Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells Sanjeeva Mohanam 1 , Sushma L Jasti 2 , Sudha R Kondraganti 3 , Nirmala Chandrasekar 1 , Sajani S Lakka 1 , Yoshiaki Kin 3 , Gregory N Fuller 4 , Alfred WK Yung 2 , Anthanassios P Kyritsis 2 , Dzung H Dinh 5 , William C Olivero 5 , Meena Gujrati 6 , Francis Ali-Osman 3 and Jasti S Rao* ,1,5 1 Division of Cancer Biology, Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, Illinois, IL 61656, USA; 2 Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, TX 77030, USA; 3 Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, TX 77030, USA; 4 Department of Neuropathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, TX 77030, USA; 5 Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois, IL 61656, USA; 6 Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, IL 61656, USA Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited signi®cant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with con- trols. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was signi®cantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any post- chemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy. Oncogene (2001) 20, 3665 ± 3673. Keywords: glioblastoma; invasion; cathepsin B Introduction Tumor cell invasion is a complex multistep process involving tumor cell attachment to the extracellular matrix (ECM) followed by degradation of host barriers (ECM and basement membranes) by proteolysis and tumor colony formation at distant sites (Duy, 1992). Several enzyme systems participate in the degradation of ECM and basement membrane (Liotta et al., 1991), among which is cathepsin B, a lysosomal cysteine proteinase (Lah and Kos, 1998). Increased or altered expression of cathepsin B occurs in many types of tumors, including those of the breast (Foekens et al., 1998; Maguire et al., 1998), colon (Hirai et al., 1999; Iacobuzio-Donahue et al., 1997), prostate (Sinha et al., 1998) and lung (Werle et al., 1999), suggesting that cathepsin B may be involved in tumor invasiveness and metastasis. It has also been demonstrated that cathepsin B not only degrades components of the ECM such as laminin, collagen, and ®bronectin and structures of basement membranes (Buck et al., 1992), but also activates other proteolytic enzyme systems (Kobayshi et al., 1993; Schmitt et al., 1992). A recent study has shown that inhibition of extracellular cathepsin B inhibits the growth and metastasis of rat colon cancer cells (Van Noorden et al., 1998), providing further evidence for the involvement of cathepsin B in tumor invasion. Cells transfected with antisense cathepsin B cDNA exhibited a decrease in cathepsin B activity, which resulted in a reduced invasion and motility of treated cells (Krueger et al., 1999). Our interest in gliomas, the most common form of brain tumors, leads us to consider cathepsin B's role in glioma invasiveness. The mechanisms underlying brain tumor cell invasion are not well understood, but it is known that malignant progression of human gliomas is associated with an increase in cysteine proteases (McCormick, 1993; Sivaparvathi et al., 1996). We earlier reported high levels of cathepsin B in tumor and endothelial cells of brain tumor tissues (Sivaparvathi et Oncogene (2001) 20, 3665 ± 3673 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: JS Rao Received 8 August 2000; revised 7 March 2001; accepted 21 March 2001