Antiproliferative Activity and Apoptosis Induction of Crude Extract and Fractions of Avicennia Marina

Authors

1 1Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran

2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

 




Objective(s): Regarding the presence of many active biological constituents in Avicennia marina, the present investigation was carried out to study cytotoxic activity of crude methanol leave extract and column chromatographic fractions of A. marina against MDA-MB 231 cell line (human breast cancer cell) and HEK (Human embryonic kidney cell) line
.
 
Materials and Methods:
The anticancer activity of crude methanol extract and sub-fractions were evaluated, using MTT assay. The induction of apoptosis was determined by analyzing DNA fragmentation in breast cancer cells treated with active fraction of crude methanol extract using agarose gel electrophoresis. To investigate molecular mechanism of apoptosis, gene expression levels of p53 and Bcl-2 were measured using quantitative real time PCR.
Results:
Fraction 10 was the most active fraction and was detected with HPLC as luteolin. The 50% cell cytotoxic concentration (CC50) of crude methanol extract and luteolin was 250 and 28 μg/ml, respectively. This fraction was found to be an apoptotic agent against MDA-MB 231 cells, which leads to causing DNA fragmentation. The mRNA expression level of Bcl-2 and p53 was significantly decreased and increased respectively in cancer cells treated by luteolin.
Conclusion:
The results suggested that Luteolin isolated from Avicennia marina could probably induce apoptosis on breast cancer cell line by the regulation of p53 and Bcl-2 pathways.

Keywords


1. Robert AS, Vilma C, Durado B, Debbie S, Otis WB.
Cancer Screening in the United States, 2010: A review
of current American cancer society guidelines and
issues in cancer screening. CA Cancer J Clin 2010;
60:99-119.
2. Rates SMK. Plants as source of drugs. Toxicon 2000;
39:603
613.
3. Graham JG, Quinn ML, Fabricant DS
 
 
.
Farnsworth NR
Plants used against cancer - an extension of the work of
 
 
0
10
20
30
40
50
60
2 h 6 h 12 h
relative mRNA expression
P53
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
2 h 6 h 12 h
relative mRNA expression
Bcl
A
B
relative mRNA expression
Antiproliferative Activity of Avicennia marina Momtazi-borojeni et al
Iran J Basic Med Sci, Vol. 16,
 
1208
No. 11, Nov 2013
Jonathan Hart well. J Ethnopharmacol 2000; 73:347
377.
4. Duke NC. A systematic revision of the mangrove genus Avicennia (Avicenniaceae) in Australasia. Austra Systema Botany 1991; 4: 299-324.
 
5. Bandaranayake WM. Survey of mangrove plants from Northern Australia for phytochemical constituents and uv-absorbing compounds. Curr Top Phytochem 1995; 14:69-78.
 
6. Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol 1999; 17:2941-2953.
 
7. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991; 351: 453-456.
 
8. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 2008; 9:47-59.
 
9. Miyashita T, Harigai M, Hanada M, Reed JE. Identification of a p53-dependent negative response element in the Bcl-2 gene. Cancer Res 1994; 54:3131-3135.
 
10. König G, Rimpler H. Iridoid glucosides in Avecinnia marina. Phytochemi stry 1985; 24:1245
 
 
1248.
11. Hogg RW, Gillan FT. Fatty acids, sterols and hydrocarbons in the leaves from eleven species of mangrove. Phytochemi stry 1984; 23: 93-97.
 
12. Khafagi I, Gab-Alla A, Salama W, Fouda M. Biological activities and phytochemical constituents of the gray mangrove Avicennia marina (Forssk.) Vierh. Egypt J Bot 2003; 5:62-69.
 
13. Sharaf M, El-Ansari MA
 
 
و
Saleh NAM. New flavonoids from Avicennia marina. Fitoterapia 2000; 71:274-277.
14. Twentyman PR, Luscombe M. A study of some variables in tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer 1987; 56: 279-85.
 
15. Suzuki K, Kazui T, Yoshida M, Uno T, Kobayashi T, Kimura T,
 
 
et al.
Drug-induced apoptosis and p53, bcl-2 and bax expression in breast cancer tissues in vivo and in fibroblast cells in vitro. Jpn J Clin Oncol 1999; 29:323-331.
16. Pau Ni IB, Lim P, Balraj P, Ui Hang ES, Zakaria Z. Quantitative analysis of the expression of p53 gene in colorectal carcinoma by using real-time PCR. Tro Biomed 2006; 23:53
 
 
59.
17. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation
 
 
. Nature 1980; 284:555
556.
18. Suffness M, Pezzuto JM. Assays related to cancer drug discovery. In: Hostettmann K. editor. Methods Plant Biochemistry: Assays for Bioactivity. London: Academic Press; 1990.p.71-133.
 
19. James T, Mukinda J, Syce A, Fisher D, Meyer M. Effect of the Plant Matrix on the Uptake of luteolin derivatives-containing
 
 
Artemisia afra
Aqueous-extract in Caco-2 cells. J Ethnopharma 2010; 130:439-449.
20. Jung HA, Eun Jin S, Min BS, Kim BW, Choi JS. Anti-inflammatory activity of Korean thistle Cirsium maackii and its major flavonoid, luteolin 5-O-glucoside. Food Chem Toxicol 2012; 50:2171-2179.
 
21. Rooban BN, Sasikala V, Gayathri Devi V, Sahasranamam V, Abraham A. Prevention of selenite induced oxidative stress and cataractogenesis by luteolin isolated from
 
 
Vitex negundo
. Chem Biol Interact 2012; 196:30-38.
22.
 
 
Orhan F, Barış O, Yanmış D, Bal T, Güvenalp Z, Güllüce M. Isolation of some luteolin derivatives from Mentha longifolia
(L.) Hudson subsp. longifolia and determination of their genotoxic potencies. Food Chem 2012; 135:764-769.
23. Bagli E, Stefaniotou M, Morbidelli L, Ziche M, Psillas K, Murphy C,
 
 
et al. Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 30-kinase activity. Cancer Res 2004; 64:7936
7946.
24. Lin Y, Shi R, Wang X, Shen HM. Luteolin, a flavonoid with potential for cancer prevention and therapy. Curr Cancer Drug Targets 2008; 8:634
 
 
646.
25. Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS,
 
 
et al
. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 1994; 262:1274-1277.
26. Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LGGC, Masucci M,
 
 
et al. Small molecule RITA binds to p53, blocks p53HDM-2 interaction and activates p53 function in tumors. Nat Med 2004; 10:1321
1328.
27. Sano H, Kawahito Y, Wilder RL, Hashiramoto A, Mukai S, Asai K,
 
 
et al.
Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res 1995; 55:3785-3789.
28. Changa JS, Hsub Y, Kuoc P, Kuob Y, Chianga L, Lin CC. Increase of Bax/ Bcl-XL ratio and arrest of cell cycle by luteolin in immortalized human hepatoma cell line. Life Sci 2005; 76:1883-1893.
 
29. Huang ST, Yangc RC, Yanga LJ, Leea PN, Pang JHS. Phyllanthus urinaria triggers the apoptosis and Bcl-2 down-regulation in Lewis lung carcinoma cells. Life Sci 2003; 72:1705-1716.