Studies on triterpene saponins from Saponaria vaccaria seed and their apoptosis-inducing effect on human cancer cell lines

Medicinal plants have provided important advances in the treatment of numerous diseases and many plant-derived drugs are currently in use or under investigation for the treatment of many ailments including cancer.A phytochemical analysis of the methanol extract from the seed of Saponaria vaccaria L. cultivated in Saskatchewan was performed which resulted in the detection of several bisdesmosidic saponins. A high-performance liquid chromatographic method using photodiode array and single quadrupole electrospray mass detection for analysis and profiling was developed. Due to their structural similarities, purification of bisdesmosidic saponins was challenging. However, monodesmosidic saponin Vaccaroside B and cyclopeptides Segetalin A, Segetalin B, and a new cyclopeptide, segetalin I [whose structure was proposed to be cyclo(Gly1-Pro2-Tyr3-Tyr4-Pro5-Phe6)], were purified employing various chromatographic techniques such as HPLC, VLC, PTLC). Crude methanol extracts of S. vaccaria seed were evaluated for cytotoxic activity using the methyl-thiazol-tetrazolium non-radioactive cell proliferation assay (MTT assay). Various concentrations of the extract (2-50 ug/mL) were tested against a series of four human cancer cell lines (WiDr, colon; MDA-MB-231, breast; NCI-417, lung and PC-3, prostate). The human foreskin (BJ)-derived normal human fibroblast cell line CRL-2522 was included as a non-cancerous control. Results showed that cytotoxic activities from the seed extract were greater than commercially available Quillaja saponaria saponin. The human cancer cell lines were also exposed to fractions containing high titers of saponins as well as semi-purified saponins (ca. 80%). All bisdesmosidic saponins and fractions thereof showed cytotoxicity against the cell lines studied. The effect was particularly strong in breast and prostate cancer cell lines with IC50 values in the range 1–4 ug/mL. Monodesmosidic saponins, phenolics and cyclopeptides did not show activity even at the highest concentration (50 ug/mL) tested in this study. Chemical modifications of the saponin structures resulted in an overall decrease in activity, but an increase in selectivity in comparison to CRL-2522. Time and concentration-dependent studies using the nuclear stains propidium iodide and Hoechst 33342, demonstrated that the stimulation of apoptosis was the mechanism of cytotoxic action. When breast and prostate cell lines were exposed to small amounts (4-7 mM) of bisdesmosidic saponins Segetalin H (MW 1448) and Segetalin I (MW 1464), it was observed that apoptosis was induced at an early incubation time (4-10 h). Activation of caspases and changes in membrane potential were determined by flow cytometry.As a result of this study, we propose that triterpene bisdesmosidic saponins from the seed of Saponaria vaccaria L. represent a new type of drug having potential antitumor/anticancer activity due to their ability to induce apoptosis in vitro in human cancer cell lines at low concentrations. These compounds are extracted from a plant that can be easily cultivated using conventional agricultural equipment in Western Canada

Characterization of an oleate 12-desaturase from Physaria fendleri and identification of 5\u2032UTR introns in divergent FAD2 family genes

Mining of an EST sequence collection representing genes expressed during seed development in Physaria fendleri identified abundant sequences encoding apparent homologues of the Arabidopsis oleate 12-desaturase (AtFAD2 At3g12120). Of the 62 sequenced clones, 59 were identified as encoding the previously characterized bifunctional oleate 12-hydroxylase/desaturase (LFAH12/PfFAH12). The remaining 3 clones encoded a second FAD2 homologue. Isolation of a full length ORF and heterologous expression in yeast revealed that this sequence, designated PfFAD2, is the first full length sequence from any Physaria species that encodes an oleate 12-desaturase. PfFAD2 was expressed in both leaf and developing seed with activity on palmitate (16:1\u3b49) and oleate (18:1\u3b49). Sequence comparison revealed that PfFAD2 shares 93% amino acid identity with Arabidopsis FAD2 and only 84% identity with PfFAH12. By comparison of EST and genomic sequences it was revealed that the PfFAD2 gene encodes a transcript with a single intron of 1120bp in the 5'-untranslated region (5'UTR). A short intron, 81bp in length, was also discovered in the 5'UTR of the PfFAH12 gene, 16bp upstream of the translation initiation codon. In silico examination of FAD2 like genes from the genome of castor (Ricinus communis) identified putative 5'UTR introns in genes encoding the castor oleate 12-desaturase (RcFAD2) and oleate 12-hydroxylase (CFAH12). By sequencing of genomic DNA the presence of single 5'UTR introns in each gene, and the size of these introns, was confirmed. These findings suggest that 5'UTR introns may be a characteristic feature of FAD2 genes and also of divergent FAD2 genes encoding fatty acid modifying enzymes, and that the selection pressure maintaining these introns is very different.Peer reviewed: YesNRC publication: Ye

Involvement of Arabidopsis acyl-coenzyme A desaturase-like2 (At2g31360) in the biosynthesis of the very-long-chain monounsaturated fatty acid components of membrane lipids

The Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A (CoA) desaturase-like (ADS) gene family contains nine genes encoding fatty acid desaturase-like proteins. The biological function of only one member of the family, fatty acid desaturase5 (AtADS3/ FAD5, At3g15850), is known, and this gene encodes the plastidic palmitoyl-monogalactosyldiacylglycerol D7 desaturase. We cloned seven members of the gene family that are predicted not to have a chloroplast transit peptide and expressed them in the yeast Saccharomyces cerevisiae. All seven have previously undescribed desaturase activity on very-long-chain fatty acid (VLCFA) substrates and exhibit diverse regiospeci\ufb01city, catalyzing introduction of double bonds relative to the methyl end of the molecule (n-x) at n-6 (AtADS4, At1g06350), n-7 (AtADS1.3, At1g06100 and AtADS4.2, At1g06360), n-9 (AtADS1, At1g06080 and AtADS2, At2g31360) or D9 (relative to the carboxyl end of the molecule) positions (AtADS1.2, At1g06090 and AtADS1.4, At1g06120). Through forward and reverse genetics it was shown that AtADS2 is involved in the synthesis of the 24:1(n-9) and 26:1(n-9) components (X:Y, where X is chain length and Y is number of double bonds) of seed lipids, sphingolipids, and the membrane phospholipids phosphatidylserine, and phosphatidylethanolamine. Plants de\ufb01cient in AtADS2 expression showed no obvious phenotype when grown under normal growing conditions, but showed an almost complete loss of phosphatidylethanolamine (42:4), phosphatidylserine(42:4), dihydroxy-monohexosylceramide(42:2)-2, trihydroxy-monohexosylceramide(42:2)-3, and trihydroxy-glycosylinositolphosphoceramide(42:2)-3, lipid species that contain the VLCFA 24:1(n-9), and trihydroxyglycosylinositolphosphoceramide(44:2)-3, a lipid containing 26:1(n-9). Acyl-CoA pro\ufb01ling of these plants revealed a major reduction in 24:1-CoA and a small reduction in 26:1-CoA. Overexpression of AtADS2 resulted in a substantial increase in the percentage of glycerolipid and sphingolipids species containing 24:1 and a dramatic increase in the percentage of very-long-chain monounsaturated fatty acids in the acyl-CoA pool. Plants de\ufb01cient in AtADS1 expression had reduced levels of 26:1(n-9) in seed lipids, but no signi\ufb01cant changes in leaf phospholipids or sphingolipids were observed. These \ufb01ndings indicate that the 24-carbon and 26-carbon monounsaturated VLCFAs of Arabidopsis result primarily from VLCFA desaturation, rather than by elongation of long chain monounsaturated fatty acids.Peer reviewed: YesNRC publication: Ye