Research Article IFN-B Is a Highly Potent Inhibitor of Gastroenteropancreatic Neuroendocrine Tumor Cell Growth In vitro Giovanni Vitale,1 Wouter W. de Herder,1 Peter M. van Koetsveld,1 Marlijn Waaijers,1 Wenda Schoordijk,1 Ed Croze,2 Annamaria Colao,3 Steven W.J. Lamberts,1 and Leo J. Hofland1 1Department of Internal Medicine, Erasmus Medical Center, Rotterdam, the Netherlands; 2Department of Immunology, Berlex Bioscience, Inc., Richmond, Richmond, California; and 3Department of Molecular and Clinical Endocrinology and Oncology, ‘‘Federico II’’ University of Naples, Naples, Italy Abstract retroperitoneal and ovarian metastases, where secreted products by-pass the liver detoxification, is commonly associated with this IFN-A controls hormone secretion and symptoms in human gastroenteropancreatic neuroendocrine tumors (GEP-NET) syndrome (4). In addition to the impaired quality of life, patients with advanced but it rarely induces a measurable tumor size reduction. The f effect of other type I IFNs, e.g., IFN-B, has not been evaluated. GEP-NETs have a dismal prognosis with a 5-year survival of 20% We compared the antitumor effects of IFN-A and IFN-B in BON to 30% (5). In these cases, there are relatively few therapeutic cells, a functioning human GEP-NET cell line. As determined options and most of them are palliative. Surgery is indicated only by quantitative reverse transcription-PCR analysis and immu- for conservative resections of the intestine, mesenteric tumors, and nocytochemistry, BON cells expressed the active type I IFN fibrotic areas to improve symptoms and quality of life (6). Hepatic receptor mRNA and protein (IFNAR-1 and IFNAR-2c subunits). resection in selected patients with metastatic functioning GEP- After 3 and 6 days of treatment, IFN-B significantly inhibited NETs is safe, provides temporary relief of symptoms, and may prolong survival (6, 7). As alternative to hepatic resection in BON cell growth in a time- and dose-dependent manner. IC50 and maximal inhibitory effect on day 6 were 8 IU/mL and 98%, patients with liver metastases, local ablative procedures may also respectively. In contrast, the effect of IFN-A resulted signifi- be considered, including chemoembolization, laser, and radio- frequency ablation, as palliative support to control symptoms cantly in a less potent effect (IC50: 44 IU/mL, maximal inhibition: 26%). IFN-A induced only cell cycle arrest, with of hypersecretion syndrome (6). The use of chemotherapeutic an accumulation of the cells in S phase. IFN-B, apart from a regimens is limited to fast growing or undifferentiated tumors (8). Few studies suggest the use of targeted radiotherapy with more potent delay in S-G2-M phase transit of the cell cycle, h also induced a strong stimulation of apoptosis, evaluated by somatostatin analogues coupled to - or Auger electron emitters flow cytometry (Annexin V and 7-AAD) and measurement in the management of GEP-NETs, but its definitive role has yet to of the DNA fragmentation. Besides, only IFN-B severely be defined (9–11). The most common medical treatment in suppressed chromogranin A levels in the medium from BON advanced GEP-NETs includes the use of somatostatin analogues a cells after 6 days of treatment. In conclusion, IFN-B is much and IFN- , alone or in combination. Somatostatin analogues are more potent, compared with IFN-A, in its inhibitory effect on used in the treatment of clinical symptoms, but tachyphylaxis is frequently recorded and these drugs are unable to control tumor GEP-NET cell proliferation in vitro through the induction of a apoptosis and cell cycle arrest. Further studies are required to progression (12, 13). IFN- induces a 42% biochemical and 14% establish whether IFN-B has comparable potent tumor growth tumor response in carcinoid tumors, and a 51% biochemical and 12% tumor response in neuroendocrine pancreatic tumors (14, 15). inhibitory effects in vivo. (Cancer Res 2006; 66(1): 554-62) However, also after the treatment with IFN-a, a measurable tumor reduction is only reported occasionally (16). Introduction Therefore, therapeutic options to inhibit the growth of Gastroenteropancreatic-neuroendocrine tumors (GEP-NET) are metastatic GEP-NET tumors are still unsatisfactory and many a heterogeneous category of tumors and represent the largest patients become refractory to the conventional palliative therapy. group of all NETs (1). GEP-NETs frequently synthesize several For all these reasons, novel treatment strategies seem mandatory. biologically active substances, including bioamines and neuro- Few authors reported that IFN-h has greater antitumor effects than peptides, leading to disease characterized by diarrhea, abdominal IFN-a on melanoma, squamous carcinoma, glioma, breast, and cramps and pain, cardiac valve disease, bronchoconstriction, hepatocellular cancer (17–21). On these bases, IFN-h seems to be of flushing, telangiectasias, and pellagra-like dermatitis, often debil- potential interest in the treatment of tumors. itating for patients (1–3). In the liver, monoamine oxidases can To further explore the possibilities of new medical therapies in detoxify the amines released by the tumor and prevent the GEP-NETs, we compared in this study the role and mechanism of occurrence of the tumor-associated hypersecretion syndrome. action of IFN-a and IFN-h in the regulation of cell growth in BON However, the presence of hepatic and occasionally large cells, a functioning human NET cell line, derived from a lymph node metastasis of a pancreatic carcinoid. Requests for reprints: Giovanni Vitale, Department of Internal Medicine, Erasmus Medical Center, Room EE 585d, Dr. Molewaterplein 50, 3015 GE Rotterdam, the Materials and Methods Netherlands. Phone: 31-10-4635952; Fax: 31-10-4635430; E-mail: [email protected]. I2006 American Association for Cancer Research. Cell lines and culture conditions. Human pancreatic carcinoid BON doi:10.1158/0008-5472.CAN-05-3043 cells were obtained from Dr. Townsend (The University of Texas Medical Cancer Res 2006; 66: (1). January 1, 2006 554 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Type I IFNs and Carcinoids Branch, Galveston, TX). The cells were cultured in a humidified incubator TAAAAA-3V;IFNAR-2cprobe,5V-FAM-TGGATTGGTTATATATGCTTAA- j containing 5% CO2 at 37 C. The culture medium consisted of a 1:1 mixture GAAATAGCCTCCCCA-TAMRA-3V;HPRTforward,5V-TGCTTTCCTTGG- of DMEM and F12K medium, supplemented with 10% FCS, penicillin (1 Â TCAGGCAGTAT-3V; HPRT reverse, 5V-TCAAATCCAACAAAGTCTGGCTTA- 5 10 units/L), fungizone (0.5 mg/L), and L-glutamine (2 mmol/L). TATC-3V;andHPRTprobe,5V-FAM-CAAGCTTGCGACCTTGAC- Periodically, cells were confirmed as Mycoplasma-free. Cells were harvested CATCTTTGGA-TAMRA-3V. with 10% trypsin EDTA and resuspended in medium. Before plating, cells The detection of HPRT mRNA was used for normalization of IFN were counted microscopically using a standard hemocytometer. Trypan receptor mRNA levels. Expression of IFNAR-2a mRNA, the soluble form of blue staining was used to assess cell viability, which always exceeded 95%. IFNAR-2 subunit, was determined indirectly by subtracting IFNAR-2b and Media and supplements were obtained from Life Technologies Bio-cult IFNAR-2c from IFNAR-2 total. Several controls were included in the RT-PCR Europe (Invitrogen, Breda, the Netherlands). experiments. To exclude contamination of the PCR reaction mixtures, the Drugs and reagents. Human recombinant IFN-a-2b (Roferon-A) was reactions were also done in the absence of DNA template in parallel with obtained from Roche (Mijdrecht, the Netherlands). Human recombinant cDNA samples. As a positive control for the PCR reactions of HPRT and type IFN-h-1a was obtained from two preparations: Serono, Inc. (Rebif, I IFN receptors, human cDNA was amplified in parallel with the cDNA Rockland, MA) and Life Technologies (Paisley, United Kingdom). All samples. compounds were stored at À20jC and the stock solution was constituted Immunocytochemistry. Cytospin preparations of BON cells were fixed in distilled water according to the instructions of the manufacturer. with acetone and subsequently incubated for 30 minutes at room Antihuman IFN-h-neutralizing antibody was purchased from Sigma-Aldrich temperature with antibodies to human IFNAR-1 (rabbit polyclonal antibody, (St. Louis, MO). Santa Cruz Biotechnology, Inc., Santa Cruz, CA), IFNAR-2b (rabbit Quantitative reverse transcription-PCR. The expression of type I IFN polyclonal antibody, Santa Cruz Biotechnology), and IFNAR-2c (monoclonal receptors (IFNAR-1, IFNAR-2 total, the short form IFNAR-2b, and the long antibody, Dr E. Croze, Berlex Biosciences, Richmond, CA) subunits. Finally, a form IFNAR-2c) and housekeeping gene hypoxanthine phosphoribosyltrans- peroxidase complex (IL Immunologic, Duiven, the Netherlands) for IFNAR-1 ferase (HPRT) mRNA was evaluated by quantitative reverse transcription- and IFNAR-2b, or standard streptavidin-biotinylated alkaline phosphatase PCR (RT-PCR) in BON cells. (IL Immunologic) for IFNAR-2c, were used according to the recommenda- Polyadenylated mRNA [poly(A)+ mRNA] was isolated using Dynabeads tions of the manufacturer to visualize the bound antibodies. f 5 Oligo (dT)25 (Dynal AS, Oslo, Norway) from cell pellets containing 5 Â 10 Negative controls for the immunohistochemistry included (a) omission cells. The cells were lysed for 2 minutes on ice in a buffer containing of the primary antibody and (b) preabsorbtion of the antibody for IFNAR-2b 100