Interleukin-6 counteracts therapy-induced cellular oxidative stress in multiple myeloma by up-regulating manganese superoxide dismutase

IL (interleukin)-6, an established growth factor for multiple myeloma cells, induces myeloma therapy resistance, but the resistance mechanisms remain unclear. The present study determines the role of IL-6 in re-establishing intracellular redox homoeostasis in the context of myeloma therapy. IL-6 treatment increased myeloma cell resistance to agents that induce oxidative stress, including IR (ionizing radiation) and Dex (dexamethasone). Relative to IR alone, myeloma cells treated with IL-6 plus IR demonstrated reduced annexin/propidium iodide staining, caspase 3 activation, PARP [poly(ADP-ribose) polymerase] cleavage and mitochondrial membrane depolarization with increased clonogenic survival. IL-6 combined with IR or Dex increased early intracellular pro-oxidant levels that were causally related to activation of NF-κB (nuclear factor κB) as determined by the ability of N-acetylcysteine to suppress both pro-oxidant levels and NF-κB activation. In myeloma cells, upon combination with hydrogen peroxide treatment, relative to TNF (tumour necrosis factor)-α, IL-6 induced an early perturbation in reduced glutathione level and increased NF-κB-dependent MnSOD (manganese superoxide dismutase) expression. Furthermore, knockdown of MnSOD suppressed the IL-6-induced myeloma cell resistance to radiation. MitoSOX Red staining showed that IL-6 treatment attenuated late mitochondrial oxidant production in irradiated myeloma cells. The present study provides evidence that increases in MnSOD expression mediate IL-6-induced resistance to Dex and radiation in myeloma cells. The results of the present study indicate that inhibition of antioxidant pathways could enhance myeloma cell responses to radiotherapy and/or chemotherapy.

For quantitative analysis of DHE oxidation products, HPLC electrochemical analysis was performed [3,4]. Briefly, 8226 cells (1×10 6 cells/condition) were preloaded with DHE (10 μM at 37 • C for 30 min), followed by treatment with IL-6 (50 ng/ml) or antimycin A (10 μM), or no treatment (control) for 30 min. Cells were centrifuged, washed with PBS, counted and stored at − 80 • C. On the day of HPLC analysis, samples were prepared as described by Zielonka et al. [3]. An ESA CoulArray system with a Phenomenex 250 mm×4.6 mm Synergi 4 μm Polar-RP 80A column was used to quantify oxidation products of DHE. Figure S1 Treatment with IL-6 or IR induces early ROS production in MM.1S myeloma cells MM.1S cells were treated for 0.5, 1 or 2 h with (A) IL-6 or (B) IR, and DHE oxidation was measured by flow cytometry. Graphs with MFI values are shown from one experiment, and are representative of two experiments. (C) Myeloma cells were pretreated without or with NAC, and then incubated with IL-6 (1, 2.5, 10 or 50 ng/ml) and H 2 DCF-DA oxidation was measured at 2 h. *P < 0.05 compared with control (no IL-6 treatment).
Quantification of 2-OH-E + was determined using the 200, 280 and 365 mV channels, and E + , using the 280, 365, 400, 450 and 500 mV channels and standard curves were prepared from authentic standards. HPLC data were adjusted for cell numbers and dilutions made during sample processing and analysis.

NF-κB assay
The transcriptional activity of NF-κB was measured using an ELISA-based assay (TransAM NF-κB p65 transcription factor assay kit) as described previously [5]. Cells were infected with or without Ad-IκBα-DN for 48 h and exposed to 6-Gy IR or sham IR. Nuclear extracts were prepared 2 h after IR and incubated with immobilized oligonucleotide containing a consensus-binding site for the p65 subunit of NF-κB or the control oligonucleotide. Binding was detected by a primary antibody specific for p65 followed by incubation with anti-IgG antibody conjugated to horseradish peroxidase. The product was quantified at 450 nm. Absorbance of the untreated cells was set to 1 and the fold activation/repression in the NF-κB activity was evaluated after normalizing the samples for cellular protein content. Representative HPLC chromatograms from 8226 cells incubated with DHE and then treated with antimycin A (Ant A) or untreated (control; Cont) for an additional 30 min. For simplicity, one channel (365 mV) of the eight channels monitored is presented to illustrate retention times and relative intensities of the various oxidation products of DHE found in cell extracts. HE is dihydroethidine, 2-OH-E + is the O 2

C. O. Brown and others
• − -specific 2-hydroxyethidium product, and E + is the non-specific two-electron oxidation product, ethidium. HPLC with electrochemical detection was performed using an ESA CoulArray system with a Phenomenex 250 mm×4.6 mm Synergi 4 μm Polar-RP 80A column. Quantification of 2-OH-E + was determined using the 200, 280 and 365 mV channels, and E + was determined using the 280, 365, 400, 450 and 500 mV channels, and standard curves were prepared from authentic standards. The Table (inset) provides estimates of net rates of accumulation of the oxidation products in cells during experiments on a per cell basis; zmol is zeptomoles (10 − 21 ).

Figure S3 Dex induces early ROS production in MM.1S myeloma cells and IL-6 protects myeloma cells against Dex-induced cytotoxicity
(A) MM.1S cells were treated for 0.5, 1 or 2 h with Dex and DHE oxidation was measured by flow cytometry. For each graph depicting MFI, the x axis and y axis show the percentage of cells and DHE oxidation respectively. MFI values are shown from one experiment. (B) The MTT cell viability assay was performed after treatment with IL-6 and/or Dex. The data are expressed as the percentage of live cells relative to the untreated control, which is set at 100. *P < 0.05 compared with control, and ζ P < 0.01 and P < 0.0001 compared with Dex.

Figure S4 IL-6 treatment increases MnSOD expression and NBD and Ad-IκBα-DN inhibit NF-κB activity in myeloma cells
MM.1S and 8226 cells were treated with (A) IL-6 (2.5 or 10 ng/ml) and whole-cell extract was probed for MnSOD expression at 24 h. The ratio between MnSOD and tubulin levels, after normalization to untreated control, is shown above each band. Myeloma cells were treated with IL-6 (50 ng/ml) and/or IR (B) after pretreatment without or with NBD (50 μM for 2 h). Luc activity was measured at 4 h. Values are depicted as the fold change in mean RLU + − S.D., n = 3, normalized to protein concentration. *P < 0.01 compared with IL-6, P < 0.01 compared with IR treatment alone or IL-6 plus IR. Myeloma cells were treated with IL-6 (50 ng/ml) and/or IR (C) after 48 h of transduction with Ad-IκBα-DN (MOI = 10). The DNA-binding activity of NF-κB was analysed at 2 h in nuclear extracts. The results are presented as the fold repression or activation of NF-κB activity compared with control. *P < 0.01 and P < 0.01 compared with IR treatment alone. (D) Myeloma cells were transduced with Ad-IκBα-DN (MOI = 10), whole-cell extract was made at 48 h and probed with an antibody against IκBα or tubulin.