Production of Aralin, a Selective Cytotoxic Lectin against Human Transformed Cells, in Callus Culture of Aralia elata

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Recently we found that aralin, a novel lectin, from Aralia elata selectively induces apoptosis in human virus-transformed and cancer cells compared with normal cells. To overcome the reduction of aralin biosynthesis according to the development of leaves, we established callus tissues from the young shoots. The N-terminal amino acid sequence and immunochemical analyses revealed that ca-aralin, an aralin synthesized in the callus, was structurally identical with aralin except for the glycosylation of the B chain. The selective cytotoxic index (ratio of IC₅₀ value against human lung normal cells versus virus-transformed cells) of ca-aralin was 2-fold higher than that of aralin. Thus, these results suggest that ca-aralin is more useful than aralin as an anticancer agent.

The young shoot of Aralia elata (the Japanese Angelica tree, Taranoki in Japanese) is a popular edible plant, especially in the spring in Japan. Additionally, the bark of root cortex is widely used in folk medicine having tonic, anti-arthritic, and anti-diabetic effects [1]. Recently, we found that aralin selectively induces apoptosis in virus-transformed and cancer cells when compared with normal cells [2]. Aralin is composed of A and B chains, and the N-terminal sequence of B chain shows homology with the ricin B chain (52 % identity) as a subunit of type II ribosome-inactivating protein (RIP) [2], [3], [4]. RIPs including ricin have been used as the cytotoxic moiety of immunotoxins against various cancer cells [5]. Therefore, aralin is also expected to act as a potent anticancer agent like other RIPs. However, aralin is actively synthesized only for a limited period in spring in the young shoots, and thereafter its biosynthesis is diminished according to the development of leaves. Therefore, to overcome the seasonal problem of aralin production, in this study we tried to produce physiologically active aralin in a callus culture of A. elata.

As summarized in Table [1], ca-aralin (0.5 mg) was homogeneously purified from 1.1 kg fresh callus by ammonium sulfate precipitation, and anion-exchange and affinity column chromatographies as previously reported [2], [6]. Stable production of ca-aralin in the callus culture was maintained for at least up to 6 months. Ca-aralin untreated with 2-mercaptoethanol (2-ME) was separated as a 62-kDa single protein on SDS-PAGE, while in the presence of 2-ME ca-aralin was divided into A (29 kDa) and B (31 kDa) chains as well as aralin (Fig. [1] A). Since the molecular weight of the ca-aralin B chain was consistent with that of the deglycosylated aralin B chain, the glycosylation levels of both B chains might differ from each other. The A and B chains of ca-aralin were substantially detected by Western blot with an anti-aralin antibody (Fig. [1] B). In accordance with this result, the N-terminal amino acid sequences of ca-aralin A and B chains were identical with those of aralin (Fig. [1] C). These results indicate that ca-aralin is structurally identical with aralin except for the glycosylation level of the B chain.

As shown in Fig. [2], the IC₅₀ values of ca-aralin and aralin for virus-transformed VA-13 cells were estimated to be 0.9 and 0.8 ng/mL, respectively. On the other hand, their IC₅₀ values for normal WI-38 cells were 20 and 10 ng/mL, respectively. Thus, the sensitivities of transformed cells for ca-aralin and aralin were strikingly higher than those of normal cells. Furthermore, it is noteworthy that the selective cytotoxic index of ca-aralin is 2-fold higher than that of aralin. Considering these results, it seems likely that ca-aralin is more suitable than aralin for use as an anticancer drug.

Materials and Methods

Callus tissues were established from young shoots of Aralia elata (Miq.) Seem. (Araliaceae) collected at Akita Prefecture (Japan) in April 2001. A voucher specimen (number Ar-01 - 04) was deposited at the herbarium of our institute. Murashige & Skoog (MS) medium [7] containing 1 - 3 mg/L 2,4-dichlorophenoxyacetic acid and 0.1 mg/L kinetin as the plant growth regulators without glycine was used for callus induction. The callus tissues were sub-cultured every 5 - 6 weeks on fresh MS medium containing 3 mg/L α-naphthaleneacetic acid with 0.1 mg/L 6-benzylaminopurine at 25 ± 1 °C in the dark. The callus tissues were harvested at five-week intervals and were stored at -20 °C until use.

Wet callus cultures (1.1 kg) were homogenized with 3.3 L of buffer A (50 mM Tris-HCl, pH 7.4, 1 mM EDTA), and centrifuged at 17,800 × g for 20 min. Subsequently ammonium sulfate was added to the supernatant at 80 % saturation. After centrifugation, the resulting precipitate was dissolved in a small volume of buffer A and dialyzed against the same buffer. The dialyzate was loaded onto a Q Sepharose HP column (2.6 × 10 cm) and eluted with a 700 mL linear gradient of 0 - 0.5 M NaCl in buffer A. The fractions exhibiting a potent cytotoxicity against virus-transformed VA-13 cells were combined and loaded onto a Sepharose 4B column (1.4 × 19 cm) pre-equilibrated with buffer B (25 mM Tris-HCl, pH 7.4, 0.1 M NaCl). After washing with buffer B, ca-aralin was eluted with 0.1 M lactose in buffer B. The resulting cytotoxic fractions were collected and the buffer exchanged to phosphate-buffered saline using a Centriplus-30 (Millipore).

SDS-PAGE was carried out as described previously [8]. Protein bands in a gel were detected by the SDS-Zn reverse staining method [9] using a Negative Gel Stain MS Kit (Wako). Deglycosylation was carried out using an Enzymatic Deglycosylation Kit (Bio-Rad). For Western blot analysis, samples were resolved by a 12 % SDS-PAGE and blotted onto a PDVF membrane. The protein bands that cross-reacted with anti-aralin IgG were detected by an ECL Plus chemiluminescence kit (Amersham) using horseradish peroxidase-conjugated anti-rabbit IgG as a secondary antibody. Precision Protein Standards (Bio-Rad) were used as size markers. Protein concentrations were determined as described previously [10]. The amino acid sequences of the N-termini of ca-aralin A and B chains were analyzed by means of a PPSQ-10 protein sequencer (Shimadzu) after separation on SDS-PAGE with 2-ME and blotting onto a PDVF membrane.

Normal human lung cells (WI-38) and SV40-transformed WI-38 cells (VA-13) were purchased from the RIKEN Cell Bank, Japan, and cultivated in Eagle’s minimal essential medium (MEM) supplemented with heat-inactivated fetal bovine serum (10 %), penicillin (100 units/mL) and streptomycin (100 μg/mL) at 37 °C under 5 % CO₂ in a humidified atmosphere. The cytotoxicity of ca-aralin against WI-38 and VA-13 cells was determined by the WST-1 assay as described previously [2]. Aralin from the intact plant was used as a positive control.

Acknowledgements

We thank Dr. T. Nakamura, Faculty of Pharmaceutical Sciences, Tokyo University of Science for plant identification and his critical reading of the manuscript.

References

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• 2 Tomatsu M, Kameyama M O, Shibamoto N. Aralin, a new cytotoxic protein from Aralia elata, inducing apoptosis in human cancer cells.  Cancer Lett. 2003;  199 19-25
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Makoto Tomatsu

Akita Research Institute of Food and Brewing (ARIF)

4-26 Sanuki

Arayamachi

Akita 010-1623

Japan

Fax: +81-18-888-2008

Email: tomatsu@arif.pref.akita.jp

References

• 1 Perry L M, Metzger J. Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses. Massachusetts; MIT Press 1980: p 41
  Search in Google Scholar
• 2 Tomatsu M, Kameyama M O, Shibamoto N. Aralin, a new cytotoxic protein from Aralia elata, inducing apoptosis in human cancer cells.  Cancer Lett. 2003;  199 19-25
  CrossrefPubMedSearch in Google Scholar
• 3 Lord J M, Roberts L M, Robertus J D. Ricin: Structure, mode of action, and some current applications.  FASEB J. 1994;  8 201-8
  PubMedSearch in Google Scholar
• 4 Barbieri L, Battelli M G, Stirpe F. Ribosome-inactivating proteins from plants.  Biochim Biophys Acta. 1993;  1154 237-82
  PubMedSearch in Google Scholar
• 5 Tang W, Hemm I, Bertram B. Recent development of antitumor agents from Chinese herbal medicines. Part II. High molecular compounds.  Planta Med. 2003;  69 193-201
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Olsnes S. Ricin and ricinus agglutinin, toxic lectins from castor bean.  Methods Enzymol. 1978;  50 330-5
  PubMedSearch in Google Scholar
• 7 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiol Plant. 1962;  15 473-97
  CrossrefPubMedSearch in Google Scholar
• 8 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.  Nature. 1970;  227 680-5
  PubMedSearch in Google Scholar
• 9 Fernandezpatron C, Hardy E, Sosa A, Seoane J, Castellanos L. Double staining of Coomassie blue-stained polyacrylamide gels by imidazole-sodium dodecyl sulfate-zinc reverse staining: Sensitive detection of Coomassie blue-undetected proteins.  Anal Biochem. 1995;  224 263-9
  CrossrefPubMedSearch in Google Scholar
• 10 Lowry O H, Rosebrough N J, Farr A L, Randall R J. Protein measurement with the Folin phenol reagent.  J Biol Chem. 1951;  193 265-75
  PubMedSearch in Google Scholar

Makoto Tomatsu

Akita Research Institute of Food and Brewing (ARIF)

4-26 Sanuki

Arayamachi

Akita 010-1623

Japan

Fax: +81-18-888-2008

Email: tomatsu@arif.pref.akita.jp