Uncovering the pharmacological response of novel sesquiterpene derivatives that differentially alter gene expression and modulate the cell cycle in cancer cells.

The present study aimed to assess the pharmacological anticancer profile of three natural and five synthetic sesquiterpenes developed by total chemical synthesis. To this end, their properties at the cellular and molecular level were evaluated in a panel of normal and cancer cell lines. The results obtained by performing cytotoxicity assays and gene expression analysis by reverse transcription-quantitative polymerase chain reaction showed that: i) Among the sesquiterpene derivatives analyzed, VDS58 exhibited a notable anticancer profile within attached (U-87 MG and MCF-7) and suspension (K562 and MEL-745) cancer cell cultures; however, U-87 MG cells were able to recover their proliferation capacity rapidly after 48 h of exposure; ii) gene expression profiling of U-87 MG cells, in contrast to K562 cells, showed a transient induction of cyclin-dependent kinase inhibitor 1A (CDKN1) expression; iii) the expression levels of transforming growth factor ß1 (TGFB1) increased after 12 h of exposure of U-87 MG cells to VDS58 and were maintained at this level throughout the treatment period; iv) in K562 cells exposed to VDS58, TGFB1 expression levels were upregulated for 48 h and decrease afterwards; and v) the re-addition of VDS58 in U-87 MG cultures pretreated with VDS58 resulted in a notable increase in the expression of caspases (CASP3 and CASP9), BCL2­associated agonist of cell death (BAD), cyclin D1, CDK6, CDKN1, MYC proto-oncogene bHLH transcription factor (MYC), TGFB1 and tumor suppressor protein p53. This upregulation persisted only for 24 h for the majority of genes, as afterwards, only the expression of TGFB1 and MYC was maintained at high levels. Through bioinformatic pathway analysis of RNA-Seq data of parental U-87 MG and K562 cells, substantial variation was reported in the expression profiles of the genes involved in the regulation of the cell cycle. This was associated with the differential pharmacological profiles observed in the same cells exposed to VDS58. Overall, the data presented in this study provide novel insights into the molecular mechanisms of action of sesquiterpene derivatives by dysregulating the expression levels of genes associated with the cell cycle of cancer cells.

Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

EST analysis and annotation of transcripts derived from a trichome-specific cDNA library from Salvia fruticosa.

Greek sage (Salvia fruticosa Mill., Syn. Salvia triloba L.) is appreciated for its essential oil which is used as an aromatic spice and active against a wide range of microorganisms and viruses. The essential oil is dominated by terpenoids and flavonoids which are produced and stored in glandular trichomes on the plant surface. The present study aims to give insights into the metabolic activities of S. fruticosa trichomes on a transcriptome level. A total of 2,304 clones were sequenced from a cDNA library from leaves' trichomes of S. fruticosa. Exclusion of sequences shorter than 100 bp resulted in 1,615 high-quality ESTs with a mean length of 592 bp. Cluster analysis indicated the presence of 197 contigs (908 clones) and 707 singletons, generating a total of 904 unique sequences. Of the 904 unique ESTs, 628 (69.5%) had significant hits in the non-redundant protein database and were annotated. A total of 517 (82.3%) sequences were functionally classified using the gene ontologies (GO) and established pathway associations to 220 (24.3%) sequences in Kyoto encyclopedia of genes and genomes (KEGG). In addition, 52 (5.8%) of the unique ESTs revealed a GO biological term with relation to terpenoid (78 ESTs), phenylpropanoid (43 ESTs), flavonoid (18 ESTs) or alkaloid (10 ESTs) biosynthesis or to P450s (26 ESTs). Expression analysis of a selected set of genes known to be involved in the pathways of secondary metabolite synthesis showed higher expression levels in trichomes, validating the tissue specificity of the analyzed glandular trichome library.