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HGF, Sil-6R and TGF-Β1 Play a Significant Role in the Progression Of Journal of Cancer 2014, Vol. 5 518 Ivyspring International Publisher Journal of Cancer 2014; 5(7): 518-524. doi: 10.7150/jca.9266 Research Paper HGF, sIL-6R and TGF-β1 Play a Significant Role in the Progression of Multiple Myeloma Artur Jurczyszyn1, Jacek Czepiel2, Grażyna Biesiada2, Joanna Gdula-Argasińska3, Dorota Cibor2, Danuta Owczarek2, William Perucki4, Aleksander B. Skotnicki1 1. Department of Haematology, University Hospital, Kraków, Poland; 2. Department of Gastroenterology, Hepatology and Infectious Diseases, Jagiellonian University Medical College, Kraków, Poland; 3. Department of Radioligands, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland; 4. Students’ Scientific Society, Jagiellonian University Medical College, Kraków, Poland. Corresponding author: Artur Jurczyszyn, MD, PhD. Oddział Hematologii, Szpital Uniwersytecki, ul. Kopernika 17, 31-501 Kraków, Polska. Phone: 12 – 424-76-00, fax: 12-424-74-26 e-mail: [email protected]. © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2014.03.31; Accepted: 2014.05.22; Published: 2014.06.11 Abstract Background. In the last few years, it has been widely reported that proinflammatory and angi- ogenic cytokines are important for the development and progression of multiple myeloma (MM). Objectives. To further validate and acquire more insight into this view we decided to check whether plasma levels of certain cytokines and their soluble receptors differ between MM patients and healthy subjects. Patients and Methods. The study was conducted in 76 MM patients aged 22 to 77 years (60±10 years) and 35 healthy controls aged 20 to 63 years (33±10 years). Plasma levels of interleukin-6 (IL-6), b-fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1), as well as soluble receptors for IL-6 (sIL-6R) and VEGF (sVEGF-R2) were measured using enzyme-linked immunosorbent assay (ELISA). Results. Significantly higher plasma levels of IL-6 (13.65±42.61 vs. 1.04±1.12 pg/ml, p=0.006), HGF (2174±2714 vs. 648±130 pg/ml, p<0.001), b-FGF (7.92±10.78 vs. 2.54±5.38 pg/ml, p<0.001) and sIL-6R (37.1±14.2 vs. 25.3±6.4 ng/ml, p=0.003) were observed in MM patients vs. healthy controls, respectively. Plasma sVEGF-R2 was significantly lower in MM patients than in controls (7518±2119 vs. 8725±1281 pg/ml, respectively; p<0.001). We observed an inverse correlation between length of treatment and the level of sIL-6R, and TGF-β1 in plasma. Conclusions. Plasma levels of HGF, b-FGF, IL-6 and sIL-6R in MM patients were higher when compared to the control group. Antineoplastic therapy leads to a time-dependent decrease in plasma levels of sIL-6R, and TGF-β1 in MM patients. Blood plasma level of HGF is an optimal measure to differentiate patients in whom disease is progressing versus patients who respond to therapy. Key words: b-FGF, cytokines, HGF, interleukin-6, multiple myeloma. Introduction Recent studies have confirmed the importance of likely the inhibition of apoptosis of myeloma cells. It soluble cytokines and their receptors in the biology of has been shown that the level of soluble receptors for multiple myeloma (MM) [1-3]. The principal mecha- IL-6 (sIL-6R) is elevated in myeloma patients com- nism of action of interleukin-6 (IL-6) in MM is most pared to healthy subjects, and its level correlates with http://www.jcancer.org Journal of Cancer 2014, Vol. 5 519 disease severity [3,4]. Currently, the serum level of women (54.3%) and 16 men (45.7%) F/M ratio = 1.2. sIL-6R is considered to be one of the prognostic fac- The diagnosis of MM was based on cytologic exami- tors in MM [4]. Vascular endothelial growth factor nation of bone marrow aspirate revealing at least 10% (VEGF) is one of the strongest drivers of angiogenesis. plasmocytes, the presence of monoclonal proteins in In MM, the binding of VEGF with its receptors, serum or urine, and bone osteolytic lesions. Patients VEGF-R1 and VEGF-R2, induces angiogenesis and with MM were divided into 2 groups, designated N leads to exponential growth of the tumour. Further- for newly diagnosed, previously untreated, and S for more, with the activation of these receptors, there is patients undergoing antiproliferative treatment. increased expression of integrins on the cell surface, Group N consisted of 29 patients (38.2%), while group which facilitates adhesion and migration [6, 7, 8]. In S consisted of 47 people (61.8%). We subdivided MM, the serum level of basic fibroblast growth factor group S into three groups, based on length of treat- (b-FGF) increases with disease progression. It was ment: S1 included patients undergoing treatment for 1 also found that myeloma cells express surface recep- to 2 years (n=13), S2 comprised patients undergoing tor FGF-R3 (which is not found on normal lymphoid treatment for 2 to 3 years (n=11), and S3 consisted of cells), divide more rapidly and undergo apoptosis less patients undergoing treatment for longer than 3 years frequently than cells without FGF-R3. Effective anti- (n=23). Additionally, any patient in group S under- proliferative treatment often causes a reduction in going treatment for more than a year was evaluated b-FGF levels in the serum of patients with MM for response to antiproliferative therapy. Patients with [1,9,10]. Transforming growth factor-β1 (TGF-β1) is MM were treated according to the guidelines of the synthesized by almost all cells of the body. The in- Polish Myeloma Group. For patients under 65 years of creased microvascular network in the bone marrow of age, first line treatment consisted of the CTD protocol patients with MM is likely the result of the plasma (cyclophosphamide, thalidomide, and dexame- secretion of TGF-β1, which stimulates the release of thasone) followed by CTX and G-CSF combined with platelet derived growth factor and thereby enhances the auto-PBSCT procedure. Throughout treatment we angiogenesis [11]. Hepatocyte growth factor (HGF) continued thalidomide. In cases of disease recurrence, has mitogenic, chemotactic, and morphogenic prop- treatment consisted of bortezomib or lenalidomide in erties. It has a significant effect on the morphogenesis many cases. Patients over 65 years of age were treated of new blood vessels and bone marrow stroma, and it either with the MPT (melphalan, prednisone, and stimulates the synthesis of angiogenic factors. Ele- thalidomide) or VMP (bortezomib, melphalan and vated levels of HGF in the serum of patients with MM prednisone) protocols. In cases of disease progression, is associated with an unfavourable prognosis. Nu- lenalidomide and dexamethasone were given [17]. We merous studies have shown that HGF is involved in evaluated whether there was progression of disease, the progression of MM, including damage to bone, resistance to treatment, or if treatment had a positive angiogenesis, and myeloma cell adhesion to bone effect. These analyses were based on: assessment of marrow stroma [12,13]. Studies indicate the im- bone marrow (the percentage of plasma cells in bone portance of the HGF-c-MET axis in the stimulation of marrow biopsy), protein parameters (serum protein proliferation and inhibition of apoptosis in MM cells electrophoresis), biochemical parameters (serum [4,15,16]. C-reactive protein - CRP, β2 microglobulin, calcium), The aim of this study was to compare the plasma and complete blood count. During our testing we levels of cytokines IL-6, VEGF, b-FGF, TGF-β1, HGF, were unable to evaluate the clinical outcome of and soluble receptors sIL-6R and sVEGF-R2 between treatment in 6 patients, and therefore testing was patients with MM and healthy volunteers. conducted on the remaining group of 41 patients. Of these remaining patients 23 positively responded to Patients and methods treatment while 18 patients showed progressive MM Clinical characteristics of the study group which was resistant to therapy. The study was ap- proved by the local Ethics Committee. The study comprised 76 patients aged 22 to 77 years (mean age 60 years, SD ± 10) with MM treated at Blood and plasma analysis the Department of Haematology, University Hospital, Blood samples were collected between 7:30 and Krakow, Poland. The control group consisted of 35 8:30 hours in sterile Vacutainer tubes (Becton Dickin- healthy volunteers aged 20 to 63 years (mean age 33 son, New Jersey, USA) which contained an anticoag- years, SD ± 10). Both test and control groups were ulant (EDTA). The tubes were then centrifuged for 10 similar with respect to gender distribution. The group minutes at 1,000 RPM. Plasma was pipetted into ster- of patients with MM included 38 women and 38 men, ile tubes (Nunc, Roskilde, Denmark) and stored at F/M ratio = 1. The control group consisted of 19 -70°C until analysis was performed. Biochemical as- http://www.jcancer.org Journal of Cancer 2014, Vol. 5 520 says of lactate dehydrogenase (LDH), blood urea, to initiation of treatment. Conversely, we observed a creatinine, total calcium concentration in both groups drop in plasma levels of sIL-6R and TGF- β1 as dura- and CRP, β2 microglobulins, serum protein electro- tion of disease and treatment increased. Decreased phoresis in MM group were performed using stand- levels of VEGF, HGF, and b-FGF were noticed after ard methods. Results of blood counts were obtained initiation of therapy. Values of sVEGF-R2 were simi- via photo-optical and conductivity methods using a lar in all analyzed subgroups.
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