Enhanced proapoptotic effects of tumor necrosis factor–related apoptosis-inducing ligand on temozolomide-resistant glioma cells

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Object

Death receptor targeting is an attractive approach in experimental treatment for tumors such as malignant gliomas, which are resistant to radiation and chemotherapy. Among the family of cytokines referred to as death li gands, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has attracted clinical interest. The aim of this study was to assess whether TRAIL can be used as an adjuvant to temozolomide (TMZ) for apoptosis induction in malignant glioma cell lines.

Methods

Six human malignant glioma cell lines (A172, U87, U251, T98, U343, and U373) were exposed to human (h)TRAIL, TMZ, or an hTRAIL/TMZ combined treatment. Cell viability was assayed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and phase-contrast microscopy. Cell apoptosis was detected using the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling technique and quantified using flow cytometric analysis. The apoptosis signaling cascade was studied with Western blotting.

The additive effects of hTRAIL and TMZ resulted in a significant decrease in cell viability and an increased apoptotic rate. Expression of the death receptors DR5 and DR4 in two cell lines (A172 and U251) upregulated significantly when they were used in combination hTRAIL/TMZ treatment (p < 0.05 compared with baseline control), leading to activation of caspase-8 and caspase-3 (p < 0.05 compared with baseline control) and confirming an extrinsic apoptotic pathway. A cell intrinsic pathway through mitochondrial cytochrome c was not activated.

Conclusions

Based on this work, one may infer that hTRAIL should be considered as an adjuvant treatment for TMZ-resistant human malignant gliomas.

Abbreviations used in this paper:hTRAIL = human tumor necrosis factor–related apoptosis-inducing ligand; MTT = 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyltetrazolium bromide; SDS = sodium dodecyl sulfate; TBST = Tris-buffered saline with Tween 20; TMZ = temozolomide; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling.

Article Information

Address reprint requests to: Isabelle Germano, M.D., Department of Neurosurgery, Mount Sinai School of Medicine, Annenberg 8-90, 1 Gustave Levy Place, Box 1136, New York, New York 10029. email: isabelle.germano@mountsinai.org.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Photomicrographs of cells (A172, U251, and T98) exposed to hTRAIL (TRAIL, 30 ng/ml), TMZ (100 μmol/ml), or both (TR+TMZ) for 24 hours. The TUNEL labeling in A172 and U251 cells showed an increased number of TUNEL-positive cells after exposure to hTRAIL/TMZ. Apoptosis was not detected in T98 cell lines after hTRAIL/TMZ exposure.

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    Expression of death receptors in human malignant glioma cells; the left panels show Western blots and the right panels show quantified data in bar graphs. The A172, U251, and T98 cell lines were exposed to hTRAIL (30 ng/ml), TMZ (100 μmol/ml), or both for 24 hours. A: A significant increase in DR4 expression was seen after exposure to hTRAIL/TMZ in A172 and U251 cell lines. Data were normalized to actin. B: Expression of DR5 in a similar setting. The A172 cells showed a significant increase in expression of DR5 when treated with combined hTRAIL/TMZ. In U251 cells there was a trend toward an increase in DR5 expression after combination treatment, but this was not statistically significant. The T98 cells treated similarly showed no change in DR4 or DR5 expression. Asterisks denote statistical significance (p < 0.05, Student t-test). CON = control.

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    Expression of activated caspase-8 in A172, U251, and T98 cell lines. The A172, U251, and T98 cells were exposed to hTRAIL (30 ng/ml), TMZ (100 μmol/ml), or both. Left: Representative Western blot. Right: Quantified data in bar graph. A significant increase (*p < 0.05, Student t-test) in activated caspase-8 expression was seen after exposure to hTRAIL/TMZ in A172 and U251 cell lines. Data were normalized to actin. In T98 cells caspase activation was not detected.

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    Expression of caspase-3 in A172, U251, and T98 cell lines. The A172, U251, and T98 cells were exposed to hTRAIL (30 ng/ml), TMZ (100 μmol/ml), or both for 24 hours. A: Representative Western blot. B: Quantified data in bar graph. A significant increase (*p < 0.05, Student t-test) in caspase-3 expression was seen after exposure to hTRAIL/TMZ in A172 and U251 cell lines. Data were normalized to actin. In T98 cells, caspase-3 activation was not detected. C: The A172 cells were fractionated after exposure to a combination of hTRAIL/TMZ, and the mitochondrial fraction was labeled for voltage-dependent anion channel (VDAC). The same samples were probed for cytochrome c (Cyto-C), but no changes were detected.

References

1

Almasan AAshkenazi A: Apo2L/TRAIL: apoptosis signaling, biology and potential for cancer therapy. Cytokines Growth Factor Rev 14:3373482003

2

Ashkenazi APai RCFong SLeung SLawrence DAMarsters SA: Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 104:1551621999

3

Bower MNewlands ESBleehen NMBrada MBegent RJCalvert H: Multicentre CRC phase II trial of temozolomide in recurrent or progressive high-grade glioma. Cancer Chemother Pharmacol 40:4844881997

4

Grataroli RVindrieux DSelva JFelsenheld CRuffion ADecaussin M: Characterization of tumor necrosis factor-alpha-related apoptosis inducing ligand and its receptors in the adult human testis. Mol Human Reprod 10:1231282004

5

Hao CBeguinot FCondorelli GTrencia AVan Meir EGYong VW: Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells. Cancer Res 61:116211702001

6

Kagawa SHe CGu JKoch PRha SJRoth JA: Anti-tumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. Cancer Res 61:333033382001

7

Kanzawa TGermano IMKomata TIto HKondo YKondo S: Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ 11:4484572004

8

Kim JHAjaz MLokshin ALee YJ: Role of antiapoptotic proteins in tumor necrosis factor-related apoptosis-inducing ligand and cisplatin-augmented apoptosis. Clin Cancer Res 9:313431412003

9

Knight MJRiffkin CDMuscat AMAshley DMHawkins CJ: Analysis of FasL and TRAIL induced apoptosis pathways in glioma cells. Oncogene 20:578957982001

10

Nagane MPan GWeddle JJDixit VMCavenee WKHuang HJ: Increased death receptor 5 expression by chemotherapeutic agents in human gliomas causes synergistic cytotoxicity with tumor necrosis factor related apoptosis-inducing ligand in vitro and in vivo. Cancer Res 60:8478532000

11

Newlands ESStevens MFWedge SRWheelhouse RTBrock C: Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev 23:35611997

12

Pan GO'Rourke KChinnaiyan AMGentz REbner RNi J: The receptor for the cytotoxic ligand TRAIL. Science 276:1111131997

13

Pitti RMMarsters SARuppert SDonahue CJMoore AAshkenazi A: Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271:12687126901996

14

Rieger JNaumann UGlaser TAshkenazi AWeller M: APO2 ligand: a novel lethal weapon against malignant glioma?. FEBS Lett 427:1241281998

15

Shankar SSrivastava RK: Enhancement of therapeutic potential of TRAIL by cancer chemotherapy and irradiation: mechanisms and clinical implications. Drug Resist Update 7:1391562004

16

Sheridan JPMarstars SAPitti RMGurney ASkubatch MBaldwin D: Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277:8188211997

17

Stevens MFHickman JAStone RGibson NWBaig GULunt E: Antitumor imidazotetrazines. 1. Synthesis and chemistry of 8-carbamoyl-3–(2-chloroethyl)imidazo[5, 1d]-1, 2, 3, 5-tetrazin-4(3H)-one, a novel broad-spectrum antitumor agent. J Med Chem 27:1962011984

18

Stupp Rvan den Bent MJHegi ME: Optimal role of temozolomide in the treatment of malignant gliomas. Curr Neurol Neurosci Rep 5:1982062005

19

Surawicz TSDavis FFreels SLaws ER JrMenck HR: Brain tumor survival: results from the National Cancer Center Data Base. J Neurooncol 40:1511601998

20

Walczak HMiller REAriail KGliniak BGriffith TSKubin M: Tumoricidal activity of tumor necrosis factor-related apoptosis-induced ligand in vivo. Nat Med 5:1571631999

21

Wang SEl-Deiry WS: TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 22:862886332003

22

Wiley SRSchooley KSmolak PJDin WSHuang CPNicholl JK: Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3:6736821995

23

Yung WKPrados MDYaya-Tur RRosenfeld SSBrada MFriedman HS: Multicenter phase II trial of temozolomide in patients with anaplastic astrocytoma or anaplastic oligoastrocytoma at first relapse. Temodal Brain Tumor Group. J Clin Oncol 17:276227711999

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