Oksidatif, Apoptotik ve İnflamatuar Sinyal Yolakları üzerinden C6 Glioblastoma Hücrelerindeki ML351'in Antiproliferatif Etkileri

Abstract

Amaç: Kanser tedavisindeki başarılı yaklaşımlardan biri de spesifik inhibitörlerin kullanılmasıdır. Çoklu doymamış yağ asitlerinin metabolizmasından sorumlu olan 12/15-lipoksijenaz (12/15-LOX) nöronlarda oksidatif stres kaynaklanan hücre ölümüne aracılık etmesinin yanı sıra, kanser gibi birçok önemli hastalıkta yer alan metabolitlerin üretemini de gerçekleştirir. Bu çalışmada, 12/15-LOX inhibitörü olan ML351'in C6 glioblastoma hücrelerin üzerindeki anti-proliferatif etkilerini çeşitli biyokimyasal süreçler üzerinden araştırılması amaçlanmıştır. Gereç ve Yöntemler: Çalışmada önce C6 hücreleri için sitotoksik ML351 konsantrasyonları metil tiazolil tetrazolyum (MTT) kullanılarak tespit edilmiştir ve ardından total oksidan kapasite (TOS), sitokrom c (CYC), kaspaz 3 (CASP3), tümör nekroz faktör alfa (TNF-α) ve interlökin-6 (IL-6) seviyeleri ölçüldü. Bulgular: Sonuç olarak, ML351 C6 hücreleri üzerinde konsantrasyon bağımlı anti-proliferatif etkilerinin olduğu görüldü. ML351 uygulamasının C6 hücrelerinde oksidatif strese neden olarak TOS seviyelerini artırdığı belirlendi. ML351 uygulanması kontrol grubu ile karşılaştırıldığında CASP3 ve CYC seviyelerini artırdı (p<0,001). Bu sonuçlara göre, ML351 uygulamasının C6 hücrelerinde apoptozu indüklediği görüldü. Öte yandan, ML351 ile tedavi edilen C6 hücrelerinde kontrol grubuna göre TNF-α ve IL-6 seviyelerinin düştüğü belirlendi (p<0,01). Sonuç: Çalışmamız, C6 glioma hücrelerinde ML 351 uygulamasının pro-oksidan/oksidan dengesini bozarak oksidatif hasar ve apoptoza neden olduğunu ve inflamasyonu azalttığını göstermektedir. Bu nedenle bir antikanser ilaç olma potansiyeline sahip ML351'in farklı kanser hücre hatlarındaki faklı metabolik yolaklar üzerindeki etkilerini araştıran daha ileri çalışmalar yapılmalıdır.

Keywords

• ML351
• C6glioblastoma
• oksidatif stres
• apoptoz
• inflamasyon

Antiproliferative Effects of ML351 on C6 Glioblastoma Cells through Oxidative, Apoptotic and İnflammatory Signaling Pathways

Abstract

Aim: One of the successful approaches in cancer treatment is the use of specific inhibitors. Responsible for the metabolism of polyunsaturated fatty acids, 12/15-lipoxygenase (12/15-LOX) not only mediates cell death caused by oxidative stress in neurons, but also produces metabolites involved in many important diseases such as cancer. In this study, it was aimed to investigate the anti-proliferative effects of ML351, 12/15-LOX inhibitor, on C6 glioblastoma cells through various biochemical processes. Material and Methods: In the study, cytotoxic ML351 concentrations for C6 cells were determined using methyl thiazolyl tetrazolium (MTT), and then total oxidant capacity (TOS), cytochrome c (CYC), caspase 3 (CASP3), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels were measured. Results: As a result, it was found to have concentration dependent anti-proliferative effects on ML351 C6 cells. It was determined that ML351 treatment increased TOS levels by causing oxidative stress in C6 cells. The ML351 treatment increased CASP3 and CYC levels compared to the control group (p<0.01). Based on these results, it was observed that ML351 treatment induced apoptosis in C6 cells. On the other hand, TNF-α and IL-6 levels were decreased in C6 cells treated with ML351 compared to the control group (p<0.01). Conclusion: Our study shows that ML 351 treatment causes oxidative damage and apoptosis by disrupting the pro-oxidant/oxidant balance and decreases inflammation in C6 glioma cells. Therefore, further studies should be conducted to investigate the effects of ML351, which has the potential to be an anticancer drug, on different metabolic pathways in different cancer cell lines.

Keywords

• ML351
• C6 glioblastoma
• oxidative stress
• apoptosis
• inflammation

References

1. Modrek AS, Bayin NS, Placantonakis DG. Brain stem cells as the cell of origin in glioma. World J Stem Cells. 2014; 6(1): 43-52.
2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007; 114(2): 97-109.
3. Chen R, Smith-Cohn M, Cohen AL, Colman H. Glioma subclassifications and their clinical significance. Neurotherapeutics. 2017;14(2): 284-97.
4. Grobben B, De Deyn PP, Slegers H. Rat C6 glioma as experimental model system for the study of glioblastoma growth and invasion. Cell Tissue Res. 2002; 310(3): 257-70.
5. Colquhoun A. Cell biology-metabolic crosstalk in glioma. Int J Biochem Cell Biol. 2017; 89: 171-81.
6. Azrad M, Turgeon C, Demark-Wahnefried W. Current evidence linking polyunsaturated Fatty acids with cancer risk and progression. Front Oncol. 2013; 3: 224.
7. Souza FDC, Ferreira MT, Colquhoun A. Influence of Lipoxygenase Inhibition on Glioblastoma Cell Biology. Int J Mol Sci. 2020; 21(21): 8395.
8. Orafaie A, Matin MM, Sadeghian H. The importance of 15-lipoxygenase inhibitors in cancer treatment. Cancer Metastasis Rev. 2018 Sep;37(2-3):397-408.

9. Rai G, Joshi N, Perry S, Yasgar A, Schultz L, Jung JE, et al. Discovery of ML351, a potent and selective inhibitor of human 15-lipoxygenase-1. Available from http://www.ncbi.nlm.nih.gov/books/NBK190602
10. Hacioglu C, Kar F, Kacar S, Sahinturk V, Kanbak G. Bexarotene inhibits cell proliferation by inducing oxidative stress, DNA damage and apoptosis via PPARγ/ NF-κB signaling pathway in C6 glioma cells. Med Oncol. 2021; 38(3): 31.
11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265-275.
12. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144(5): 646-74.
13. Catalano A, Procopio A. New aspects on the role of lipoxygenases in cancer progression. Histol Histopathol. 2005; 20(3): 969-75.
14. Tang DG, Bhatia B, Tang S, Schneider-Broussard R. 15-lipoxygenase 2 (15-LOX2) is a functional tumor suppressor that regulates human prostate epithelial cell differentiation, senescence, and growth (size). Prostaglandins Other Lipid Mediat. 2007; 82(1-4): 135-46.
15. Pidgeon GP, Lysaght J, Krishnamoorthy S, Reynolds JV, O'Byrne K, Nie D, et al. Lipoxygenase metabolism: roles in tumor progression and survival. Cancer Metastasis Rev. 2007; 26(3-4): 503-24.
16. DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv. 2016; 2(5): e1600200.
17. Schneider C, Pozzi A. Cyclooxygenases and lipoxygenases in cancer. Cancer Metastasis Rev. 2011; 30(3-4): 277-94.
18. Hyde CA, Missailidis S. Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis. Int Immunopharmacol. 2009; 9(6): 701-15.
19. Hosseinymehr M, Matin MM, Sadeghian H, Bahrami AR, Kaseb-Mojaver N. 8-Farnesyloxycoumarin induces apoptosis in PC-3 prostate cancer cells by inhibition of 15-lipoxygenase-1 enzymatic activity. Anticancer Drugs. 2016; 27(9): 854-62.
20. Jun M, Bacay AF, Moyer J, Webb A, Carrico-Moniz D. Synthesis and biological evaluation of isoprenylated coumarins as potential anti-pancreatic cancer agents. Bioorg Med Chem Lett. 2014; 24(19): 4654-58.
21. Durackova Z. Some current insights into oxidative stress. Physiol Res. 2010; 59(4): 459-69.
22. Azad MB, Chen Y, Gibson SB. Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid Redox Signal. 2009; 11(4): 777-90.
23. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med. 2010; 49(11): 1603-16.
24. Chang J, Tang N, Fang Q, Zhu K, Liu L, Xiong X, et al. Inhibition of COX-2 and 5-LOX regulates the progression of colorectal cancer by promoting PTEN and suppressing PI3K/AKT pathway. Biochem Biophys Res Commun. 2019; 517(1): 1-7.
25. Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D'Orazi G. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging (Albany NY). 2016; 8(4): 603-19.
26. Jeong SY, Seol DW. The role of mitochondria in apoptosis. BMB Rep. 2008; 41(1): 11-22.
27. Avis I, Hong SH, Martinez A, Moody T, Choi YH, Trepel J, et al. Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions. FASEB J. 2001; 15(11): 2007-9.
28. Avis IM, Jett M, Boyle T, Vos MD, Moody T, Treston AM, et al. Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling. J Clin Invest. 1996; 97(3): 806-13.
29. Wong BC, Wang WP, Cho CH, Fan XM, Lin MC, Kung HF, et al. 12-Lipoxygenase inhibition induced apoptosis in human gastric cancer cells. Carcinogenesis. 2001; 22(9): 1349-54.
30. Sarveswaran S, Thamilselvan V, Brodie C, Ghosh J. Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon. Biochim Biophys Acta. 2011; 1813(12): 2108-17.
31. Mantovani A. Cancer: Inflaming metastasis. Nature. 2009; 457(7225): 36-7.
32. Rauert H, Stühmer T, Bargou R, Wajant H, Siegmund D. TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms. Cell Death Dis. 2011; 2(8): e194.
33. Martin M, Wei H, Lu T. Targeting microenvironment in cancer therapeutics. Oncotarget. 2016; 7(32): 52575-83.
34. Pidgeon GP, Lysaght J, Krishnamoorthy S, Reynolds JV, O'Byrne K, Nie D, Honn KV. Lipoxygenase metabolism: roles in tumor progression and survival. Cancer Metastasis Rev. 2007; 26(3-4): 503-24.
35. Gunning WT, Kramer PM, Steele VE, Pereira MA. Chemoprevention by lipoxygenase and leukotriene pathway inhibitors of vinyl carbamate-induced lung tumors in mice. Cancer Res. 2002; 62(15): 4199-201.