Vol. 4, 171-176, January 1998 Clinical Cancer Research 171 Antitumor Activity of Actinonin in Vitro and in Vivo Yang Xu, Lawrence T. Lai, Janice L. Gabrilove, 8), mye!oid and monocytic cells, and most myeloblastic leuke- and David A. Scheinberg’ mias as well as on cells and tissues outside the hematopoietic system including fibroblasts, intestinal epithelium, renal tubular Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 epithelium, and synaptic membranes of the central nervous system (I ). APN occurs as a homodimer, and the molecule is a 150-kDa, transmembrane glycoprotein with an intracellular ABSTRACT amino terminus (1). F23, an antihuman CD13/APN mAb, is able Actinonin, an antibiotic and CD13/aminopeptidase N to completely block the active site of the enzyme (9). (APN) inhibitor, has been shown to be cytotoxic to tumor Bestatin, a CD 1 3/APN inhibitor, was examined in preclin- cell lines in vitro. We investigated the antiproliferative ef- ical and clinical studies; bestatin could inhibit lymph node fects of actinonin on human and murine leukemia and lym- metastasis of P388 leukemia in mice (10) and was used in phoma cells. Actinonin inhibited growth of NB4 and HL6O clinical trials in malignant skin tumors (1 1), in head and neck human cell lines and AKR mouse leukemia cells in vitro with cancer (12), in esophageal cancer (13), and in gynecologic an IC50 of about 2-S g/ml. The inhibitory effect on CD13- tumors (14). High doses of bestatin resulted in the significant positive cells was not blocked by pretreatment with the inhibition of preexisting experimental and spontaneous metas- anti-CD13/APN monoclonal antibody F23, which binds with tasis in mice (15). Results with bestatin prompted us to examine high affinity to the active site of CD13/APN and blocks its the actions of actinonin as an anticancer agent. Actinonin, a enzymatic activity. Moreover, F23 alone was not inhibitory naturally occurring antibiotic derivative of L-prolinol and a to CD13-positive cells. Furthermore, a similar inhibitory potent CD13/APN inhibitor, is obtained from the culture fib- IC50 of actinonin was seen in the CD13-negative cell lines trates of a Streptomyces species classified as Streptomyces Cut- RA.JI and DAUDI human lymphoma. These data suggest ter C/2 NCIB 8845 (16). Actinonin has been shown to be that the inhibitory effect of actinonin is not mediated by generally active against Gram-positive bacteria. The action of inhibition of CD13/APN. Cell cycle analysis showed that the antibiotic involves disruption of RNA synthesis in bacteria. actinonin induces a G1 arrest in HL6O and NB4 cells; apop- In vivo, it has no apparent toxicity to mice in doses up to 400 tosis was observed in 20-35% of the cells as measured by mg/kg body weight (16). Actinonin is eight times more potent intracellular flow cytometry. To assess whether these effects than bestatin (9). could be seen in vivo, the effect of actinonin on the syngeneic We report here the antiproliferative effects of actinonin on AKR mouse leukemia model was evaluated. Actinonin human leukemia and bymphoma cells in vitro and the treatment showed dose-dependent antitumor effects on AKR leukemia of syngeneic AKR leukemia in an AKR mouse model using in vivo, resulting in a survival advantage. In conclusion, actinonin. apoptosis, growth inhibition, and therapeutic effects in vivo are induced by actinonin and are not likely to be mediated by CD13/APN. MATERIALS AND METHODS Materials. Actinonin, amastatin, and bestatin were pur- INTRODUCTION chased from Sigma (St. Louis, MO). mAbs 4B4, OKT9, Leu- CD 1 3/APN2 (EC 3.4. 1 1.2) is a ubiquitous cell surface zinc 1 la, Leu-15, Leu-Ml, MY4, control IgG1, and IgG2a were aminopeptidase involved in down-regulation of regulatory pep- purchased from Coulter (Hialeah, FL) or Becton Dickinson (San tide signals (1). Recently, APN has been shown to be the major Jose, CA). Fluoresceinated, affinity-purified goat antiserum to receptor for the enteropathogenic coronavirus TGEV (2) and for mouse immunoglobulins (GAM-FITC) was purchased from human coronavirus 229E (3) and to be involved in tumor cell Kirkegaard & Perry (Gaithersburg, MD). mAbs F23, TA99, invasion (4, 5). Human APN is identical to the myeboid differ- JD12, and M195 were produced in Memorial Sloan-Kettering entiation antigen CD13 (6, 7) found on HL6O leukemia cells (7, Cancer Center. Recombinant human IL-3 was obtained from Amgen (Thousand Oaks, CA). Stock vials of human IL-3 were stored at 4#{176}C.For each experiment, all factors were diluted in serum-containing medium on the day of use. Received 8/14/97; revised 10/6/97; accepted 10/13/97. Cell Separation Techniques. Bone marrow cells were The costs of publication of this article were defrayed in part by the obtained from healthy volunteers after informed consent. The payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mononuclear cells were isolated by centrifugation on Ficoll- indicate this fact. Hypaque gradients (1.077 g/mb; Pharmacia Fine Chemicals, I To whom requests for reprints should be addressed, at 1275 York Piscataway, NJ), washed twice in PBS and suspended in Avenue, New York, NY 10021. Phone: (212) 639-5010; Fax: (212) Iscove’s modified Dulbecco’s medium containing 10% FCS 717-3068. (Hycbone, Logan, UT) supplemented with penicillin (100 units/ 2 The abbreviations used are: APN. aminopeptidase N; mAb, mono- clonal antibody; IL, interleukin; CFU-GM, colony forming unit-granu- ml; Life Technologies, Inc., Grand Island, NY), streptomycin bocyte macrophage. (100 p.g!ml; Life Technologies, Inc.), and 3 mg/ml glutamine Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 1998 American Association for Cancer Research. 172 Antitumor Activity of Actinonin (Life Technologies, Inc.). These cells were used as target cell A populations for the CFU-GM progenitor cell assay. U 1. Cl CFU-GM. Low-density (I X l0 cells/mi) or CD34 e V (5 X l0 ce!!s/ml) bone marrow cells were cultured in 35-mm U tissue culture dishes (Coming, Corning, NY) in McCoy’s mod- ified assay medium containing 0.3% agar (DIFCO Laboratories, Detroit, MI) and 10% FCS (17). Cultures were stimulated by the U I- addition of 100 ng/ml IL-3. U 0. I- Animals. Five-week-old female AKR mice were pur- U V chased from Jackson Laboratory (Bar Harbor, ME). All bedding Cl material was sterilized before use; the cages were covered with = an air filter and maintained in isolation cabinets. Animal han- dbing and experiments were performed in a sterile atmosphere using a laminar flow hood following institutional care guide- Actinonin (.tg/ml) lines. Cell Lines and Culture Conditions. AKR leukemia cells were obtained from the spleen of old AKR mice who spontaneously developed leukemia at 10 months. HL6O (acute myeloid leukemia, CD13 positive), NB4 (acute promyelocytic leukemia, CD13 positive, from Dr. M. Lanotte of the Louis U I. Pasteur Institute, Paris, France), and RAJI and DAUDI (B Cl VU lineage Burkitt’s lymphomas, CD13 negative) were maintained U in culture using RPMI 1640 supplemented with 10% Serum Plus (JRH Biosciences, Lenexa, KS) and 10% heat-inactivated FCS (Intergen, Purchase, NY) at 37#{176}Cina humidified atmosphere of .0 5% CO2. Cell viability was always higher than 90%, and cells Cl were free of mycoplasma contamination. Transplantation of AKR Leukemia Cells into AKR Mice and Therapy. A 0.l-ml aliquot containing 2 X 106 AKR leukemia cells from suspension culture was transplanted .001 .01 .1 1 10 100 Actinonin (.tg/ml) s.c. into AKR mice. Tumors grew s.c., and the cutaneous tumor size was measured as a cross product to derive surface area. To Fig. 1 Cytotoxicity and inhibition of protein synthesis in cell lines by protect animals, mice were considered dead and sacrificed when actinonin. A, inhibition of protein synthesis in cells by actinonin. Ce!! lines (5 x l0 cells/mb) were incubated for 5 days at 37#{176}Cinthe the tumor surface area reached more than 400 mm2. The test presence of actinonin. Levels of protein synthesis were determined by animals were treated i.p. with actinonin in a final volume of 0.1 5-h incorporation of tritiated beucine into trichloroacetic acid-precipita- ml. Control mice were treated with 0.1 ml of saline. ble protein. B, cell viability determined by trypan blue exclusion. Cell Flow Cytometry Assays. Cells were washed and resus- lines (5 x l0 cells/mi) were incubated for 5 days at 37#{176}Cinthe pended in 2% rabbit serum (Pci Freeze, Rogers, AK) to reduce presence of actinonin. Trypan blue was added, and live and dead cells were enumerated. nonspecific binding. Cells (5 X 10) in a final volume of 0.1 ml were incubated for 1 h on ice in the presence of primary antibody. Cells were washed twice, incubated for 30 ruin on ice with a secondary FITC-labeled antibody (goat antimouse im- munoglobubin; Kirkegaard & Perry), washed twice, and fixed PBS and fixed in 2% paraformaldehyde for 10 mm, and then with 0.5% paraformaldehyde. FITC fluorescence intensity was exposed to 0.1% Triton X-lOO for 10 mm. After washing with measured on an EPICS Profile H flow cytometer (Coulter). PBS and blocking with 1% human AB serum, cells were incu- Inhibition of Tritiated Thymidine or Leucine Incorpo- bated with FITC-conjugated anti-BCL-2 mAb (DAKO A/S. ration. An aliquot containing 200 p.! of cells was washed and Carpinteria, CA) on ice for 30 mm and then analyzed on an incubated at 37#{176}Cin96-well plates in the presence or absence of EPICS Profile II flow cytometer (Coulter).