[CANCER RESEARCH 64, 2105–2112, March 15, 2004] Human -Lactalbumin Made Lethal to Tumor Cells (HAMLET) Kills Human Glioblastoma Cells in Brain Xenografts by an Apoptosis-Like Mechanism and Prolongs Survival Walter Fischer, 1,2 Lotta Gustafsson, 1 Ann-Kristin Mossberg, 1 Janne Gronli, 3 Sverre Mork, 4 Rolf Bjerkvig, 5 and Catharina Svanborg 1 1 Institute of Laboratory Medicine, Department of Microbiology, Immunology and Glycobiology, University of Lund, Sweden; 2 Department of Neurosurgery, Haukeland University Hospital, Bergen, Norway; 3 Department of Physiology, University of Bergen, Bergen, Norway; 4 Department of Pathology, Haukeland University Hospital, Bergen, Norway; and 5 NorLux Neuro-Oncology, Department of Biomedicine, University of Bergen and Centre de Reserche Public de la Sante ´ Luxembourg, Bergen, Norway ABSTRACT Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human -lactalbumin made lethal to tu- mor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from -lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofac- tor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n  20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and -lactal- bumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all -lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance im- aging scans revealed large differences in tumor volume (456 versus 63 mm 3 ). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be addi- tionally explored as a novel approach to controlling GBM progression. INTRODUCTION Most intracranial neoplasms originate from neuroglial cells and form the heterogeneous group known as gliomas (1). They account for more than 60% of all primary brain tumors and have an unfavorable prognosis (2– 4). Glioblastomas (GBMs) are the most malignant of the gliomas with a mean survival time of less than 1 year (4), and they constitute approximately one-fourth of all intracranial tumors. In recent years, the surgical treatment of brain tumors has made significant technical advances. Microsurgery, neuro-navigation, and new high resolution imaging techniques have reduced surgical morbidity, but the survival time has not improved. The GBMs remain inaccessible to complete surgical removal due to their invasive nature and diffuse infiltrating growth. As a consequence, the current treatment of these patients is palliative, involving partial tumor resection, radiotherapy, and chemotherapy (5). Gene therapy, antisense treatment (6), immunoliposomes (7), 125 I- labeled epidermal growth factor receptor 425 monoclonal antibod- ies (8), and defective viruses may be efficient in brain tumor models, but those few candidates that have made it to clinical trials have been disappointing (9, 10), except regional infusion of a transferrin-diphtheria toxin complex (11). Ideally, new treatment strategies should aim to reach and selectively destroy malignant glioma cells without damaging the intact brain. Human -lactalbumin made lethal to tumor cells (HAMLET) is a molecular complex of -lactalbumin and oleic acid. It is formed when the protein unfolds upon release of the tightly bound Ca 2+ ion. The fatty acid then stabilizes the altered fold (Refs. 12 and 13; Fig. 1A). HAMLET induces apoptosis in a wide variety of tumor cell lines in vitro, but nontransformed differentiated cells are resistant to this effect (14). The lymphoid tumor cells are the most sensitive (LD 50 = 0.01 mM), but carcinomas of different origins also succumb to HAMLET at LD 50 concentrations around 0.02 mM. This study investigated the therapeutic efficacy of HAMLET in a human GBM xenograft model. We show that HAMLET maintains the ability to selectively induce apoptosis in GBMs in vivo and that HAMLET limits tumor progression and prolongs survival of tumor- bearing rats with no signs of toxicity. MATERIALS AND METHODS Preparation of HAMLET. HAMLET was produced from apo -lactalbu- min by ion exchange chromatography on a DEAE-Trisacryl M (BioSepra, Cergy-Saint-Christophe, France) column preconditioned with the C18:1, 9 cis fatty acid (13). 125 I labeling of HAMLET (1 mg/ml) was by the lactoperoxi- dase method (12). For real-time confocal microscopy, HAMLET was conju- gated to Alexa Fluor 568 (Molecular Probes Inc., Eugene, OR). Cellular Interactions. The cell lines were cultured as described previ- ously (12), detached, harvested, washed, and exposed to HAMLET or -lactalbumin. Cell viability was determined by trypan blue exclusion (percentage of dead cells/100 counted cells). Glioma cell line D54 was a gift from Darrel D-Bigner (Duke University, Durham, NC). U251 and CRL2365 were obtained from American Type Culture Collection. A single cell suspension of differentiated murine brain cells was prepared by placing tissue in DMEM (Life Technologies, Inc. Ltd., Paisley, Scotland, United Kingdom) with 1% trypsin and 0.25% DNase in 1% FCS for 30 min at room temperature. After repeated washing, the cells were suspended in DMEM at 4  10 6 /ml. The viability was 99%. Confocal microscopy was in an MRC-1024 confocal system attached to a Eclipse 800 upright micro- scope (Nikon, Kanagawa, Japan). Nontransformed human astrocytes CC-2565 (Cambrex, La Jolla, CA) were cultured according to the manufacturer’s instructions. After harvesting from tissue culture flasks, cells were allowed to attach to 8-well glass slides (Nalge Nunc, Naperville, IL) or 24-well cell culture plates for 2 h in astrocyte basal medium medium (Cambrex) supplemented with ascorbic acid, recombinant human epidermal growth factor (rhEGF), GA-1000, insulin, L-glutamin, and 3% FBS. The adherent cells were washed once with PBS and then exposed to 0.03 mM of Alexa Fluor labeled HAMLET in RPMI 1640 without FCS. Confocal microscopy was in an LSM 510 META confocal system (Carl Zeiss, Received 8/26/03; revised 12/15/03; accepted 1/20/04. Grant support: American Cancer Society; The Swedish Cancer Society; The Swedish Pediatric Cancer Foundation; The Medical Faculty (Lund University); The Segerfalk Foundation; The Royal Physiographic Society; The Anna-Lisa, Sven-Eric Lundgren Foundation (Lund, Sweden); The Norwegian Cancer Society; and Centre de Reserche Public de la Sante ´ Luxembourg. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: W. Fischer and L. Gustafsson contributed equally to this work. Requests for reprints: Catharina Svanborg, Department of Microbiology, Immunol- ogy and Glycobiology, So ¨lvegatan 23, 223 62 Lund, Sweden. E-mail: Catharina. Svanborg@mig.lu.se. 2105 Downloaded from http://aacrjournals.org/cancerres/article-pdf/64/6/2105/2523307/zch00604002105.pdf by guest on 19 June 2022