Neslihan TEKİN, Barış KERİMOĞLU, Mehtap TARHAN, Kamile ÖZTÜRK

İNSAN LÖSEMİ HÜCRE HATLARINDA S-ALLİL-L-SİSTEİN’İN APOPTOZ VE OTOFAJİ ÜZERİNE OLASI ETKİLERİNİN ARAŞTIRILMASI

AMAÇ: S-Allil-L-sistein (SAC), sarımsağın biyolojik olarak aktif bir organosülfür bileşenidir ve çeşitli farmakolojik etkilere sahiptir. SAC anti-kanser aktivite göstermektedir, ancak mekanizması belirlenememiştir. Bu çalışma, SAC'nin iki insan lösemi hücre dizisi üzerindeki olası apoptotik ve otofajik etkilerini araştırmaya odaklanmıştır: akut promiyelositik lösemi (HL-60) ve kronik miyeloid lösemi (K562).GEREÇ VE YÖNTEM: Hücre sitotoksisitesi MTT testi ile değerlendirildi. Bax, Bcl-2, kaspaz 3, mTOR, AKT ve PI3K gen ekspresyon miktarları, kantitatif gerçek zamanlı ters transkripsiyon polimeraz zincir reaksiyonu (qRT-PCR) yoluyla tanımlandı. HL-60 ve K562 hücreleri, sırasıyla üç farklı dozda (5 mM, 10 mM ve 20 mM) (3,75 mM, 7,5 mM ve 15 mM) SAC ile inkübe edildi.BULGULAR: SAC, sırasıyla yaklaşık 11.525 mM ve 10.025 mM IC50 değerleri ile HL-60 ve K562 hücreleri üzerinde sitotoksik etkiye neden olmuştur. HL-60 hücrelerinde, Bax ekspresyon seviyelerinde 5 mM ve 10 mM SAC dozlarında artış tespit edildi (p = 0.027, p = 0.000). 10 mM SAC ile tedavi, kontrol ve 5 mM SAC ile tedavi edilen hücrelere kıyasla HL-60 hücrelerinde kaspaz 3 ekspresyon seviyesini artırdı (p = 0.020, p = 0.000). K562 hücrelerinde SAC, tüm dozlarda mTOR, AKT ve PI3K ekspresyon seviyelerinde önemli bir düşüşe neden oldu (p = 0.000, p = 0.000, p = 0.000).SONUÇ: Sonuç olarak, verilerimiz SAC'nin K562 hücrelerinde PI3K/AKT/mTOR sinyal yolunu down-regüle ederek otofajiyi indüklediğini göstermektedir. Ayrıca, artan Bax ve kaspaz 3 gen ekspresyon seviyeleri, SAC'nin HL-60 hücrelerinde apoptozu indüklemek için etkili bir aktif bileşen olabileceğini düşündürmektedir.

Anahtar Kelimeler:

S-Alil-L-sistein, Bax, kaspaz 3, Bcl-2, mTOR

INVESTIGATION OF THE POSSIBLE EFFECT OF S-ALLYL-L-CYSTEINE ON APOPTOSIS AND AUTOPHAGY IN HUMAN LEUKEMIA CELL LINE

OBJECTIVE: S-Allyl-L-cysteine (SAC) is a biological active organosulfur component of garlic and has various pharmacological effects. SAC has displayed anti-cancer activity but the mechanism is unresolved. This study has focused on investigating the possible apoptotic and autophagic effects of SAC on two human leukemia cell lines: acute promyelocytic leukemia (HL-60) and chronic myeloid leukemia (K562).MATERIAL AND METHODS: Cell cytotoxicity was evaluated via MTT test. Bax, Bcl-2, caspase 3, mTOR, AKT, and PI3K gene expression amounts were identified via Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). HL-60 and K562 cells were incubated with SAC at three diverse doses (5 mM, 10 mM, and 20 mM) (3,75 mM, 7,5 mM, and 15 mM), respectively.RESULTS: SAC caused a cytotoxic effect on HL-60 and K562 cells with IC50 values of approximately 11.525 mM and 10.025 mM, respectively. In HL-60 cells, an increase in Bax expression levels was detected at doses of 5 mM and 10 mM SAC (p=0.027, p=0.000). Treatment with 10 mM SAC increased the expression level of caspase 3 in HL-60 cells as compared with the control and 5 mM SAC treated cells (p=0.000, p=0.020). In K562 cells, SAC induced a significant decrease in mTOR, AKT, and PI3K expression levels in at all doses (p=0.000, p=0.000, p=0.000).CONCLUSIONS: In conclusion, our data indicates that SAC induces autophagy in K562 cells by downregulating the PI3K/AKT/mTOR signaling pathway. Furthermore, increased Bax and caspase 3 gene expression levels suggest that SAC may be an effective active ingredient with which to induce apoptosis in HL-60 cells.

Keywords:

S-Allyl-L-cysteine, Bax, caspase 3, Bcl-2, mTOR,

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1. Bounid D, Haouach K. Acute Leukemias in Marrakech: Epidemiology and Cytological Profile. Int J Biol Med Res. 2019;10:6685-9.
2.Yan H, Zhao RM, Wang ZJ, Zhao FR, Wang SL. Knockdown of PRAME enhances adriamycin-induced apoptosis in chronic myeloid leukemia cells. Eur Rev Med Pharmacol Sci. 2015;19:4827-34.
3. Su WC, Chang SL, Chen TY, Chen JS, Tsao CJ. Comparison of in vitro growth-inhibitory activity of carboplatin and cisplatin on leukemic cells and hematopoietic progenitors: the myelosuppressive activity of carboplatin may be greater than its antileukemic effect. Jpn J Clin Oncol. 2000;30:562-7.
4. Pearce A, Haas M, Viney R, et al. Incidence and severity of self-reported chemotherapy side effects in routine care: A prospective cohort study. PLoS One. 2017;12:e0184360.
5. Sawadogo WR, Schumacher M, Teiten MH, Cerella C, Dicato M, Diederich M. A Survey of Marine Natural Compounds and Their Derivatives with Anti-Cancer Activity Reported in 2011. Molecules. 2013;18:3641-73.
6. Mojarraba M, Lagzianb MJ, Emamic SA, Asilic J, Najaranb ZT. In vitro anti-proliferative and apoptotic activity of different fractions of Artemisia armeniaca. Rev Bras Farmacogn. 2013;23:783-8.
7. Xu YS, Feng JG, Zhang D, et al. S-allylcysteine, a garlic derivative, suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro. Acta Pharmacol Sin. 2014;35:267-74.
8. Cho O, Hwang HS, Lee BS, Oh YT, Kim CH, Chun M. Met inactivation by S-allylcysteine suppresses the migration and invasion of nasopharyngeal cancer cells induced by hepatocyte growth factor. Radiat Oncol J. 2015;33:328-36.
9. Sun HJ, Meng LY, Shen Y, Zhu YZ, Liu HR. S-benzyl-cysteine-mediated Cell Cycle Arrest and Apoptosis Involving Activation of Mitochondrial-dependent Caspase Cascade through the p53 Pathway in Human Gastric Cancer SGC-7901 Cells. Asian Pac J Cancer Prev. 2013;14:6379-84.
10. Ho JN, Kang M, Lee S, et al. Anticancer effect of S allyl L cysteine via induction of apoptosis in human bladder cancer cells. Oncol Lett. 2018;15:623-9.
11. Platini F, Pérez-Tomás R, Ambrosio S, Tessitore L. Understanding Autophagy in Cell Death Control. Curr Pharm Des. 2010;16:101-13.
12. Heras-Sandoval D, Pérez-Rojas JM, Hernández-Damián J, Pedraza-Chaverri J. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal. 2014;26:2694-701.
13. Wang Z, Zhou L, Zheng X, et al. Autophagy protects against PI3K/Akt/mTOR-mediated apoptosis of spinal cord neurons after mechanical injury. Neurosci Lett. 2017;656:158-64.
14. Xu K, Liu P, Wei W. mTOR signaling in tumorigenesis. Biochim Biophys Acta. 2014;1846:638-54.
15. Rahmani F, Ferns GA, Talebian S, Nourbakhsh M, Avan A, Shahidsales S. Role of regulatory miRNAs of the PI3K/AKT signaling pathway in thepathogenesis of breast cancer. Gene. 2020;737:1444592.
16. Tang FY, Chiang EP, Pai MH. Consumption of S-allylcysteine inhibits the growth of human non-small-cell lung carcinoma in a mouse xenograft model. J Agric Food Chem. 2010;58:11156-64.
17. Tekin N, Öztürk K, Baran T, Kerimoğlu B, Tarhan M, Menteş A. Cytotoxic and apoptotic activities of novel Pd(II) complexes against human leukemia cell lines in vitro. Journal of Macromolecular Science, Part A. 2017;54:263-70.
18. Ma L, Li W, Wang R, et al. Resveratrol enhanced anticancer effects of cisplatin on non-small cell lung cancer cell lines by inducing mitochondrial dysfunction and cell apoptosis. Int J Oncol. 2015;47:1460-8.
19. Ng KT, Guo DY, Cheng Q, et al. A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma. PLoS One. 2012;7:31655.
20. Gapter LA, Yuin OZ, Ng KY. S-Allylcysteine reduces breast tumor cell adhesion and invasion. Biochem Biophys Res Commun. 2008;367:446-51.
21. Shirin H, Pinto JT, Kawabata Y, et al. Antiproliferative Effects of S-Allylmercaptocysteine on Colon Cancer Cells When Tested Alone or in Combination with Sulindac Sulfide. Cancer Res. 2001;61:725-31.
22. John GB, Anjum R, Khar A, Nagaraj R. Subcellular localization and physiological consequences of introducing a mitochondrial matrix targeting signal sequence in bax and its mutants. Exp Cell Res. 2002;278:198-208.
23. Zhang H, Wang K, Lin G, Zhao Z. Antitumor mechanisms of S-allyl mercaptocysteine for breast cancer therapy. BMC Complement Altern Med. 2014;14:270.
24. Upadhyay RK. Garlic Induced Apoptosis, Cell Cycle Check Points and Inhibition of Cancer Cell Proliferation. J Cancer Res Treat. 2017;5:35-54.
25. Izdebska M, Grzanka D, Gagat M, Hałas-Wiśniewska M, Grzanka A. Downregulation of importin-9 protects MCF-7 cells against apoptosis induced by the combination of garlic-derived alliin and paclitaxel. Oncol Rep. 2016;35:3084-93.
26. QM Chen, J Liu and JB Merrett. Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts. Biochem J. 2000;347:543-51.
27. CA Schmitt. Senescence, apoptosis and therapy--cutting the lifelines of cancer. Nat Rev Cancer. 2003;3:286-95.
28. Raouf AA, Evoy DA, Carton E, et al. Loss of Bcl-2 expression in Barrett's dysplasia and adenocarcinoma is associated with tumor progression and worse survival but not with response to neoadjuvant chemoradiation. Dis Esophagus. 2003;16:17-23.
29. Stoklosa T, Glodkowska-Mrowka E, Hoser G, et al. Diverse mechanisms of mTOR activation in chronic and blastic phase of chronic myelogenous leukemia. Exp Hematol. 2013;41:462-9.
30. Zhang CZ, Wang XD, Wang HW, et al. Sorafenib inhibits liver cancer growth by decreasing mTOR, AKT, and PI3K expression. J BUON 2015;20:218-22.