless of cell type, makes Dexra a promising tool as an ovarian shield.
DXR can cause cellular toxicity via two main mechanisms: oxidative stress and TOP2-mediated dsDNA breaks following DXR intercalation into DNA. If DNA damage occurs via the TOP2-dependent mechanism, we predict that Dexra-afforded protection will be solely dependent upon Dexra's inhibitory constant for TOP2 and therefore independent of DXR concentration. In agreement with this prediction, we found that DNA damage caused by 50 or 500 nM DXR was prevented by Dexra under identical pretreatment conditions ( and ). If the tested Dexra dose is sufficient to completely inhibit TOP2 activity, the observed protection will also be independent of Dexra concentration. While we could not copurify TOP2-Dexra complexes from treated KK-15 cells, studies from multiple groups have demonstrated that Dexra and other members of the bisdioxopiperazine family inhibit the enzymatic activity of purified TOP2 [48
] and have identified residues in the N-terminus of TOP2 critical for Dexra binding through mutation and crystallography studies [25
]. Our data fit the model of TOP2 dependency for dsDNA breaks because every tested Dexra concentration afforded similar protection from DXR-induced DNA damage (). In contrast, Dexra prevented oxidative stress-induced DNA damage in a dose-dependent manner. In addition, doses of 2 and 20 μM Dexra were sufficient to completely prevent DXR-induced DNA damage in KK-15 and murine granulosa cells, but either failed or only partially decreased H2
-induced DNA damage. We therefore propose a model in which DXR causes acute DNA damage in granulosa cells primarily in a TOP2-dependent manner and KK-15 cells are protected from that insult mechanism by Dexra pretreatment. Dexra-afforded protection from DXR-induced cytotoxicity, however, may be more complicated. In primary murine granulosa cells, 20 and 200 μM Dexra increased cell viability in response to DXR, where 2 μM Dexra did not. This dose dependence suggests that DXR cell demise may include an oxidative stress response or other unknown ovarian-specific mechanisms. In addition, Dexra did not completely prevent DXR-induced cell demise at DXR concentrations above 100 nM, consistent with the possible role of multiple DXR-toxicity mechanisms or indicating that higher doses of Dexra may be required to completely eliminate DXR-induced cytotoxicity.
Dexra pretreatment is required to afford protection from DXR in our cell system, just as in the heart and skin; adding Dexra and DXR to the cells simultaneously did not provide protection. While DXR is labeled a TOP2 poison, Dexra is an inhibitor of TOP2 catalytic activity, and the two drugs act at different points in the enzymatic cycle of TOP2 as illustrated in steps 1 and 2 of the model (). DXR, and other TOP2 poisons, act early in the enzymatic cycle, preventing resealing of TOP2 dsDNA breaks based on the drug's presence in the DNA strands (step 1 of the model, ) [16
]. During normal DNA processing, in the absence of TOP2 poisons, TOP2 seals the strand breaks and then releases the DNA (step 2 of the model, ). Dexra acts by stabilizing the closed-clamp conformation in which TOP2 holds the DNA after resealing breaks [16
]. In this model, pretreating cells with Dexra locks TOP2 in the closed-clamp conformation, preventing DNA release from the TOP2 complex and making the enzyme unavailable to cleave the DNA when cells are subsequently treated with DXR (step 3 of the model, ). We hypothesize that this allows the cells to clear DXR, providing long-term increase in cell viability. The effects of Dexra are reversible, making it well-tolerated in slow- or nondividing cells.
FIG. 7. Model for TOP2-mediated DXR insult and Dexra protection. Red ball structures represent DXR intercalated into the DNA. Step 1 illustrates DXR intercalation into DNA; the structure is from Protein Data Bank 1D12 . The presence of DXR prevents resealing (more ...)
Whether only one of both of the DXR-insult mechanisms occurs in the intact ovary in vivo remains to be seen. The ovary is a unique, heterogeneous organ within which follicles reside in various developmental stages ranging from rapidly growing antral follicles to quiescent primordial follicles. It is possible that these distinct follicle populations respond differently to DXR based on cell division rates and metabolic activity. One model is that primordial follicles may respond to DXR via the oxidative stress pathway, in a manner similar to nondividing heart cells, while growing follicles may succumb to TOP2-mediated DXR insult. Given this inherent heterogeneity of the organ, however, our data indicate that Dexra may still be effective in protecting the ovary as a whole. Dexra similarly protected granulosa and stroma/theca cells with no evidence for separate populations exhibiting differential responses. These data therefore suggest Dexra provides a unique tool that may be well-suited to protecting a heterogeneous organ like the ovary because it can prevent both mechanisms of DXR toxicity and appears effective across granulosa and stroma/theca cell types. Future studies may also determine whether other members of the bisdioxopiperazine family can similarly protect the ovary from chemotherapy insult.
Dexra may provide a time- and cost-efficient way to attenuate DXR insult in the ovary and prevent premature menopause and associated health risks without decreasing the effectiveness of cancer therapy as evidenced by its clinical application to prevent both DXR-induced cardiotoxicity and extravasation [17
]. Dexra does not diminish DXR antitumor activity in pediatric leukemia cases; while it may slow the response, it does not diminish survival in breast cancer cases; and nor does Dexra increase the risk for secondary malignant neoplasm in leukemia cases [17
]. One possible explanation for this dichotomy, protecting healthy tissue but not cancer cells, was provided in a mechanistic study by Yan et al. [51
]. Their work demonstrated that while Dexra blocked DXR-induced dsDNA breaks in a human fibrosarcoma cell line, it did not prevent DXR-induced apoptosis that occurred via glutathione depletion in a TOP2-independent manner. Dexra can have synergistic effects when combined with other chemotherapy agents in cancer treatment. Dexra impairs the development of DXR resistance in the leukemia cell line K562 [52
] and is synergistic with docetaxel or docetaxel plus DXR in treating MCF7 wild-type or resistant breast cancer cells and BT474 breast cancer cells [53
]. Combined therapy incorporating Dexra with DXR allows an increase in the maximal DXR dosage used [40
]. This may be beneficial for patients with triple-negative breast cancer, whose cancer is aggressive and DXR is part of the frontline chemotherapy regime [38
]. Similarly, Dexra pretreatment allows increased doses of etoposide in a mouse model by preventing not only cadiotoxicity, but preventing etoposide-induced decreases in white blood cell, platelet, and absolute neutrophil cell counts [55
], suggesting that Dexra has the potential to prevent insult across a range of TOP2 poisons.
Utilizing a chemical ovarian shield like Dexra presents a clear advantage over traditional hormone replacement therapy and current fertility preservation approaches. Traditional fertility preservation options are both time and cost prohibitive. While they can provide the option for a female cancer survivor to have biologically related children in the future, they do not protect endocrine function of the ovary. This leaves survivors susceptible to premature ovarian insufficiency and associated health complications, including osteoporosis and cardiovascular disease. Ovarian hyperstimulation and in vitro fertilization cycles required for oocyte and embryo banking for cancer patients can delay cancer therapy and promote the growth of tumors that are hormone responsive. In addition, oocyte and embryo cryopreservation are not treatment options for prepubescent patients. Pretreating patients with Dexra prior to DXR may provide a time- and cost-effective way to preserve not only fertility, but endocrine function as a whole. It is a therapy that should be equally effective in pediatric as well as adult cancer patients and bypass the pitfalls of hormone stimulation. This therapy therefore has the potential to increase the quality of life for female cancer patients without compromising their cancer treatment.