Hammerhead ribozyme against γ-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins

Cancer Gene Therapy volume 8, pages803–814(2001)Cite this article

Abstract

Multidrug resistance in cancer cells is often associated with an elevation in the concentration of glutathione (GSH) and the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme for GSH. We constructed a hammerhead ribozyme against a γ-GCS heavy subunit (γ-GCSh) mRNA transcript and transfected it to human colonic cancer cells (HCT8DDP) resistant to cisplatin (CDDP). The effect of the ribozyme transfection on the drug resistance of cancer cells was studied. (a) Transfection of the ribozyme decreased the GSH level and the efflux of CDDP–GSH adduct, resulting in higher sensitivity of the cells to CDDP. (b) The transfection suppressed the expression of ATP-binding cassette (ABC) family of transporters such as MRP1, MRP2, and MDR1, and stimulated the expression of mutant p53. (c) An electrophoretic mobility shift assay showed that mutant p53 suppresses the SP1–DNA binding activity, suggesting that this mutant p53 is functional and it, in turn, suppresses the expression of ABC transporters. Collectively, transfection of anti–γ-GCSh ribozyme reduced the synthesis of GSH and the expression of ABC transporters, which causes an increase in the sensitivity of cancer cells to anticancer drugs. Suppression of the SP1–DNA binding activity by p53 may be a factor of down-regulation of ABC transporters. Cancer Gene Therapy (2001) 8, 803–814

Download PDF

References

  1. 1

    Meister A . Methods for the selective modification of glutathione metabolism and study of glutathione transport Methods Enzymol 1985 113: 571–585

    CAS  PubMed  Google Scholar 

  2. 2

    Kondo T, Yoshida K, Urata Y, et al . Gamma-glutamylcysteine synthetase and active transport of glutathione S-conjugate are responsive to heat shock in K562 erythroid cells J Biol Chem 1993 268: (27) 20366–20372

    CAS  PubMed  Google Scholar 

  3. 3

    Urata Y, Yamamoto H, Goto S, et al . Long exposure to high glucose concentration impairs the responsive expression of gamma-glutamylcysteine synthetase by interleukin-1 beta and tumor necrosis factor-alpha in mouse endothelial cells J Biol Chem 1996 271: (25) 15146–15152

    CAS  PubMed  Google Scholar 

  4. 4

    Beutler E, Gelbart T, Kondo T, et al . The molecular basis of a case of gamma-glutamylcysteine synthetase deficiency Blood 1999 94: (8) 2890–2894

    CAS  PubMed  Google Scholar 

  5. 5

    Yan N, Meister A . Amino acid sequence of rat kidney γ-glutamylcysteine synthetase J Biol Chem 1990 265: 1588–1593

    CAS  PubMed  Google Scholar 

  6. 6

    Iida T, Mori E, Mori K, et al . Co-expression of gamma-glutamylcysteine synthetase subunits in response to cisplatin and doxorubicin in human cancer cells Int J Cancer 1999 82: (3) 405–411

    CAS  PubMed  Google Scholar 

  7. 7

    Goto S, Yoshida K, Morikawa T, et al . Augmentation of transport for cisplatin–glutathione adduct in cisplatin-resistant cancer cells Cancer Res 1995 55: (19) 4297–4301

    CAS  PubMed  Google Scholar 

  8. 8

    Goto S, Iida T, Cho S, et al . Overexpression of glutathione S-transferase pi enhances the adduct formation of cisplatin with glutathione in human cancer cells Free Radical Res 1999 31: (6) 549–558

    CAS  Google Scholar 

  9. 9

    Godwin AK, Meister A, O'Dwyer PJ, et al . High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis Proc Natl Acad Sci USA 1992 89: (7) 3070–3074

    CAS  PubMed  Google Scholar 

  10. 10

    Yao KS, Godwin AK, Johnson SW, et al . Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines Cancer Res 1995 55: (19) 4367–4374

    CAS  PubMed  Google Scholar 

  11. 11

    Kuo MT, Bao J, Furuichi M, et al . Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in drug-resistant cells, untreated tumor cells, and normal mouse tissues Biochem Pharmacol 1998 55: (5) 605–615

    CAS  PubMed  Google Scholar 

  12. 12

    Ogretmen B, Bahadori HR, McCauley MD, et al . Co-ordinated over-expression of the MRP and gamma-glutamylcysteine synthetase genes, but not MDR1, correlates with doxorubicin resistance in human malignant mesothelioma cell lines Int J Cancer 1998 75: (5) 757–761

    CAS  PubMed  Google Scholar 

  13. 13

    Cole SP, Bhardwaj G, Gerlach JH, et al . Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line Science 1992 258: (5088) 1650–1654

    CAS  Google Scholar 

  14. 14

    Kool M, de Haas M, Scheffer GL, et al . Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance–associated protein gene (MRP1), in human cancer cell lines Cancer Res 1997 57: (16) 3537–3547

    CAS  Google Scholar 

  15. 15

    Hour TC, Chen J, Huang CY, et al . Characterization of chemoresistance mechanisms in a series of cisplatin-resistant transitional carcinoma cells lines Anticancer Res 2000 20: (5A) 3221–3225

    CAS  PubMed  Google Scholar 

  16. 16

    Borst P, Evers R, Kool M, et al . A family of drug transporters: the multidrug resistance–associated proteins J Natl Cancer Inst 2000 92: (16) 1295–1302

    CAS  PubMed  Google Scholar 

  17. 17

    Hinoshita E, Uchiumi T, Taguchi K, et al . Increased expression of an ATP-binding cassette superfamily transporter, multidrug resistance protein 2, in human colorectal carcinomas Clin Cancer Res 2000 6: (6) 2401–2407

    CAS  Google Scholar 

  18. 18

    Schrenk D, Baus PR, Ermel N, et al . Up-regulation of transporters of the MRP family by drugs and toxins Toxicol Lett 2001 120: (1–3) 51–57

    CAS  PubMed  Google Scholar 

  19. 19

    Materna V, Holm PS, Dietel M, et al . Kinetic characterization of ribozymes directed against the cisplatin resistance–associated ABC transporter cMOAT/MRP2/ABCC2 Cancer Gene Ther 2001 8: (3) 176–184

    CAS  PubMed  Google Scholar 

  20. 20

    Manna SK, Kuo MT, Aggarwal BB . Overexpression of gamma-glutamylcysteine synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappa B and activator protein-1 Oncogene 1999 18: 4371–4382

    CAS  PubMed  Google Scholar 

  21. 21

    Gomi A, Shinoda S, Masuzawa T, et al . Transient induction of the MRP/GS-X pump and gamma-glutamylcysteine synthetase by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea in human glioma cells Cancer Res 1997 57: (23) 5292–5299

    CAS  PubMed  Google Scholar 

  22. 22

    Yamane Y, Furuichi M, Song R, et al . Expression of multidrug resistance protein/GS-X pump and gamma-glutamylcysteine synthetase genes is regulated by oxidative stress J Biol Chem 1998 273: (47) 31075–31085

    CAS  PubMed  Google Scholar 

  23. 23

    Scanlon KJ, Ishida H, Kashani-Sabet M . Ribozyme-mediated reversal of the multidrug-resistant phenotype Proc Natl Acad Sci USA 1994 91: (23) 11123–11127

    CAS  PubMed  Google Scholar 

  24. 24

    Kashani-Sabet M, Funato T, Florenes VA, et al . Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme Cancer Res 1994 54: (4) 900–902

    CAS  PubMed  Google Scholar 

  25. 25

    Haseloff J, Gerlach WL . Simple RNA enzymes with new and highly specific endoribonuclease activities Nature 1988 334: (6183) 585–591

    CAS  Google Scholar 

  26. 26

    Scanlon KJ, Ohta Y, Ishida H, et al . Oligonucleotide-mediated modulation of mammalian gene expression FASEB J 1995 9: (13) 1288–1296

    CAS  PubMed  Google Scholar 

  27. 27

    Kijima H, Tsuchida T, Kondo H, et al . Hammerhead ribozymes against gamma-glutamylcysteine synthetase mRNA down-regulate intracellular glutathione concentration of mouse islet cells Biochem Biophys Res Commun 1998 247: (3) 697–703

    CAS  PubMed  Google Scholar 

  28. 28

    Beutler E . A manual of biochemical methods In: Beutler E, ed., Red Cell Metabolism, 3rd edn Orlando: Grune and Stratton 1984 72–136

  29. 29

    Abbaszadegan MR, Futscher BW, Klimecki WT, et al . Analysis of multidrug resistance–associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells Cancer Res 1994 54: (17) 4676–4679

    CAS  PubMed  Google Scholar 

  30. 30

    Narasaki F, Oka M, Nakano R, et al . Human canalicular multispecific organic anion transporter (cMOAT) is expressed in human lung, gastric, and colorectal cancer cells Biochem Biophys Res Commun 1997 240: (3) 606–611

    CAS  PubMed  Google Scholar 

  31. 31

    Narasaki F, Matsuo I, Ikuno N, et al . Multidrug resistance–associated protein (MRP) gene expression in human lung cancer Anticancer Res 1996 16: (4A) 2079–2082

    CAS  PubMed  Google Scholar 

  32. 32

    Wang Q, Beck WT . Transcriptional suppression of multidrug resistance–associated protein (MRP) gene expression by wild-type p53 Cancer Res 1998 58: (24) 5762–5769

    CAS  PubMed  Google Scholar 

  33. 33

    Sambrook J, Fritsch EF, Maniatis T . Molecular Cloning: A Laboratory Manual 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory 1989

    Google Scholar 

  34. 34

    Abmayr SM, Warkman JL . Current Protocols in Molecular Biology New York: Greene Publishing Associates and Wiley-Interscience 1987

    Google Scholar 

  35. 35

    Sen R, Baltimore D . Multiple nuclear factors interact with the immunoglobulin enhancer sequences Cell 1986 46: (5) 705–716

    CAS  PubMed  Google Scholar 

  36. 36

    Zhu Q, Center MS . Cloning and sequence analysis of the promoter region of the MRP gene of HL60 cells isolated for resistance to adriamycin Cancer Res 1994 54: (16) 4488–44892

    CAS  PubMed  Google Scholar 

  37. 37

    Grant CE, Valdimarsson G, Hipfner DR, et al . Overexpression of multidrug resistance–associated protein (MRP) increases resistance to natural product drugs Cancer Res 1994 54: (2) 357–361

    CAS  PubMed  Google Scholar 

  38. 38

    Redinbaugh MG, Turley RB . Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions Anal Biochem 1986 153: (2) 267–271

    CAS  PubMed  Google Scholar 

  39. 39

    Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays J Immunol Methods 1983 65: (1–2) 55–63

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Caserini C, Pratesi G, Tortoreto M, et al . Apoptosis as a determinant of tumor sensitivity to topotecan in human ovarian tumors: preclinical in vitro / in vivo studies Clin Cancer Res 1997 3: (6) 955–961

    CAS  PubMed  Google Scholar 

  41. 41

    Koike K, Kawabe T, Tanaka T, et al . A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells Cancer Res 1997 57: (24) 5475–5479

    CAS  PubMed  Google Scholar 

  42. 42

    Tipnis SR, Blake DG, Shepherd G, et al . Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance Biochem J 1999 337: 559–566

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Zhang K, Chew M, Yang EB, et al . Modulation of cisplatin cytotoxicity and cisplatin-induced DNA cross-links in HepG2 cells by regulation of glutathione-related mechanisms Mol Pharmacol 2001 59: (4) 837–843

    CAS  PubMed  Google Scholar 

  44. 44

    Hour TC, Chen J, Huang CY, et al . Characterization of chemoresistance mechanisms in a series of cisplatin-resistant transitional carcinoma cell lines Anticancer Res 2000 20: (5A) 3221–3225

    CAS  PubMed  Google Scholar 

  45. 45

    Loe DW, Deeley RG, Cole SP . Biology of the multidrug resistance–associated protein, MRP Eur J Cancer 1996 32A: (6) 945–957

    CAS  PubMed  Google Scholar 

  46. 46

    Ishikawa T, Bao JJ, Yamane Y, et al . Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells J Biol Chem 1996 271: (25) 14981–14988

    CAS  PubMed  Google Scholar 

  47. 47

    Kuo MT, Bao J, Furuichi M, et al . Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in drug-resistant cells, untreated tumor cells, and normal mouse tissues Biochem Pharmacol 1998 55: (5) 605–615

    CAS  PubMed  Google Scholar 

  48. 48

    Cornwell MM, Smith DE . SP1 activates the MDR1 promoter through one of two distinct G-rich regions that modulate promoter activity J Biol Chem 1993 268: (26) 19505–19511

    CAS  PubMed  Google Scholar 

  49. 49

    Zhu Q, Center MS . Evidence that SP1 modulates transcriptional activity of the multidrug resistance–associated protein gene DNA Cell Biol 1996 15: (2) 105–111

    CAS  PubMed  Google Scholar 

  50. 50

    Ohga T, Koike K, Ono M, et al . Role of the human Y box-binding protein YB-1 in cellular sensitivity to the DNA-damaging agents cisplatin, mitomycin C, and ultraviolet light Cancer Res 1996 56: (18) 4224–4228

    CAS  PubMed  Google Scholar 

  51. 51

    Ohga T, Uchiumi T, Makino Y, et al . Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene J Biol Chem 1998 273: (11) 5997–6000

    CAS  PubMed  Google Scholar 

  52. 52

    Yu L, Cohen D, Piekarz RL, et al . Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene J Biol Chem 1993 268: (10) 7520–7526

    CAS  PubMed  Google Scholar 

  53. 53

    Sullivan GF, Yang JM, Vassil A, et al . Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells J Clin Invest 2000 105: (9) 1261–1267

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Ishikawa T, Kuo MT, Furuta K, et al . The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design Clin Chem Lab Med 2000 38: (9) 893–897

    CAS  PubMed  Google Scholar 

  55. 55

    Torgeman A, Mor-Vaknin N, Zelin E, et al . SP1–p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12- O -tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C Virology 2001 281: (1) 10–20

    CAS  PubMed  Google Scholar 

  56. 56

    Levine AJ . p53, the cellular gatekeeper for growth and division Cell 1997 88: (3) 323–331

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Horikoshi N, Usheva A, Chen J, et al . Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression Mol Cell Biol 1995 15: (1) 227–234

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Lane DP, Benchimol S . p53: oncogene or anti-oncogene? Genes Dev 1990 4: (1) 1–8

    CAS  PubMed  Google Scholar 

  59. 59

    Nicoletti ML, Myers TG, Fojo T, et al . Wild-type p53 marginally induces endogenous MDR-1 mRNA without causing a measurable drug resistance in human cancer cells Int J Oncol 2001 18: (2) 375–381

    CAS  PubMed  Google Scholar 

  60. 60

    Hirose M, Kuroda Y . p53 may mediate the mdr-1 expression via the WT1 gene in human vincristine-resistant leukemia/lymphoma cell lines Cancer Lett 1998 129: (2) 165–171

    CAS  PubMed  Google Scholar 

  61. 61

    Galimberti S, Marchetti A, Buttitta F, et al . Multidrug resistance–related genes and p53 expression in human non small cell lung cancer Anticancer Res 1998 18: (4C) 2973–2976

    CAS  PubMed  Google Scholar 

  62. 62

    Bahr O, Wick W, Weller M . Modulation of MDR/MRP by wild type and mutant p53 J Clin Invest 2001 107: (5) 643–646

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Wartenberg M, Ling FC, Schallenberg M, et al . Down-regulation of intrinsic p-glycoprotein expression multicellular prostate tumor spheroids by reactive oxygen species J Biol Chem 2001 276: (20) 17420–17428

    CAS  PubMed  Google Scholar 

  64. 64

    Kirch HC, Flaswinkel S, Rumpf H, et al . Expression of human p53 requires synergistic activation of transcription from the p53 promoter by AP-1, NF-kappaB and Myc/Max Oncogene 1999 18: (17) 2728–2738

    CAS  PubMed  Google Scholar 

  65. 65

    Aruoma OI, Halliwell B (eds.) . Molecular Biology of Free Radicals in Human Diseases London: Oica International 1998

    Google Scholar 

  66. 66

    Cho S, Urata Y, Iida T, et al . Glutathione downregulates the phosphorylation of I kappa B: autoloop regulation of the NF-kappa B–mediated expression of NF-kappa B subunits by TNF-alpha in mouse vascular endothelial cells Biochem Biophys Res Commun 1998 253: (1) 104–108

    CAS  PubMed  Google Scholar 

  67. 67

    Kohno T, Yamada Y, Hata T, et al . Relation of oxidative stress and glutathione synthesis to CD95(Fas/APO-1)–mediated apoptosis of adult T cell leukemia cells J Immunol 1996 156: (12) 4722–4728

    CAS  PubMed  Google Scholar 

  68. 68

    Klatt P, Molina EP, De Lacoba MG, et al . Redox regulation of c-Jun DNA binding by reversible S-glutathiolation FASEB J 1999 13: (12) 1481–1490

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported, in part, by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.

Author information

Affiliations

  1. Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Nagasaki, 852-8523, Japan

    Tetsuya Iida, Yoshishige Urata, Shinji Goto, Yoshito Ihara & Takahito Kondo

  2. Department of Pathology, Tokai University School of Medicine, Isehara, 259-1193, Japan

    Hiroshi Kijima

  3. The Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, 852-8523, Japan

    Mikio Oka & Shigeru Kohno

  4. Cancer Research Department, Berlex Biosciences, Richmond, California, USA

    Kevin J Scanlon

Authors
  1. Tetsuya Iida
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroshi Kijima
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoshishige Urata
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shinji Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoshito Ihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mikio Oka
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shigeru Kohno
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kevin J Scanlon
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Takahito Kondo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takahito Kondo.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Iida, T., Kijima, H., Urata, Y. et al. Hammerhead ribozyme against γ-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins. Cancer Gene Ther 8, 803–814 (2001). https://doi.org/10.1038/sj.cgt.7700371

Download citation

Keywords

  • γ-Glutamylcysteine synthetase
  • hammerhead-ribozyme
  • glutathione
  • cisplatin
  • multidrug resistance protein-1 (MRP1)
  • p53

Further reading

Search

Quick links