Journals / SDÜ Tıp Fakültesi Dergisi / 2026 / Cilt: 30 Sayı: 1

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

K2 VİTAMİNİNİN OSTEOSARKOMA HÜCRELERİNDE ANTİPROLİFERATİF ETKİLERİ: FARKLI SİTOTOKSİSİTE ANALİZLERİNİN KARŞILAŞTIRILMASI

Büşranur Özalper(MUS ALPARSLAN UNIVERSITY)0000-0002-1599-3637
Tuba Özdemir Sancı(ANKARA YILDIRIM BEYAZIT UNIVERSITY)0000-0002-9468-4719
Habibe Özgüner(ANKARA YILDIRIM BEYAZIT UNIVERSITY)0000-0003-0364-5907
Pages
1–8
Publication Date
2023-03-14
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AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

It has been concluded that incubations at different concentrations were required to prevent misinterpretation of results in cytotoxicity analyses, and tetrazolium salt-based tests should be supplemented with other non-metabolic tests.

📌 3 Key Takeaways

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Objective & Hypothesis:Objective The aim of this study was to compare four commonly used cytotoxicity assays (XTT, neutral red uptake, crys- tal violet assay, and propidium iodide staining) which analyzed the antiproliferative effects of vitamin K2.
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Key Findings & Data:Material and Method Saos-2 cells, an osteosarcoma cell line, were exposed to vitamin K2 at different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM and 200 μM) for 48 and 72 hours.
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Conclusion & Impact:It has been concluded that incubations at different concentrations were required to prevent misinterpretation of results in cytotoxicity analyses, and tetrazolium salt-based tests should be supplemented with other non-metabolic tests.
🔬Design:Araştırma Makalesi
📍Scope:Comprehensive Sample
⚙️Variables:İn vitro sitotoksisite · Hücre canlılık testleri · Osteosarkoma

Abstract

Objective The aim of this study was to compare four commonly used cytotoxicity assays (XTT, neutral red uptake, crys- tal violet assay, and propidium iodide staining) which analyzed the antiproliferative effects of vitamin K2. Material and Method Saos-2 cells, an osteosarcoma cell line, were exposed to vitamin K2 at different concentrations (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM and 200 μM) for 48 and 72 hours. Tetrazolium salt test (XTT), neutral red uptake (NR) assay, crystal violet assay, and propidium iodide (PI) staining were performed to determine cytotoxic potential of vitamin K2 in terms of the cell viability and IC50 values. The results were evaluated with one-way analysis of variance (ANOVA) and the Tukey test. Results Cytotoxic effects of vitamin K2 on osteosarcoma cells were analyzed with XTT, neutral red, crystal violet assay, and propidium iodide, respectively. IC50 values were 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. Conclusion Statistical analysis revealed that there is a significant statistical difference between four tests used in this study. In addition, it was determined that the viability rates in propidium iodide staining were higher than other tests for cytotoxicity analyses. It has been concluded that incubations at different concentrations were required to prevent misinterpretation of results in cytotoxicity analyses, and tetrazolium salt-based tests should be supplemented with other non-metabolic tests.

Özet

Amaç Bu çalışmanın amacı, K2 vitamininin antiproliferatif etkilerini saptamak için yaygın olarak kullanılan dört sitotoksisite analizinin (XTT, nötral kırmızı alım, kristal viyole ve propidyum iyodür boyama) doğruluk potansiyelini karşılaştırmaktır. Gereç ve Yöntem Osteosarkoma hücre hattı olan Saos-2 hücreleri, 48 ve 72 saat boyunca K2 vitaminine (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM ve 200 μM) maruz bırakıldı. Tetrazolyum tuzu testi (XTT), nötral kırmızı alım testi (NR), kristal viyole testi ve propidyum iyodür (PI) boyama yapılarak hücrelerin canlılık oranlarına göre K2 vitamini sitotoksisitesi belirlendi ve farklı dozların IC50 değerleri hesaplandı. Analiz sonuçları tek yönlü varyans analizi (ANOVA) ve Tukey testiyle değerlendirildi. Bulgular K2 vitamininin osteosarkoma hücreleri üzerinde sitotoksik etkileri olduğu sırasıyla XTT, NR, kristal viyole testi ve PI yöntemleri kullanılarak belirlendi ve IC50 değerleri 48 saatlik maruziyet için sırasıyla 61,93; 40,21; 62,11; 70,57 μM ve 72 saatlik maruziyet için sırasıyla 75,44; 68,22; 41,66; 88,01 μM olarak saptandı. Sonuç İstatistiksel analizler çalışmada uygulanan testlerden elde edilen sonuçların farklılık gösterdiğini ortaya koymuştur. Ayrıca propidyum iyodür boyama sonucundaki canlılık oranlarının diğer testlerle elde edilen sonuçlara kıyasla çarpıcı bir şekilde daha yüksek olduğu belirlenmiştir. Sitotoksisite analizlerinde sonuçların yanlış yorumlanmasını önlemek için farklı zamanlarda ve farklı konsantrasyonlarda inkübasyonlar gerektiği ve ayrıca tetrazolyum tuzu temelli testlerin metabolik olmayan diğer testlerle desteklenmesi gerektiği sonucuna varılmıştır.

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Cite This Article

Özalper, B., Sancı, T. Ö., Özgüner, H. (2023). ANTIPROLIFERATIVE EFFECTS OF VITAMIN K2 IN OSTEOSARCOMA CELLS: COMPARISON OF DIFFERENT CYTOTOXICITY ANALYZES. SDÜ Tıp Fakültesi Dergisi, Cilt: 30 Sayı: 1, 1–8. https://doi.org/10.17343/sdutfd.1099504

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References (32)

  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.