Identification and characterization of riproximin, a new type II ribosome-inactivating protein with potent anticancer activity, from aqueous Ximenia americana extracts used by African traditional medicine

1918

The antineoplastic activity of a plant powder used in African traditional medicine for treating cancer was investigated by analyzing the activity of various extracts in vitro. The most active, aqueous extract was subsequently subjected to a detailed investigation in a panel of 21 cell lines, showing a median IC₅₀ of 27 mg raw powder /ml medium. The sensitivity of the cell lines varied from 1.7 mg/ml in MCF7 breast cancer cells to 170 mg/ml in AR230 chronic-myeloid leukemia cells. The in vivo antitumor activity was determined in the CC531 rat colorectal liver metastasis model. Treatment started one week after implanting 4x10⁶ tumor cells intra-portally and was administered every second day for three weeks. Significant anticancer activity was found following administration of equitoxic total doses of 1000 (po) and 50 (ip) mg raw powder/kg, indicating reduced activity following intestinal absorption. By sequencing the mitochondrial gene for the large subunit of the ribulose bis-phosphate carboxylase (rbcL) in DNA extracted from the plant material, the source plant was identified as Ximenia americana. A physico-chemical characterization showed that the active antineoplastic component(s) of the plant material are proteins with galactose-affinity. By a combination of pre-extraction, extraction, ion-exchange and affinity chromatography, a mixture of two cytotoxic proteins of 50-60 kDa (non-reducing SDS-PAGE) was isolated. Reducing SDS-PAGE revealed that both proteins consisted of two subunits of 25-30 kDa. By mass spectrometry one of these proteins was shown to contain a stretch of 11 amino acids identical to a tryptic peptide from the ribosome-inactivating protein ricin. In addition, a second peptide with homology to the ricin B chain was identified by de novo sequencing. Using degenerated primers designed on these peptides, a DNA fragment was amplified and the sequence obtained was used to determine the complete cDNA sequence by the RACE method. Sequence analysis showed the new protein, termed riproximin, to belong to the family of type II ribosome inactivating proteins. These results are in good agreement with the ability of riproximin to inhibit protein synthesis in a cell free system, as well as with its cytotoxicity, as demonstrated by IC₅₀ value of 0.5 pM in MCF7, and 0.6 pM in CC531 cells. To assess the antineoplastic efficacy of the purified riproximin in vivo, the CC531 rat colorectal liver metastasis model was used as for the aqueous plant material extract. Significant anticancer activity was found following administration of total doses of 100 (po) and 10 (ip) pmol riproximin/kg. The favorable activity following peroral administration of low dosages is uncommon for other type II RIPs and suggests that riproximin has distinct potential for cancer treatment.