Cancer Chemother Pharmacol (2014) 73:223–229 DOI 10.1007/s00280-013-2344-1
ORIGINAL ARTICLE
Changes of cytokines in patients with liver cirrhosis and advanced hepatocellular carcinoma treated by sorafenib Hidenari Nagai · Takenori Kanekawa · Kojiro Kobayashi · Takanori Mukozu · Daigo Matsui · Teppei Matsui · Masahiro Kanayama · Noritaka Wakui · Kouichi Momiyama · Mie Shinohara · Koji Ishii · Yoshinori Igarashi · Yasukiyo Sumino
Received: 27 March 2013 / Accepted: 29 October 2013 / Published online: 13 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose Recently, the oral multikinase inhibitor sorafenib has been used to treat advanced hepatocellular carcinoma (aHCC). Tumor necrosis factor (TNF) induces apoptosis of tumor cells by binding to TNF-related apoptosis-inducing ligand, while binding of the Fas ligand on cytotoxic T lymphocytes to the Fas receptor on hepatocytes also causes apoptosis. The aim of this study was to retrospectively evaluate changes of cytokines in patients with liver cirrhosis (LC) and aHCC receiving sorafenib therapy. Methods Fifty-seven adult Japanese LC patients received sorafenib for aHCC (200–800 mg/day for 4 weeks) between 2009 and 2012 at our hospital. Blood samples were collected in the early morning before and after treatment, and the serum levels of soluble TNF-alpha (sTNFalpha), soluble TNF receptor (sTNF-R), soluble Fas ligand (sFas L), and soluble Fas (sFas) were evaluated. Results Ten patients were treated with sorafenib at 200 mg/day (200 mg group), 37 patients were given 400 mg/day (400 mg group), and 10 patients received 800 mg/day (800 mg group). The serum level of sTNFalpha was significantly increased after treatment compared with before treatment in the 400 and 800 mg groups. The serum level of sTNF-R also showed a significant increase after treatment in the 400 mg group, although there was no significant difference of sTNF-R between before and after treatment in the 200 and 800 mg groups. sFas showed a H. Nagai (*) · T. Kanekawa · K. Kobayashi · T. Mukozu · D. Matsui · T. Matsui · M. Kanayama · N. Wakui · K. Momiyama · M. Shinohara · K. Ishii · Y. Igarashi · Y. Sumino Division of Gastroenterology and Hepatology, Department of Internal Medicine (Omori), Faculty of Medicine, School of Medicine, Toho University, 6‑11‑1, Omorinishi, Ota‑ku, Tokyo 143‑8541, Japan e-mail: [email protected]
significant decrease after treatment compared with before treatment in the 400 and 800 mg groups, although the serum level of sFas L never exceeded 0.15 ng/ml. Conclusions These findings suggest that treatment with sorafenib at doses ≥400 mg/day might promote TNFrelated or Fas-related apoptosis by increasing the circulating level of TNF-alpha or decreasing that of sFas. Keywords Fas · TNF · Apoptosis · Advanced HCC · Sorafenib
Introduction Sorafenib has revolutionized the treatment of advanced hepatocellular carcinoma (aHCC) in patients with liver cirrhosis (LC). In the Sorafenib HCC Assessment Randomised Protocol (SHARP) study, 602 patients (mainly Europeans) were randomized to receive either sorafenib or placebo therapy. They had an Eastern Cooperative Oncology Group performance status of 0–2 and were in Child-Pugh class A. The median overall survival time was 10.7 months in the sorafenib group versus 7.9 months in the placebo group [1]. Sorafenib has also demonstrated significant clinical activity against HCC in phase II and phase III studies [2, 3], achieving both a longer median survival time and longer time to radiologic progression compared with placebo. Sorafenib is the first systemic agent to be approved for the treatment of HCC and is a multikinase inhibitor with activity against VEGFR2, PDGFR, c-Kit receptor, b-RAF, and p38 [4], which are signal transduction pathways that may be involved in the pathogenesis of HCC [5]. This agent simultaneously inhibits several components of the Raf-MEKERK signaling pathway, thus preventing tumor growth and the production of VEGFR-1, VEGFR-2, VEGFR-3, and
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PDGFR-b, so that it inhibits neoangiogenesis [6]. Sorafenib has also been shown to induce apoptosis of multiple cancer cell lines by downregulating and inhibiting the translation of Mcl-1, a member of the Bcl-2 family [7]. Fas is an important member of a family of receptors that transduce apoptotic signals which lead to programmed cell death. It belongs to the tumor necrosis factor (TNF) receptor superfamily, which includes TNF receptor I (TNFr-I) and TNF receptor II (TNFr-II), the first members to be discovered and characterized [8, 9]. These receptors are expressed on the surface of various cells, while their soluble forms are detected in the serum after cleavage of the extra-cytoplasmic domains or alternative splicing [10]. It has been suggested that detection of soluble TNF receptors may reflect activation of the TNF system [11]. In addition, the clinical outcome could be directly influenced if these receptors interfere with apoptotic cell death by competitive binding with the corresponding ligand [12], and a similar inhibitory effect has already been described for soluble Fas [10]. Several previous studies have shown the cytotoxic synergy between sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in various types of cancer cells and have indicated that the inhibition of Mcl-1 is a target of sorafenib on TRAIL sensitivity [13–15]. However, in patients with LC and aHCC receiving sorafenib therapy, it has not been clear how sorafenib affects cytokines in such patients. It is hypothesized that change of the serum levels of cytokines after treatment in patients with LC and aHCC might relate to the objective response to sorafenib. Accordingly, the aim of this study was to retrospectively evaluate changes of cytokines in patients with LC and aHCC receiving sorafenib therapy.
Materials and methods Patients Between 2009 and 2012, 57 adult Japanese liver cirrhosis (LC) patients were treated for aHCC with sorafenib at our hospital. An oral dose of sorafenib at 200 or 400 mg was administered twice daily, after breakfast and dinner (400 or 800 mg/day), although an oral dose of sorafenib at 200 mg was administered once daily after breakfast (200 mg/day) on the basis of a physician’s decision in case of patients who had low body weight. Blood samples were collected in the early morning before and after the 4-week treatment period. Cytokines assays Soluble TNF-alpha (sTNF-alpha) was measured in duplicate using a commercially available enzyme immunoassay
13
Cancer Chemother Pharmacol (2014) 73:223–229
(Quantikine, R&D Systems Inc, Mineapolis, USA). The sensitivity of this assay was 2.6 pg/mL, the intra-assay coefficient of variation was ±8.8 %, and the inter-assay coefficient of variation was ±16.7 %. Serum sTNF-alpha receptor I (sTNFr-I) levels were quantified with a commercially available ELISA (Quantikine, R&D Systems Inc, Mineapolis, USA). Serum soluble Fas (sFas) was measured by a commercially available quantitative sandwich enzyme immunoassay (Quantikine, R&D Systems Inc, Mineapolis, USA), while sFas ligand (sFas L) was quantified by using an immunoassay kit for sFas L (MBL, Tokyo, Japan). Concomitant measurement of seven sFas L standards with known concentrations (0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 ng/mL) was performed together with the patient samples. The detection limit for sFas L was 400 mg/day might have induced TNFrelated or Fas-related apoptosis by increasing sTNF-alpha or decreasing sFas in the PR + SD group, while sorafenib might not have promoted TNF-related apoptosis in the PD group. The Th1 subset is responsible for activation of cell-mediated immunity and cytotoxic CD8+ T lymphocytes (CTLs), while the Th2 subset primarily functions as helper cells for B cell activation [18]. The direction in which naive CD4+ cells differentiate depends on their first encounter with various
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228
agents. The factors that determine the process of differentiation are not fully understood, although the cytokine environment during differentiation of antigen-primed CD4+ T helper cells is thought to determine the subset which emerges [19]. Kohga et al. [20] reported that sorafenib therapy enhances the sensitivity of HCC cells to NK activity via inhibition of ADAM9 protease and alternation of MICA expression. We have also reported that sorafenib induces Th1-dominant host immunity in LC patients with aHCC [21]. It might be possible that antigen-primed CD4+ T helper cells recognize MICA expression on HCC cells and that generation of subsets among naive CD4+ cells induces Th1 dominance in LC patients with aHCC receiving sorafenib therapy. In the present study, the serum level of sTNF-alpha was significantly increased after sorafenib treatment, so Th1 dominance induced by sorafenib might have increased the sTNF-alpha level. sTNF-R was also significantly increased after treatment in the 400 mg group. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L), a type II transmembrane protein from the tumor necrosis factor superfamily, has been shown to strongly induce apoptosis in a manner suitable for cancer therapy [22, 23]. Several studies have demonstrated the synergy of cytotoxicity between sorafenib and TRAIL against various types of cancer cells [13, 14]. It is possible that Th1 dominance led to increased activity and NK cells released TNF-alpha, while increased TNF-R expression resulted in release of sTNF-R into the circulation of patients receiving sorafenib therapy. Furthermore, an increase in serum TNF-alpha could activate TRAIL and induce apoptosis of HCC. Fernando et al. [24] have reported that HCC cells regain the capacity to respond to TNF when treated with sorafenib through an extrinsic mechanism involving caspase-8 activation and cleavage of BID. They also reported that sorafenib sensitizes resistant HCC cells to Fig. 6 Possible mechanism for induction of Fas-related apoptosis in liver cirrhosis patients with HCC receiving sorafenib treatment. Sorafenib might release Fas from the Fas-c-Met complex by inhibiting Raf and thus restore the binding of Fas to Fas ligand, while inhibiting the binding of HGF to c-Met in HCC
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Cancer Chemother Pharmacol (2014) 73:223–229
TRAIL-induced apoptosis [25] and that such sensitization to TNF-induced death might be related to STAT-3 inhibition and a decline in the induction of MCLI by TNF [26]. These reports support our findings of an increase in sTNFalpha after sorafenib therapy in the PR + SD group. It was reported that sFas is generated by alternative mRNA splicing and antagonizes FasL-mediated cell killing in a concentration-dependent manner [27–30]. sFas levels are increased in patients with solid tumors, and its elevation reflects the disease stage and tumor burden. In HCC patients, elevation of the serum sFas level has been reported [31], and it has been hypothesized that escape of tumor cells from Fasmediated apoptosis could occur by 3 different mechanisms. First, loss of cell surface Fas expression would make tumor cells resistant to Fas-mediated apoptosis. Second, neutralization of FasL by sFas could prevent the ligand from triggering apoptosis. The third possible mechanism of resistance to Fas-mediated apoptosis could involve blocking of Fas signal transduction. Nagao et al. [32] reported that HCC cells escape from host immune surveillance in two ways, which are the loss of cell surface Fas expression and an increase in serum sFas. In the present study, the serum level of sFas showed a significant decrease after sorafenib treatment, although the sFas L level never exceeded 0.15 ng/ ml. These results indicate that treatment with sorafenib at daily doses >400 mg might promote Fas-mediated apoptosis by decreasing sFas in LC patients with aHCC. Wang et al. [33] reported that the Fas-c-Met complex played an important role in mediating the survival of liver cells. C-Met is the cellular receptor for hepatocyte growth factor (HGF), and it is known to have a cytoprotective effect mediated by the induction of signaling cascades that lead to the activation of PI3K/AKT. Under physiological conditions, c-Met acts as a scavenger and binds the Fas receptor to prevent its activation
Cancer Chemother Pharmacol (2014) 73:223–229
by Fas ligand. sFas is generated by alternative splicing of the intact exon 6 encoding the transmembrane domain of Fas, so detection of a smaller DNA fragment than the anticipated full-length Fas cDNA on RT-PCR implies the generation of sFas. The present study showed that the serum level of sFas decreased after sorafenib treatment in LC patients with aHCC. This could suggest that sorafenib released sFas from the Fas-c-Met complex by inhibiting Raf and thus restored Fas–Fas ligand binding, with a resultant decrease in the serum level of sFas (Fig. 6). In conclusion, treatment with sorafenib at doses >400 mg/day might promote induced by TNF or Fas by increasing TNF-alpha levels or decreasing sFas levels in patients with aHCC. In particular, an increase in the sTNFalpha might be an important indicator of the efficacy of sorafenib therapy for aHCC. Conflict of interest The authors have no conflicts of interest to declare.
References 1. Llover JM, Ricci S, Mazzaferro V et al (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:378–390 2. Moreno-Aspitia A, Morton RF, Hillman DW et al (2009) Phase II trial of sorafenib in patients with metastatic breast cancer previously exposed to anthracyclines or taxanes: North Central Cancer Treatment Group and Mayo Clinic Trial N0336. J Clin Oncol 27:11–15 3. Abou-Alfa GK, Schwartz L, Ricci S et al (2006) Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol 24:4293–4300 4. Wilhelm SM, Carter C, Lynch M et al (2006) Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 5:835–844 5. Avila MA, Berasain C, Sangro B, Prieto J (2006) New therapies for hepatocellular carcinoma. Oncogene 25:3866–3884 6. Wilhelm SM, Crter C, Tang L et al (2004) BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/ MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 64:7099–7109 7. Liu L, Cao Y, Chen C et al (2006) Sorafenib blocks the RAF/ MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 66:11851–11858 8. Nagata S, Golstein P (1995) The Fas death factor. Science 267:1449–1455 9. Armitage RJ (1994) Tumor necrosis factor receptor superfamily members and their ligands. Curr Opin Immunol 6:407–413 10. Ruberti G, Cascino I, Papoff G, Eramo A (1996) Fas splicing variants and their effect on apoptosis. Adv Exp Med Biol 406:125–134 11. Van Zee KJ, Kohno T, Fischer E, Rock CS et al (1992) Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor alpha in vitro and in vivo. Proc Natl Acad Sci USA 89:4845–4849 12. Seckinger P, Isaaz S, Dayer JM (1989) Purification and biologic characterization of specific tumor necrosis factor alpha inhibitor. J Biol Chem 264:11966–11973
229 13. Meng XW, Lee SH, Dai H et al (2007) Mcl-1 as a buffer for proapoptotic Bcl-2 family members during TRAIL-induced apoptosis: a mechanistic basis for sorafenib (Bay 43-9006)-induced TRAIL sensitization. J Biol Chem 282:29831–29846 14. Ricci MS, Kim SH, Ogi K et al (2007) Reduction of TRAILinduced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer Cell 12:66–80 15. Roasato PR, Almenara JA, Coe S et al (2007) The multikinase inhibitor sorafenib potentiates TRAIL lethality in human leukemia cells in association with Mcl-1 and cFLIPL down-regulation. Cancer Res 67:9490–9500 16. Llovet JM, Di Bisceglie AM, Bruix J et al (2008) Design and endpoints of clinical trials in hepatocellular carcinoma. J Natl Cancer Inst 100:698–711 17. Lencioni R, Llovet JM (2010) Modified RECIST (mRECIST) assessment for hepatocellular carcinoma. Semin Liver Dis 30:52–60 18. Arai KI, Lee F, Miyajima A et al (1990) Cytokines: coordinators of immune and inflammatory responses. Annu Rev Biochem 59:783–836 19. Seder RA, Paul WE (1994) Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol 12:635–673 20. Kohga K, Takehara T, Tatsumi T et al (2010) Sorafenib inhibits the shedding of major histocompatibility complex class I-related chain A on hepatocellular carcinoma cells by down-regulating a disintegrin and metalloproteinase 9. Hepatology 51:1264–1273 21. Nagai H, Mukozu T, Matsui D et al (2012) Sorafenib prevents escape from host immunity in liver cirrhosis patients with advanced hepatocellular carcinoma. Clin Dev Immunol 2012:607851 22. Takeda K, Stagg J, Yagita H et al (2007) Targeting death-inducing receptors in cancer therapy. Oncogene 26:3745–3757 23. Wang S (2008) The promise of cancer therapeutics targeting the TNF-related apoptosis-inducing ligand and TRAIL receptor pathway. Oncogene 27:6207–6215 24. Fernando J, Sancho P, Fernández-Rodriguez CM et al (2012) Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli. J Cell Physiol 227:1319–1325 25. Ricci MS, Kim SH, Ogi K et al (2007) Reduction of TRAILinduced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer Cell 12:66–80 26. Chen KF, Tai WT, Liu TH et al (2010) Sorafenib overcomes TRAIL resistance of hepatocellular carcinoma cells through the inhibition of STAT3. Clin Cancer Res 1(16):5189–5199 27. Chang J, Zhou T, Liu C et al (1994) Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 263:1759–1762 28. Owen-Schaub LB, Angelo LS, Radinsky R et al (1995) Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis? Cancer Lett 94:1–8 29. Cascino I, Fiucci G, Papohh G, Ruberti G (1995) Three functional soluble alternative splicing. J Immunol 154:2706–2713 30. Natoli G, Ianni A, Costanzo A et al (1995) Resistance to Fasmediated apoptosis in human hepatoma cells. Oncogene 10:1157–1164 31. Jodo S, Kobayashi S, Nakajima Y et al (1998) Elevated serum levels of soluble Fas/APO-1 (CD95) in patients with hepatocellular carcinoma. Clin Exp Immunol 112:166–171 32. Nagao M, Nakajima Y, Hisanaga M et al (1999) The alternation of Fas receptor and ligand system in hepatocellular carcinomas: how do hepatoma cells escape from the host immune surveillance in vivo? Hepatology 30:413–421 33. Wang X, deFrances M, Dai Y et al (2002) A mechanism of cell survival: Sequestration of Fas by the HGF receptor Met. Mol Cell 9:411–421
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ORIGINAL ARTICLE
Changes of cytokines in patients with liver cirrhosis and advanced hepatocellular carcinoma treated by sorafenib Hidenari Nagai · Takenori Kanekawa · Kojiro Kobayashi · Takanori Mukozu · Daigo Matsui · Teppei Matsui · Masahiro Kanayama · Noritaka Wakui · Kouichi Momiyama · Mie Shinohara · Koji Ishii · Yoshinori Igarashi · Yasukiyo Sumino
Received: 27 March 2013 / Accepted: 29 October 2013 / Published online: 13 November 2013 © Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose Recently, the oral multikinase inhibitor sorafenib has been used to treat advanced hepatocellular carcinoma (aHCC). Tumor necrosis factor (TNF) induces apoptosis of tumor cells by binding to TNF-related apoptosis-inducing ligand, while binding of the Fas ligand on cytotoxic T lymphocytes to the Fas receptor on hepatocytes also causes apoptosis. The aim of this study was to retrospectively evaluate changes of cytokines in patients with liver cirrhosis (LC) and aHCC receiving sorafenib therapy. Methods Fifty-seven adult Japanese LC patients received sorafenib for aHCC (200–800 mg/day for 4 weeks) between 2009 and 2012 at our hospital. Blood samples were collected in the early morning before and after treatment, and the serum levels of soluble TNF-alpha (sTNFalpha), soluble TNF receptor (sTNF-R), soluble Fas ligand (sFas L), and soluble Fas (sFas) were evaluated. Results Ten patients were treated with sorafenib at 200 mg/day (200 mg group), 37 patients were given 400 mg/day (400 mg group), and 10 patients received 800 mg/day (800 mg group). The serum level of sTNFalpha was significantly increased after treatment compared with before treatment in the 400 and 800 mg groups. The serum level of sTNF-R also showed a significant increase after treatment in the 400 mg group, although there was no significant difference of sTNF-R between before and after treatment in the 200 and 800 mg groups. sFas showed a H. Nagai (*) · T. Kanekawa · K. Kobayashi · T. Mukozu · D. Matsui · T. Matsui · M. Kanayama · N. Wakui · K. Momiyama · M. Shinohara · K. Ishii · Y. Igarashi · Y. Sumino Division of Gastroenterology and Hepatology, Department of Internal Medicine (Omori), Faculty of Medicine, School of Medicine, Toho University, 6‑11‑1, Omorinishi, Ota‑ku, Tokyo 143‑8541, Japan e-mail: [email protected]
significant decrease after treatment compared with before treatment in the 400 and 800 mg groups, although the serum level of sFas L never exceeded 0.15 ng/ml. Conclusions These findings suggest that treatment with sorafenib at doses ≥400 mg/day might promote TNFrelated or Fas-related apoptosis by increasing the circulating level of TNF-alpha or decreasing that of sFas. Keywords Fas · TNF · Apoptosis · Advanced HCC · Sorafenib
Introduction Sorafenib has revolutionized the treatment of advanced hepatocellular carcinoma (aHCC) in patients with liver cirrhosis (LC). In the Sorafenib HCC Assessment Randomised Protocol (SHARP) study, 602 patients (mainly Europeans) were randomized to receive either sorafenib or placebo therapy. They had an Eastern Cooperative Oncology Group performance status of 0–2 and were in Child-Pugh class A. The median overall survival time was 10.7 months in the sorafenib group versus 7.9 months in the placebo group [1]. Sorafenib has also demonstrated significant clinical activity against HCC in phase II and phase III studies [2, 3], achieving both a longer median survival time and longer time to radiologic progression compared with placebo. Sorafenib is the first systemic agent to be approved for the treatment of HCC and is a multikinase inhibitor with activity against VEGFR2, PDGFR, c-Kit receptor, b-RAF, and p38 [4], which are signal transduction pathways that may be involved in the pathogenesis of HCC [5]. This agent simultaneously inhibits several components of the Raf-MEKERK signaling pathway, thus preventing tumor growth and the production of VEGFR-1, VEGFR-2, VEGFR-3, and
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224
PDGFR-b, so that it inhibits neoangiogenesis [6]. Sorafenib has also been shown to induce apoptosis of multiple cancer cell lines by downregulating and inhibiting the translation of Mcl-1, a member of the Bcl-2 family [7]. Fas is an important member of a family of receptors that transduce apoptotic signals which lead to programmed cell death. It belongs to the tumor necrosis factor (TNF) receptor superfamily, which includes TNF receptor I (TNFr-I) and TNF receptor II (TNFr-II), the first members to be discovered and characterized [8, 9]. These receptors are expressed on the surface of various cells, while their soluble forms are detected in the serum after cleavage of the extra-cytoplasmic domains or alternative splicing [10]. It has been suggested that detection of soluble TNF receptors may reflect activation of the TNF system [11]. In addition, the clinical outcome could be directly influenced if these receptors interfere with apoptotic cell death by competitive binding with the corresponding ligand [12], and a similar inhibitory effect has already been described for soluble Fas [10]. Several previous studies have shown the cytotoxic synergy between sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in various types of cancer cells and have indicated that the inhibition of Mcl-1 is a target of sorafenib on TRAIL sensitivity [13–15]. However, in patients with LC and aHCC receiving sorafenib therapy, it has not been clear how sorafenib affects cytokines in such patients. It is hypothesized that change of the serum levels of cytokines after treatment in patients with LC and aHCC might relate to the objective response to sorafenib. Accordingly, the aim of this study was to retrospectively evaluate changes of cytokines in patients with LC and aHCC receiving sorafenib therapy.
Materials and methods Patients Between 2009 and 2012, 57 adult Japanese liver cirrhosis (LC) patients were treated for aHCC with sorafenib at our hospital. An oral dose of sorafenib at 200 or 400 mg was administered twice daily, after breakfast and dinner (400 or 800 mg/day), although an oral dose of sorafenib at 200 mg was administered once daily after breakfast (200 mg/day) on the basis of a physician’s decision in case of patients who had low body weight. Blood samples were collected in the early morning before and after the 4-week treatment period. Cytokines assays Soluble TNF-alpha (sTNF-alpha) was measured in duplicate using a commercially available enzyme immunoassay
13
Cancer Chemother Pharmacol (2014) 73:223–229
(Quantikine, R&D Systems Inc, Mineapolis, USA). The sensitivity of this assay was 2.6 pg/mL, the intra-assay coefficient of variation was ±8.8 %, and the inter-assay coefficient of variation was ±16.7 %. Serum sTNF-alpha receptor I (sTNFr-I) levels were quantified with a commercially available ELISA (Quantikine, R&D Systems Inc, Mineapolis, USA). Serum soluble Fas (sFas) was measured by a commercially available quantitative sandwich enzyme immunoassay (Quantikine, R&D Systems Inc, Mineapolis, USA), while sFas ligand (sFas L) was quantified by using an immunoassay kit for sFas L (MBL, Tokyo, Japan). Concomitant measurement of seven sFas L standards with known concentrations (0.16, 0.31, 0.63, 1.25, 2.5, 5, and 10 ng/mL) was performed together with the patient samples. The detection limit for sFas L was 400 mg/day might have induced TNFrelated or Fas-related apoptosis by increasing sTNF-alpha or decreasing sFas in the PR + SD group, while sorafenib might not have promoted TNF-related apoptosis in the PD group. The Th1 subset is responsible for activation of cell-mediated immunity and cytotoxic CD8+ T lymphocytes (CTLs), while the Th2 subset primarily functions as helper cells for B cell activation [18]. The direction in which naive CD4+ cells differentiate depends on their first encounter with various
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228
agents. The factors that determine the process of differentiation are not fully understood, although the cytokine environment during differentiation of antigen-primed CD4+ T helper cells is thought to determine the subset which emerges [19]. Kohga et al. [20] reported that sorafenib therapy enhances the sensitivity of HCC cells to NK activity via inhibition of ADAM9 protease and alternation of MICA expression. We have also reported that sorafenib induces Th1-dominant host immunity in LC patients with aHCC [21]. It might be possible that antigen-primed CD4+ T helper cells recognize MICA expression on HCC cells and that generation of subsets among naive CD4+ cells induces Th1 dominance in LC patients with aHCC receiving sorafenib therapy. In the present study, the serum level of sTNF-alpha was significantly increased after sorafenib treatment, so Th1 dominance induced by sorafenib might have increased the sTNF-alpha level. sTNF-R was also significantly increased after treatment in the 400 mg group. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L), a type II transmembrane protein from the tumor necrosis factor superfamily, has been shown to strongly induce apoptosis in a manner suitable for cancer therapy [22, 23]. Several studies have demonstrated the synergy of cytotoxicity between sorafenib and TRAIL against various types of cancer cells [13, 14]. It is possible that Th1 dominance led to increased activity and NK cells released TNF-alpha, while increased TNF-R expression resulted in release of sTNF-R into the circulation of patients receiving sorafenib therapy. Furthermore, an increase in serum TNF-alpha could activate TRAIL and induce apoptosis of HCC. Fernando et al. [24] have reported that HCC cells regain the capacity to respond to TNF when treated with sorafenib through an extrinsic mechanism involving caspase-8 activation and cleavage of BID. They also reported that sorafenib sensitizes resistant HCC cells to Fig. 6 Possible mechanism for induction of Fas-related apoptosis in liver cirrhosis patients with HCC receiving sorafenib treatment. Sorafenib might release Fas from the Fas-c-Met complex by inhibiting Raf and thus restore the binding of Fas to Fas ligand, while inhibiting the binding of HGF to c-Met in HCC
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Cancer Chemother Pharmacol (2014) 73:223–229
TRAIL-induced apoptosis [25] and that such sensitization to TNF-induced death might be related to STAT-3 inhibition and a decline in the induction of MCLI by TNF [26]. These reports support our findings of an increase in sTNFalpha after sorafenib therapy in the PR + SD group. It was reported that sFas is generated by alternative mRNA splicing and antagonizes FasL-mediated cell killing in a concentration-dependent manner [27–30]. sFas levels are increased in patients with solid tumors, and its elevation reflects the disease stage and tumor burden. In HCC patients, elevation of the serum sFas level has been reported [31], and it has been hypothesized that escape of tumor cells from Fasmediated apoptosis could occur by 3 different mechanisms. First, loss of cell surface Fas expression would make tumor cells resistant to Fas-mediated apoptosis. Second, neutralization of FasL by sFas could prevent the ligand from triggering apoptosis. The third possible mechanism of resistance to Fas-mediated apoptosis could involve blocking of Fas signal transduction. Nagao et al. [32] reported that HCC cells escape from host immune surveillance in two ways, which are the loss of cell surface Fas expression and an increase in serum sFas. In the present study, the serum level of sFas showed a significant decrease after sorafenib treatment, although the sFas L level never exceeded 0.15 ng/ ml. These results indicate that treatment with sorafenib at daily doses >400 mg might promote Fas-mediated apoptosis by decreasing sFas in LC patients with aHCC. Wang et al. [33] reported that the Fas-c-Met complex played an important role in mediating the survival of liver cells. C-Met is the cellular receptor for hepatocyte growth factor (HGF), and it is known to have a cytoprotective effect mediated by the induction of signaling cascades that lead to the activation of PI3K/AKT. Under physiological conditions, c-Met acts as a scavenger and binds the Fas receptor to prevent its activation
Cancer Chemother Pharmacol (2014) 73:223–229
by Fas ligand. sFas is generated by alternative splicing of the intact exon 6 encoding the transmembrane domain of Fas, so detection of a smaller DNA fragment than the anticipated full-length Fas cDNA on RT-PCR implies the generation of sFas. The present study showed that the serum level of sFas decreased after sorafenib treatment in LC patients with aHCC. This could suggest that sorafenib released sFas from the Fas-c-Met complex by inhibiting Raf and thus restored Fas–Fas ligand binding, with a resultant decrease in the serum level of sFas (Fig. 6). In conclusion, treatment with sorafenib at doses >400 mg/day might promote induced by TNF or Fas by increasing TNF-alpha levels or decreasing sFas levels in patients with aHCC. In particular, an increase in the sTNFalpha might be an important indicator of the efficacy of sorafenib therapy for aHCC. Conflict of interest The authors have no conflicts of interest to declare.
References 1. Llover JM, Ricci S, Mazzaferro V et al (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:378–390 2. Moreno-Aspitia A, Morton RF, Hillman DW et al (2009) Phase II trial of sorafenib in patients with metastatic breast cancer previously exposed to anthracyclines or taxanes: North Central Cancer Treatment Group and Mayo Clinic Trial N0336. J Clin Oncol 27:11–15 3. Abou-Alfa GK, Schwartz L, Ricci S et al (2006) Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. J Clin Oncol 24:4293–4300 4. Wilhelm SM, Carter C, Lynch M et al (2006) Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 5:835–844 5. Avila MA, Berasain C, Sangro B, Prieto J (2006) New therapies for hepatocellular carcinoma. Oncogene 25:3866–3884 6. Wilhelm SM, Crter C, Tang L et al (2004) BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/ MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 64:7099–7109 7. Liu L, Cao Y, Chen C et al (2006) Sorafenib blocks the RAF/ MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 66:11851–11858 8. Nagata S, Golstein P (1995) The Fas death factor. Science 267:1449–1455 9. Armitage RJ (1994) Tumor necrosis factor receptor superfamily members and their ligands. Curr Opin Immunol 6:407–413 10. Ruberti G, Cascino I, Papoff G, Eramo A (1996) Fas splicing variants and their effect on apoptosis. Adv Exp Med Biol 406:125–134 11. Van Zee KJ, Kohno T, Fischer E, Rock CS et al (1992) Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor alpha in vitro and in vivo. Proc Natl Acad Sci USA 89:4845–4849 12. Seckinger P, Isaaz S, Dayer JM (1989) Purification and biologic characterization of specific tumor necrosis factor alpha inhibitor. J Biol Chem 264:11966–11973
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