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Carcinogenesis response modulation induced by gelonin encapsulated in liposome

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Abstract

The effectiveness of gelonin to arrest protein synthesis, thereby limiting the growth of cancer cells was studied by encapsulating it into liposomes. The protein was extracted from the seeds of Indian plant Gelonium multiflorum by ammonium sulfate precipitation and purified using cation-exchange and gel-filtration chromatography. Biological activity of purified gelonin was determined using a rabbit reticulocyte lysate assay in the cell-free translational experiments. Gelonin was encapsulated in conventional liposomes prepared by the dry film method in order to retain biological activity of the entrapped protein. Carcinogenesis was induced in Swiss albino mice by intravenous administration of DBN (10 mg kg−1 body weight) at weekly intervals. Marker enzyme assays (GGT, AChE, and GST), GSH levels, cell proliferation assay, hepatocyte DNA analysis, histological examination of micro sections of liver tissues were parameters used to monitor carcinogenesis induction, and regression in mice. From the in vitro experiments conducted, it was observed that gelonin upon its encapsulation into liposome, resulted in significant destruction of the transformed liver cells by its cytotoxic effects that arrest protein synthesis. Various parameters studied to monitor regression also suggested mass cell destruction to liver upon administration of liposomal gelonin in mice exposed to DBN.

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Abbreviations

DBN:

N,N nitrosodibutylamine

GGT:

γ-Glutamyl transpeptidase

AchE:

Acetylcholine esterase

GSH:

Glutathione (reduced)

GST:

Glutathione-S-transferase

RIP:

Ribosome-inactivating protein

ED:

Effective dose

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

RES:

Reticulo-endothelial system

FCS:

Fetal Calf Serum

DMEM:

Dulbecco’s Modified Eagle Medium

DPPC:

Dipalmitoylphosphatidylcholine

Chol:

Cholesterol

PC:

Phosphatidylcholine

PE:

Phosphatidyl ethanolamine

PS:

Phosphatidyl serine

PG:

Phosphatidyl glycerol

PEG:

Polyethylene glycol

BrdU:

Bromodeoxyuridine

References

  1. Barbeieri L, Battelli MG, Stripe F (1993) Ribosome-inactivating proteins from plants. Biochim Biophys Acta 1154:237–282

    Google Scholar 

  2. Nicolas E, Beggs JM, Haltiwanger BM, Taraschi TF (1997) Direct evidence for the deoxyribonuclease activity of the plant ribosome inactivating protein gelonin. FEBS Lett 406:162–164. doi:10.1016/S0014-5793(97)00267-6

    Article  PubMed  CAS  Google Scholar 

  3. Gao W, Ling J, Zhong X, Liu W, Zhang R, Yang H et al (1994) Luffin S–a small novel ribosome-inactivating protein from Luffa cylindrical. Characterization and mechanism studies. FEBS Lett 347:257–260. doi:10.1016/0014-5793(94)00554-0

    Article  PubMed  CAS  Google Scholar 

  4. Dallal JA, Ivin JD (1978) Enzymatic inactivation of eukaryotic ribosomes by the pokeweed antiviral protein. FEBS Lett 89:257–259. doi:10.1016/0014-5793(78)80230-0

    Article  PubMed  CAS  Google Scholar 

  5. Barbeiri L, Stripe F (1982) Ribosome-inactivating proteins from plants: properties and possible uses. Cancer Surv 1:489–520

    Google Scholar 

  6. Jiminez A, Andvasquez D (1985) Plant and fungal protein and glycoprotein toxins inhibiting eukaryote protein synthesis. Annu Rev Microbiol 39:649–672

    Google Scholar 

  7. Lord JM, Hartely MR, Roberts LM (1991) Ribosome inactivating proteins of plants. Semin Cell Biol 2:15–22

    PubMed  CAS  Google Scholar 

  8. Endo Y, Tsurugi K (1987) RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem 262:8128–8130

    PubMed  CAS  Google Scholar 

  9. Endo Y (1988) Immunotoxins. In: Frankel AE (ed) Kluwer, Boston, p 75

  10. Endo Y, Mitsui K, Motizuki M, Tsurugi K (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes: the site and the characteristics of the modification in 28S ribosomal RNA caused by toxins. J Biol Chem 262:5908–5912

    PubMed  CAS  Google Scholar 

  11. Atkinson SF, Bettinger T, Seymour LW, Behr JF, Ward CM (2001) Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells. J Biol Chem 276:27930–27935. doi:10.1074/jbc.M102825200

    Article  PubMed  CAS  Google Scholar 

  12. Better M (1996) T-cell-targeted immunofusion proteins from E. coli. Ann N Y Acad Sci 782:544–554

    Article  PubMed  CAS  Google Scholar 

  13. Lambert JM, Senter PD, Yau-Young A, Battler WA, Goldmacher VS (1985) Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins. J Biol Chem 260:12035–12041

    PubMed  CAS  Google Scholar 

  14. Singh V, Sairam MR (1989) Hormonotoxins: conjugation of human choriogonadotropin with the ribosome inactivating protein gelonin and comparison with lutropin conjugates. Mol Cell Endocrinol 67:217–229. doi:10.1016/0303-7207(89)90212-8

    Article  PubMed  CAS  Google Scholar 

  15. Singh V, Sairam MR, Bhagavai GN, Akharas RG (1989) Hormonotoxins preparation and characterization of Ovine leutinizing hormone-gelonin conjugates. J Biol Chem 264:3084–3095

    Google Scholar 

  16. Alam A, Bhuri SRK, Mavila AK, Singh V (1992) Design of liposome to improve encapsulation efficiency of gelonin and its effect on immunoreactivity and ribosome inactivating property. Mol Cell Biochem 112:97–107. doi:10.1007/BF00227566

    Article  PubMed  CAS  Google Scholar 

  17. Provoda CJ, Stier EM, Lee KD (2003) Tumor cell killing by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin. J Biochem 278:35102–35108

    CAS  Google Scholar 

  18. Stripe F, Olsnes S, Pihl A (1980) Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A. J Biol Chem 255:6947–6953

    Google Scholar 

  19. Singh V, Singh RC, Dubey RK, Alam A (1999) Purification and characterization of gelonin from sees of Gelonium multiflorum. Indian J Biochem Biophys 36:258–265

    PubMed  CAS  Google Scholar 

  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:12863

    Article  Google Scholar 

  21. Kirby CJ, Gregoriadis G (1984) A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions. In Gregoriadis G (ed) Liposome technology, vol 1. CRC Press, Boca Raton, pp 19–35

    Google Scholar 

  22. Meister A, Tate SS, Griffith OW (1981) γ-Glutamyl transpeptidase. Methods Enzymol 16:307

    Google Scholar 

  23. Ellman GL, Courtney DK, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95

    Article  PubMed  CAS  Google Scholar 

  24. Habig WH, Jakoby WB (1981) Glutathione-S-transferases (rat and human). Methods Enzymol 77:218

    Article  PubMed  CAS  Google Scholar 

  25. Ellman GL (1959) Tissue sulfhydryl group. Arch Biochem Biophys 82:70

    Article  PubMed  CAS  Google Scholar 

  26. Ratcliffe NA (1983) Practical illustrated histology. The Macmillan Press, pp 32–24

  27. Fry JR (1981) Preparation of mammalian hepatocytes. Methods Enzymol 77:130

    PubMed  CAS  Google Scholar 

  28. Seglen PO (1994) Isolation of hepatocytes. In: Cell biology: a laboratory handbook, Academic Press Inc., p 96

  29. Alam A, Singha LI, Singh V (2005) Molecular characterization of tumour associated antigen in mice exposed to a hepatocarcinogen. Mol Cell Biochem 271:177–188

    Article  PubMed  CAS  Google Scholar 

  30. Gratzner GH (1982) Monoclonal antibodies to 5-bromo and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474–475

    Article  PubMed  CAS  Google Scholar 

  31. Blin N, Stafford DW (1976) A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 3:2303–2308

    PubMed  CAS  Google Scholar 

  32. Singh V, Kar SK (1992) Properties of a ribosome-inactivating protein, gelonin, purified using three different methods. Indian J Biochem Biophys 29(1):31–41

    PubMed  CAS  Google Scholar 

  33. Papahadjopoulos D (ed) (1978) Liposomes and their uses in biology and medicine. Ann N Y Acad Sci 308

  34. Vertut-Doi, Ishiwata H, Miyajima K (1996) Binding and uptake of liposomes containing poly (ethylene glycol) derivative of cholesterol (stealth liposomes) by a macrophage cell line J774: influence of PEG content and its molecular weight. Biochem Biophys Acta 1278:19

  35. Ying TS, Sarma DSR, FarberE (1982) Effects of the delays in the cell cycle on the initiation of preneoplastic lesions in rat liver by 1,2-dimethylhydrazine. Cancer Res 42:876

    PubMed  CAS  Google Scholar 

  36. Naoyuki T (1974) Purification and some properties of γ-glutamyl transpeptidase from azodye-induced hepatoma. J Biochim 75:473

    Google Scholar 

  37. Fiala S, Reuber MD (1970) Gann High glutathiome activity in “minimum deviation” Reuber hepatoma H-139 61:275

    CAS  Google Scholar 

  38. Kosower ES, Kosower EM (1978) The glutathione status of cells. Int Rev Cytosol 54:109

    CAS  Google Scholar 

  39. Columbano A, Rajalakhsmi S, Sarma DSR (1980) Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures. Cancer Res 41:2079

    Google Scholar 

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Authors and Affiliations

  1. Immunology Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong, 793022, Meghalaya, India

    Anis Alam, K. S. Nakhuru & L. I. Singha

  2. Department of Biochemistry, St. Anthony’s College, Shillong, 793001, Meghalaya, India

    L. I. Singha

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  1. Anis Alam
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  2. K. S. Nakhuru
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  3. L. I. Singha
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Correspondence to Anis Alam.

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Alam, A., Nakhuru, K.S. & Singha, L.I. Carcinogenesis response modulation induced by gelonin encapsulated in liposome. Mol Cell Biochem 315, 85–95 (2008). https://doi.org/10.1007/s11010-008-9792-7

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