Skip to main content
Log in

Synergistic Effects of Arsenic Trioxide and Silibinin on Apoptosis and Invasion in Human Glioblastoma U87MG Cell Line

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

Patients with glioblastoma multiforme (GBM) have poor therapeutic outcomes despite their current therapy. In an attempt to increase the efficacy of therapy for GBM, we studied the efficacy of arsenic trioxide (ATO), a newly introduced treatment for glioma, combined with silibinin, a natural polyphenolic flavonoid, in the GBM cell line, U87MG. The combination therapy synergically inhibited metabolic activity, cell proliferation, and gelatinase A and B activities; it also increased apoptosis. Additionally, it decreased the mRNA level of cathepsin B, uPA, matrix metalloproteinase-2 and 9, membrane type 1-MMP, survivin, BCL2, CA9; it increased mRNA level of caspase-3. Altogether, these results showed that ATO and silibinin in some cases improved and/or complemented the anticancer effects. This study may supply insight into the design of new combination cancer therapies to cells intrinsically less sensitive to routine therapies and suggested a new combination therapy for the highly invasive human glioma treatment.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A et al (2007) Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev 21:2683–2710

    Article  PubMed  CAS  Google Scholar 

  2. Khasraw M, Lassman AB (2010) Advances in the treatment of malignant gliomas. Curr Oncol Rep 12:26–33

    Article  PubMed  CAS  Google Scholar 

  3. Clarke J, Butowski N, Chang S (2010) Recent advances in therapy for glioblastoma. Arch Neurol 67:279–283

    Article  PubMed  Google Scholar 

  4. Lamszus K, Kunkel P, Westphal M (2003) Invasion as limitation to anti-angiogenic glioma therapy. Acta Neurochir Suppl 88:169–177

    PubMed  CAS  Google Scholar 

  5. Cheng L, Bao S, Rich JN (2010) Potential therapeutic implications of cancer stem cells in glioblastoma. Biochem Pharmacol 80:654–665

    Article  PubMed  CAS  Google Scholar 

  6. Ghavamzadeh A, Alimoghaddam K, Ghaffari SH, Rostami S, Jahani M, Hosseini R et al (2006) Treatment of acute promyelocytic leukemia with arsenic trioxide without ATRA and/or chemotherapy. Ann Oncol 17:131–134

    Article  PubMed  CAS  Google Scholar 

  7. Ning S, Knox SJ (2004) Increased cure rate of glioblastoma using concurrent therapy with radiotherapy and arsenic trioxide. Int J Radiat Oncol Biol Phys 60:197–203

    Article  PubMed  CAS  Google Scholar 

  8. Dilda PJ, Hogg PJ (2007) Arsenical-based cancer drugs. Cancer Treat Rev 33:542–564

    Article  PubMed  CAS  Google Scholar 

  9. Zhao S, Tsuchida T, Kawakami K, Shi C, Kawamoto K (2002) Effect of As2O3 on cell cycle progression and cyclins D1 and B1 expression in two glioblastoma cell lines differing in p53 status. Int J Oncol 21:49–55

    PubMed  Google Scholar 

  10. Carre M, Carles G, Andre N, Douillard S, Ciccolini J, Briand C et al (2002) Involvement of microtubules and mitochondria in the antagonism of arsenic trioxide on paclitaxel-induced apoptosis. Biochem Pharmacol 63:1831–1842

    Article  PubMed  CAS  Google Scholar 

  11. Sturlan S, Baumgartner M, Roth E, Bachleitner-Hofmann T (2003) Docosahexaenoic acid enhances arsenic trioxide-mediated apoptosis in arsenic trioxide-resistant HL-60 cells. Blood 101:4990–4997

    Article  PubMed  CAS  Google Scholar 

  12. Deep G, Agarwal R (2010) Antimetastatic efficacy of silibinin: molecular mechanisms and therapeutic potential against cancer. Cancer Metastasis Rev 29:447–463

    Article  PubMed  CAS  Google Scholar 

  13. Flaig TW, Glode M, Gustafson D, van Bokhoven A, Tao Y, Wilson S et al (2010) A study of high-dose oral silybin-phytosome followed by prostatectomy in patients with localized prostate cancer. Prostate 70:848–855

    PubMed  CAS  Google Scholar 

  14. Flaig TW, Gustafson DL, Su LJ, Zirrolli JA, Crighton F, Harrison GS et al (2007) A phase I and pharmacokinetic study of silybin-phytosome in prostate cancer patients. Invest New Drugs 25:139–146

    Article  PubMed  CAS  Google Scholar 

  15. Hoh C, Boocock D, Marczylo T, Singh R, Berry DP, Dennison AR et al (2006) Pilot study of oral silibinin, a putative chemopreventive agent, in colorectal cancer patients: silibinin levels in plasma, colorectum, and liver and their pharmacodynamic consequences. Clin Cancer Res 12:2944–2950

    Article  PubMed  CAS  Google Scholar 

  16. Momeny M, Malehmir M, Zakidizaji M, Ghasemi R, Ghadimi H, Shokrgozar M et al (2010) Silibinin inhibits invasive properties of human glioblastoma U87MG cells through suppression of cathepsin B, nuclear factor kappa B-mediated induction of matrix metalloproteinase 9. Anti-Cancer Drugs 21:252

    Article  PubMed  CAS  Google Scholar 

  17. Gazitt Y, Akay C (2005) Arsenic trioxide: an anti cancer missile with multiple warheads. Hematology 10:205–213

    Article  PubMed  CAS  Google Scholar 

  18. Carney DA (2008) Arsenic trioxide mechanisms of action–looking beyond acute promyelocytic leukemia. Leuk Lymphoma 49:1846–1851

    Article  PubMed  CAS  Google Scholar 

  19. Ramasamy K, Agarwal R (2008) Multitargeted therapy of cancer by silymarin. Cancer Lett 269:352–362

    Article  PubMed  CAS  Google Scholar 

  20. Chou TC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58:621–681

    Article  PubMed  CAS  Google Scholar 

  21. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C (2006) Clonogenic assay of cells in vitro. Nat Protoc 1:2315–2319

    Article  PubMed  CAS  Google Scholar 

  22. Hawkes SP, Li H, Taniguchi GT (2010) Zymography and reverse zymography for detecting MMPs and TIMPs. Methods Mol Biol 622:257–269

    Article  PubMed  CAS  Google Scholar 

  23. Schmittgen T, Livak K (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108

    Article  PubMed  CAS  Google Scholar 

  24. Belloc F, Dumain P, Boisseau MR, Jalloustre C, Reiffers J, Bernard P et al (1994) A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells. Cytometry 17:59–65

    Article  PubMed  CAS  Google Scholar 

  25. Shen Z, Chen G, Ni J, Li X, Xiong S, Qiu Q et al (1997) Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy, pharmacokinetics in relapsed patients. Blood 89:3354

    PubMed  CAS  Google Scholar 

  26. Chen G, Shi X, Tang W, Xiong S, Zhu J, Cai X et al (1997) Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells. Blood 89:3345

    PubMed  CAS  Google Scholar 

  27. Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I (2003) Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res 63:2103–2108

    PubMed  CAS  Google Scholar 

  28. Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA et al (2003) Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 22:8271–8282

    Article  PubMed  CAS  Google Scholar 

  29. Mohamed MM, Sloane BF (2006) Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer 6:764–775

    Article  PubMed  CAS  Google Scholar 

  30. Vasiljeva O, Turk B (2008) Dual contrasting roles of cysteine cathepsins in cancer progression: apoptosis versus tumour invasion. Biochimie 90:380–386

    Article  PubMed  CAS  Google Scholar 

  31. Chwieralski C, Welte T, Bühling F (2006) Cathepsin-regulated apoptosis. Apoptosis 11:143–149

    Article  PubMed  CAS  Google Scholar 

  32. Pucer A, Castino R, Mirkovic B, Falnoga I, Slejkovec Z, Isidoro C et al (2010) Differential role of cathepsins B and L in autophagy-associated cell death induced by arsenic trioxide in U87 human glioblastoma cells. Biol Chem 391:519–531

    Article  PubMed  CAS  Google Scholar 

  33. Podgorski I, Sloane BF (2003) Cathepsin B and its role(s) in cancer progression. Biochem Soc Symp 70:263–276

    PubMed  CAS  Google Scholar 

  34. Sloane BF, Yan S, Podgorski I, Linebaugh BE, Cher ML, Mai J et al (2005) Cathepsin B and tumor proteolysis: contribution of the tumor microenvironment. Semin Cancer Biol 15:149–157

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  36. Kroemer G, Jaattela M (2005) Lysosomes and autophagy in cell death control. Nat Rev Cancer 5:886–897

    Article  PubMed  CAS  Google Scholar 

  37. Weller M, Malipiero U, Aguzzi A, Reed JC, Fontana A (1995) Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation. J Clin Invest 95:2633–2643

    Article  PubMed  CAS  Google Scholar 

  38. Fels C, Schafer C, Huppe B, Bahn H, Heidecke V, Kramm CM et al (2000) Bcl-2 expression in higher-grade human glioma: a clinical and experimental study. J Neurooncol 48:207–216

    Article  PubMed  CAS  Google Scholar 

  39. Wick W, Wagner S, Kerkau S, Dichgans J, Tonn JC, Weller M (1998) BCL-2 promotes migration and invasiveness of human glioma cells. FEBS Lett 440:419–424

    Article  PubMed  CAS  Google Scholar 

  40. Chakravarti A, Zhai G, Zhang M, Malhotra R, Latham D, Delaney M et al (2004) Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms. Oncogene 23:7494–7506

    Article  PubMed  CAS  Google Scholar 

  41. Shirai K, Suzuki Y, Oka K, Noda SE, Katoh H, Itoh J et al (2009) Nuclear survivin expression predicts poorer prognosis in glioblastoma. J Neurooncol 91:353–358

    Article  PubMed  CAS  Google Scholar 

  42. Cardone RA, Casavola V, Reshkin SJ (2005) The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer 5:786–795

    Article  PubMed  CAS  Google Scholar 

  43. Brahimi-Horn MC, Chiche J, Pouyssegur J (2007) Hypoxia and cancer. J Mol Med 85:1301–1307

    Article  PubMed  Google Scholar 

  44. Swietach P, Vaughan-Jones RD, Harris AL (2007) Regulation of tumor pH and the role of carbonic anhydrase 9. Cancer Metastasis Rev 26:299–310

    Article  PubMed  CAS  Google Scholar 

  45. Martinez-Zaguilan R, Seftor EA, Seftor RE, Chu YW, Gillies RJ, Hendrix MJ (1996) Acidic pH enhances the invasive behavior of human melanoma cells. Clin Exp Metastasis 14:176–186

    Article  PubMed  CAS  Google Scholar 

  46. Chiang Y, Chou CY, Hsu KF, Huang YF, Shen MR (2008) EGF upregulates Na+/H+ exchanger NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness. J Cell Physiol 214:810–819

    Article  PubMed  CAS  Google Scholar 

  47. Kim J, Shin H, Kim T, Cho K, Shin K, Kim B et al (2006) Tumor-associated carbonic anhydrases are linked to metastases in primary cervical cancer. J Cancer Res Clin Oncol 132:302–308

    Article  PubMed  CAS  Google Scholar 

  48. McLean LA, Roscoe J, Jorgensen NK, Gorin FA, Cala PM (2000) Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes. Am J Physiol Cell Physiol 278:C676–C688

    PubMed  CAS  Google Scholar 

  49. Said H, Staab A, Hagemann C, Vince G, Katzer A, Flentje M et al (2007) Distinct patterns of hypoxic expression of carbonic anhydrase IX (CA IX) in human malignant glioma cell lines. J Neurooncol 81:27–38

    Article  PubMed  CAS  Google Scholar 

  50. Said H, Hagemann C, Staab A, Stojic J, Kühnel S, Vince G et al (2007) Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF, HIF-1alpha in human glioma in vitro, in vivo. Radiother Oncol J Eur Soc Therap Radiol Oncol 83:398

    Article  CAS  Google Scholar 

  51. Madshus IH (1988) Regulation of intracellular pH in eukaryotic cells. Biochem J 250:1–8

    PubMed  CAS  Google Scholar 

  52. Nakada M, Okada Y, Yamashita J (2003) The role of matrix metalloproteinases in glioma invasion. Front Biosci 8:e261–e269

    Article  PubMed  CAS  Google Scholar 

  53. Wang M, Wang T, Liu S, Yoshida D, Teramoto A (2003) The expression of matrix metalloproteinase-2 and-9 in human gliomas of different pathological grades. Brain Tumor Pathology 20:65–72

    Article  PubMed  Google Scholar 

  54. Sawaya R, Yamamoto M, Gokaslan Z, Wang S, Mohanam S, Fuller G et al (1996) Expression and localization of 72 kDa type IV collagenase (MMP-2) in human malignant gliomas in vivo. Clin Exp Metastasis 14:35–42

    Article  PubMed  CAS  Google Scholar 

  55. Rao J, Yamamoto M, Mohaman S, Gokaslan Z, Fuller G, Stetler-Stevenson W et al (1996) Expression and localization of 92 kDa type IV collagenase/gelatinase B (MMP-9) in human gliomas. Clin Exp Metastasis 14:12–18

    Article  PubMed  CAS  Google Scholar 

  56. Lin TH, Kuo HC, Chou FP, Lu FJ (2008) Berberine enhances inhibition of glioma tumor cell migration, invasiveness mediated by arsenic trioxide. BMC Cancer 8:58

    Article  PubMed  CAS  Google Scholar 

  57. Belien AT, Paganetti PA, Schwab ME (1999) Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter. J Cell Biol 144:373–384

    Article  PubMed  CAS  Google Scholar 

  58. Forsyth PA, Wong H, Laing TD, Rewcastle NB, Morris DG, Muzik H et al (1999) Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas. Br J Cancer 79:1828–1835

    Article  PubMed  CAS  Google Scholar 

  59. Rao JS (2003) Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 3:489–501

    Article  PubMed  CAS  Google Scholar 

  60. Lakka SS, Bhattacharya A, Mohanam S, Boyd D, Rao JS (2001) Regulation of the uPA gene in various grades of human glioma cells. Int J Oncol 18:71–79

    PubMed  CAS  Google Scholar 

  61. Gondi CS, Lakka SS, Yanamandra N, Siddique K, Dinh DH, Olivero WC et al (2003) Expression of antisense uPAR and antisense uPA from a bicistronic adenoviral construct inhibits glioma cell invasion, tumor growth, and angiogenesis. Oncogene 22:5967–5975

    Article  PubMed  CAS  Google Scholar 

  62. Gondi CS, Lakka SS, Dinh DH, Olivero WC, Gujrati M, Rao JS (2004) Downregulation of uPA, uPAR and MMP-9 using small, interfering, hairpin RNA (siRNA) inhibits glioma cell invasion, angiogenesis and tumor growth. Neuron Glia Biol 1:165–176

    Article  PubMed  Google Scholar 

  63. Chen PN, Hsieh YS, Chiang CL, Chiou HL, Yang SF, Chu SC (2006) Silibinin inhibits invasion of oral cancer cells by suppressing the MAPK pathway. J Dent Res 85:220–225

    Article  PubMed  CAS  Google Scholar 

  64. Chu SC, Chiou HL, Chen PN, Yang SF, Hsieh YS (2004) Silibinin inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Mol Carcinog 40:143–149

    Article  PubMed  CAS  Google Scholar 

  65. Hsieh YS, Chu SC, Yang SF, Chen PN, Liu YC, Lu KH (2007) Silibinin suppresses human osteosarcoma MG-63 cell invasion by inhibiting the ERK-dependent c-Jun/AP-1 induction of MMP-2. Carcinogenesis 28:977–987

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

The authors would like to acknowledge Majid Momeny for his assistances and advices. This study was supported by a grant from Hematology, Oncology and stem cell therapy, Tehran University of Medical Sciences, Tehran, Iran.

Conflict of interest

None declared.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Seyed H. Ghaffari.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dizaji, M.Z., Malehmir, M., Ghavamzadeh, A. et al. Synergistic Effects of Arsenic Trioxide and Silibinin on Apoptosis and Invasion in Human Glioblastoma U87MG Cell Line. Neurochem Res 37, 370–380 (2012). https://doi.org/10.1007/s11064-011-0620-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-011-0620-1

Keywords

Navigation