sdü tıp fak derg

Medical Journal of Süleyman Demirel University

2602-2109

Süleyman Demirel Üniversitesi

10.17343/sdutfd.1099504

Clinical Sciences

Klinik Tıp Bilimleri

K2 VİTAMİNİNİN OSTEOSARKOMA HÜCRELERİNDE ANTİPROLİFERATİF ETKİLERİ: FARKLI SİTOTOKSİSİTE ANALİZLERİNİN KARŞILAŞTIRILMASI

ANTIPROLIFERATIVE EFFECTS OF VITAMIN K2 IN OSTEOSARCOMA CELLS: COMPARISON OF DIFFERENT CYTOTOXICITY ANALYZES

https://orcid.org/0000-0002-1599-3637

Özalper

Büşranur

MUS ALPARSLAN UNIVERSITY

https://orcid.org/0000-0002-9468-4719

Özdemir Sancı

Tuba

ANKARA YILDIRIM BEYAZIT UNIVERSITY

https://orcid.org/0000-0003-0364-5907

Özgüner

Habibe

ANKARA YILDIRIM BEYAZIT UNIVERSITY

03 14 2023

30 1 1 8 04 06 2022 02 22 2023

1994

Süleyman Demirel Üniversitesi Tıp Fakültesi Dergisi

AmaçBu çalışmanın amacı, K2 vitamininin antiproliferatifetkilerini saptamak için yaygın olarak kullanılan dörtsitotoksisite analizinin (XTT, nötral kırmızı alım, kristalviyole ve propidyum iyodür boyama) doğruluk potansiyelinikarşılaştırmaktır.Gereç ve YöntemOsteosarkoma hücre hattı olan Saos-2 hücreleri, 48ve 72 saat boyunca K2 vitaminine (10 μM, 20 μM, 30μM, 40 μM, 50 μM, 100 μM ve 200 μM) maruz bırakıldı.Tetrazolyum tuzu testi (XTT), nötral kırmızı alımtesti (NR), kristal viyole testi ve propidyum iyodür (PI)boyama yapılarak hücrelerin canlılık oranlarına göreK2 vitamini sitotoksisitesi belirlendi ve farklı dozlarınIC50 değerleri hesaplandı. Analiz sonuçları tek yönlüvaryans analizi (ANOVA) ve Tukey testiyle değerlendirildi.BulgularK2 vitamininin osteosarkoma hücreleri üzerinde sitotoksiketkileri olduğu sırasıyla XTT, NR, kristal viyoletesti ve PI yöntemleri kullanılarak belirlendi ve IC50 değerleri48 saatlik maruziyet için sırasıyla 61,93; 40,21;62,11; 70,57 μM ve 72 saatlik maruziyet için sırasıyla75,44; 68,22; 41,66; 88,01 μM olarak saptandı.Sonuçİstatistiksel analizler çalışmada uygulanan testlerdenelde edilen sonuçların farklılık gösterdiğini ortaya koymuştur.Ayrıca propidyum iyodür boyama sonucundakicanlılık oranlarının diğer testlerle elde edilen sonuçlarakıyasla çarpıcı bir şekilde daha yüksek olduğubelirlenmiştir. Sitotoksisite analizlerinde sonuçlarınyanlış yorumlanmasını önlemek için farklı zamanlardave farklı konsantrasyonlarda inkübasyonlar gerektiğive ayrıca tetrazolyum tuzu temelli testlerin metabolikolmayan diğer testlerle desteklenmesi gerektiği sonucunavarılmıştır.

ObjectiveThe aim of this study was to compare four commonlyused cytotoxicity assays (XTT, neutral red uptake, crys-tal violet assay, and propidium iodide staining) whichanalyzed the antiproliferative effects of vitamin K2.Material and MethodSaos-2 cells, an osteosarcoma cell line, wereexposed to vitamin K2 at different concentrations (10μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM and 200μM) for 48 and 72 hours. Tetrazolium salt test (XTT),neutral red uptake (NR) assay, crystal violet assay,and propidium iodide (PI) staining were performed todetermine cytotoxic potential of vitamin K2 in termsof the cell viability and IC50 values. The results wereevaluated with one-way analysis of variance (ANOVA)and the Tukey test.ResultsCytotoxic effects of vitamin K2 on osteosarcoma cellswere analyzed with XTT, neutral red, crystal violetassay, and propidium iodide, respectively. IC50 valueswere determined exposure to 61.93; 40.21; 62.11;70.57 μM vitamin K2 for 48 and 75.44; 68.22; 41.66;88.01 μM vitamin K2 for 72 hours.ConclusionStatistical analysis revealed that there is a significantstatistical difference between four tests used in thisstudy. In addition, it was determined that the viabilityrates in propidium iodide staining were higher thanother tests for cytotoxicity analyses. It has beenconcluded that incubations at different concentrationswere required to prevent misinterpretation of results incytotoxicity analyses, and tetrazolium salt-based testsshould be supplemented with other non-metabolictests.

In vitro cytotoxicity Cell viability tests Osteosarcoma Vitamin K.

İn vitro sitotoksisite Hücre canlılık testleri Osteosarkoma K vitamini.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

1. Ando K, Heymann MF, Stresing V, Mori K, Rédini F, Heymann D. Current therapeutic strategies and novel approaches in osteosarcoma. Cancers. 2013;5(2):591-616.

2. Roberts RD, Lizardo MM, Reed DR, Hingorani P, Glover J, Allen-Rhoades W, Fan T, et al. Provocative questions in osteosarcoma basic and translational biology: A report from the Children's Oncology Group. Cancer. 2019;125(20): 3514-3525.

3. Hoyt M, Reger M, Marley A, Fan H, Liu Z, Zhang J. Vitamin K intake and prostate cancer risk in the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) Screening Trial. Am J Clin Nutr. 2019;109(2):392-401.

4. Xia J, Ozaki I, Matsuhashi S, Kuwashiro T, Takahashi H, Anzai K, Mizuta T. Mechanisms of PKC-mediated enhancement of HIF-1α activity and its inhibition by Vitamin K2 in hepatocellular carcinoma cells. Int J Mol Sci. 2019;20(5):1022.

5. Duan F, Yu Y, Guan R, Xu Z, Liang H, Hong L. Vitamin K2 induces mitochondria-related apoptosis in human bladder cancer cells via ROS and JNK/p38 MAPK signal pathways. PLoS One. 2016;11(8):1-20.

6. Jiang Y, Yang J, Yang C, Meng F, Zhou Y, Yu B, Khan M, Yang H. Vitamin K4 induces tumor cytotoxicity in human prostate carcinoma PC-3 cells via the mitochondria-related apoptotic pathway. Pharmazie. 2013;68:442-448.

7. Baran I, Ganea C, Scordino A, Musumeci F, Barresi V, Tudisco S, Privitera S, Grasso R, Condorelli DF, Ursu I, Baran V, Katona E, Mocanu MM et al. Effects of menadione, hydrogen peroxide, and quercetin on apoptosis and delayed luminescence of human leukemia Jurkat T-cells. Cell Biochem Biophys. 2010;58(3):169-179.

8. Akiyoshi T, Matzno S, Sakai M, Okamura N, Matsuyama K. The potential of vitamin K3 as an anticancer agent against breast cancer that acts via the mitochondria-related apoptotic pathway. Cancer Chemother Pharmacol. 2009;65(1):143-150.

9. Demirtaş F, Evim MS, Baytan B, Güneş AM. Vitamin K ve Hemostaz. Güncel Pediatri. 2010;8: 113-8.

10. Chatron N, Hammed A, Benoit E, Lattard V. Structural Insights into Phylloquinone (Vitamin K1), Menaquinone (MK4, MK7), and Menadione (Vitamin K3) Binding to VKORC1. Nutrients. 2019;11(1):67.

11. Owen R, Bahmaee H, Claeyssens F, Reilly GC. Comparison of the anabolic effects of reported osteogenic compounds on human mesenchymal progenitor-derived osteoblasts. Bioengineering. 2020;7(12):1-19.

12. Tokur O, Aksoy A. In vitro sitotoksisite testleri. Harran Üniversitesi Veteriner Fakültesi Dergisi. 2017;6(1):112-118.

13. Riss TL, Moravec RA. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay and drug development Technologies. 2004;2(1):51-62.

14. Aslantürk ÖS. In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages. Genotoxicity-A predictable risk to our actual World. 2018;2:64-80.

15. Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nogziger TH, Currens MJ, Seniff D, Boyd MR. Evaluation of a soluble tetrazoliun/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research. 1988;48:4827-4833.

16. Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Prot. 2008;3(7):1125–1131.

17. Babich H, Borenfreund E. Applications of the neutral red cytotoxicity assay to in vitro toxicology. Altern Lab Anim. 1990;18:129-144.

18. Winckler J. Vital staining of lysosomes and other cell organelles of the rat with neutral Red. Prog Histochem Cytochem. 1974;6(3):1–91.

19. Nemes Z, Dietz R, Luth JB. The pharmacological relevance of vital staining with neutral red. Experientia. 1979;35:1475–1476.

20. Zwolak I. Comparison of three different cell viability assays for evaluation of vanadyl sulphate cytotoxicity in a Chinese hamster ovary K1 cell line. Toxicology and industrial health. 2016;32(6):1013-1025.

21. Feoktistova M, Geserick P, Leverkus M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harbor Protocols. 2016;(4), pdb-prot087379.

22. Atale N, Gupta S, Yadav UCS, Rani V. Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. Journal of microscopy. 2014;255(1),7-19.

23. Crowley LC, Scott AP, Marfell BJ, Boughaba JA, Chojnowski G, Waterhouse NJ. Measuring cell death by propidium iodide uptake and flow cytometry. Cold Spring Harbor Protocols. 2016;(7), pdb-prot087163.

24. Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death & Differentiation. 2009;16(8):1093-1107.

25. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 2009;16:3–11.

26. Di W, Khan M, Gao Y, Cui J, Wang D, Qu M, Feng L, Maryam A, Gao H. Vitamin K4 inhibits the proliferation and induces apoptosis of U2OS osteosarcoma cells via mitochondrial dysfunction. Mol Med Rep. 2017;15(1),277-284.

27. Zenmyo M, Komiya S, Hamada T, Hiraoka K, et al. Transcriptional activation of p21 by vitamin D(3) or vitamin K(2) leads to differentiation of p53-deficient MG-63 osteosarcoma cells. Hum Pathol. 2001;32(4),410-6.

28. Otsuka M, Kato N, Shao RX, Hoshida Y, Ijichi H, Koike Y, et al. Vitamin K2 inhibits the growth and invasiveness of hepatocellular carcinoma cells via protein kinase A activation. Hepatology. 2004;40(1), 243-251.

29. Yoshida T, Miyazawa K, Kasuga I, Yokoyama T, Minemura K, Ustumi K, et al. Apoptosis induction of vitamin K2 in lung carcinoma cell lines: the possibility of vitamin K2 therapy for lung cancer. International journal of oncology. 2003;23(3),627-632.

30. Yokoyama T, Miyazawa K, Yoshida T, Ohyashiki K. Combination of vitamin K2 plus imatinib mesylate enhances induction of apoptosis in small cell lung cancer cell lines. International journal of oncology. 2005;26(1),33-40.

31. Li L, Qi Z, Qian J, Bi F, Lv J, Xu L, et al. Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K2 is associated with p53 and independent of the intrinsic apoptotic pathway. Molecular and cellular biochemistry. 2010;342(1),125- 131.

32. Fotakis G, Timbrell JA. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett. 2006;160:171–177.