431-440 4/7/07 11:31 Page 431 INTERNATIONAL JOURNAL OF ONCOLOGY 31: 431-440, 2007 431 Activation of polyamine catabolism by N1,N11-diethylnorspermine leads to cell death in glioblastoma RONGCAI JIANG1,7*, WOONYOUNG CHOI1*, ASAD KHAN2,8, KENNETH HESS3, EUGENE W. GERNER5, ROBERT A. CASERO Jr6, W.K. ALFRED YUNG4, STANLEY R. HAMILTON1 and WEI ZHANG1 Departments of 1Pathology, 2Pediatric Oncology, 3Biostatistics, and 4Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030; 5Department of Cell Biology and Anatomy, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724; 6Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA Received February 5, 2007; Accepted March 26, 2007 Abstract. Glioblastoma multiforme (GBM) is one of the with N1-acetylpolymine oxidase (APAO)/spermine oxidase most therapeutically refractory human cancers. Elevated (SMO) inhibitor. Though mitochondrial damage was induced, cellular polyamine levels are a common feature of cancer neither activation of the caspase cascade nor cytochrome c cells, including GBM cells, and the polyamine pathway redistribution between the mitochondria and cytoplasm has been explored as a potential therapeutic target to inhibit was observed. Systemic DENSPM treatment of mice with polyamine biosynthesis or activate polyamine catabolism. intracerebral GBM led to longer survival. Taken together, In this study, we investigated the effect of N1,N11-diethyl- our studies indicate that DENSPM kills GBM cells through norspermine (DENSPM), a spermine analog that activates induction of SSAT coupled with H2O2 production, which is a polyamine catabolism, in GBM cells. The in vitro cell culture potential target for GBM therapy. experiments showed that DENSPM increased the sub-G1 apoptotic cell population in GBM cell lines but caused minimal Introduction cytotoxicity in normal astrocytes. Prior to apoptosis induction, DENSPM caused the elevation of spermidine/spermine N1- Polyamines are natural factors that are important for cell acetyltransferase (SSAT) expression accompanied by a growth. It has long been recognized that polyamine levels decrease in polyamine levels and an increase of acetylated are highly elevated in a broad spectrum of human cancers, polyamine levels, which temporally coincided with the onset including glioblastoma multiforme (GBM) (1-5). GBM is the of hydrogen peroxide (H2O2) induction in the cells. The most advanced and frequently occurring form of glioma. It is cytotoxic effects of DENSPM in the GBM cells could be also a notoriously refractory cancer, with little improvement partially attenuated by either turning down SSAT mRNA in median survival (less than a year) over the past few decades with small interference RNA or inhibiting H2O2 production (6,7). The polyamine pathway has been explored as a potential target for cancer therapy through down-regulation of the polyamine biosynthetic pathway or up-regulation of _________________________________________ the polyamine catabolic pathway. The initial focus has been on the development of drugs that inhibit polyamine Correspondence to: Dr Wei Zhang, Department of Pathology, biosynthesis. α-Difluoromethylornithine (DFMO), an inhibitor Unit 85, Cancer Genomics Core Laboratory, The University of of the polyamine biosynthesis-limiting enzyme ornithine Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., decarboxylase (ODC), has been tested in various cancer Houston, TX 77030, USA types and shown to exert potent antiproliferative effects as a E-mail: [email protected] single agent (8-11). However, DFMO as a single agent was not sufficiently effective in several clinical settings, which has 7 Present addresses: Tianjin Medical University General Hospital, led to the use of combination therapy, with DFMO coupled 8 Tianjin, P.R. China; Virginia Commonwealth University, Richmond, with methylglyoxal bis-guanylhydrazone or the nitrosourea VA 23298, USA alkylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), procarbazine, N-(2-chloroethyl)-N'-cyclohexyl-N- * Contributed equally nitrosurea, and vincristine (PVC) (12-17). Though DFMO combined with BCNU or PVC showed encouraging results in 1 11 Abbreviations: DENSPM, N ,N -diethylnorspermine; GBM, patients with anaplastic gliomas, it had minimal effects on 1 glioblastoma multiforme; SSAT, spermidine/spermine N -acetyl- GBM (16,18-20). One possible mechanism for the reduced 1 transferase; APAO, N -acetylpolyamine oxidase; NHA, normal effectiveness of DFMO alone or in combination therapy in human astrocyte; SMO, spermine oxidase GBM might be compensatory polyamine generation from other sources: increased uptake through the specific polyamine 1 11 Key words: glioblastoma, polyamine, N ,N -diethylnorspermine transporter or activation of alternative polyamine biosynthesis 431-440 4/7/07 11:31 Page 432 432 JIANG et al: TARGETING POLYAMINE CATABOLISM pathways (21,22). In contrast to DFMO, polyamine analogs instructions. CM-H2DCFDA and rhodamine 123 were pur- have been developed to activate the polyamine catabolic chased from Molecular Probes (Eugene, OR) and dissolved pathway. One of these analogs, N1, N11-diethylnorspermine in dimethyl sulfoxide. (DENSPM), can strongly deplete polyamine pools and elevate the expression of spermidine/spermine N1-acetyltransferase Cell lines. The U87 and LN229 GBM cell lines from the (SSAT) mRNA, resulting in apoptosis with minimal cyto- American Type Culture Collection (Manassas, VA) were toxicity (23,24). DENSPM has also been shown to be effective maintained in Dulbecco's modified Eagle's medium (DMEM)/ in combination therapy. When combined with other chemo- F12 with 10% dialyzed serum (Hyclone, Logan, UT) at 37˚C therapy drugs, such as 5-fluorouracil and oxaliplatin, DENSPM in a 5% CO2 incubator. Dialyzed serum was used because can markedly elevate SSAT mRNA expression (25,26). The fetal bovine serum has abundant thymine and polyamine. induction of SSAT is accompanied by a synergistic cell kill Normal human astrocytes (NHAs) were purchased from effect in colon and ovarian cancer cell culture systems (25,27). Cambrex Corporation (East Rutherford, NJ) and cultured in DENSPM is currently being evaluated as a single agent in AGM™ BulletKit medium according to the manufacturer's several cancers, including lung, breast, and liver cancers instructions. (28-31). The mechanisms of polyamine analog-induced apoptosis Cell viability assay. Cell viability was evaluated using the MTS have been investigated in a number of studies (23,32-35). (3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-methoxyphenyl]- SSAT activation is one of the key steps in this process for the 2-[4-sul-fophenyl]-2H-tetrazolium compound) assay (Promega majority of polyamine analogs (22). Transfection with SSAT Corporation, Madison, WI). For the MTS assay, we seeded can enhance the apoptosis response to DENSPM (36,37). 3,000 GBM cells per well in 100 μl of medium in a 96-well Reduction of SSAT by small interference RNA (siRNA) plate. On the second day, various concentrations of DENSPM attenuates analog-induced apoptosis by inhibiting the depletion were added to the wells. For the H2O2 effect, GBM cells of polyamine (23). However, some polyamine analogs deplete treated with DENSPM were co-cultured in the presence of polyamine pools in the absence of SSAT activation (38,39), the polyamine oxidase inactivator MDL 72527, with a final which could lead to apoptosis without SSAT induction (40,41). concentration of 25 μM. After cells were incubated with These findings suggest that polyamine analogs can induce DENSPM alone or with MDL 72527 for the indicated time, apoptosis in cancer cells via several mechanisms (42). the MTS assay was used to measure cell viability. After 20 μl One key regulator of apoptosis caused by polyamine- of MTS solution had been added to each well and mixed, analogs that induce SSAT is APAO, which utilizes acetylated the cells were incubated at 37˚C in the 5% CO2 incubator spermine/spermidine to produce high levels of H2O2. The according to the product instructions. Absorbance at 490 nm increased H2O2 leads to damage in the mitochondrial mem- was measured with a microplate reader (MRX, Danatech branes, which is believed to be the main cause of the release Laboratory, Houston, TX). All MTS assays were performed at of cytochrome c. Cytochrome c release is an upstream activator least in triplicate for each treatment condition, and experiments of the cascade of caspases, which in turn initiates the caspase were repeated at least twice. 9-controlled extrinsic cascade of apoptosis (32). However, previous studies have observed inconsistent patterns of caspase Cell cycle analysis by flow cytometry. Cells were seeded in involvement in DENSPM-induced cell death (25,34). The a 10-cm2 dish (1.5x105 cells/dish) in 10 ml of medium sup- application of APAO inhibitor significantly reduced apoptosis plemented with 10% dialyzed fetal bovine serum. Twenty- induced by polyamine analogs (43). Another key regulator four hours later, 10 μM DENSPM was added. After being is spermine oxidase (SMO), which converts spermine to incubated with the drug for 48 h (U87 cells) or 72 h (LN229 spermidine, a reaction that also produces H2O2. A small cells), adherent and non-adherent cells were harvested by molecule inhibitor of APAO was shown to inhibit SMO as trypsinization and centrifugation, washed with ice-cold well (44). Thus, it is likely that the balance of these three key phosphate-buffered saline (PBS), fixed with ethanol, stained enzymes of polyamine