Bioactive diterpenoid metabolism and cytotoxic activities of genetically transformed Euphorbia lathyris roots

Abstract

Plants in the genus Euphorbia produce a wide variety of pharmacologically active diterpenoids with anticancer, multidrug resistance reversal, and antiviral properties. Some are the primary industrial source of ingenol mebutate, which is approved for treatment of the precancerous skin condition actinic keratosis. Similar to other high value phytochemicals, Euphorbia diterpenoids accumulate at low concentrations in planta and chemical synthesis produces similarly low yields. We established genetically transformed root cultures of Euphorbia lathryis as a strategy to gain greater access to diterpenoids from this genus. Transformed roots produced via stem explant infection with Agrobacterium rhizogenes strain 15834 recapitulated the metabolite profiles of field-grown plant roots and aerial tissues. Several putative diterpenoids were present in transformed roots, including ingenol and closely related structures, indicating that root cultures are a promising approach to Euphorbia-specific diterpenoid production. Treatment with methyl jasmonate led to a significant, albeit transient increase in mRNA levels of early diterpenoid biosynthetic enzymes (farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and casbene synthase), suggesting that elicitation could prove useful in future pathway characterization and metabolic engineering efforts. We also show the potential of transformed E. lathyris root cultures for natural product drug discovery applications by measuring their cytotoxic activities using a panel of human carcinoma cell lines derived from prostate, cervix, breast, and lung.

Keywords: Agrobacterium; Anticancer; Diterpene; Euphorbia; Ingenol; Specialized metabolism.

Fig.1.

Establishment and culture maintenance of transformed E. lathyris roots. (a) Stem explants of 3-week-old greenhouse grown plants were used for co-culture with A. rhizogenes. (b) Roots emerged from callus at the site of infection after 2 to 3 weeks.(c) Adventitious roots displaying the characteristic of the “hairy root” phenotype. (d) Growth characteristics of the isogenic root line used in this study on agar media and in (e) liquid media.

Fig.2.

Base peak chromatograms of transformed E. lathyris roots compared to wild-type plant roots and aerial parts with putatively assigned metabolites that are structurally related to ingenol.

Fig.3.

The MS/MS (fragmentation) data of m/z 477.2732 aided in the putative assignment of compound 8 as 15-O-acetyl-3-O-iso-butyryljolkinol-5β,6β-oxide. The fragmentation structures and m/z values correspond to each other (i.e. structures and peaks A-D).

Fig.4.

Bioinformatic analysis and chemical elicitation of early diterpenoid biosynthetic genes expressed in transformed E. lathyris roots. Comparison of the deduced amino acid sequences of (a) ElFPS and (b) ElGGPS highlighting two conserved aspartate-rich domains [DDxx(xx)D]. (c) Comparison of the deduced amino acid sequence of ElCS highlighting a conserved [DDxxD] motif that is essential to the cyclization functionalities of terpene synthases. (d) Time course of E. lathyris diterpenoid biosynthetic gene transcript levels in transformed root cultures treated with 100 μM methyl jasmonate. Asterisks indicate statistical significance in comparison to 0 h control assessed by one-way ANOVA (**,P< 0.01; *,P< 0.05)

Fig.5.

Cytotoxic activities of transformed E. lathyris root extract in human carcinoma and embryonic cell lines. a) DU-145 (prostate) b) HeLa (cervix) c) MCF-7 (breast) d) MDA-MB-231 (breast) e) and H2347 (lung) were treated with DMSO (carrier), or increasing concentrations of transformed root MeOH extracts (31.3 ug/ml, 52.5 ug/ml, 125 ug/ml, 250 ug/ml). Titer-Glo^® (Promega) was used to count cell lines 48 h post-treatment. Each dose and timepoint was performed in triplicate. Asterisks indicate statistical significance in comparison to carrier (DMSO) control assessed by one-way ANOVA (**,P< 0.01; *,P< 0.05)