Abstract
Arginine deprivation is a promising strategy for treating ASS-negative malignant tumors including melanoma. However, autophagy can potentially counteract the effectiveness of this treatment by acting as a pro-survival pathway. By combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with arginine deprivation using ADI-PEG20 (pegylated arginine deiminase), we achieved enhanced apoptosis and accelerated cell death in melanoma cell lines. This implies a switch from autophagy to apoptosis. In our current investigation, we found that TRAIL could induce the cleavage of two key autophagic proteins, Beclin-1 and Atg5, in the combination treatment. Using specific inhibitors for individual caspases, we found that caspase-8 inhibitor could completely abolish the cleavage. Furthermore, caspase-8 inhibitor was able to fully reverse the enhanced cytotoxicity induced by TRAIL. Inhibitors for caspase-3, 6, 9, and 10 were able to block the cleavage of these two autophagic proteins to some extent and correspondingly rescue cells from the cytotoxicity of the combination of TRAIL and arginine deprivation. In contrast, calpain inhibitor could not prevent the cleavage of either Beclin-1 or Atg5, and was unable to prevent cell death. Overall, our data indicate that the cleavage of Beclin-1 and Atg5 by TRAIL-initiated caspase activation is one of the mechanisms that lead to the enhancement of the cytotoxicity in the combination treatment.
Similar content being viewed by others
Abbreviations
- ADI:
-
Arginine deiminase
- ASS:
-
Argininosuccinate synthetase
- DMSO:
-
Dimethyl sulfoxide
- DPBS:
-
Dulbecco’s phosphate-buffered saline
- FBS:
-
Fetal bovine serum
- FITC:
-
Fluorescein isothiocyanate
- HIF1α :
-
Hypoxia-inducible factor 1 alpha
- kDa:
-
Kilodalton
- MEM:
-
Minimum essential media
- PEG:
-
Polyethylene glycol
- PI:
-
Propidium iodide
- PVDF:
-
Polyvinylidene fluoride
- SD:
-
Standard deviation
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- siRNA:
-
Small interfering ribonucleic acid
- TBS:
-
Tris(hydroxymethyl)aminomethane-buffered saline
- TRAIL:
-
Tumor necrosis factor-related apoptosis-inducing ligand
References
Bhatia S, Tykodi SS, Thompson JA (2009) Treatment of metastatic melanoma: an overview. Oncology (Williston Park) 23(6):488–496
Ascierto PA, Scala S, Castello G, Daponte A, Simeone E, Ottaiano A, Beneduce G, De Rosa V, Izzo F, Melucci MT, Ensor CM, Prestayko AW, Holtsberg FW, Bomalaski JS, Clark MA, Savaraj N, Feun LG, Logan TF (2005) Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies. J Clin Oncol 23(30):7660–7668
Feun L, Savaraj N (2006) Pegylated arginine deiminase: a novel anticancer enzyme agent. Expert Opin Investig Drugs 15(7):815–822
Takaku H, Takase M, Abe S, Hayashi H, Miyazaki K (1992) In vivo anti-tumor activity of arginine deiminase purified from Mycoplasma arginini. Int J Cancer 51(2):244–249
Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA (2002) Pegylated arginine deiminase (ADI-SS PEG20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo. Cancer Res 62(19):5443–5450
Wheatley DN, Campbell E (2003) Arginine deprivation, growth inhibition and tumour cell death: 3. Deficient utilisation of citrulline by malignant cells. Br J Cancer 89(3):573–576
Feun LG, Marini A, Walker G, Elgart G, Moffat F, Rodgers SE, Wu CJ, You M, Wangpaichitr M, Kuo MT, Sisson W, Jungbluth AA, Bomalaski J, Savaraj N (2012) Negative argininosuccinate synthetase expression in melanoma tumours may predict clinical benefit from arginine-depleting therapy with pegylated arginine deiminase. Br J Cancer 106:1481–1485
Feun L, You M, Wu CJ, Kuo MT, Wangpaichitr M, Spector S, Savaraj N (2008) Arginine deprivation as a targeted therapy for cancer. Curr Pharm Des 14(11):1049–1057
You M, Savaraj N, Wangpaichitr M, Wu C, Kuo MT, Varona-Santos J, Nguyen DM, Feun L (2010) The combination of ADI-PEG20 and TRAIL effectively increases cell death in melanoma cell lines. Biochem Biophys Res Commun 394(3):760–766. doi:10.1016/j.bbrc.2010.03.066
Tsai WB, Aiba I, Lee SY, Feun L, Savaraj N, Kuo MT (2009) Resistance to arginine deiminase treatment in melanoma cells is associated with induced argininosuccinate synthetase expression involving c-Myc/HIF-1alpha/Sp4. Mol Cancer Ther 8(12):3223–3233. doi:10.1158/1535-7163.MCT-09-0794
Savaraj N, Wu C, Kuo M, You M, Wangpaichitr M, Robles C, Spector S, Feun L (2007) The relationship of arginine deprivation, argininosuccinate synthetase and cell death in melanoma. Drug Target Insights 2:119–128
Huang KT, Chen YH, Walker AM (2004) Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids. Biotechniques 37 (3):406, 408, 410–402
Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3(6):542–545
Shvets E, Fass E, Elazar Z (2008) Utilizing flow cytometry to monitor autophagy in living mammalian cells. Autophagy 4(5):621–628
Luo S, Rubinsztein DC (2010) Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL. Cell Death Differ 17(2):268–277. doi:10.1038/cdd.2009.121
Bhutia SK, Dash R, Das SK, Azab B, Su ZZ, Lee SG, Grant S, Yacoub A, Dent P, Curiel DT, Sarkar D, Fisher PB (2010) Mechanism of autophagy to apoptosis switch triggered in prostate cancer cells by antitumor cytokine melanoma differentiation-associated gene 7/interleukin-24. Cancer Res 70(9):3667–3676. doi:10.1158/0008-5472.CAN-09-3647
Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I, Roelandt R, De Rycke R, Verspurten J, Declercq W, Agostinis P, Vanden Berghe T, Lippens S, Vandenabeele P (2010) Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis 1(1):e18. doi:10.1038/cddis.2009.16
Cho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim JC (2009) Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer Lett 274(1):95–100. doi:10.1016/j.canlet.2008.09.004
Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L, Brunner T, Simon HU (2006) Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 8(10):1124–1132. doi:10.1038/ncb1482
Li H, Wang P, Sun Q, Ding WX, Yin XM, Sobol RW, Stolz DB, Yu J, Zhang L (2011) Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase 8-mediated cleavage of Beclin 1. Cancer Res 71(10):3625–3634. doi:10.1158/0008-5472.CAN-10-4475
Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, Jin H, Xu H, Chen Q (2010) Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein Cell 1(5):468–477. doi:10.1007/s13238-010-0048-4
Xia HG, Zhang L, Chen G, Zhang T, Liu J, Jin M, Ma X, Ma D, Yuan J (2010) Control of basal autophagy by calpain1 mediated cleavage of ATG5. Autophagy 6(1):61–66
Mizushima N, Klionsky DJ (2007) Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 27:19–40
Huang J, Lam GY, Brumell JH (2011) Autophagy signaling through reactive oxygen species. Antioxid Redox Signal 14(11):2215–2231. doi:10.1089/ars.2010.3554
O’Donovan TR, O’Sullivan GC, McKenna SL Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics. Autophagy 7 (5):509–524
Levine B, Yuan J (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115(10):2679–2688. doi:10.1172/JCI26390
Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 306(5698):990–995. doi:10.1126/science.1099993
Cao Y, Klionsky DJ (2007) Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res 17(10):839–849
Savaraj N, You M, Wu C, Wangpaichitr M, Kuo MT, Feun LG (2010) Arginine deprivation, autophagy, apoptosis (AAA) for the treatment of melanoma. Curr Mol Med 10 (4):405–412
Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132(1):27–42
Martinez-Outschoorn UE, Pavlides S, Whitaker-Menezes D, Daumer KM, Milliman JN, Chiavarina B, Migneco G, Witkiewicz AK, Martinez-Cantarin MP, Flomenberg N, Howell A, Pestell RG, Lisanti MP, Sotgia F (2010) Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors. Cell Cycle 9(12):2423–2433
Dillon BJ, Prieto VG, Curley SA, Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA (2004) Incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation. Cancer 100(4):826–833
Szlosarek PW, Klabatsa A, Pallaska A, Sheaff M, Smith P, Crook T, Grimshaw MJ, Steele JP, Rudd RM, Balkwill FR, Fennell DA (2006) In vivo loss of expression of argininosuccinate synthetase in malignant pleural mesothelioma is a biomarker for susceptibility to arginine depletion. Clin Cancer Res 12(23):7126–7131. doi:10.1158/1078-0432.CCR-06-1101
Curley SA, Bomalaski JS, Ensor CM, Holtsberg FW, Clark MA (2003) Regression of hepatocellular cancer in a patient treated with arginine deiminase. Hepatogastroenterology 50(53):1214–1216
Kim RH, Coates JM, Bowles TL, McNerney GP, Sutcliffe J, Jung JU, Gandour-Edwards R, Chuang FY, Bold RJ, Kung HJ (2009) Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent apoptosis. Cancer Res 69(2):700–708
Nicholson LJ, Smith PR, Hiller L, Szlosarek PW, Kimberley C, Sehouli J, Koensgen D, Mustea A, Schmid P, Crook T (2009) Epigenetic silencing of argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotrophy in ovarian cancer. Int J Cancer 125(6):1454–1463
Acknowledgments
We thank Polaris, Inc. for providing ADI-PEG20 for our investigation. This study was supported by grants from the National Institutes of Health R01CA109578 and R01CA152197.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
You, M., Savaraj, N., Kuo, M.T. et al. TRAIL induces autophagic protein cleavage through caspase activation in melanoma cell lines under arginine deprivation. Mol Cell Biochem 374, 181–190 (2013). https://doi.org/10.1007/s11010-012-1518-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-012-1518-1