Abstract
Background/Aim: Although numerous cytokines influence proliferation and progression of multiple myeloma (MM), a relevant action in the onset of the disease also seems to be played by the oxidative state. Patients and Methods: In the present study we evaluated the concentrations of interleukin-8 (IL-8) and soluble receptor of advanced glycation end products (sRAGE) in patients with MM, assessing the existing variations with respect to a control group and the possible existence of correlations between these molecules and the biological variables or the presence of a correlation between IL-8 and sRAGE. The study was conducted on 33 patients affected by MM compared to 39 healthy subjects. Results: IL-8 and sRAGE levels were significantly higher in MM patients compared to healthy subjects. sRAGE and IL-8 evidence no significant linear correlation. Furthermore, IL-8 was significantly increased in both sexes, but we found a slight variation for females compared to males. Conclusion: IL-8 could play an important role in the onset of MM and the progression of bone disease, while the increased sRAGE values would seem to have a protective action in MM patients. Further studies on animal models may clarify the real impact of introducing modulation of IL-8 and sRAGE levels.
- Multiple myeloma
- interleukin-8
- sRAGE
- oxidative stress
- cancer
- gender
Multiple myeloma (MM) is the second most common hematological malignancy after non-Hodgkin's lymphoma, with a greater occurrence in the elderly. Several cytokines regulate proliferation and evolution of the disease (1, 2), while a fundamental role in the onset and progression of the disease also appears to be played by the oxidative state (3). Among the cytokines, a particularly relevant action could be performed by interleukin 8 (IL-8), a component of the CXCL group of chemokines which bind to the receptors CXCR1 and CXCR2 (4). It is a pro-inflammatory molecule that is modified to give rise to a functionally active protein of 77 amino acids generated by parenchymal cells and 72 amino acids in the case of the one generated by macrophages and monocytes (5).
The presence of receptors for IL-8 in endothelial cells, tumor cells, and tumor-associated macrophages indicates that the production of IL-8 by tumor cells should have a potent action on the tumor microenvironment (6, 7). IL-8 exhibits pleiotropic actions on tumor cells and can influence several moments of cancer diffusion, comprising cell growth, survival, and angiogenesis (8, 9). Furthermore, IL-8 also has powerful pro-osteoclastogenic action and has been recognized as an osteolytic element (10). Overexpression of IL-8 has been detected in breast cancer (BC) cells and an increased serum IL-8 concentration is correlated with osteolytic lesions and bone metastasis in BC subjects (11, 12).
Several researchers reported the existence of elevated circulating amounts of IL-8 in subjects with advanced tumors with respect to control subjects (12, 13), while it was stated that the production of IL-8 is very abundant in cancer cells both in vitro and in vivo (14-16). As for oxidative stress, glycative stress caused by advanced glycation end products (AGEs) can considerably influence tumor progression. High levels of oxidative stress promote the accumulation of damaged biomolecules, the impairment of cell signalling pathways and the increase of oncogenic hits (17). AGEs are a various set of irreversible abducts resultant from non-ezymatic glycation and oxidation of proteins, lipids, and nucleic acids (18, 19).
AGEs include chemical components such as argpyrimidine, pentosidine, N-ε-car-boxymethyl-lysine, pyrraline, and N-ε-carboxyethyl-lysine. There are four diverse receptors for AGEs (RAGE): full-length RAGE, C-truncated RAGE which has two different isoforms, soluble Receptor of AGE (sRAGE) and endogenous secretory receptor (esRAGE), and N-truncated RAGE. Connection of AGEs and RAGE has unfavourable effects on cell activity and provokes progression of several diseases, including aging, diabetes, nephropathy, retinopathy, obesity, neuropathy, and cardiovascular diseases (20). In fact, binding of AGE and RAGE is able to modify numerous intracellular signalling, stimulation of nuclear-factor kappa-B (NF-kB), modification of gene expression and increase of reactive oxygen species (ROS) (21, 22).
Finally, glycative stress can substantially affect cancer evolution. Actually, oxidative stress can modify the activity of numerous transcription components, such as -catenin/Wnt, hypoxia-inducible factor-1, protein-53, activating protein-1, and nuclear factor erythroid-2-related factor.
In a previous research performed to study the oxidative status of MM subjects, we valued the amounts of advanced oxidation protein products (AOPPs) and protein nitrosylation in untreated MM subjects (23). We demonstrated that plasma concentrations of AOPPs and S-nitrosylated proteins were significantly increased in MM subjects with respect to subjects affected by monoclonal gammopathy of uncertain significance (MGUS) and to healthy subjects. In a diverse study, we also evaluated serum levels of carbonyl groups, and these were also increased in MM patients with respect to controls, especially in the more advanced stages of the disease (24).
Furthermore, a recent study has reported that the levels of AGEs and RAGE are increased in the saliva of MM patients with lytic lesions (25), while from our researches emerges the role of the sRAGE/AGE axis in MM. Circulating sRAGE levels may reflect RAGE concentration and may be increased in parallel with AGE levels as a counter-system against AGE-provoked tissue damage (26, 27).
Finally, some studies performed in vitro and on patients suffering from infectious diseases have revealed the presence of a correlation between oxidative stress and IL-8. In human models of pulmonar tuberculosis, sRAGE was statistically diminished, whereas gene expression of RAGE was increased. RAGE expression positively correlated with IL-8 cytokine concentrations and clinico-radiographical gravity. Moreover, AGEs augmented IL-8 production from Sw.71 cells (28).
On the basis of these data, in the present study we evaluated the concentrations of IL-8 and sRAGE in patients with MM, evaluating the existing variations with respect to a control group and the possible existence of correlations between these molecules and the biological variables of these patients and the possible presence of a correlation between IL-8 and sRAGE.
Patients and Methods
Patients and control subjects. The study was conducted at the Division of Hematology of the University Hospital of Messina “Policlinico G. Martino”, Italy. It was performed according to good clinical and laboratory practice rules and according to the principles of the Declaration of Helsinki. Informed written consent was obtained after potential risks were explained to the subjects. The study was approved by the local ethic committee (Prot. 0066794 of 2 May 2018. The company approval resolution is n. 887 of 7 June 2018).
Blood samples were obtained from 33 patients (PTS) affected by MM (14 males – 19 females; mean age 70.33±10.42 years) compared to 39 sex-matched and age matched healthy subjects (CTRL).
MM subjects were characterized using the Durie Salmon system (9 patients were in stage I, 9 stage II and 15 stage III), and the International Staging System (ISS) (10 patients were in stage I, 12 stage II and 11 stage III). All but one patient had lytic lesions. Routine laboratory studies consisted of complete blood count with differential, platelet count, and blood chemistry including beta-2 microglobulin, renal and liver function tests, calcemia, serum albumin, and lactate dehydrogenase (LDH), as well as immunoglobulin, erythrocyte sedimentation rate (ESR) and bone marrow aspiration. Physical examination and skeletal X-rays were always performed. Blood samples were immediately centrifuged at 14,000 rpm for 20 min and aliquoted into 1.5 ml centrifugation tubes. Samples were stored at −80°C until tested.
sRAGE analysis. Blood samples for the determination of sRAGE (R&D System human RAGE Quantikine ELISA Kit DRG00, USA) have been analyzed by ELISA (enzyme-linked immunosorbent assay) according to the manufacturer's instruction. ELISA assay was run in duplicate.
IL-8 analysis. The release of IL-8 was evaluated by the ELISA method (enzyme-linked immunosorbent assays; (DuoSet R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.
Statistical analysis. Data are expressed as mean±(SD) standard deviation or as median and interquartile range (IQR=0.75-0.25) when the variable were not-normally distributed. Before the analysis, sRAGE and IL-8 variables were log transformed for normal distribution. The statistical significance was evaluated using unpaired Student's t-test. ANCOVA analysis was conducted to determine a statistically significant difference between Group (PTS and CTRL) respectively on RAGE and IL-8 controlling for age and sex. Results were considered statistically significant with p<0.05.
Results
sRAGE levels were significantly different in patients compared to controls (Table I). IL-8 was significantly elevated in PTS compared to CTRL (Table I). Values are [F (1.67)=4.62, p=0.035 partial η2=0.065] for sRAGE and [F (1.47)=6.59, p=0.013 partial η2=0.123] for IL-8, respectively after adjusting for age and sex. Moreover, sRAGE and IL-8 evidence no significant linear correlation (Figure 1).
Discussion
Our results revealed increased IL-8 and sRAGE values in MM patients compared to the control group. However, this increase has a completely different meaning for the two variables examined. In fact, the increase in IL-8 would seem to have a negative impact on the onset, progression and complications of myelomatous disease, while, on the contrary, the increase in sRAGE could have a protective role against neoplasia.
Previous in vitro studies have reported that stromal cells, co-cultured with supernatants from bone marrow (BM) cells of MM, delivered increased concentrations of IL-8 (29), and BM cells from MM subjects produced greater levels of IL-8 than control subjects. Finally, IL-8 generation was demonstrated in cultures of CD138 plasma cells and CD138 cells separated from BM of MM subjects, and in three human myeloma cell lines (30).
The actions by which IL-8 could facilitate the onset of MM are manifold, but an action on immunosurveillance seems to be the main mechanism. For instance, IL-8 stimulates granulocytic myeloid suppressor cells to generate the development of neutrophil extracellular traps (NETs). These actions mediated by IL-8 could be crucial in the creation of a tumour microenvironment that aid the tumour to escape the immune system (31), as the generation of NETs has an important action in the inhibition of the immune response against the tumour cells. NETs which protect several powerful proteases could act by degradation of the extracellular matrix, and NETs would be able to facilitate MM progression by creating an obstacle between MM cells and the immune system (32).
All our patients except one had lytic lesions, and IL-8 seems to have a relevant action in the development of bone disease in patients with MM. In fact, IL-8 augments RANKL expression in osteoblastic cells, so modifying the RANKL/OPG ratio in the cells and increasing osteoclast formation (33). Moreover, IL-8 augments the differentiation of blood mononuclear cells into osteoclasts.
A large amount of research on the activity of IL-8 on osteoclast function has been performed employing BC cells. MDA-MB-231 cells modified to overexpress IL-8 present an increased capability to augment osteoclast maturation and proliferation. BC cells also utilize osteoblasts to produce more IL-8 within the bone microenvironment (34). Moreover, xenografts of bone-metastatic PC tumors express increased concentration of IL-8 (35). In a diverse study inhibition of IL-8 in PC cells overexpressing the N -myc downstream regulated gene 2 caused a reduction in bone osteolysis (36).
Another interesting finding emerges from our study. IL-8 was significantly increased in both sexes but we found a slight variation for females compared to males. This high expression of IL-8 in women could be useful data to explain the increase in IL-8 concentrations detected in the course of neoplastic diseases such as ovarian cancer. In these pathologies the IL-8 would seem able to influence progression through its induction of cancer cell growth, survival, angiogenesis, and metastasis (37-39).
Moreover, we confirmed the presence of high concentrations of sRAGE in patients affected by MM compared to healthy subjects, as demonstrated in our previous study (26).
It is well known that the AGE/RAGE axis has a pro-tumor activity. In fact, the action of AGE/RAGE ligand in the mitogen-activated protein kinase (MAPK)/NF-kB cascade signaling pathway is of particular significance (40). The activation of STAT3, augmented by RAGE-dependent pathways, stimulates NF-kB. NF-kB and STAT3 signalling pathways cause tumor-stimulating actions by the generation of interleukin-6 (a well-known growth factor for myelomatous plasma cells), matrix metalloproteinases, and prostaglandin E2 in the microenvironment of a tumor (41-44).
Levels of sRAGE have been suggested to be a protective factor against neoplasms. As for the action of sRAGE in solid neoplasms, sRAGE was shown to decrease motility and pro-metastatic stimulation of A-375 melanoma cells by blocking S100A4-RAGE signaling axis (45). Moreover, in a study of Finnish male smokers, serum levels of sRAGE were found inversely correlated with pancreatic cancer risk (46).
The body is provided with anti-AGE–RAGE defending mechanisms such as degradation of AGE with metabolizing enzymes and AGE receptor and circulating sAGE-receptor. sRAGE counteracts the effect of AGE and RAGE by binding with AGEs. sRAGE neutralizes the AGE and RAGE-mediated damage by operating as a decoy molecule. The augment in sRAGE amounts would, therefore, seem to have a protective action in MM subjects.
Conclusion
The direct interplay between MM cells and BM cell components has a crucial role in disease pathogenesis and skeletal destruction. In fact, BM cells interact with MM cells and secrete high levels of cytokines, while as a result of genomic instability, tumour cells typically have elevated oxidative stress.
Our data seem to confirm those that appear to be common features to neoplastic diseases. In fact, in patients with MM, we found an increase in IL-8 and sRAGE concentrations. However, the data in the literature do not appear to be unique. In fact, although other researchers have found an increase in IL-8 concentration in MM patients, different studies have shown that IL-8 was not significantly associated with PFS and OS in MM patients (47). It is likely that the action of cytokines is be considered in an integrated manner within metabolic or proliferative pathways. In this respect we tried to evaluate whether the production of interleukin 8 could have a relationship with oxidative stress in patients with MM.
Previous studies performed on patients with pulmonary tuberculosis have shown the existence of a correlation between IL-8, RAGE and sRAGE and clinico-radiographical severity of the disease. Moreover, ex vivo, recombinant-RAGE potentiated cytokine release when combined with some substances like lipoarabinomannan (28). In our study we were unable to identify a similar correlation. However, the quantitative alteration of the two substances could affect the natural history of the disease with different mechanisms. Il-8 could worsen the immunological dysregulation present in MM patients and reduce the already compromised immunosurveillance, while the increase in sRAGE could reduce the negative action exerted by AGE. Actually, the actions by which IL-8 could facilitate the onset of MM are manifold, but an effect on immunosurveillance seems to be the main mechanism.
Although our study had a small number of participants, the data provided herein could have a translational interest. Today, the availability of novel agents, such as the proteasome inhibitors, the immunomodulatory drugs, monoclonal antibodies, inhibitors of heat shock protein, vaccine therapy or the use of adoptive immunotherapy has significantly improved the clinical outcome of these patients (48-53). However, MM remains currently an incurable disease and the identification of new therapeutic targets appears indispensable. Both IL-8 and sRAGE could represent two possible moments of therapeutic intervention. The possibility of inducing a reduction in IL-8 concentrations or increasing sRAGE levels could exert a positive effect in patients with MM.
The generation of humanized monoclonal antibodies against CXC chemokines (such as IL-8), as well as drugs that block CXCR1/2 receptors, permitted to evaluate the result of repressing the signalling of these receptors by IL-8 in the cancer growth. Thus, it has been reported that the dispensation of ABX-IL-8 to animals carrying xenografts of bladder, melanoma or prostate tumour reduces the number of metastases (54-57).
Similarly, Ace inhibitors, perindopril, rosiglitazone, metformin and statins augmented the serum concentrations of sRAGE (57-60). Exogenous administration of sRAGE could also represent a potential therapeutic approach. This method could allow to augment the serum concentrations of sRAGE that will bind to AGE, provoking a decreased interaction of AGE with RAGE and consequently a modification in the pathophysiology of MM. In different areas of research, experimental models demonstrated that exogenous administration of sRAGE reduced the progress of restenosis and atherosclerosis (61).
Experimental studies on MM animal models may clarify the real impact of such a therapeutic intervention and the possibility of introducing modulation of IL-8 and sRAGE levels in clinical practice.
Footnotes
* These Authors contributed equally to this study.
Authors' Contributions
Conceptualization: Alessandro Allegra, Elisabetta Pace, Caterina Musolino and Sebastiano Gangemi; Data curation: Vanessa Innao, Eleonora Di Salvo, Andrea Gaetano Allegra and Maria Ferraro; Formal analysis: Gennaro Tartarisco; Investigation, Vanessa Innao, Eleonora Di Salvo, Andrea Gaetano Allegra and Maria Ferraro; Software: Gennaro Tartarisco; Writing – original draft: Alessandro Allegra; Writing – review & editing: Alessandro Allegra, Elisabetta Pace, Caterina Musolino and Sebastiano Gangemi.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received January 25, 2020.
- Revision received February 11, 2020.
- Accepted February 12, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved