Dex is among one of the most clinically effective drugs in myeloma therapy. In lymphoid malignancies, Dex-induced apoptotic cell death pathways have been partially deciphered [20,21
]. Our results show that Dex treatment results in the activation of the mitochondrial apoptotic cascade in myeloma cells (), further substantiating the role of mitochondrial dysfunction in Dex-mediated apoptosis. Studies have linked Dex-induced apoptosis and oxidative stress using a thymic lymphoma cell line where clones resistant to H2
(by catalase overexpression or selection of H2
-resistant clones) showed Dex cross-resistance [51
]. Dex treatment has been shown to upregulate the messenger RNA expression of glutathione S
-transferase and down-regulate the messenger RNA of thioredoxin, glutathione peroxidase, catalase, and superoxide dismutase in T lymphocytes [33,34
]. However, in MM, a causal role for oxidative stress in Dex-induced tumor cell death has not been firmly established. Chauhan et al. [36
] showed that Dex-induced apoptosis of myeloma cells is independent of O•-2
production. However, it was recently shown that newly diagnosed myeloma patients treated with Dex exhibit gene expression changes indicative of oxidative stress [37
]. In the current report, we show for the first time that besides various other known myeloma cell death mechanism(s), Dex increases oxidative stress-induced clonogenic cell killing in myeloma cells ().
Newly diagnosed MM patients have higher circulating levels of malondialdehyde indicative of lipid peroxidation and lower levels of antioxidant enzymes (superoxide dismutases, glutathione peroxidases, and catalase) in erythrocytes [30–32
]. Because myeloma patients may be under systemic oxidative stress at diagnosis, treatment with agents that selectively induce oxidative stress (chemotherapy and radiotherapy) in tumor cells may significantly enhance their oxidative cytolysis. In support of this hypothesis, our in vitro
studies with combined exposure to Dex and radiation clearly show an increase in Dex-induced oxidative stress (, B
) that seems to be causally related to a significant decrease in clonogenic survival of myeloma cells (). Furthermore, Dex-induced increases in oxidative stress and cell killing were not noted in BMSCs, thus not worsening hematopoietic recovery after radiotherapy as also seen by peripheral blood cell counts (, B
). This leads to the speculation that the combination of Dex with radiation may increase the therapeutic index of skeletal targeted radiotherapy in MM by selectively inducing oxidative stress in cancer cells.
Our studies show that in contrast to myeloma cells, Dex inhibited radiation-induced oxidative stress in normal hematopoietic stem and progenitor cells (). We hypothesize that Dex attenuates radiation-induced cytotoxicity to HSCs and HPCs by decreasing steady-state levels of pro-oxidants to a threshold level that may be aiding in improved hematopoietic reconstitution. Our in vivo
studies show that Dex cotreatment results in less severe and faster peripheral blood reconstitution of blood cell types (granulocytes and platelets) compared with radiotherapy alone (, B
). In erythroid cells, a hematoprotective role of Dex with chemotherapeutic drugs has been shown where it stimulates a sustained proliferation of immature cells and delays their differentiation [52
]. Recently, ROS has been shown to act as a positive regulator of hematopoietic reconstitution by upregulating vascular cell adhesion molecule 1 expression on endothelial cells [53
]. To assess whether Dex was protecting hematopoietic stem/progenitor cells from radiation-induced damage, clonogenic survival of HSC/HPC was performed where the progenitor number of different lineages (myeloid and lymphoid lineages, common myeloid progenitor, and common lymphoid progenitor subsets) was collectively analyzed. Compared with radiation alone, Dex cotreatment showed higher clonogenic survival of both HSCs and HPCs (), suggesting that the improved in vivo
restoration of normal hematopoiesis after combination treatment with Dex plus 153-Sm-EDTMP could be partially rendered by Dex-mediated radioprotection of HSCs and HPCs.
In the present study, we have focused on the myeloma cell proproliferative cytokine IL-6 [54
] that is released by irradiated BMSCs (). It is well established that radiation treatment upregulates redox-sensitive transcriptional factors (i.e., NF-κB and AP-1) [55
] that have been shown to increase IL-6 production in MM cells [27
]. Dex has also been shown to inhibit radiation-induced IL-6 up-regulation in fibroblasts [56
]. Mechanistically, Dex can inhibit constitutive and radiation-induced IL-6 production by binding and inhibiting NF-κB [57
] or upregulating the transcription of IκB [58
] or glucocorticoid-induced leucine zipper that can inhibit NF-κB and AP-1 [59–61
]. Using myeloma-BMSCs coculture studies, we show that IL-6 as well as other radiation-induced pro-inflammatory cytokines may be stimulating myeloma growth and proliferation, and this paracrine cytokine stimulation can be effectively inhibited by Dex () with selective radiosensitization of myeloma cells.
To evaluate the efficacy of the proposed combination therapy for Dex plus radiation, studies were extended in an animal model ( and ). We have previously shown that the in vitro
cell killing of a chemoradiotherapy regimen with γ-radiation can be effectively reproduced in vivo
with 153-Sm-EDTMP [42
]. Cotreatment of Dex with 153-Sm-EDTMP resulted in inhibition of myeloma disease progression and improved survival (). With 153-Sm-EDTMP, 87.5% of the radiation dose would be delivered to the BM in three half-lives (>6 days). We therefore tested a two-cycle protocol where 153-Sm-EDTMP was administered 10 days apart that also gave time for Dex-mediated recovery of the peripheral blood counts. A two-cycle protocol of Dex plus 153-Sm-EDTMP prolonged the survival over one-cycle protocol (), indicating that cotreatment with Dex may be used for designing a repeat dosing regimen of bone-seeking radionuclide and/or for escalation of radiation dose to improve the therapeutic outcome of MM.
Dex is among one of the most clinically effective drugs in myeloma therapy [62
] that is used in chemotherapy protocols with novel molecularly targeted agents such as bortezomib and thalidomide [63
]. When combined with radiation treatment, an increased myeloma cell killing of thalidomide [13
] and bortezomib [14,15
] has been noted. In our previous study, the combination of bortezomib with 153-Sm-EDTMP resulted in the prolongation of survival without increasing the myelosuppressive effects of 153-Sm-EDTMP [42
]. The current report shows that the combination of 153-Sm-EDTMP with Dex selectively radiosensitizes myeloma cells in vivo
and partially alleviates radiotoxicity toward the BM hematopoietic system. Mechanistically, we show that the combination of radiotherapy and Dex selectively enhances killing of myeloma cells while protecting normal BM hematopoiesis through a mechanism that involves selective increases in oxidative stress in myeloma cells. The results of this study can be extended for myeloma therapy in combination with bortezomib, thalidomide, or other novel biologic agents that may enhance the potency of Dex and/or radiation in killing myeloma cells. To our knowledge, this is the first report showing that Dex significantly and specifically enhances radiation-induced oxidative stress and killing of myeloma cells in monocultures, direct cocultures, and in vivo
in a myeloma model. Our findings in normal tissues suggest that Dex decreases hematotoxicity and increases the overall therapeutic index of radiotherapy in MM. This study provides a potential new avenue for therapeutic use of Dex in designing protocols for myeloma therapy. Furthermore, the proposed combination of Dex and radiation can be tested in other malignancies because both these treatment modalities are widely used in various cancers.