The Induction Studies Of Oligogalacturonides On Paclitaxel Biosynthesis In Taxus Yunnanensis Cell Cultures

Paclitaxel, one of taxanes secondary metabolite in various Taxus (yew) species, is an excellent anticancer drug of special mechanism. Meanwhile, 10-deacetyl baccatin III (10-DAB III) viewed as a synthetic precursor of paclitaxel, also has biological activity itself. Since the supply of paclitaxel and 10-DAB III from the Taxus tree is in great shortage, the production of them in Taxus cells has been a research focus recently. According to the mechanism of plant hypersensitivity reaction to bio-elicitors, oligogalacturonides (OGA) were used to induce paclitaxel and 10-DAB III biosynthesis in Taxus yunnanensis cell suspension cultures. Moreover, its induction mechanism was also studied prelimeliry here.Firstly, we established a new HPLC method to determinate the production of paclitaxel and 10-DAB III simultaneously. 10-DAB III in the cell suspension cultures elicited by OGA was analyzed by RP-HPLC with UV detection at 232 nm, using ethanol-acetonitrile-water (2:3:8) as mobile phase. The results showed that a good linear relation with peak area in the range of 0.0515².06μg (Y=89.146X+27.067, R2 =0.9996). Average recoveries of the intracellular and extracellular concentration were 94.96% and 100.72% with RSD 0.44% and 0.81%, respectively. This is a simple, credible and high sensitivity method.Oligogalacturonides elicitor was hydrolyzed from polygalacturonic acid with pectinase for 20 min at room temperature. Then the enzymatic hydrolysates were treated by Sephdex G-10 column. The higher molecular weight parts were kept as OGA elicitor, whose average degree of polymerization was 13.231The optimum induction conditions were compared. Under the optimum treatment condition with 40μg/mL OGA to T. yunnanensis cells after 12-day culture, the production of paclitaxel and 10-DAB III reached 0.51 mg/L and 0.98 mg/L, respectively after OGA treatment of 3 days, 2.92- and 2.62-folds of control (0.13 mg/L and 0.27 mg/L, respectively). Our results showed that OGA was a good elicitor for inducing paclitaxel and 10-DAB III in T. yunnanensis cell cultures.Programmed cell death (PCD) of T. yunnanensis cells by OGA was proved by AO/EB dying, TUNEL and DNA ladder. PCD cells increased and the apoptosis ratio reached 28.5% after 24 h-treatment by AO/EB dying. The similar results were also showed by TUNEL assay. DNA ladders appeared after 3 day-treatment, indicating the occuring of cells apoptosis.Finally, oxidation-reduction status of T. yunnanensis cells treated by OGA was measured. OGA brought about H₂O₂ burst and then led to auumulation of secondary metabolites such as paclitaxel biosynthesis in T. yunnanensis cells. A fast and transitory H₂O₂ burst happened after 1.5² h-treatment by OGA, simultaneously, superoxide dismutase (SOD) and polyphenol oxidase (PPO) activity increased while catalase (CAT) and peroxidase (POD) activity decreased. Phenylalanine ammonia lyase (PAL) activity reached highest (39.01 U/h mg protein) 2 days after the treatment. The oxide enzymes were activated and so was PAL——the key enzyme of secondary metabolite. PAL activity and production of taxanes were all decreased when H₂O₂ was inhabited by CAT and DPI. It's indicate that H₂O₂ in cells maintained at a certain level is necessary and there is a closely connection among H₂O₂ burst, cell apoptosis and taxanes biosynthesis.In this study, we established an efficient and stable cell suspension cultures system of T. yunnanensis for paclitaxel and 10-DAB III production. The regulatory effect of OGA on taxanes biosynthesis in T. yunnanensis cells was studied. Both PCD appearance and oxidation-reduction situation changes indicated the elicited mechanism of OGA, in which a new regulated way for producing natural medicine by plant cells was found. We provided some basis for the technique of high-paclitaxel-production by cell suspension cultures. A potential application prospect of taxanes producing by large-scale cell cultures is expected.