Production of biologically active taxoids by callus culture of Taxus cuspidata, chemical modification and biotransformation of the products, and their application as anticancer agents and anti-MDR cancer reversal agents
Liyan Wang, Jiao Bai, Mutumi Kitabatake, Jungui Dai, Junichi Sakai, Jinlan Wang, Shujun Zhang, Wanxia Tang, Liming Bai, Katsutoshi Hirose, Minjie Li, Toshiaki Hasegawa, Masayoshi Ando
Pages: 17 - 36
Number of pages: 20
Production of taxoids by callus culture of Japanese yew tree, Taxus cuspidata was investigated. In standard conditions (dark, NAA 0.5 mg/L, modified Gamborg’s B5 medium) taxuyunnanine C and its analogs (1–5), compound 15, one of the biosynthetic intermediates of paclitaxel, and paclitaxel (taxol®) and its analogs (16, 17, 19, and 20) were produced in 0.486%, 0.0005%, and 0.016%, respectively based on the weight of dry callus. By the addition of methyl jasmonate (100 μM/L) to standard conditions, taxuyunnanine C and its analogs (1–5), biosynthetic intermediates of paclitaxel (10, 12–15), and paclitaxel and its analogs (16, 18–20) are produced in 1.505%, 0.108%, and 0.042% yields, respectively. In light irradiation conditions, the callus culture of T. cuspidata gave taxuyunnanine C and its analogs (1–7) in 0.552% yield in addition to abietanes and abeo-abietanes (22, 27, 28, and 32), but paclitaxel and its analogs and biosynthetic intermediates of paclitaxel were not produced. In the presence of β-cyclodextrin, the callus culture of T. cuspidata gave taxuyunnanine C and its analogs (1–5 and 7) in 1.131% yield and abietanes and abeo-abietanes (21, 22, 26, and 28) in 0.252% yield. The yields of taxol (0.0041%) were remarkably decreased. The biosynthetic precursors of paclitaxel, phenylalanine and acetic acid were added to the medium of standard conditions in the presence and absence of methyl jusmonate, but no improvement of the yields of paclitaxel and its analogs were observed. Interestingly, the bad growth callus culture in standard conditions produced taxuyunnanine C (1) and six its analogs (2–7), six biosynthetic intermediates of paclitaxel (8, 9, 12–15), four paclitaxel and its analogs (16, 17, 19, and 20), five abietanes (23–27), and five abeo-abietanes (28–32) along two possible paths of taxanes and abietanes biosyntheses. Compounds 2, 4, 9, and 10 showed effects on the accumulation of Vincristine (VCR) in multidrug-resistant (MDR) cancer cells 2780AD. The callus culture cultivated on the medium containing 0.3 mg/L KTOS and 1.0 mg/L NAA in the presence of 200 mM methyl jasmonate gave the maximum content of 10 (0.0631%). Bio-transformation of taxuunnanine C (1) by Absidia coerulea IFO 4011 in the presence of β-cyclodextrin gave the biosynthetic intermediates (36 and 37) of paclitaxel. Chemical transformation of taxuyunnanine C (1) gave the corresponding mono-hydroxy derivatives (42–44), a dihydroxy derivative (45), the acylation of which with different kinds of acylchlorides gave the substrates (42a–d, 43a, 44a, and 45a–c). Substrate (42f) was derived by Chugaev reaction from 42 and substrate (46a) was derived from 46 by benzoylation with PhCOCl. Effects of these compounds on the accumulation of calcein in MDR 2780AD cells and their cell growth inhibitory activities against WI-38, VA-13 and HepG2 cells were examined. Compound 45a showed most effective activity on the accumulation of calcein in MDR 2780AD cells. Effect of the compounds on accumulation of VCR in MDR 2780AD cells was examined and compounds (43a, 45a, and 45b) were effective. The effects of compounds 42 and 45a on the cytotoxicity of paclitaxel, adriamycin (ADM), and vincristine (VCR) toward MDR 2780AD cells and its parental cells (A2780) were tested comparing with those of verapamil, which is a well-known MDR reversal agent. Compounds 42 and 45a showed modulating activity toward MDR cancer cells in this experiment.
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