A tuber lectin from Arisaema helleborifolium Schott with anti-insect activity against melon fruit fly, Bactrocera cucurbitae (Coquillett) and anti-cancer effect on human cancer cell lines

https://doi.org/10.1016/j.abb.2005.10.021Get rights and content

Abstract

An anti-insect and anti-cancer lectin has been isolated from Arisaema helleborifolium Schott by affinity chromatography using asialofetuin-linked amino activated silica beads. The bound A. helleborifolium lectin (AHL) was eluted with 100 mM glycine–HCl buffer, pH 2.5. It gave a single band on SDS–PAGE, pH 8.3, and PAGE, pH 4.5. However, multiple bands were obtained in PAGE at pH 8.3 and isoelectric focusing. The lectin was a homotetramer having subunit molecular mass 13.4 kDa while its native molecular mass was 52 kDa. It was a glycoprotein with 3.40% carbohydrate and was stable up to 60 °C for 30 min. It showed anti-insect activity towards second instar larvae of Bactrocera cucurbitae (Coquillett) with LC50 value of 16.4 μg/ml. Larvae fed on artificial diet containing sub-lethal dose of AHL showed a significant decrease in acid phosphatase and alkaline phosphatase activity while esterase activity markedly increased as compared to larvae fed on diet without lectin. AHL was also found to inhibit in vitro proliferation of some well established human cancer cell lines viz HOP-62 (95%), HCT-15 (92%), HEP-2 (66%), HT-29 (68%), PC-3 (39.4%), and A-549 (20.7%).

Section snippets

Chemicals and reagents

Fetal calf serum from Sera Lab (GB) and RPMI 1640 from Gibco-BRL, New York, USA, were procured and stored at 4 °C. All sugars/derivatives, glycoproteins, bovine serum albumin, Freund’s complete adjuvant, adriamycin, 5-fluorouracil, and paclitaxel were obtained from Sigma Chemical, St. Louis, USA. Standard molecular weight markers, gel filtration markers and ampholine of pI range 3.0–9.5 were from Amersham Pharmacia, New Jersey, USA. Amino activated silica beads used were from Clifmar, UK. Biogel

Protein and neutral sugar content analyses

Protein concentration in the crude and purified lectin samples was determined by the method of Lowry et al. [13] using bovine serum albumin as standard while neutral sugar content of the purified lectin preparation was estimated by anthrone method [14] using d-glucose as standard.

Polyacrylamide gel electrophoresis of native and denatured lectin

Native polyacrylamide gel electrophoresis (PAGE) was carried out using 7.5% (w/v) gel at pH 4.5 [15] and 10% (w/v) gel at pH 8.3 [16]. SDS–PAGE was performed according to Laemmli [17] using 11% (w/v) separating gel.

Anti-insect activity

Effect of AHL on development of melon fruit fly, B. cucurbitae (Coquillett) along with its effect on the activity of some of the hydrolytic enzymes was studied. Cultures of B. cucurbitae were maintained at 25 ± 2 °C, photoperiod (L10:D14) and 70–80% relative humidity. Diet was prepared according to the method described by Gupta et al. [24].

AHL at 10, 20, 40, 80, and 160 μg/ml was incorporated in artificial diet prepared according to the method described by Srivastava [25]. Diet was poured into

Results and discussion

The present study describes the purification and characterization of an anti-cancer and anti-insect lectin from the tubers of A. helleborifolium. The lectin was purified by affinity chromatography and asialofetuin, which was one of the hemagglutination inhibitor, was used as a ligand. The results of isolation procedure are summarized in Table 1. The unbound fractions in PBS were devoid of any lectin activity indicating that there was complete adsorption of AHL to the column. The lectin was

Acknowledgments

This work was supported by a research grant from the CSIR. First and second authors are recipients of Senior Research Fellowship from CSIR, New Delhi, India. We are also thankful to sophisticated instrumentation research center, Punjab University, Chandigarh to carry out metal ion analysis.

References (35)

  • C.R. Carlini et al.

    Toxicon

    (2002)
  • M.L.R. Macedo et al.

    Biochim. Biophys. Acta

    (2002)
  • S. Shangary et al.

    Phytochemistry

    (1995)
  • J. Singh et al.

    Biochem. Biophys. Res. Commun.

    (2004)
  • O.H. Lowry et al.

    J. Biol. Chem.

    (1951)
  • R.G. Spiro

    Methods Enzymol.

    (1966)
  • E.F. Robertson et al.

    Ann. Biochem.

    (1987)
  • M. Paulova et al.

    Biochem. Biophys. Acta

    (1971)
  • F. Naseem et al.

    Arch. Biochem. Biophys.

    (2004)
  • B. Katzenellenbogen et al.

    J. Insect Physiol.

    (1971)
  • E.D. Green et al.

    J. Biol. Chem.

    (1988)
  • C.E. Hayes et al.

    J. Biol. Chem.

    (1974)
  • H.B.F. Dixon

    Nature

    (1981)
  • F.I. Abdullaev et al.

    Nat. Toxins

    (1997)
  • U. Schumacher et al.

    Cancer

    (1994)
  • R. Parslew et al.

    Br. J. Dermatol.

    (1999)
  • W.J. Peumans et al.

    Plant Physiol.

    (1995)
  • Cited by (0)

    View full text