[Frontiers in Bioscience E5, 706-719, January 1, 2013]

Thymoquinone induces apoptosis in malignant T-cells via generation of ROS

Eileen Manasse Dergarabetian1, Khaled Imad Ghattass1, Sally Boulos El-Sitt1, Rasha Mahmoud Al Mismar1, Chirine Omar El-Baba4, Wafica Sami Itani1, Nada Mohamad Melhem2, Hiba Ahmad El-Hajj3, Ali Abdul Hamid Bazarbachi3, Regine Schneider-Stock4, Hala Uthman Gali-Muhtasib1

1Department of Biology, American University of Beirut, Beirut, Lebanon, 2Medical Laboratory Sciences Program, American University of Beirut, Beirut, Lebanon, 3Internal Medicine, American University of Beirut, Lebanon, 4Experimental Tumor Pathology, Institute for Pathology, University Erlangen-Nuremberg, Germany


1. Abstract
2. Introduction
3. Methods
3.1. Cell culture and treatments
3.2. Viability assays
3.3. Cell cycle analysis
3.4. Hoechst staining
3.5. Evaluation of mitochondrial transmembrane potential
3.6. Cytochrome c
3.7. Caspase activity
3.8. Measurement of intracellular Glutathione levels
3.9. Measurement of ROS
3.10. Protein extraction and immunoblot analysis
3.11. Annexin V staining
3.12. Statistical analysis
4. Results
4.1.TQ reduces viability and increases sub-G1 population and apoptosis in malignant T cells, while PBMCs are more resistant
4.2. TQ disrupts mitochondrial potential and releases cytochrome c in malignant T cells
4.3. TQ induces caspase-dependent apoptosis
4.4. TQ depletes GSH levels and increases ROS generation in malignant T cells
4.5. Pretreatment with NAC and CAT protected malignant T cells against TQ-induced ROS generation and apoptosis
5. Discussion
6. Acknowledgements
7. References


We show that HTLV-1 negative leukemia cells are more sensitive to TQ due to higher levels of drug-induced reactive oxygen species (ROS). PreG1 population in HTLV-1 negative Jurkat and CEM was higher than HTLV-1 transformed HuT-102 and MT-2 cells. Peripheral blood mononuclear cells were more resistant. Hoechst staining indicated more features of apoptosis, namely nuclear blebs and shrunken nuclei in HuT-102 than Jurkat. A greater depletion of the antioxidant enzyme glutathione occurred in Jurkat, which consequently led to an increase in ROS, loss of mitochondrial membrane potential, cytochrome c release, activation of caspases 3 and 9, and cleavage of PARP. Treatment with z-VAD-fmk partially reversed TQ-induced apoptosis, suggesting a caspase-dependent mechanism. N-acetyl cysteine prevented apoptosis providing evidence that cell death is ROS-dependent. Catalase prevented apoptosis to a lesser extent than NAC. In summary, TQ induces apoptosis in adult T cell leukemia/lymphoma by decreasing glutathione and increasing ROS, and levels of ROS underlie the differential cellular response to TQ. Our data suggest a potential therapeutic role for TQ in sensitizing HTLV-I-negative T-cell lymphomas.