The Effect of the Crocus Sativus L. Carotenoid, Crocin, on the Polymerization of Microtubules, in Vitro

Document Type : Original Article

Authors

1 Department of Biochemistry, Institute of Biochemistry and Biophysics (IBB), Tehran University, Tehran, Iran

2 School of Biology, University College of Sciences, University of Tehran, Tehran, Iran

Abstract

Objective(s): Crocin, as the main carotenoid of saffron, has shown anti-tumor activity both in vitro and in vivo. Crocin might interact with cellular proteins and modulate their functions, but the exact target of this carotenoid and the other compounds of the saffron have not been discovered yet. Microtubular proteins, as one of the most important proteins inside the cells, have several functions in nearly all kinds of cellular processes. The aim of this study was to investigate whether crocin affects microtubule polymerization and tubulin structure.
Materials and Methods: Microtubules were extracted from sheep brains after two cycles of temperature-dependant assembly-disassembly in the polymerization buffer (PMG). Then phosphocellulose P11 column was used to prepare MAP-free tubulin. Turbidimetric assay of microtubules was performed by incubation of tubulins at 37 ºC in PIPES buffer. To investigate the intrinsic fluorescence spectra of tubulins, the emission spectra of tryptophans was monitored. To test the interaction of crocin with tubulin in more details, ANS has been used.
Results: Crocin extremely affected the tubulin polymerization and structure. Ultraviolet spectroscopy indicated that crocin increased polymerization of microtubules by nearly a factor of two. Fluorescence spectroscopic data also pointed to significant conformational changes of tubulin.
Conclusion: We showed that crocin increased tubulin polymerization and microtubule nucleation rate and this effect was concentration dependant. After entering cell, crocin can modulate cellular proteins and their functions. Concerning the results of this study, crocin would be able to affect several cell processes through interaction with tubulin proteins or microtubules.

Keywords


Abdullaev FI. Cancer chemopreventive and tumoricidal properties of saffron (Crocus sativus L.). Exp Biol Med (Maywood ) 2002; 227:20-25.
2. Nair SC, Pannikar B, Panikkar KR. Antitumour activity of saffron (Crocus sativus). Cancer Lett 1991; 57:109-114.
3. Tarantilis PA, Morjani H, Polissiou M, Manfait M. Inhibition of growth and induction of differentiation of promyelocytic leukemia (HL-60) by carotenoids from Crocus sativus L. Anticancer Res 1994; 14:1913-1918.
4. Chryssanthi DG, Lamari FN, Iatrou G, Pylara A, Karamanos NK, Cordopatis P. Inhibition of breast cancer cell proliferation by style constituents of different Crocus species. Anticancer Res 2007; 7:357-362.
5. Zhao P, Luo CL, Wu XH, Hu HB, Lv CF, Ji HY. [Proliferation apoptotic influence of crocin on human bladder cancer T24 cell line]. Zhongguo Zhong Yao Za Zhi 2008; 33:1869-1873.
6. Dhar A, Mehta S, Dhar G, Dhar K, Banerjee S, Van VP, et al. Crocetin inhibits pancreatic cancer cell proliferation and tumor progression in a xenograft mouse model. Mol Cancer Ther 2009; 8:315-323.
7. Premkumar K, Thirunavukkarasu C, Abraham SK, Santhiya ST, Ramesh A. Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agents in mice. Hum Exp Toxicol 2006; 25:79-84.
8. Aung HH, Wang CZ, Ni M, Fishbein A, Mehendale SR, Xie JT, et al. Crocin from Crocus sativus possesses significant anti-proliferation effects on human colorectal cancer cells. Exp Oncol 2007; 29:175-180.
9. Abdullaev FI, Frenkel GD. The effect of saffron on intracellular DNA, RNA and protein synthesis in malignant and non-malignant human cells. Biofactors 1992; 4:43-45.
10. Escribano J, Alonso GL, Coca-Prados M, Fernandez JA. Crocin, safranal and picrocrocin from saffron         (Crocus sativus L.) inhibit the growth of human cancer cells in vitro. Cancer Lett  1996; 100:23-30.
11. Abdullaev JF, Caballero-Ortega H, Riveron-Negrete L, Pereda-Miranda R, Rivera-Luna R, Manuel HJ, et al. [In vitro evaluation of the chemopreventive potential of saffron]. Rev Invest Clin 2002; 54:430-436.
12. McKeithan TW, Rosenbaum JL. The biochemistry of microtubules. A review. Cell Muscle Motil 1984; 5:255-288.
13. Snyder JA, McIntosh JR. Biochemistry and physiology of microtubules. Annu Rev Biochem 1976; 45:699-720.
14. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov 2010; 9:790-803.
15.  Yue QX, Liu X, Guo DA. Microtubule-binding natural products for cancer therapy. Planta Med 2010; 76:1037-1043.
16.           Weingarten MD, Lockwood AH, Hwo SY, Kirschner MW. A protein factor essential for microtubule assembly. Proc Natl Acad Sci USA 1975; 72:1858-1862.
17. Weinert T, Cappuccinelli P, Wiche G. Potent microtubule inhibitor protein from dictyostelium discoideum. Biochemistry 1982; 21:782-789.
18. Dubikovskaya EA, Thorne SH, Pillow TH, Contag CH, Wender PA. Overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters. Proc Natl Acad Sci USA 2008; 105:12128-12133.
19. Zhou J, Panda D, Landen JW, Wilson L, Joshi HC. Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint. J Biol Chem 2002; 277:17200-1728.
20. Gheshlaghi ZN, Riazi GH, Ahmadian S, Ghafari M, Mahinpour R. Toxicity and interaction of titanium dioxide nanoparticles with microtubule protein. Acta Biochim Biophys Sin (Shanghai) 2008; 40:777-782.
21. Solomaha E, Palfrey HC.Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence. Biochem J 2005; 391:601-611.