Real time monitoring of cytotoxicity of Callistemon citrinus against Colo-205 cell line

Abstract

DOI: 10.26650/IstanbulJPharm.2019.418892

Callistemon citrinus is a member of Myrtaceae family that thrives under different ecological conditions. The leaves, flowers, stem backs and roots of the plant contain various phytochemicals that are useful in   folk medicine for different remedies such as antimicrobial, anti-nociceptive, fungicide and anti-inflammatory purposes. In this study, we investigated the cytotoxic effect of Callistemon citrinus leaf and flower methanolic extracts against human Colo-205 Cell Line using real time cell analyzer device for monitoring in time-dependent manner. To determine the mechanism of cytotoxicity of the extracts, Western blotting assay was used for measuring evocation of Akt pathway. Extracts were found to exert cytotoxic effect at a dose dependent manner. IC50 values of leaves and flowers extract were 6.49 µg/mL and 5.22 µg/mL, respectively. At the early stages of the experiment, Akt pathway was triggered at high extract concentrations. Although, high extract concentrations showed proliferative effect at early stages, this effect reversed after 5 and 8 h resulting in low cell viability. Findings from this study therefore showed that extracts of leaf and flower from Callistemon citrinus demonstrated cytotoxic effect against Colo-205 but seems not to be related Akt signaling pathway.

Cite this article as: Dokumacı AH, Fayemi PO, Aycan Yerer B. (2019). Real time monitoring of cytotoxicity of Callistemon citrinus against Colo-205 cell line. Istanbul J Pharm 49 (1): 25-32.

Keywords

• Callistemon citrinus,Akt/p-Akt,colon cancer,xCELLigence

References

1. 1. Malvezzi, M., et al., European cancer mortality predictions for the year 2015: does lung cancer have the highest death rate in EU women. Ann Oncol, 2015. 26(4): p. 779-786.
2. 2. Harden, G.J., Flora of New South Wales. Vol. 4. 1990: UNSW Press.
3. 3. Oyedeji, O.O., et al., Chemical composition and antibacterial activity of the essential oils of Callistemon citrinus and Callistemon viminalis from South Africa. Molecules, 2009. 14(6): p. 1990-1998.
4. 4. Sudhakar, M., et al., Antinociceptive and anti-inflammatory effects of the standardized oil of Indian Callistemon lanceolatus leaves in experimental animals. East and Central African Journal of Pharmaceutical Sciences, 2004. 7(1): p. 10-15.
5. 5. Paluri, V., et al., Phytochemical composition and in vitro antimicrobial activity of methanolic extract of Callistemon lanceolatus DC. Int J Pharm Pharm Sci, 2012. 4(2): p. 699-2.
6. 6. Brodbeck, D., P. Cron, and B.A. Hemmings, A human protein kinase Bγ with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain. Journal of Biological Chemistry, 1999. 274(14): p. 9133-9136.
7. 7. Altomare, D.A., et al., Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin. Oncogene, 1998. 16(18): p. 2407-2411.
8. 8. Altomare, D.A., et al., Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene. Oncogene, 1995. 11(6): p. 1055-1060.

9. 9. COFFER, P.J. and J.R. WOODGETT, Molecular cloning and characterisation of a novel putative protein‐serine kinase related to the cAMP‐dependent and protein kinase C families. European Journal of Biochemistry, 1991. 201(2): p. 475-481.
10. 10. Murthy, S., et al., Mapping of AKT3, encoding a member of the Akt/protein kinase B family, to human and rodent chromosomes by fluorescence in situ hybridization. Cytogenetic and Genome Research, 2000. 88(1-2): p. 38-40.
11. 11. Cheng, J.Q., et al., AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proceedings of the National Academy of Sciences, 1992. 89(19): p. 9267-9271.
12. 12. Staal, S.P., et al., The AKT1 proto-oncogene maps to human chromosome 14, band q32. Genomics, 1988. 2(1): p. 96-98.
13. 13. Vivanco, I. and C.L. Sawyers, The phosphatidylinositol 3-kinase–AKT pathway in human cancer. Nature Reviews Cancer, 2002. 2(7): p. 489-501.
14. 14. Datta, S.R., A. Brunet, and M.E. Greenberg, Cellular survival: a play in three Akts. Genes & development, 1999. 13(22): p. 2905-2927.
15. 15. Blume-Jensen, P. and T. Hunter, Oncogenic kinase signalling. Nature, 2001. 411(6835): p. 355-365.
16. 16. Brognard, J., et al., Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer research, 2001. 61(10): p. 3986-3997.
17. 17. Kreisberg, J.I., et al., Phosphorylation of Akt (Ser473) is an excellent predictor of poor clinical outcome in prostate cancer. Cancer research, 2004. 64(15): p. 5232-5236.
18. 18. Grille, S.J., et al., The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer research, 2003. 63(9): p. 2172-2178.
19. 19. Sun, M., et al., Phosphatidylinositol-3-OH kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor α (ERα) via interaction between ERα and PI3K. Cancer research, 2001. 61(16): p. 5985-5991.
20. 20. Roy, H.K., et al., AKT proto-oncogene overexpression is an early event during sporadic colon carcinogenesis. Carcinogenesis, 2002. 23(1): p. 201-205.
21. 21. Itoh, N., et al., Phosphorylation of Akt/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colorectal carcinoma. Cancer, 2002. 94(12): p. 3127-3134.
22. 22. Ke, N., et al., The xCELLigence system for real-time and label-free monitoring of cell viability. Mammalian Cell Viability: Methods and Protocols, 2011: p. 33-43.
23. 23. Bird, C. and S. Kirstein, Real-time, label-free monitoring of cellular invasion and migration with the xCELLigence system. Nature methods, 2009. 6(8).
24. 24. Kumar, D., et al., Chemical composition and in vitro cytotoxicity of essential oils from leaves and flowers of Callistemon citrinus from western Himalayas. PloS one, 2015. 10(8): p. e0133823.