Gnidilatimonoein from Daphne mucronata induced differentiation and apoptosis in leukemia cell lines

Authors

Abstract

Among various treatments, plants play a crucial role in cancer chemotherapy. Base on literature data, different species of the thymeleaeceae family have been found to be good sources for anticancer agents. Therefore, our laboratory has initiated a research project on evaluating the anticancer properties of the Iranian medical plant, Daphne mucronata (Thymeleaeceae).
Gnidilatimonoein is a new diterpene ester compound that is purified from Daphne mucronata with antiproliferative and anti-metastatic effects in different tumor cell lines. Based on the inhibitory role of this agent on the cell cycle progression of K562 cell line, we were interested to check gnidilatimonoein for induction of differentiation and apoptosis in different human leukemia ML-1, HL-60 and K562 cells.
Cell viability was determined by trypan blue exclusion method. Cell differentiation was studied in terms of morphology, NBT reduction in leukemia cells (HL-60 and ML-1) and benzidine staining in erythroleukemia (K562) cells. Apoptosis was detected by fluorescence microscopy (Acridine orange/Ethedium bromide double staining), annexineV flow cytometry and DNA electrophoresis.
After 48 hours of treatment with 10 µM gnidilatimonoein, 80-70% of HL-60 and ML1 cells showed NBT reduction. Indeed gnidilatimonoein induced monocyte differentiation of myeloid leukemia HL-60 and ML-1 cells but not K562 erythroluekemia cells. In this condition, we observed the growth inhibitory effect (60%) without substantial loss of viability (<10%). However, at 25 µM, gnidilatimonoein was a strong cytotoxic agent. Morphology of HL-60, ML1 and K562 cells treated with 25 µM of gnidilatimonoein for 48 h indicated that, more than 50% of HL-60 and K562 cells and 31% of ML1 cells showed apoptosis. DNA degradation was also confirmed in all of cell lines. Both cell cycle and annexin V flow cytometry analysis indicated that early induction of apoptosis without cell cycle arrest occurs in all of cells after 25 µM drug treatment.
The traditional way of leukemia treatment is eradication of every tumor cells, but hemorrhagic diathesis often occurs rapidly with fatal outcomes. Differentiation therapy may be an alternative approach to the treatment of leukemia. Our results indicate that genidilatimonoein, at low concentrations (<10 µM) is a potent differentiation inducer and at high concentrations, it early induces apoptosis. Therefore, based on the dual apoptotic and differentiation effects of gnidilatimonoein, it may be concluded that this new anticancer agent is a powerful candidate for leukemia chemotherapy.

Iranian Journal of Pharmaceutical Research (2004): Supplement 2

Iranian Journal of Pharmaceutical Research (2004): Supplement 2: 76-76
Poster Presentations
/Biological Effect of Medicinal Plants

2nd International Congress on Traditional Medicine and Materia Medica
October 4-7, 2004, Tehran, Iran

228

Gnidilatimonoein from Daphne mucronata induced differentiation and apoptosis in leukemia cell lines

Yazdanparast R., Moosavi M.A.

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

Among various treatments, plants play a crucial role in cancer chemotherapy. Base on literature data, different species of the thymeleaeceae family have been found to be good sources for anticancer agents. Therefore, our laboratory has initiated a research project on evaluating the anticancer properties of the Iranian medical plant, Daphne mucronata (Thymeleaeceae).

Gnidilatimonoein is a new diterpene ester compound that is purified from Daphne mucronata with antiproliferative and anti-metastatic effects in different tumor cell lines. Based on the inhibitory role of this agent on the cell cycle progression of K562 cell line, we were interested to check gnidilatimonoein for induction of differentiation and apoptosis in different human leukemia ML-1, HL-60 and K562 cells.

Cell viability was determined by trypan blue exclusion method. Cell differentiation was studied in terms of morphology, NBT reduction in leukemia cells (HL-60 and ML-1) and benzidine staining in erythroleukemia (K562) cells. Apoptosis was detected by fluorescence microscopy (Acridine orange/Ethedium bromide double staining), annexineV flow cytometry and DNA electrophoresis.

After 48 hours of treatment with 10 µM gnidilatimonoein, 80-70% of HL-60 and ML1 cells showed NBT reduction. Indeed gnidilatimonoein induced monocyte differentiation of myeloid leukemia HL-60 and ML-1 cells but not K562 erythroluekemia cells. In this condition, we observed the growth inhibitory effect (60%) without substantial loss of viability (<10%). However, at 25 µM, gnidilatimonoein was a strong cytotoxic agent. Morphology of HL-60, ML1 and K562 cells treated with 25 µM of gnidilatimonoein for 48 h indicated that, more than 50% of HL-60 and K562 cells and 31% of ML1 cells showed apoptosis. DNA degradation was also confirmed in all of cell lines. Both cell cycle and annexin V flow cytometry analysis indicated that early induction of apoptosis without cell cycle arrest occurs in all of cells after 25 µM drug treatment.

The traditional way of leukemia treatment is eradication of every tumor cells, but hemorrhagic diathesis often occurs rapidly with fatal outcomes. Differentiation therapy may be an alternative approach to the treatment of leukemia. Our results indicate that genidilatimonoein, at low concentrations (<10 µM) is a potent differentiation inducer and at high concentrations, it early induces apoptosis. Therefore, based on the dual apoptotic and differentiation effects of gnidilatimonoein, it may be concluded that this new anticancer agent is a powerful candidate for leukemia chemotherapy.

Presenting Author: Moosavi, M.A. moosav_m@ibb.ut.ac.ir