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

Final Technical Report

The best summary of our results is probably provided by the list of publications based on work supported by this grant, which is given below. In general, the objectives were realized, and we have demonstrated, for the first time, that radiolabeled Abs can kill single tumor cells very effectively, and that they can also be effective in treating models of human tumors growing as xenografts in SCID mice. Our original work, as proposed in the application, was with Abs to B-lymphoma cells, namely anti-CD20 and anti-HLA-DR. After our successful efforts with these Abs, we decided to extend the results to other tumor types. Accordingly, carcinomas of the breast, ovary and other tissues were treated with radiolabeled Abs to EGFr and HER-2. These tumors cells were also effectively killed in vitro with radiolabeled Abs. This is significant because these Abs are widely used, and successful, in the clinic (unlabeled) and because the flattened shape of the cells, in vitro, is expected to make them considerably more difficult to kill than the spherical lymphoma cells. A major goal was to compare radionuclides emitting different types of radiation, namely low energy electrons (Auger and conversion electrons), {beta}-particles, and {alpha}-particles. All three types could effectively kill cells in vitro with considerable specificity. However, the low energy electrons, which we abbreviate LEEs, have significant advantages, mainly due to their lower level of non-specific toxicity. This was demonstrated both in vitro and in vivo. Thus, {beta}-particle emitters conjugated to anti-CD20 could protect mice against the growth of B-lymphoma tumor cells, but the therapeutic effect was limited by the maximum dose that could be administered, without killing the mouse. In contrast, the LEE emitters were more effective, largely because the toxicity was much less, allowing an approximately 10-fold higher {mu}Ci dose to be injected. Conjugates with {alpha}-particle emitters were also less effective than the LEE emitters, probably because of the much shorter half-life of the available {alpha}-particle emitter (less than 1 hr). Of the LEE emitters tested, {sup 67}Ga was considerably more potent than {sup 111}In, per decay, but {sup 111}In has major advantages due to the fact that better chelators are available, and the purity of the commercial radionuclide is much higher. Both were better than {sup 125}I because of their more suitable half-lives. Therefore, 111In remains the optimal LEE emitter at the current time, although it is useful to continue to consider the use of other radionuclides. In fact, we have emphasized that there are many LEE-emitting radionuclides that would be much more potent than {sup 111}In; these are not available at all, or not available carrier-free, or suitable conjugation methods have not been developed. Our results indicate that the development of such radionuclides, at the DOE reactors or at other facilities, would be likely to have substantial medical applications. In therapy, we have thus far been able to treat micrometastatic tumors (injected i.v.) and only small s.c. tumors, barely visible by eye, thin disks with a diameter of 1-2 mm. Because some of the tumors used grow slowly, we are able to obtain effective therapy as late as one month after tumor injection. While this is a limitation, perhaps due to the short tissue path-length of the LEEs, it does not mean that they are not clinically useful: many patients have microscope disease, and such tumors are probably the most difficult, and important, to treat. If we can effectively eliminate such micrometastases, there is a prospect of curing patients in whom the tumor would otherwise recur. Also, it is still possible that we could use this approach to treat larger tumor, if multiple doses are administered. It should also be pointed out that my laboratory is virtually the only one doing experiments of this type. Previous theoretical calculations had suggested that it should be possible to kill single cells with Abs conjugated to LEE emitters, and that the level of Ab binding, with high-density antigens, should be sufficient to achieve this effect. But we were the first to put this idea into practice. Therefore, we feel that we have essentially opened up a new area of research, and that future investigators will be able to build on the solid foundation than we have laid

Induction of Apoptosis by Cross-Linking Antibodies Bound to Human B-Lymphoma Cells: Expression of Annexin V Binding Sites on the Antibody Cap

There are many reports that cross-linking antibodies (Abs) bound to the surface of B-lymphoma cells can induce apoptosis and/or cell death, especially with anti-CD20 Abs. This study was intended to extend our understanding of these effects. To determine if CD20 is a unique target in this respect, or whether Abs to other antigens would have similar effects, six Abs were tested, with and without cross-linking with a secondary Ab, on three target cell lines. We utilized assays that distinguish between apoptotic, dead, and viable cells. Two assays were used: Annexin V plus propidium iodide, and JC-1 plus SYTOX® green (Molecular Probes, Eugene, OR). Most of the Abs tested induced a low level of apoptosis and cell death in Ramos cells, but not in the other two cell lines (Raji and RL). In general, the level of toxicity was correlated with the level of antigen expression, with Abs to high-density antigens having the strongest effects. However, since the majority of Ramos cells continued to multiply, it is questionable whether toxicity at this level can provide a significant clinical benefit. Unexpectedly, there was also a population of cells that stained weakly with Annexin V. These cells were distinct from classical apoptotic cells, and appeared to belong to the viable cell population. In these cells, Annexin V stained the region of the Ab cap, in contrast to the ringed staining of classical apoptotic cells. In conclusion: 1) Low-level induction of apoptosis was not unique for anti-CD20 Abs, but occurred similarly with other Abs, and 2) results of Annexin V staining experiments may need to be reevaluated. Further studies are required to explain why Annexin V binding sites are exposed in the region of an Ab cap