Georgi Momekov1, Deyan Yossifov2, Margarita Guenova3, Albena Michova4, Nikolay Stoyanov3, Spiro Konstantinov2, Todor Ionkov5, Pavlina Sacheva6, Iliana Ionkova6. 1. Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria. Electronic address: gmomekov@gmail.com. 2. Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria. 3. Laboratory of Haematopathology and Immunology, National Specialised Hospital for Active Treatment of Haematological Diseases, Sofia, Bulgaria. 4. Laboratory of Haematopathology and Immunology, National Specialised Hospital for Active Treatment of Haematological Diseases, Sofia, Bulgaria; Department of Immunology, National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria. 5. Department of Automation, Faculty of Automatics, Technical University of Sofia, Sofia, Bulgaria. 6. Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria.
Abstract
BACKGROUND: The present study aimed at optimization of the biotechnological production of the lignan justicidin B by genetically transformed cultures of Linum leonii and the pharmacological evaluation of the pro-apoptotic effects of the compound in HL-60 cells. METHODS: A rapidly growing selected root line of L. leonii was grown in 2-L bioreactor for period of 40 days and the protocols for obtaining of the compound have been optimized. The pharmacological study included evaluation of the cytotoxicity of the compound in HL-60 cells (MTT-assay), its apoptogenic effects and its effects on caspase 3,8 and 9 activation. RESULTS: After 40 days of sterile run scale up of hairy root culture in bioreactor, 27.2g/L dry weight of root biomass was harvested from the bioreactor culture vessel, recording about nine times increase over initial inoculum (3.0g), with 1.55%±0.07 Justicidin B, greater than yields from 300ml flasks. Our findings are the first work toward the scale up of L. leonii hairy roots-based biotechnological production of Justicidin B, employing bioreactors for high biomass production to meet the industrial requirement. The results from the pharmacological evaluation have shown that the tested arylnaphtalene lignan is a potent cytotoxic and proapoptotic agent against HL-60. The induction of apoptosis proceeds via activation of the intrinsic mitochondrial cell-death signaling pathways. CONCLUSION: The potent activity at low micromolar concentration and the feasibility of biotechnological production of justicidin B implies that there is enormous scope in its further evaluation as possible antineoplastic drug candidate.
BACKGROUND: The present study aimed at optimization of the biotechnological production of the lignanjusticidin B by genetically transformed cultures of Linum leonii and the pharmacological evaluation of the pro-apoptotic effects of the compound in HL-60 cells. METHODS: A rapidly growing selected root line of L. leonii was grown in 2-L bioreactor for period of 40 days and the protocols for obtaining of the compound have been optimized. The pharmacological study included evaluation of the cytotoxicity of the compound in HL-60 cells (MTT-assay), its apoptogenic effects and its effects on caspase 3,8 and 9 activation. RESULTS: After 40 days of sterile run scale up of hairy root culture in bioreactor, 27.2g/L dry weight of root biomass was harvested from the bioreactor culture vessel, recording about nine times increase over initial inoculum (3.0g), with 1.55%±0.07 Justicidin B, greater than yields from 300ml flasks. Our findings are the first work toward the scale up of L. leonii hairy roots-based biotechnological production of Justicidin B, employing bioreactors for high biomass production to meet the industrial requirement. The results from the pharmacological evaluation have shown that the tested arylnaphtalene lignan is a potent cytotoxic and proapoptotic agent against HL-60. The induction of apoptosis proceeds via activation of the intrinsic mitochondrial cell-death signaling pathways. CONCLUSION: The potent activity at low micromolar concentration and the feasibility of biotechnological production of justicidin B implies that there is enormous scope in its further evaluation as possible antineoplastic drug candidate.