Abstract

Bu çalışmada, insan akut miyeloid lösemisinde timokinonun sitotoksik, apoptotik ve otofajik etkilerinin araştırılması amaçlandı. Timokinonun sitotoksik etkileri 3-(4,5-dimetiltiyazol2-yl)-2,5-difeniltetrazolyum-bromür (MTT) testi ile belirlendi. B hücre lenfoma 2 ile ilişkili X proteini (Bax), B hücre lenfoma 2 (Bcl-2), kaspaz 3, rapamisinin memeli hedefi (mTOR), fosfatidilinositol-3-kinaz (PI3K) ve protein kinaz B (AKT) gen ekspresyon analizleri kantitatif gerçek zamanlı polimeraz zincir reaksiyonu (qRT-PCR) ile yapıldı. Bax, Bcl-2, kaspaz 3, mTOR, AKT ve PI3K proteinlerinin aktif bölgesindeki timokinonun potansiyel bağlanmasını araştırmak için AutoDock Tools 4.2 yazılımı kullanıldı. Timokinon, HL-60 hücrelerinde (İnsan lösemi hücre hattı) 16.35 µM değerinde sitotoksik etkiye neden olmuştur. Uygulanan tüm dozlarda kontrole göre Bcl-2 ekspresyonu azalmıştır. 10 µM, 15 µM ve 25 µM timokinon ile tedavi edilen hücrelerde kontrole göre kaspaz 3 ekspresyon seviyesinde bir azalma tespit edildi. Timokinon, tüm dozlarda kontrole kıyasla mTOR ve PI3K ifadelerinde önemli bir azalmaya neden olurken, AKT 15 µM'lik bir dozda azaldı. Yerleştirme sonuçları, timokinonun apoptotik ve otofajik proteinlerin aktif bölge amino asitleri ile hidrofobik etkileşimler ve hidrojen bağı yoluyla etkileşime girdiğini belirtti. Bulgularımız, timokinonun HL-60 hücrelerinde PI3K/AKT/mTOR yolunu önleyerek otofajiyi uyarabildiğini ve akut miyeloid lösemi tedavisi için yeni bir hedef olabileceğini düşündürmektedir.

Project Number

2017-058

In vitro AND in silico EVALUATION OF THYMOQUINONE AS POTENTIAL ANTICANCER AGENT IN HUMAN ACUTE MYELOID LEUKEMIA HL-60 CELLS

Abstract

This study aims to explore the cytotoxic, apoptotic and autophagic effects of thymoquinone on human acute myeloid leukemia. The cytotoxic effects of thymoquinone were determined with 3-(4, 5-dimethylthiazol-2-yl)-2 and 5-diphenyltetrazolium bromide (MTT) tests. B-cell lymphoma 2 associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), caspase 3, mammalian target of rapamycin (mTOR), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (AKT) gene expression analyzes were studied with quantitative real-time polymerase chain reaction (qRT-PCR). AutoDock Tools 4.2 software was applied to research the potential binding of thymoquinone in the active sites of Bax, Bcl-2, caspase 3, mTOR, PI3K, and AKT proteins. Thymoquinone caused a cytotoxic effect on HL-60 cells (Human leukemia cell line) with a value of 16.35 µM. Bcl-2 expression was decreased in all concentrations applied compared to the control. A decrease in caspase 3 expression level was detected in the cells treated with 10 µM, 15 µM, and 25 µM thymoquinone compared to the control. Thymoquinone induced an important decrease in mTOR and PI3K expressions compared to the control at all doses, while AKT decreased at a dose of 15 µM. The docking outcomes showed that thymoquinone interacts with the active site amino acids of apoptotic and autophagic proteins via hydrophobic interactions and hydrogen bonding. The present findings suggest that thymoquinone can stimulate autophagy by prevention of PI3K/AKT/mTOR pathway in HL-60 cells and may become a new target for the therapy of acute myeloid leukemia.

Keywords

• AML
• Apoptosis
• Autophagy
• Molecular docking
• PI3K/AKT/mTOR
• Thymoquinone

Supporting Institution

Aksaray üniversitesi

Project Number

2017-058

Thanks

This study was financed by Aksaray University Scientific Research Fund (grant number 2017-058). We are thankful to Aksaray University Scientific and Technological Application and Research Center for the use of the Molecular Biology and Metabolism Laboratory. I would like to thank Biotechnologist Mehmet KARATAŞ for his kind help in the determination of molecular docking study.

References

1. 1. Ahmad, A., Husain, A., Mujeeb, M., Khan, S.A., Najmi, A.K., Siddique, N.A., Damanhouri, Z.A. & Anwar, F. 2013. A review on therapeutic potential of Nigella sativa: A miracle herb. Asian Pacific Journal of Tropical Biomedicine, 3: 337-352.
2. 2. Al-Otaibi, N.A.S., Cassoli, J.S., Slater, N.K.H. & Rahmounc, H. 2019. Molecular characterization of human leukemia 60 (HL-60) cells as a model of acute myelogenous leukemia post cryopreservation. Methods in Molecular Biology, 1916: 239-247.
3. 3. Artun, F.T. & Karagöz, A. 2021. Antiproliferative and apoptosis inducing effect of the methanolic extract of Centaurca hermannii in human cervical cancer cell line. Biotechnic and Histochemistry, 96:1-10.
4. 4. Aykaç, A., Gören, M.Z. & Cabadak, H. 2015. Altered ratio of proapoptotic and antiapoptotic proteins in different brain regions of female rats in model of post-traumatic stress disorder. Turkish Journal of Biochemistry, 40: 1-7.
5. 5. Banerjee, S., Kascb, A.O., Wang, Z., Kong, D., Mohammed, M., Padhye, S., Sarkar, F.H. & Mohammed, R.M. 2009. Antitumor activity of gemcitabine and oxaliplatin is augmented by thymoquinone in pancreatic cancer. Cancer Research, 69: 5575-5583.
6. 6. Bashmail, H.A., Alamoudi, A.A., Noorwali, A., Hegazy, G.A., AJabnoor, G.M. & Al-Abd, A.M. 2020. Thymoquinone enhances paclitaxel anti-breast cancer activity via inhibiting tumor-associated stem cells despite apparent mathematical antagonism. Molecules, 25: 426.
7. 7. Bashmail, H.A., Alamoudi, A.A., Noorwali, A., Hegazy, G.A., AJabnoor, G.M., Choundhry, H. & Al-Abd, A.M. 2018. Thymoquinone synergizes gemcitabine anti-breast cancer activity via modulating its apoptotic and autophagic activities. Scientific Reports, 8: 11674.
8. 8. Chang, C.H., Lee, C.H., Lu, C.C., Tsai, F.J., Hsu, Y.M., Tsao, J., Juan, Y.N., Chiu, H.Y., Yang, J.S. & Wang, C.C. 2017. Resveratrol-induced autophagy and apoptosis in cisplatin-resistant human oral cancer CAR cell: A key role of AMPK and AKT/mTOR signaling. International Journal of Oncology, 50: 873-882.

9. 9. Chen, A., Wang, H., Zhang, Y., Wang, X., Yu, L., Xu, W., Xu, W. & Lin, Y. 2017. Paeoniflorin exerts neuroprotective effects against glutamate-induced PC12 cellular cytotoxicity by inhibiting apoptosis. International Journal of Molecular Medicine, 40: 825-883.
10. 10. Christodoulou, M.I., Kontos, C.K., Halabalaki, M., Skaltsounis, A.L. & Scorilas, A. 2014. Nature promises new anticancer agents: Interplay with the apoptosis-related BCL2 gene family. Anti-Cancer Agents in Medicinal Chemistry, 14: 375-399.
11. 11. Effenberger-Neidnicht, K. & Schobert, R. 2011. Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin. Cancer Chemotherapy and Pharmacology, 67: 867-874.
12. 12. El-Mahdy, M.A., Zhu, Q., Wang, Q., Wani, G. & Wani, A.A. 2005. Thymoquinone induces apoptosis through activation of caspase-8 and mithocondrial events in p53-null myloblastic leukemia HL-60 cells. International Journal of Cancer, 117: 409-417.
13. 13. Fan, Y., Chiu, J.F., Liu, J., Deng, Y., Xu, C., Zhang, J. & Li, G. 2018. Resveratrol induces autophagy-dependent apoptosis in HL-60 cells. BioMed Central Cancer Cancer, 18: 582.
14. 14. Fricdrich, K., Wicder, T., Haefen, C.V., Radetzki, S., Janicke, R., Schulze-Osthoff, K., Dörken, B. & Danie, P.T. 2001. Overexpression of caspase-3 restores sensitivity for drug-induced apoptosis in breast cancer cell lines with acquired drug resistance. Oncogene, 20: 2749-2760.
15. 15. Gali-Muhtasib, H., Diab-Assaf, M., Boltze, C., Al-Hmaira, J., Harting, R., Roessner, A. & Schneider-Stock, R. 2004a. Tyhmoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cell via a p53-dependent mechanism. International Journal of Oncology, 25: 857-866.
16. 16. Gali-Muhtasib, H.U., Abou-Kehr W.G., Kehr, L.A., Darreiche, N. & Crooks, P.A. 2004b. Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes. Anti-Cancer Drugs, 15: 389-99.
17. 17. Ge, J., Liu, Y., Li, Q., Guo, X., Gu, L., Ma, Z.G. & Zhu, Y.P. 2013. Resveratrol induces apoptosis and autophagy in T cell acute lymphoblastic leukemia cells by inhibiting Akt/mTOR and acting p38 MAPK. Biomedical and Environmental Sciences, 26: 902-911.
18. 18. Gupta, R.K., Banerjee, A., Pathak, S., Sharma, C. & Singh, N. 2013. Induction of mithocondrial-mediated apoptosis by Morinda citrifolia (Noni) in human cervical cancer cells. Asian Pacific Journal of Cancer Prevention, 14: 237-242.
19. 19. Gurung, R.L., Lim, S.N., Khaw, A.K., Soon, J.F., Shenoy, K., Mohammed-Ali, S., Jayapal, M., Sethu, S., Baskar, R. & Hande, M. 2010. Tyhmoquinone induces telomere shortening, DNA damage and apoptosis in human glioblastoma cells. Plos One, 5: e12124.
20. 20. Huang, H., Zhang, X.F., Zhou, H.J., Xue, Y.H., Dong, Q.Z., Yc, Q.H. & Qin, L.X. 2010. Expression and prognostic significance of osteopontin and caspase-3 in hepatocellular carcinoma patients after curative resection. Cancer Science, 101: 1314-1319.
21. 21. Iskender, B., Izgi, K. & Canatan, H. 2016a. Novel anti-cancer agent myrtucommulonc-A and thymoquinone abrogate epithelialmesenchymal transition in cancer cells mainly through the inhibition of PI3K/AKT signaling axis. Molecular and Cellular Biochemistry, 416: 71-84.
22. 22. Iskender, B., Izgi, K., Hizar, E., Arslanhan, A., Yüksek, E.H. & Canatan, H. 2016b. Inhibition of epithelial-mesenchymal transition in bladder cancer cells via modulation of mTOR signalling. Tumor Biology, 37: 8281-8291.
23. 23. Kahl, M., Brioli, A., Bens, M., Perner, F., Kreisky, A., Schnetzle, U., Hinze, A., Sbirkov, Y., Stengel, S., Simonetti, G., Petrie, K., Zelent, A., Böhmer, F.D., Groth, M., Ernst, T., Heidel, F.H., Scholl, S., Hochhaus, A. & Schenk, T. 2019. The acetyltransferase GCN5 maintains ATRA-resistance in non-APL AML. Leukemia, 33: 2628-2639.
24. 24. Krumar, D., Das, B., Sen, R., Kundu, P., Manna, A., Sarkar, A., Chowdhury, C., Chatterje, M. & Das, P. 2015. Andrographolide analoge induces apoptosis and autophagy mediated cell death in U937 cells by inhibition of PI3K/Akt/mTOR pathway. Plos One, 10: e0139657.
25. 25. Kuttikrishnan, S., Siveen, K.S., Prabhu, K.S., Khan, A.Q., Ahmed, E.I., Akhtar, S., Ali, T.A., Merhi, M., Dermime, S., Steinhoff, M. & Uddin, S. 2019. Curcumin Induces Apoptotic Cell Death via Inhibition of PI3-Kinase/AKT Pathway in B-Precursor Acute Lymphoblastic Leukemia. Frontiers in Oncology, 9: 484.
26. 26. Liu, F., Gao, S., Yang, Y., Zhao, X., Fan, Y., Ma, W., Yang, D., Yang, A. & Yu, Y. 2018. Antitumor activity of curcumin by modulation of apoptosis and autophagy in human lung cancer A549 cells through inhibiting PI3K/Akt/mTOR pathway. Oncology Reports, 39: 1523-1531.
27. 27. Maha, A., Cheong, S.K., Leong, C.F. & Show H.F. 2008. Cell viability of acute myeloid leukaemia blast in culture correlates with treatment outcome. Hematology, 13: 13-20.
28. 28. Majdalawieh, A.F., Fayyad, M.W. & Nasrallah, G.K. 2017. Anti-cancer properties and mechanism of action of thymoquinone, the major active ingredient of Nigella sativa. Critical Reviews in Food Science and Nutrition, 57: 3911-3928.
29. 29. Majoloab, F., Knabbende, L., Delwinga, L.K.O.B., Marnitta, D.J., Bustamente-Filhoc, I.C. & Goettert, M.I. 2019. Medicinal plants and bioactive natural compounds for cancer treatment: Important advances for drug discovery. Phytochemistry Letters, 31: 196-207.
30. 30. Meng, X, Xia, C., Ye, Q. & Nie, X. 2020. Tert-Butyl-p-benzoquinone induces autophagy by inhibiting the Akt/mTOR signaling pathway in RAW 264.7 cells. Food and Function, 1: 4193-4201.
31. 31. Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S. & Olson, A.J. 2009. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. Journal of Computational Chemistry, 30: 2785-27891.
32. 32. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65: 55-63.
33. 33. Ng, W.K., Yazan, L.S. & İsmail, M. 2011. Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cell via apoptosis with down-regulation of Bcl-2 protein. Toxicology in Vitro, 25: 1392-1398.
34. 34. Paramasivam, A., Sambantham, S., Shabnam, J., Raghunandhakumar, S., Anandan, B., Rajiv, R., Priyadharsini, J.V. & Jayaraman, G. 2012. Anti-cancer effect of thymoquinone in mouse neuroblastoma (Neuro-2a) cells through caspase-3 activation with down-regulation of XIAP. Toxicology Letters, 213: 151-159.
35. 35. Pathania, A.S., Guru, S.K., Verma, M.K., Sharma, C., Abdullah, S.T., Malik, F., Chandra, S., Katoch, M. & Bhushan, S. 2013. Disruption of the PI3K/AKT/mTOR signaling cascade and induction of apoptosis in HL-60 cells by an essential oil from Manarda citriodora. Food and Chemical Toxicology, 62: 246-254.
36. 36. Peng, L., Liu, A., Shen, Y., Xu, H.Z., Yang, S.Z., Ying, X.Z., Liao, W., Liu, H.X. & Lin, Z.Q. Chen, Q.Y., Cheng, S.W. & Shen, W.D. 2013. Antitumor and anti-angiogenesis effects of thymoquinone on osteosarcoma through the NF-κB pathway. Oncology Reports, 29: 571-578.
37. 37. Racoma, I.O., Meisen, W.H., Wang, Q.E., Kaur, B. & Wani, A.A. 2013. Thymoquinone inhibits autophang and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells. Plos One, 8: e72882.
38. 38. Saiki, S., Sasazawa, Y., Imamichi, Y., Kawajiri, S., Fujimaki, T., Tanida, I., Kobayashi, H., Sato, F., Sato, S., Ishikawa, K., Imoto, M. & Hattori, N. 2011. Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy, 7: 176-187.
39. 39. Salim, L..ZA., Othman, R., Abdulla, M.A., Al-Jashamy, K., Ali, H.M., Hassandarvish, P., Dehghan, F., Ibrahim, M.Y., Omer, F.A. & Mohan, S. 2014. Thymoquinone inhibits murine leukemia WEHI-3 cells in vivo and in vitro. Plos One, 9: e115340.
40. 40. Samarghandian, S., Nezhad-Azimi, M. & Farkhondeh, T. 2019. Thymoquinone-induced antitumor and apoptosis in human lung adenocarcinoma cells. Journal of Cellular Physiology, 234: 10421-10431.
41. 41. Sitheek, M.A., Sivakumari, K., Rajesh, S. & Ashok, K. 2020. Molecular docking studies of apoptotic proteins Caspase-3, Caspase-9, Bax, Bcl-2 and Bcl-Xl with Ethyl (2s)-2-methyl butanoate and 1-(ethylsulfanyl) ethane-1-thiol from durian fruit. International Journal of Biology, Pharmacy and Applied Sciences, 9: 2513-2523.
42. 42. Sun, J., Feng, Y., Wang, Y., Ji, Q., Cai, G., Shi, L., Wang, Y., Huang, Y., Zhang, J. & Li, Q. 2019. a-hederin induces autophagic cell death in colorectal cancer cells through reactive oxygen species dependent AMPK/mTOR signaling pathway activation. International Journal of Oncology, 54: 1601-1612.
43. 43. Woo, C.C., Loo, S.Y., Gee, V., Yap, C.W., Sethi, G., Kumar, A.P. & Tan, K.H. 2011 Anticancer activity of thymoquinone in breast cancer cells: possible involvement of PPAR gamma pathway. Biochemical Pharmacology, 82: 464-475.
44. 44. Zhang, C.L., Zeng, T., Zhao, X.L., Yu, L.H., Zhu, Z.P. & Xie, K.Q. 2012. Protective effects of garlic oil on hepatocarcinoma ınduced by n-nitrosodieethylamine in rats. International Journal of Biological Sciences, 8: 363-374.
45. 45. Zhang, M., Du, H., Huang, Z., Zhang, P., Yue, Y., Wang, W., Liu, W., Zeng, J., Ma, J., Chen, G., Wang, X. & Fan, J. 2018a. Thymoquinone induces apoptosis in bladder cancer cell via endoplasmic reticulum stress-dependent mitochondrial pathway. Chemico-Biological Interactions, 292: 65-75.
46. 46. Zhang, Y., Fan, Y., Huang, S., Wang, G., Han, R., Lei, F., Luo, A., Jing, X., Zhao, L., Gu, S. & Zhao, X. 2018b. Thymoquinone inhibits the metastasis of renal cell cancer cell by inducing auto-handy via AMPK/mTOR signaling pathway. Cancer Science, 109: 3865-3873.
47. 47. Zhou, Z.W., Li, X.X., He, Z.X., Pan, S.T., Yang, Y., Zhang, X., Chow, K., Yang, T., Qui, J.X., Zhou, Q., Tan, J., Wang, D. & Zhou, S.F. 2015. Induction of apoptosis and autophagy via sirtuin1 and PI3K/Akt/mTOR mediated pathways by plumbagin in human prostate cancer cells. Drug Design, Development and Therapy, 9: 1511-54.