Inhibition of cystine uptake disrupts the growth of primary brain tumors

Abstract

Glial cells play an important role in sequestering neuronally released glutamate via Na+-dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumors (gliomas). Instead, gliomas release glutamate, causing excitotoxic death of neurons in the vicinity of the tumor. We now show that glutamate release from glioma cells is an obligatory by-product of cellular cystine uptake via system xc-, an electroneutral cystine-glutamate exchanger. Cystine is an essential precursor for the biosynthesis of glutathione, a major redox regulatory molecule that protects cells from endogenously produced reactive oxygen species (ROS). Glioma cells, but not neurons or astrocytes, rely primarily on cystine uptake via system xc- for their glutathione synthesis. Inhibition of system xc- causes a rapid depletion of glutathione, and the resulting loss of ROS defense causes caspase-mediated apoptosis. Glioma cells can be rescued if glutathione status is experimentally restored or if glutathione is substituted by alternate cellular antioxidants, confirming that ROS are indeed mediators of cell death. We describe two potent drugs that permit pharmacological inhibition of system xc-. One of these drugs, sulfasalazine, is clinically used to treat inflammatory bowel disease and rheumatoid arthritis. Sulfasalazine was able to reduce glutathione levels in tumor tissue and slow tumor growth in vivo in a commonly used intracranial xenograft animal model for human gliomas when administered by intraperitoneal injection. These data suggest that inhibition of cystine uptake into glioma cells through the pharmacological inhibition of system xc- may be a viable therapeutic strategy with a Food and Drug Administration-approved drug already in hand.

Figures

Figure 1.

System is highly expressed in gliomas. a, Expression of mRNA transcripts of xCT, 4F2hc, and β-actin in human glioma cell lines (D54-MG, STTG-1, U251-MG, and U87-MG) and a glioma primary culture (GBM62) is demonstrated by RT-PCR. The protein expression of 4F2hc determined by Western blot with anti-4F2hc antibodies is illustrated below. b, RT-PCR was performed with RNA extracts from patient biopsies and illustrates expression of mRNA transcripts of xCT in tumors (ID47, ID34, ID20) as well as in comparison brain tissues, which were operated on for other reasons and did not show any evidence of malignancy (ID59, ID56, ID78). c, The expression of 4F2hc, the regulatory subunit of system and of the Na+-dependent glutamate transporter GLT-1, was compared between glioma biopsies and comparison brain tissues by Western blot analysis. The expression of GLT-1 was almost completely missing in tumor biopsies, all of which prominently expressed 4F2hc. The origin of human biopsies was as follows: ID61, 22-year-old white male; ID56, unknown; ID57, 1.5-year-old Hispanic male; ID59, 26-year-old white female; ID78, 48-year-old white female; ID20, 45-year-old black female; ID21, 55-year-old white female; ID25, 58-year-old unknown male; ID34, 47-year-old unknown male; ID47, 38-year-old white female. Tissues were obtained in compliance with the Institutional Review Board of the University of Alabama at Birmingham. GBM, Glioblastoma multiforme.

Figure 2.

Inhibition of system selectively reduces cystine uptake and depletes intracellular glutathione in glioma cells. a, Coincubation with (S)-4-CPG and sulfasalazine (SAS) reduced cystine uptake in glioma cell lines and in a primary glioma cell culture (GBM62). b, Sulfasalazine affects cystine uptake into cortical astrocytes to a much lesser extent than up take into D54-MG glioma cells. c, Cystine uptake in cortical neurons was negligible compared with astrocytes orglioma cells. d, Sulfasalazine caused a time- and dose-dependent decrease in intracellular glutathione in D54-MG cells. e, Intracellular level of glutathione in astrocytes is significantly less affected by sulfasalazine. f, Treatment with (S)-4-CPG sulfasalazine depleted intracellular glutathione in glioma cells but not cortical astrocytes or neurons. Error bars indicate SE.

Figure 3.

Inhibition of cystine uptake via system compromises glioma cell growth. a, b, Treatment of glioma cells with (S)-4-CPG (a) or sulfasalazine (SAS) (b) shows dose-dependent growth inhibition in glioma cell lines and primary cultured glioma cells. c, However, (S)-4-CPG does not inhibit the growth of cultured cortical astrocytes or cortical neurons. d, The broad-spectrum mGluR antagonist E4CPG (0.25 mm) does not inhibit cell growth, nor can 1 mm glutamate reverse (S)-4-CPG (0.25 mm)-induced growth inhibition, suggesting that these effects were not mediated via mGluRs. e, Exogenous l-cystine in a dose-dependent manner overcomes the growth inhibition exerted by (S)-4-CPG. f, Similarly, incubation with glutathione ethyl ester, a membrane-permeable form of glutathione, overcomes the growth inhibition by (S)-4-CPG or sulfasalazine in D54-MG cells. Error bars indicate SE.

Figure 4.

System inhibitors block DNA synthesis and increase the percentage of S phase cells. a, b, (S)-4-CPG and sulfasalazine (SAS) inhibit BrdU incorporation into chromosomal DNA in D54-MG cells as assessed by ELISA (a) or immunohistochemistry (b) (representative 20× magnification images). One millimolar cystine (Cys) restored DNA synthesis in the continued presence of either (S)-4-CPG or sulfasalazine. DAPI, 4′,6-Diamidino-2-phenylindole. c, DNA content was determined using a FACS-based assay of propidium iodide. Treatment with either (S)-4-CPG or sulfasalazine significantly increased the cell population in S phase. Error bars indicate SE.

Figure 5.

Cystine uptake inhibition induces apoptotic cell death. a, Treatment with free radical scavengers including vitamin E, TMPO ( scavenger), and PBN ( scavenger) partially restores glioma cell growth in the presence of (S)-4-CPG. b, (S)-4-CPG causes DNA fragmentation as indicated by flow cytometric analysis, which is indicative of apoptotic cell death. c, (S)-4-CPG and sulfasalazine (SAS) increase the activated form of caspase-3. Caspase-3 antibodies recognize both the proform (32 kDa) and the activated form (17 kDa). d, The panspecific caspase-3 inhibitor Boc-D-FMK (100 μm) blocked cell death induced by 0.25 mm (S)-4-CPG, further suggesting that cell death invoked caspase-3 activity and hence is presumed to be apoptotic. e, Cell death was also confirmed using the Live/Dead assay kit (Molecular Probes), which, when cells are analyzed by fluorescence-activated cell sorting, show an enhanced number of dead cells that are ethidium homodimer positive, as opposed to live cells that express primarily calcein. Chronic treatment with either (S)-4-CPG or sulfasalazine induced cell death, and exogenously added cystine can overcome cell death in the presence of (S)-4-CPG (c) or sulfasalazine (data not shown). Error bars indicate SE.

Figure 6.

Sulfasalazine (SAS) slows tumor growth in vivo. a, Experimental brain tumors were induced in CB-17 scid mice by xenografting D54-ffLuc cells stably expressing the luciferase gene into the brain. Tumor size was determined in vivo every 7 d using a bioluminescence imaging system (IVIS system; Xenogen). Animals were randomized into three groups of 12 animals each. The control group received 1 ml of saline intraperitoneally twice daily, and the two test groups received 8 mg of sulfasalazine (in 1 ml of saline) for either 1 or 3 weeks. Both treatment regimens slowed tumor growth with near-complete inhibition of growth with a 3 week treatment protocol. Animals were retreated for 3 d at 52 d because tumor growth picked up in the animals that received only 1 week of treatment to evaluate continued responsiveness to the drug. b, In a second set of experiments, brain tumors were similarly induced in nude mice by xenografting U87-ffLuc cells that stably expressed the luciferase gene into the brain. The control group received 1 ml of saline intraperitoneally twice daily, the test group received 8 mg of sulfasalazine (in 1 ml of saline) for 3 weeks, followed by one daily dose thereafter. c, d, Representative hematoxylin-eosin staining of mouse brain sections from the same experiment obtained at 30 d. Examples of sections are from saline control (c) and 8 mg/ml SAS treated (d) after 3 weeks of twice-daily injections for both (1.25× magnifications). e-g, Immunocytochemistry using an anti-luciferase conjugated to FITC. Human U87-ffLuc cells transfected with luciferin still showed luciferase expression after 49 d of tumor growth as determined by antibody staining. e is the image of anti-luciferase alone, f is the image of the 4′,6-diamidino-2-phenylindole (DAPI) alone, and g is a merged image of anti-luciferase in green and DAPI-stained nuclei in blue. Error bars indicate SE.

Figure 7.

Immunohistochemical analysis of the sulfasalazine effects on in vivo tumors. a, b, DAB-reactive immunohistochemistry showing areas of tissue necrosis using an ApopTag kit based on TUNEL technology (20× magnification). a, A brown reaction product in single cells in the tumor and the background brown signify necrosis in the saline-control mouse (50 d survival). The green color comes from nuclei stained with methyl green. b, The same staining from a sulfasalazine (SAS)-treated mouse brain tumor (56 d survival). H&E, Hematoxylin and eosin. c, d, Representative examples of TUNEL staining (green) of a saline-treated (c) and 8 mg/ml SAS-treated (d) mouse, costained with 4′,6-diamidino-2-phenylindole (DAPI) (40× magnification). e, f, Immunocytochemistry for Ki67 in saline control and sulfasalazine-treated brain tumor sections (20× magnification). Ki-67 staining shows a clear demarcation around the tumor abutted by normal astrocytes stained by GLT-1 antibody shown in green. The Ki67-positive cells are red, and DAPI stains all of the nuclei blue.