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Project's title

 

 

 

by Marina J. Sagnou

  • Evaluation of current and novel protocols of Taxus baccata tissue culture in terms of taxoid and baccatinoid production.
  • Development of efficient isolation, separation and identification techniques of the secondary metabolites produced in tissue culture of that species, including HPLC, NMR, MS, HRMS techniques.
  • Chemical enrichment of tissue and/or plant extracts for enhanced taxoid and/or baccatinoid composition.
  • Biochemical regulation of toxin production in tissue and cell cultures.
  • Biotechnological tissue culture manipulation for maximum preparation of the desired products.

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Our approach aims in the optimisation of T. baccata tissue culture conditions for a fast, economical and easy to manage source of baccatinoid and/or taxoid compounds. A variety of tissue ex-plants, growth media, hormone mixtures etc are under investigation in order to correlate these parameters with rate of "callus" growth and metabolite production. Taxus callus cultures are developed and grown under the desired conditions. After successive subculturing and final harvesting, lyophilised material was extracted and analysed for taxoid and baccatinoid compounds by means of NMR, HPLC, MS etc. Chemical enrichment of plants extracts is also employed. Elicitors, such as jasmonic acid, have also been employed to evaluate their potential to increase of metabolite production. Further investigation in the signaling pathway, defense mechanisms and key-intermediates are anticipated to give rise to interesting scientific results.

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"TAXOL STORY"...the beginning

Taxus baccata Taxus brevifolia is a slow growing tree which is primarily found in the coastal areas of the West Coast states. A screening program for potential anti-tumour agents in the plant kingdom, initiated in 1960, identified the strong anti-tumour effect of bark, leaf and fruit extracts of that plant. In the late 1960's this profound cytotoxic activity of the plant extracts was finally related to a secondary metabolite, namely paclitaxel (TaxolTM) which was further purified and characterised structurally. Paclitaxel was found to have a unique way of preventing the growth of cancer cells through inhibiting microtubule dissociation after cell division due to its binding affinity to tubulin, the major protein component of microtubules. Breast, overian, prostate, lung and pancreas cancer are some of the "hotest" clinical trials attempted since 1984.

"TAXOL STORY"......today

Taxus cycleDue to the high demand for more testing and clinical use of the new drug, in combination with the low actual concentration of the tree in paclitaxel, the ecological impact of a massive harvest campaign and the difficulty in drying the desired tissues, isolating and purifying the active compound, a number of alternative approaches where considered including semi-synthesis from 10-deacetylbaccatin (10-DAB) (Bristol-Myer Suibb), total synthesis (K.C. Nikolaou; R. A. Holton), biosynthesis and genetic engineering, plant cell cultures etc. 10-deacetylbaccatin has been identified as the key-intermediate in the semi-synthetic approach leading to further need for increased 10-DAB production. It is worth mentioning that a species variation of T. brevifolia, namely T. Baccata, has been found localised in the area of Mediterranean countries including Greece and shows high 10-DAB and Baccatin III content.

Paclitaxel Biosynthesis

Bio - taxolA very simplified approach of paclitaxel biosynthesis can be considered to be the comprised of the conversion of phenylalanine to phenylisoserine and its subsequent "coupling" with baccatin III. The resulting intermediate can be forseen to undergo benzoylation yielding paclitaxel. The biosynthesis of the main core of the molecule has been suggested to be initiated by the cyclisation of geranylgeranyl diphosphate.

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  • For general information see:
1. G.I. Georg, T.T Chen, I. Ojima, D.M. Vyas, In "Taxane Anticancer Agents, Basic Science and Current Status", ACS Semposium Series, 1995, Vol. 583.

  • For the most important biological properties of paclitaxel see:
  1. M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, J. Am. Chem. Soc, 1971, Vol. 93, 2325.
  2. W.P. McGuire, E.K. Rowinsky, N.B. Rosenheim, F.C. Grumbine, D.S. Ettinger, D.K. Armostrong, R.C. Donehower, Ann. Intern. Med., 1989, Vol. 111, 273.
  3. M. Suffness, Ann. Rep. Med. Chem., 1993, Vol. 28, 305.
  4. E.K. Rowinsky, L.A. Cazenave, R.C. Donehower, J. Natl. Cancer Inst., 1990, Vol. 82, 1247.

  • For information about the synthesis of paclitaxel see:
  1. K.C. Nicolaou, Z. Yang, J. J. Liu, H. Ueno, P. G. Nantermet, R. K. Guy, C. F. Claiborne, J. Renaud, E. A. Couladouros, K. Paulvannan, E. J. Sorensen., Nature, 1994, Vol. 367, 630.
  2. R.A. Holton et al., J. Am. Chem. Soc, 1994, Vol. 116, 1597 J. Am. Chem. Soc, 1971, Vol. 93,2325.

  • For important information about molecular biology and biosynthesis see:
  1. M. Hezari, R.E.B. Ketchum, D.M. Gibson, R. Croteau, Arch. Biochem. Biophys., 1997, Vol. 337, 185.
  2. M. Hezari, N.L. Lewis, R. Croteau, Arch. Biochem. Biophys., 1995, Vol. 322, 437.
  3. M.R. Wildung, R. Croteau, J. Biol. Chem., 1996, Vol. 271, 9201.
  4. J. Hefner, R.E.B. Ketchum, R. Croteau, Arch. Biochem. Biophys., 1998, Vol. 360, 62.
  • For important information about tissue culture and elicitation see:
  1. A. Zhiri, M. Jaziri, Y. Guo, R. Vanhaelen-Faste, M. Vanhaelen, J. Homes, K. Yoshimatsu, K. Shimomura, Biol. Chem. Hoppe-Seyler, 1995, Vol. 376, 583.
  2. R.E.B. Ketchum, D.M. Gibson, R. Croteau, M.L. Shuler, Biotech. Bioeng., 1999, Vol. 62, 97

 

 

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  • · E. A. Couladouros, PhD; M. Sagnou, PhD; A. Michaelakis, BSc. -Agricultural University of Athens, Athens & NCSR "Demokritos", Athens, Greece
  • P. Hatzopoulos, PhD -Agricultural University of Athens, Athens, Greece
  • V. Roussis, PhD -School of Pharmacy, University of Athens, Athens, Greece

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General Secretariate of Research and Technology, Athens, Greece

Unipharma

Microkalliergies

 

Edited by Marina J. Sagnou

 

Prof. Elias A. Couladouros
E-Mail: ecoula@mail.demokritos.gr

Last Updated: [1-6-2000]