MM remains a fatal disease, primarily owing to acquired resistance to anti-myeloma chemotherapeutic drugs [1
]. Our previous studies have shown that combining targeted radiation with chemotherapeutic drugs enhances the effectiveness of radiotherapy in MM [10
]. IL-6 is known to induce myeloma cell proliferation and confer aggressive growth properties and drug resistance in myeloma cells [1
]. In myeloma and B-cell lymphoma, radioresistance has been correlated with the presence of IL-6-secreting tumour cells [42
]. We previously reported that IR induces NF-κB activation in myeloma cells [23
], and that paracrine IL-6 secretion confers radioresistance to myeloma cells [20
]. Oxidative stressors such as IR and H2
have been shown to enhance IL-6 secretion by activating NF-κB transcription [43
]. Thus IL-6 expression in the BM microenvironment is robust, and our published findings show that inhibition of constitutive and therapy-induced IL-6 synthesis is essential for effective killing of myeloma cells by IR. In the present study we evaluated the roles of myeloma-promoting cytokines, with a major focus on IL-6, in adaptation to endogenous and therapy-induced oxidative stress in myeloma and BM accessory cells.
Pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 have been reported to induce O2•−
production and also trigger downstream signalling events [3
]. Using fluorigenic probes (H2
DCF-DA and DHE), the present study shows that IL-6 or TNF-α treatments induce a rapid rise in pro-oxidants in myeloma cells. With IL-6 treatment, HPLC analysis showed considerable formation of E+
and other DHE oxidation products. Studies are ongoing to determine whether, in myeloma cells, DHE and 2-OH-E+
rapidly reacts with different oxidants (such as H2
in the presence of haem proteins or peroxidases) and whether utilizing SOD2
KD myeloma cells may aid in better identifying IL-6-induced O2•−
production. Since both IL-6 and TNF-α directly induce myeloma cell proliferation and IL-1β drives paracrine IL-6 secretion, an increase in the production of ROS could mediate enhanced proliferation and the development of resistance to oxidative stressors. Notably, when IL-6 was combined with IR or Dex, a concerted increase in early ROS production was observed. Since IL-6 treatment inhibited IR- and Dex-mediated myeloma cell death, IL-6 may play a role in the induction of redox-regulated antioxidant pathways in MM cells. Consistent with these findings, IL-6 has been shown to protect normal cells from injuries induced by oxidative stress [4
IL-6 is a pro-proliferative cytokine for myeloma cells, and increased plasma levels of IL-6 correlates with poor prognosis [1
]. On the basis of the disease stage, the serum levels of IL-6 in myeloma patients can range from 0.01 to 0.4 ng/ml; the actual IL-6 serum concentration can be even higher, since circulating IL-6 also binds to soluble IL-6 receptors [47
]. In myelomatous BM, a local high concentration of IL-6 can be reached, given that myeloma cells produce IL-6 via the autocrine loop [48
] and by adhesion with BM stromal cells, leading to paracrine IL-6 secretion by stromal cells [49
]. In the present study we have combined exogenous IL-6 (50 ng/ml) with irradiation; however, lower concentrations of IL-6 (2.5 and 10 ng/ml) also protected myeloma cells from irradiation-induced apoptosis, resulted in pro-oxidant production and induced MnSOD protein expression. The protective effects of IL-6 on cell death induced by oxidative stressors were assessed after IL-6 pre-treatment for 6 h mainly because IL-6 showed maximum NF-κB activation at 4 h. Furthermore, as IL-6 is constantly present in the myeloma cell microenvironment, IL-6 treatment was continued until samples were collected for analysis. We plan to extend the present study to primary human myeloma samples in the near future. However, for ex vivo
cultured primary myeloma cells, the culture medium is supplemented with exogenous IL-6 (5 ng/ml), making the experimental design difficult.
GSH provides a major source of thiol homoeostasis and constitutes the first line of the cellular defence mechanism against oxidative stressors. In cancer cells, increased GSH levels has been correlated with chemoresistance and radioresistance [33
], and depletion of intracellular GSH can reverse drug resistance and improve the outcome of cancer therapies, including those for haematological malignancies [51
]. GSH kinetics over time with IL-6 and TNF-α in myeloma cells show that IL-6 treatment results in a transient early decrease in GSH levels, whereas TNF-α treatment caused a more sustained decrease in total GSH levels until 24 h. Along similar lines, Nakajima et al. [36
] showed that, in neuronal cells, IL-6 treatment results in increased γ-glutamylcysteine synthetase activity, which is the rate-limiting enzyme of GSH synthesis. Also, TNF-α treatment has been shown to decrease the intracellular levels of GSH by formation of mixed disulfides and inhibition of the glutathione reductase system [53
]. Our ongoing studies will delineate how the thiol pool in myeloma cells is perturbed in the presence of various pro-inflammatory cytokines and if GSH depletion by buthionine sulfoximine would alter NF-κB activation and the overall therapy responses in multiple myeloma.
Previous studies have shown that under normal physiological conditions, i.e. when cells are able to maintain redox homoeostasis, ROS may function as intracellular signalling molecules [55
] and ROS-mediated NF-κB activation regulates cellular redox homoeostasis [56
]. NF-κB activation by oxidants is cell-context dependent and can be increased or decreased, depending on sequential events and concentrations of oxidants and antioxidants [57
]. Studies have shown that a reduced intracellular environment favours NF-κB activation [38
], and redox changes mediated by TNF-α treatment can inhibit NF-κB activity [39
]. In myeloma cells, simultaneous treatment with IL-6 and H2
showed increased NF-κB activation, whereas TNF-α in combination with H2
inhibited NF-κB activity. TNF-α plays a role in MM pathogenesis by up-regulating NF-κB-induced expression of adhesion molecules on myeloma and BM stem cells, thus increasing paracrine IL-6-mediated MM cell growth and survival. In the present study, TNF-α was not as effective as IL-6 in protecting myeloma cells from H2
-mediated oxidative myeloma cell death. Further studies are warranted to determine how TNF-α treatment regulates NF-κB activation in myeloma cells in the presence of oxidative stress-inducing therapies.
Mammalian cells possess a well-co-ordinated enzymatic antioxidant defence system comprised principally of SODs, catalase, peroxiredoxins and GPxs, and this system aids in rapid defence against oxidative stress. In myeloma cells, the present study provides evidence for NF-κB to play a role in driving IL-6-mediated up-regulation of MnSOD expression, and shows that IL-6 treatment leads to increases in the enzymatic activity of MnSOD. Furthermore, IL-6-induced NF-κB activation rendered radioresistance in myeloma cells. Indeed, MnSOD is a potent protector of cancer cells against both chemotherapy and radiotherapy [58
]. Also, TNF-α, IL-1β and IL-6 treatments can increase MnSOD expression [60
], and NF-κB-mediated SOD
2 transcription has been associated with cytokine and IR treatments [18
]. Previous studies have identified a clear role of NF-κB in IL6
gene expression [64
]. Further studies will determine whether a feed-forward loop of NF-κB on IL-6 signalling is responsible for a stronger effect on MnSOD activity. Indeed, improved myeloma cell cytotoxicity was seen when 2-methoxyestradiol, a proposed inhibitor of MnSOD, was combined with ROS-generating drugs, such as bortezomib [65
] and arsenic trioxide [66
]. In myeloma cells, IL-6-mediated MnSOD up-regulation was partially coupled with increases in the mRNA levels of CAT
. Thus induction of MnSOD by IL-6 treatment may potentially lead to a transient accumulation of H2
in myeloma cells that may be alleviated by the up-regulation of hydroperoxide-metabolizing systems.
During basal steady-state metabolism, ROS are generated primarily from mitochondrial respiratory chains, and mitochondria are believed to serve as a nodal control point that regulates apoptosis. Furthermore, antioxidant systems, such as glutathione/GPx and MnSOD, work dynamically to regulate any endogenous and/or therapy-induced elevation of mitochondrial ROS, and to inhibit apoptosis initiated by oxidative damage to mitochondria. The present study shows that treatment with IL-6 decreases IR-induced late mitochondrial pro-oxidant generation in myeloma cells, suggesting that, besides providing pro-proliferative signalling, IL-6 may inhibit IR- and chemotherapy-induced oxidative damage to the mitochondria of myeloma cells.
In conclusion, the resistance of several haemopoietic cancers to cytotoxic chemotherapy and radiotherapy is well established but not understood. The present study provides evidence to support a role for IL-6 in myeloma therapy resistance and proposes a mechanism involving NF-κB-dependent up-regulation of MnSOD. Our results provide a novel biochemical rationale for combining MnSOD inhibition with oxidative stress-inducing therapeutic agents, which paradoxically also stimulate IL-6 and thereby counteract oxidative stress, for the development of an effective new therapeutic strategy for treating MM.