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* German Cancer Research Center, Toxicology and Chemotherapy Unit, E100, Heidelberg, Germany; and
German Cancer Research Center, Central Spectroscopic Department, B090, Heidelberg, Germany
1Correspondence: Toxicology and Chemotherapy Unit, E100, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, Heidelberg 69120, Germany. E-mail: m.berger{at}dkfz.de
SPECIFIC AIMS
We recently showed that powdered material used in African traditional medicine exerts highly potent anticancer activity in vitro and in vivo. The source of the material was identified as the semiparasitic plant Ximenia americana. An initial physical and chemical characterization of the active component(s) strongly hinted to protein(s) belonging to the family of the type II ribosome-inactivating proteins (RIPs). The aim of this study was to identify and characterize the active component(s).
PRINCIPAL FINDINGS
1. Purification
Purification of the biologically active protein(s) was based on the initial characterization of an aqueous Ximenia americana extract and included four steps: 1) preextraction of the raw material with 70% acetone, to deplete tannins; 2) extraction with extraction buffer (20 mM Tris-HCl, pH=7.0); 3) ion exchange chromatography on diethylaminoethyl (DEAE) cellulose; 4) affinity chromatography on partially hydrolyzed Sepharose.
Reducing SDS-PAGE of this extract as well as of the one-step DEAE eluate (500 mM NaCl) revealed a complex pattern of protein bands that was overlaid by a Schiff’s reagent-positive smear (Fig. 1
a, lanes 1, 2). Subsequent affinity purification of the DEAE eluate resulted in a biologically active sample containing two proteins, each consisting of two subunits (Fig. 1a
, lane 3). A stepwise elution with increasing NaCl concentrations followed by affinity purification resulted in samples differing in the relative ratios of the two proteins (Fig. 1b, c
) and demonstrating a comparable cytotoxic activity. In a typical preparation with one-step elution from DEAE-cellulose, 90 µg affinity purified proteins with cytotoxic activity (see below) were obtained from 20 g raw material. This fraction was used for all subsequent experiments.
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2. Cytotoxicity
The antiproliferative activity of the protein sample was determined by the MTT method in MCF7 human breast cancer, HELA cervix carcinoma and CC531-lacZ rat colon cancer cells (Fig. 2
). The protein sample showed a distinct cytotoxic effect in all three cell lines, as evidenced by IC50 values of 0.5 pM (MCF7), 1.1 pM (HELA), and 0.6 pM (CC531-lacZ). Despite the similar IC50, the dose-response curves of the three cell lines differed in their slope, as reflected by their IC90/IC10 ratios of 83 (MCF7), 6.6 (HELA), and 20 (CC531-lacZ).
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3. Inhibition of protein synthesis
The protein sample clearly inhibited the synthesis of luciferase in an in vitro reticulocyte lysate transcription/translation assay. A clear concentration-dependent translation inhibition was seen in response to 0.17 to 50 nM nonreduced or reduced riproximin, with IC50 values of 5.5 nM (nonreduced proteins) or 2.6 nM (reduced proteins). These values reflect the activity of the heterodimeric protein and the separated A chain, respectively.
4. In vivo experiments
The protein sample was administered to male Wag Rij rats, implanted with 4 x 106 CC531-lacZ cells via the portal vein (day 0).
Experiments with crude extract had shown that the in vitro IC50 in CC531-lacZ cells (3.3 µg/ml) corresponded to an effective in vivo dose of 5 mg/kg for intraperitoneal (i.p.) therapy. Therefore, a similar pharmacodynamic relationship was assumed for selecting the in vivo doses for i.p. therapy with the affinity-purified protein sample (0.25, 0.5, and 1 pmol/kg) from the respective in vitro IC50 (0.5 pM). The dose for the peroral treatment was selected 20-fold higher (10 pmol/kg), to compensate for losses associated with gastrointestinal resorption. No toxicity was observed following these dosages.
Intraperitoneal administration of 0.25, 0.5, and 1 pmol/kg every second day dose-dependently reduced the increase in tumor cell number seen in untreated tumor bearing rats as indicated by T/C % ratios of 47.4, 34.9, and 22.6 (P<0.05), respectively. The effect on the net increase in liver weight was similarly effective as shown by T/C % ratios of 39.1, 29.0, and 30.7, respectively.
Peroral administration of 10 pmol/kg every second day was surprisingly effective: compared to the untreated controls, the net increase in mean liver weight of the treated animals was significantly lower (T/C %=0.3, P<0.05), and their mean tumor cell number was reduced by two orders of magnitude (T/C %=1.0, P<0.05). The actual tumor cell number corresponds to an 8-fold increase related to the initial tumor cell implant, which is formally equivalent to three cell divisions.
Identification of the protein, cDNA sequencing, sequence analysis, and molecular modeling
Two peptides showing homology to B chain sequences of type II ribosome-inactivating proteins (RIPs) were identified by ESI-MS-MS mass spectrometry and subsequent database search and de novo sequencing, respectively. Degenerated primers designed on these peptides amplified a 400 bp DNA fragment containing a continuous open reading frame (ORF). Additional 500 bp 5' sequence were obtained by subsequent amplification with a degenerate primer matching the RIP A chain active site. The complete sequence of the respective cDNA (1990 bp) containing an ORF of 1814 bp was obtained by the RACE method.
The homology of the translated protein sequence to known type II RIP precursors demonstrates that the new protein, termed "riproximin," is so far an unknown member of this class. As typical for type II RIPs, the riproximin precursor contains sequences corresponding to the type II RIP A and B chains, both showing highest homology to toxic members of this family. This high homology (45–55%) to sequences of the crystallographically characterized proteins ricin and viscum lectin I allowed the molecular modeling of a 3D structure of riproximin following the approach of comparative protein modeling on the SWISS-MODEL Server (Fig. 3
, center). A "blind docking" approach using AUTODOCK 3.05 was applied to screen the surface of the B chain for potential Gal-ßbeta; binding sites, with two strategies: one using the rigid homology model and a second introducing protein flexibility into the docking protocol by generating an ensemble of protein conformations using molecular dynamics simulation with the homology model as starting structure.
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The riproximin protein sequence contains several amino acid variations, resulting from single nucleotide polymorphism at 28 positions. However, by comparison to the sequence of ricin or viscum lectin I as well as modeling of the 3D structure, it became obvious that only one of these 10 amino acids is involved in the function or structure of the protein. The only exception is the asn/thr variation at position 159, which, when expressing asn159, could serve as a glycosylation site on the riproximin A chain. Because of its singularity, the presence of this glycosylation site contributes to a major difference between the A chains alleles.
All of the invariant amino acids involved in the N-glycosidase activity of a RIP A chain are conserved and cluster in the 3D model within a cleft likely to be the active site of the riproximin A chain. It can therefore be assumed that the A chain of riproximin is a fully active RNA N-glycosidase. Similar to other type II RIP A chains, the A chain of riproximin has a very low lysine content (two of 263 amino acid residues). This feature probably confers to the A chain resistance to ubiquitination and degradation in the cytosol.
The B chain of type II RIPs consists of two homologous domains termed 1 and 2, each containing three subdomains termed 1
, 1ßbeta;, 1
, and 2, 2ßbeta;, 2. The subdomains 1 and 2 are reported to be responsible for the sugar-affinity of the B chains of most type II RIPs. The B chain of riproximin also shows the typical two-domain structure, including the conserved disulfide bridges and glycosylation sites. In addition, most of the amino acids responsible for sugar binding in the ricin and viscum lectin I subdomains 1 and 2 are also present in the respective riproximin B chain subdomains. The blind ßbeta;-Gal docking calculation using the rigid homology model resulted in three binding sites with maximum energy, two corresponding to the 1 and 2 subdomains, respectively. A third docking site was located close to gln583. Surprisingly, analysis of the docking calculation with a flexible protein showed that the binding affinity of the trp370 binding site (1 subdomain) is significantly reduced compared to trp587 (2 subdomain).
CONCLUSIONS AND SIGNIFICANCE
A mixture of two new type II ribosome-inactivating proteins, one of which we termed riproximin, was identified to be the active principle of the antineoplastic activity contained in Ximenia americana plant material. Figure 3
gives a schematic overview of the set of methods used and the main findings of this study.
Interest in type II RIPs as anticancer agents rose as early as 1970, when it was shown that ricin and abrin are more toxic to tumor than to normal cells. However, ricin’s high unspecific toxicity prevented its clinical use. Recently, a recombinantly engineered viscum lectin I, rViscumin, has been developed for application in cancer treatment and is being tested in phase II clinical studies.
Our results extend the group of type II RIPs with antineoplastic potential by a new protein, riproximin. In addition to its in vitro cytotoxicity, riproximin showed potent antitumor activity in a rat metastasis model, with highest efficacy after p.o. application. This finding is surprising, since proteins are commonly characterized by a low to very low bioavailability, as known for the viscum lectin I, which, despite a low systemic LD50 in mice (5–10 µg/kg), is well tolerated at dietary dosages as high as 500 mg/kg (mice) and 200 mg/kg (rats). In line with this, cancer treatment with the recombinant rViscumin is based on systemic administration. Nevertheless, high doses of p.o. administered viscum lectin I are associated with some systemic activity: a diet containing up to 10 mg viscum lectin I per day/mouse was effective in reducing the tumor mass of a non-Hodgkin lymphoma. Notably, this dose is by more than six orders of magnitude higher than the effective p.o. dose of riproximin (0.6 µg/kg administered every 2nd day).
The differences in toxicity and specificity of type II RIPs are probably related to the sugar specificity of the respective B chains and their intracellular fate. It is interesting that only one of the two presumed sugar binding domains of the riproximin B chain is supposed to bind galactose according to a flexible docking simulation assay. Whether this could be a reason for the high therapeutic efficacy of riproximin at a relatively low and well tolerated p.o. dose remains to be elucidated.
In conclusion, these results suggest that riproximin differs considerably in its pharmacological properties from other type II RIPs, thus indicating distinct potential for cancer treatment.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5231fje
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