Multiple Myeloma Regression Mediated by Bruceantin Muriel Cuendet, 1 Konstantin Christov, 2 Daniel D. Lantvit, 1 Yunfan Deng, 3 Samad Hedayat, 3 Lawrence Helson, 4 James D. McChesney, 4 and John M. Pezzuto 5 1 Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, 2 Department of Surgical Oncology, College of Medicine, and 3 Department of Mathematics, College of Liberal Arts and Sciences, University of Illinois at Chicago, Chicago, Illinois; 4 NaPro BioTherapeutics Inc., Boulder, Colorado; and 5 Purdue University, Schools of Pharmacy, Nursing, and Health Sciences, West Lafayette, Indiana ABSTRACT Purpose: Bruceantin has been shown to induce cell differentiation in a number of leukemia and lymphoma cell lines. It also down-regulated c-MYC, suggesting a correla- tion of down-regulation with induction of cell differentiation or cell death. In the present study, we focused on multiple myeloma, using the RPMI 8226 cell line as a model. Experimental Design: The effects of bruceantin on c- MYC levels and apoptosis were examined by immunoblot- ting, 4,6-diamidino-2-phenylindole staining, evaluation of caspase-like activity, and 3,3-dihexyloxacarbocyanine io- dide staining. The potential of bruceantin to inhibit primary tumor growth was assessed with RPMI 8226 xenografts in SCID mice, and apoptosis in the tumors was evaluated by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Results: c-MYC was strongly down-regulated in cul- tured RPMI 8226 cells by treatment with bruceantin for 24 h. With U266 and H929 cells, bruceantin did not regulate c-MYC in this manner. Apoptosis was induced in the three cell lines. In RPMI 8226 cells, apoptosis occurred through proteolytic processing of procaspases and degradation of poly(ADP-ribose) polymerase. The mitochondrial pathway was also involved. Because RPMI 8226 cells were the most sensitive, they were used in a xenograft model. Bruceantin treatment (2.5–5 mg/kg) resulted in a significant regression of tumors without overt toxicity. Apoptosis was significantly elevated in tumors derived from animals treated with bruceantin (37%) as compared with the control tumors (14%). Conclusions: Bruceantin interferes with the growth of RPMI 8226 cells in cell culture and xenograft models. These results suggest that bruceantin should be reinvestigated for clinical efficacy against multiple myeloma and other hema- tological malignancies. INTRODUCTION Multiple myeloma is a tumor of the hematopoietic system, accounting for 12,000 deaths per year in the United States (1). There is a rapid increase in incidence with age and a significant excess at all ages of males. In addition, geographical and racial differences play an important role in the incidence of myeloma. The disease is much more common in African-American pop- ulations than in Caucasians, and there is a low incidence in Chinese (1, 2). The roles of genetic background and environ- ment are poorly defined (3). Multiple myeloma is usually preceded by an age-dependent premalignant disease termed monoclonal gammopathy of unde- termined significance. Numeric chromosomal abnormalities are present in virtually all multiple myelomas, and in most, if not all, cases of monoclonal gammopathy of undetermined signifi- cance (4, 5). Translocations are common. The incidence of IgH translocations increases with the stage of tumorigenesis (6). Most immunoglobulin translocations involve just three groups of genes: Cyclins D1–3 (7), MMSET, and FGFR3 (4), and two B-zip transcription factors (c-MAF and MAFB; Ref. 8). Com- plex translocations dysregulate c-myc as a late progression event that is associated with enhanced proliferation. c-MYC is rear- ranged in 15% of multiple myelomas representing all stages, but this fraction correlates with the severity of disease and is often heterogeneous among cells within the tumor (9). Studies that address the expression of c-MYC RNA and protein in multiple myeloma samples from patients are consistent with increased expression of c-MYC late in the disease (10). Secondary trans- locations contribute to subsequent progression. Progression from monoclonal gammopathy of undetermined significance to myeloma is associated with activating mutations of RAS or FGFR3. This progression seems to flip a molecular switch that results in osteolytic bone lesions that are mediated by osteoclas- togenesis, neo-angiogenesis and enhanced growth of the my- eloma clone (11). Additional tumor progression, and especially extramedullary growth, is associated with increased prolifera- tion, mutations of p53, and secondary translocations that dys- regulate c-MYC. The current therapy for myeloma has relied predominantly on glucocorticoids, such as prednisone, and alkylating agents, primarily melphalan. Neither therapy is curative, and the median survival has remained fixed at 3 years for the past decade. Although monoclonal gammopathy of undetermined signifi- cance can be diagnosed efficiently by a simple blood test, it is not possible to prevent progression or even predict when pro- gression to myeloma will occur. Clearly, there is a need for new drugs that may be useful for the control, treatment, and/or cure of this disease. Received 3/11/03; revised 10/22/03; accepted 10/22/03. Grant support: National Cancer Institute Grant P01 CA48112. 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. Requests for reprints: John M. Pezzuto, Purdue University, Schools of Pharmacy, Nursing, and Health Sciences, Heine Pharmacy Building, Room 104, 575 Stadium Mall Drive, West Lafayette, IN 47907-2051. 1170 Vol. 10, 1170 –1179, February 1, 2004 Clinical Cancer Research Research. on June 11, 2020. © 2004 American Association for Cancer clincancerres.aacrjournals.org Downloaded from