Skip to main content
SpringerLink
Log in

TRAIL induces autophagic protein cleavage through caspase activation in melanoma cell lines under arginine deprivation

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

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

  1. Bhatia S, Tykodi SS, Thompson JA (2009) Treatment of metastatic melanoma: an overview. Oncology (Williston Park) 23(6):488–496

    Google Scholar 

  2. 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

    Article  PubMed  CAS  Google Scholar 

  3. Feun L, Savaraj N (2006) Pegylated arginine deiminase: a novel anticancer enzyme agent. Expert Opin Investig Drugs 15(7):815–822

    Article  PubMed  CAS  Google Scholar 

  4. 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

    Article  PubMed  CAS  Google Scholar 

  5. 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

    PubMed  CAS  Google Scholar 

  6. 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

    Article  PubMed  CAS  Google Scholar 

  7. 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

    Article  PubMed  CAS  Google Scholar 

  8. 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

    Article  PubMed  CAS  Google Scholar 

  9. 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

    Article  PubMed  CAS  Google Scholar 

  10. 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

    Article  PubMed  CAS  Google Scholar 

  11. 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

    PubMed  Google Scholar 

  12. 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

    Google Scholar 

  13. Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3(6):542–545

    PubMed  CAS  Google Scholar 

  14. Shvets E, Fass E, Elazar Z (2008) Utilizing flow cytometry to monitor autophagy in living mammalian cells. Autophagy 4(5):621–628

    PubMed  CAS  Google Scholar 

  15. 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

    Article  PubMed  CAS  Google Scholar 

  16. 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

    Article  PubMed  CAS  Google Scholar 

  17. 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

    Article  PubMed  CAS  Google Scholar 

  18. 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

    Article  PubMed  CAS  Google Scholar 

  19. 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

    Article  PubMed  CAS  Google Scholar 

  20. 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

    Article  PubMed  CAS  Google Scholar 

  21. 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

    Article  PubMed  CAS  Google Scholar 

  22. 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

    Article  PubMed  CAS  Google Scholar 

  23. Mizushima N, Klionsky DJ (2007) Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 27:19–40

    Article  PubMed  CAS  Google Scholar 

  24. 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

    Article  PubMed  CAS  Google Scholar 

  25. 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

  26. Levine B, Yuan J (2005) Autophagy in cell death: an innocent convict? J Clin Invest 115(10):2679–2688. doi:10.1172/JCI26390

    Article  PubMed  CAS  Google Scholar 

  27. Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 306(5698):990–995. doi:10.1126/science.1099993

    Article  PubMed  CAS  Google Scholar 

  28. Cao Y, Klionsky DJ (2007) Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res 17(10):839–849

    Article  PubMed  CAS  Google Scholar 

  29. 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

    Google Scholar 

  30. Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132(1):27–42

    Article  PubMed  CAS  Google Scholar 

  31. 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

    Article  PubMed  CAS  Google Scholar 

  32. 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

    Article  PubMed  CAS  Google Scholar 

  33. 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

    Article  PubMed  CAS  Google Scholar 

  34. 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

    PubMed  Google Scholar 

  35. 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

    Article  PubMed  CAS  Google Scholar 

  36. 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

    Google Scholar 

Download references

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

  1. Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, 1475 NW 12th Avenue, Miami, FL, 33136, USA

    Min You, Niramol Savaraj, Javier Varona-Santos, Dao M. Nguyen & Lynn Feun

  2. Division of Hematology and Oncology, Miami Veterans Affairs Healthcare System, 1201 NW 16th Street, Miami, FL, 33125, USA

    Niramol Savaraj, Medhi Wangpaichitr & Chunjing Wu

  3. Department of Molecular Pathology, M.D. Anderson Cancer Center, University of Texas, 7435 Fannin Street, Houston, TX, 77054, USA

    Macus T. Kuo

  4. Department of Surgery, University of Miami Miller School of Medicine, 1120 NW 14th Street, Miami, FL, 33136, USA

    Medhi Wangpaichitr & Dao M. Nguyen

Authors
  1. Min You
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Niramol Savaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Macus T. Kuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Medhi Wangpaichitr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Javier Varona-Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chunjing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dao M. Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lynn Feun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Niramol Savaraj.

Rights and permissions

Reprints 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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-012-1518-1

Keywords

Search

Navigation