Biotechnologically production of anticancer taxanes in hazelnut (Corylus avellana) cell cultures

Paclitaxel is an important therapeutic that was obtained in Taxus brevifolia (Yew tree) barks in 1971, and the FDA approved it, which is under the brand name Taxol® for use as an anticancer drug. Taxol® (Paclitaxel) is used for the treatment of various types of cancer with success. Paclitaxel is a secondary metabolite belonging to diterpene alkaloid. Secondary metabolites are generally low molecular weight compounds that are responsible for combating stress factors in plants. Production and purification of secondary metabolites require hard and complex processes. Many methods have been tried to perform paclitaxel extraction with an efficient protocol. The most important of these methods are chemical synthesis, semi-synthesis, metabolism engineering, use of plant tissue culture techniques. The method currently used in Taxol® production is cell cultures of Taxus species. However establishment of culture systems of alternative plants promises great hope for purposes like increasing the production speed, increasing efficiency, production of paclitaxel precursors and eliminating unwanted byproduct formation. Hazelnut (Corylus avellana) being produced and exported of high quantities in Turkey is an important plant also due to its nutritional value for the human diet. As well as its economic importance, it has been shown that hazelnut produces paclitaxel-derived taxanes. In this thesis, it was aimed to investigate the potentials intended biotechnological production of taxane-derived anticancer substances (paclitaxel, baccatin III, 10-deacetlylbaccatin III, and cephalomannine) by using plant tissue culture systems of five different varieties (Tombul, Sivri, Çakıldak, Palaz, Kalinkara) and two different genotypes (Ham ve Sivri Yağlı) of hazelnut grown in our country. For this purpose, callus cultures in all hazelnut varieties and genotypes, and cell suspension cultures in Tombul and Kalinkara hazelnuts have been successfully established. In order to increase the production of taxane-derived anticancer agents in cell suspension cultures, elicitation was applied to cell suspension cultures with Methyl Jasmonate (MeJa) and Phenylalanine (Phe). In cultures applied elicitation, the amounts of paclitaxel, baccatin III, 10-deacetylbaccatin III and cephalomannine that were released total and extracellular were analyzed by HPLC. 2 mg/L 2,4-D+ 0,2 mg/L BAP was determined to be the most effective combination in all hazelnut types (Sivri Yağlı- 50%, Ham- 46%, Tombul- 36%, Sivri- 26%, Çakıldak- 40%, Palaz- 40% ve Kalinkara- 36%) of plant growth regulators (2 mg/L 2,4-D + 0,4 mg/L KİN, 2 mg/L 2,4-D + 1,6 mg/L KİN ve 2 mg/L 2,4-D+ 0,2 mg/L BAP) added to MS medium for the establishment of callus cultures. The growth curve of the cell suspension cultures belonged to Kalinkara and Tombul hazelnut types were made by counting the number of cells and measuring wet and dry weight. Based on these results, the 17th day of cultivation was determined as the middle of the logarithmic growth phase of the cells for the application of MeJa (100 μM) and Phe (3 μM) elicitors. The extraction of taxanes released into the total and extracellular medium was performed on the 30th day of cultivation that growth in cell suspension cultures stopped. From obtained extracts, qualitative and quantitative analysis of 10-deacetylbaccatin III, baccatin III, cephalomannine and paclitaxel molecules, which are taxane-derived diterpene alkaloids, were performed by HPLC. Hazelnut cell suspension culture types in which the amount of paclitaxel, baccatin III, cephalomannine and 10-DAP, which are taxane-derived substances analyzed in this thesis study, are successfully increased by elicitation application are given below. The amount of total paclitaxel contained in Tombul hazelnuts was determined to increase at the rate of 42,5% (5,43 ± 0,31 μg/g) and 56,1% (5,95 ± 0,17 μg/g) in cultures treated with respectively 100 μM MeJa and 3 μM Phe compared to control group (3,81 ± 0,30 μg/g). Similarly, the amount of baccatin III that was released into the extracellular medium in Tombul hazelnut was increased by 43,6% (28,23 ± 1,54 μg/g) and 78.3% (35,05 ± 0,74 μg/g) in cultures treated with respectively 100 μM MeJa and 3 μM Phe compared to control group (19,65 ± 0,17 μg/g). The amount of cephalomannine that was released into the extracellular medium in Tombul hazelnut was increased by 26,2% (2,69 ± 0,14 μg/g) and 27,2% (2,71 ± 0,13 μg/g) in cultures treated with respectively 100 μM MeJa and 3 μM Phe compared to control group (2,13 ± 0,03 μg/g). The amount of 10- DAP that was released into the extracellular medium in Kalinkara hazelnut was increased by 9,8 % (45,83 ± 1,49 μg/g) in cultures treated with 100 μM MeJa compared to control group (41,71 ± 0,15 μg/g). According to these results, Tombul hazelnut gave better results in terms of the production of anticancer taxane derivatives in cell suspension cultures than Kalinkara hazelnut. As a result of elicitation applications made, the rate of increase in the production of paclitaxel contained in intracellular and baccatin III, cephalomannine released into extracellular medium was found to be higher in Tombul hazelnut than in Kalinkara hazelnut. In this thesis, callus cultures and cell suspension cultures of hazelnut types grown in Turkey were established for the first time for the production of secondary metabolites. Besides, Paclitaxel molecule, the raw material of Taxol® drug that is used widely as a primary chemotherapeutic drug, has been shown to be produced in cell cultures of Tombul hazelnut (Corylus avellana) plant grown in Turkey. The production of paclitaxel and other precursors of taxane-derived substances were increased by elicitations made in cell suspension cultures of hazelnut types. This thesis suggests that it can be considered as an alternative in the production of paclitaxel and baccatin III, cephalomannine, precursors used in chemical synthesis of paclitaxel, of hazelnut types grown in our country.