Biosynthesis of ¹⁴C-Phytoene from Tomato Cell Suspension Cultures (Lycopersicon esculentum) for Utilization in Prostate Cancer Cell Culture Studies

Abstract

This work describes the development and utilization of a plant cell culture production approach to biosynthesize and radiolabel phytoene and phytofluene for prostate cancer cell culture studies. The herbicide norflurazon was added to established cell suspension cultures of tomato (Lycopersicon esculentum cv. VFNT cherry), to induce the biosynthesis and accumulation of the lycopene precursors, phytoene and phytofluene, in their natural isomeric forms (15-cis-phytoene and two cis-phytofluene isomers). Norflurazon concentrations, solvent carrier type and concentration, and duration of culture exposure to norflurazon were screened to optimize phytoene and phytofluene synthesis. Maximum yields of both phytoene and phytofluene were achieved after 7 days of treatment with 0.03 mg norflurazon/40 mL fresh medium, provided in 0.07% solvent carrier. Introduction of 14C-sucrose to the tomato cell culture medium enabled the production of 14C-labeled phytoene for subsequent prostate tumor cell uptake studies. In DU 145 prostate tumor cells, it was determined that 15-cis-phytoene and an oxidized product of phytoene were taken up and partially metabolized by the cells. The ability to biosynthesize, radiolabel, and isolate these carotenoids from tomato cell cultures is a novel, valuable methodology for further in vitro and in vivo investigations into the roles of phytoene and phytofluene in cancer chemoprevention.

A comprehensive review on the colorless carotenoids phytoene and phytofluene

“…This subject has been studied in detail cyanobacteria, algae and plants [82][83][84][85][86][87]. In a novel approach, norflurazon has been used in tomato cell cultures containing 14C-sucrose in the medium to produce 14C-labeled phytoene to gain further insight into the bioavailability and biological actions of this carotenoid [88].…”

Phytoene, phytofluene, and lycopene from tomato powder differentially accumulate in tissues of male Fisher 344 rats

“…LYC was the predominant carotenoid in the seminal vesicles, ventral, dorsolateral, and anterior prostate lobes when rats were fed tomato powder, yet measurable quantities of PE, PF, and ζ-carotene were also present. Recently, we have also shown that 14 C-phytoene accumulates in human DU 145 prostate cancer cells after a 2 d incubation period [17]. In human prostate, LYC is the most abundant carotenoid [10,27], but a variety of other tomato carotenoids also accumulate [10,14].…”

“…Future studies should focus on the in vivo metabolism of PE and PF in a similar manner to studies that have been accomplished for LYC metabolism [16,46]. As 14 C-PE and 14 C-PF sources are not available, biosynthesis of these and other carotenoids through in vitro plant cell culture methodologies is necessary [17]. Precise qualitative and quantitative identification of 14 Ccarotenoid metabolites throughout the body, with an emphasis on the prostate [16,46], are essential to evaluate potential mechanisms of actions by which tomato carotenoids may act in prostate cancer prevention.…”

“…Carotenoid concentrations in tissue and serum samples were quantified by a previously described HPLC-PDA system [16][17][18], and the HPLC mobile phases and gradient procedure utilized in this study have been previously described [19]. In brief, samples from each carotenoid-enriched tissue extract were dissolved in 32-40 µL methyl-tert-butyl ether (MTBE) and injected onto a C30, 4.6 × 150 mm analytical column (YMC, Wilmington, NC, USA) maintained at 25 °C.…”

An improved high performance liquid chromatography–diode array detection–mass spectrometry method for determination of carotenoids and their precursors phytoene and phytofluene in human serum

“…Separate quantification of lutein and zeaxanthin is essential to investigate their individual roles in age-related macular degeneration [7]. The polymeric C30 columns possess unique abilities to resolve cis/trans-carotenoids and are widely adopted for analysis in biological extracts [6,[15][16][17][18]. In a study dealing with quantitation of cis-and trans-carotenoids in human blood of Taiwan subjects, Rajendran et al [6] developed a HPLC method and separated 21 cis plus trans carotenoids within 51 min by using a polymeric C30 column and a gradient mobile phase.…”