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

Year 2023, Volume: 30 Issue: 1, 1 - 8, 14.03.2023
https://doi.org/10.17343/sdutfd.1099504

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.

Supporting Institution

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

Thanks

The Saos-2 cell line was a kind gift of Assoc. Prof. Dr. Mustafa Güngörmüş Ankara Yıldırım Beyazıt University Faculty of Dentistry. This study was carried out using the facilities of Ankara Yıldırım Beyazıt University, Central Research Laboratory Application and Research Center (MERLAB).

References

  • 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.

K2 VİTAMİNİNİN OSTEOSARKOMA HÜCRELERİNDE ANTİPROLİFERATİF ETKİLERİ: FARKLI SİTOTOKSİSİTE ANALİZLERİNİN KARŞILAŞTIRILMASI

Year 2023, Volume: 30 Issue: 1, 1 - 8, 14.03.2023
https://doi.org/10.17343/sdutfd.1099504

Abstract

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.

References

  • 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.
There are 32 citations in total.

Details

Primary Language English
Subjects Clinical Sciences
Journal Section Araştırma Makaleleri
Authors

Büşranur Özalper 0000-0002-1599-3637

Tuba Özdemir Sancı 0000-0002-9468-4719

Habibe Özgüner 0000-0003-0364-5907

Publication Date March 14, 2023
Submission Date April 6, 2022
Acceptance Date February 22, 2023
Published in Issue Year 2023 Volume: 30 Issue: 1

Cite

Vancouver Özalper B, Özdemir Sancı T, Özgüner H. ANTIPROLIFERATIVE EFFECTS OF VITAMIN K2 IN OSTEOSARCOMA CELLS: COMPARISON OF DIFFERENT CYTOTOXICITY ANALYZES. Med J SDU. 2023;30(1):1-8.

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