Back
 AJPS  Vol.3 No.10 , October 2012
Identifying Combinatorial Growth Inhibitory Effects of Various Plant Extracts on Leukemia Cells through Systematic Experimental Design
Abstract: Plant extracts are widely studied for their anti-cancer and cancer preventive effects. In this study, we compared the leukemia growth inhibition effects of seven different plant extracts, theaflavin, epigallocatechin gallate (EGCG), epicathechin (EC), apigenin, quercetin, chrysin and tannic acid, in vitro using the K562 erythroleukemia cell line and application of the design of experiments (DoE) methodology. Our systematic approach enabled us to isolate the main factor contribution, two-factor interactions and produced interaction relationships and/or models to describe growth inhibitory effects of different plant extracts when they are used in combination. The results identified tannic acid as the most significant inhibitor in this group and had synergistic effects with EGCG at specific concentrations. The fitted model of their combined effects showed that the most potent combination is at low concentrations of tannic acid (10 - 20 μM) and high concentrations of EGCG (80 - 100 μM). We further showed that tannic acid induced both growth inhibition and apoptosis in K562 cells in ranges between 10 - 100 μM. The polyphenol caused cell cycle arrest at G2- phase under the higher concentrations. In summary, use of DoE techniques effectively identified the most prominent inducer in this group of plant bioactive compounds and produced combinatorial bioactivity of various polyphenols and flavonoids over the entire range of concentrations under study. This study exemplifies the usefulness of DoE and serves as a guide in its utility for in vitro assessment of bioactivity in plant constituents.
Cite this paper: K. Cheong, K. Htay, R. Tan and M. Lim, "Identifying Combinatorial Growth Inhibitory Effects of Various Plant Extracts on Leukemia Cells through Systematic Experimental Design," American Journal of Plant Sciences, Vol. 3 No. 10, 2012, pp. 1390-1398. doi: 10.4236/ajps.2012.310168.
References

[1]   M. Roy, et al., “Anticlastogenic, Antigenotoxic and Apoptotic Activity of Epigallocatechin Gallate: A Green Tea Polyphenol,” Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 523-524, 2003, pp. 33-41.

[2]   T. Kundu, et al., “Induction of Apoptosis in Human Leukemia Cells by Black Tea and Its Polyphenol Theaflavin,” Cancer Letters, Vol. 230, No. 1, 2005, pp. 111-121. doi:10.1016/j.canlet.2004.12.035

[3]   T. Otsuka, et al., “Growth Inhibition of Leukemic Cells by (?)-Epigallocatechin Gallate, the Main Constituent of Green Tea,” Life Sciences, Vol. 63, No. 16, 1998, pp. 1397-1403. doi:10.1016/S0024-3205(98)00406-8

[4]   I. K. Wang, S. Y. Lin-Shiau and J. K. Lin, “Induction of Apoptosis by Apigenin and Related Flavonoids through Cytochrome C Release and Activation of Caspase-9 and Caspase-3 in Leukaemia HL-60 Cells,” European Journal of Cancer, Vol. 35, No. 10, 1999, pp. 1517-1525. doi:10.1016/S0959-8049(99)00168-9

[5]   S. J. Lee, J.-H. Yoon and K. S. Song, “Chrysin Inhibited Stem Cell Factor (SCF)/c-Kit Complex-Induced Cell Proliferation in Human Myeloid Leukemia Cells,” Biochemical Pharmacology, Vol. 74, No. 2, 2007, pp. 215-225. doi:10.1016/j.bcp.2007.04.011

[6]   K. J. Woo, et al., “Chrysin-Induced Apoptosis Is Mediated through Caspase Activation and Akt Inactivation in U937 Leukemia Cells,” Biochemical and Biophysical Research Communications, Vol. 325, No. 4, 2004, pp. 1215-1222. doi:10.1016/j.bbrc.2004.09.225

[7]   Y. H. Siddique, T. Beg and M. Afzal, “Antigenotoxic Effect of Apigenin against Anti-Cancerous Drugs,” Toxicology in Vitro, Vol. 22, No. 3, 2008, pp. 625-631. doi:10.1016/j.tiv.2007.12.002

[8]   D. Montgomery, “Design and Analysis of Experiments,” 6th Edition, Wiley, Berlin, 2005, p. 672.

[9]   K. Hinkelmann and O. Kempthorne, “Design and Analysis of Experiments,” Advanced Experimental Design, John Wiley & Sons, Inc., Hoboken, 2005. doi:10.1002/0471709948

[10]   J. P. Groten, E. D. Schoen and V. J. Feron, “Use of Factorial Designs in Combination Toxicity Studies,” Food and Chemical Toxicology, Vol. 34, No. 11? 1996, pp. 1083-1089.

[11]   F. K. H. Phoa, H. Xu and W. K. Wong, The Use of Non-regular Fractional Factorial Designs in Combination Toxicity Studies,” Food and Chemical Toxicology, Vol. 47, No. 9, 2009, pp. 2183-2188. doi:10.1016/j.fct.2009.06.003

[12]   N. F. Kolachi, et al., “Multivariate Optimization of Cloud Point Extraction Procedure for Zinc Determination in Aqueous Extracts of Medicinal Plants by Flame Atomic Absorption Spectrometry,” Food and Chemical Toxicology, Vol. 49, No. 10, 2011, pp. 2548-2556. doi:10.1016/j.fct.2011.06.065

[13]   M. Lim and A. Mantalaris, “Development of a Design of Experiment Methodology: Applications to the Design and Analysis of Experiments,” In: J. M. Polak, Ed., Advances in Tissue Engineering, Imperial College Press, London, 2008. doi:10.1142/9781848161832_0011

[14]   R. H. Myers and D. C. Montgomery, “Two-Level Fractional Factorial Designs, in Response Surface Methodology: Process and Product Optimization Using Designed Experiments,” John Wiley & Sons, Inc., Hoboken, 2002.

[15]   K. Hinkelmann and O. Kempthorne, “Confounding in 2n Factorial Designs, in Design and Analysis of Experiments,” John Wiley & Sons, Inc., Hoboken, 2005. doi:10.1002/0471709948.ch12

[16]   M. J. Anderson and P. J. Whitcomb, “DOE Simplified: Practical Tools for Effective Experimentation,” 2nd Edition, Productivity Press, London, 2007, p. 241.

[17]   R. W. Mee and M. Peralta, “Semifolding 2k-p Designs,” Technometrics, Vol. 42, No. 2, 2000, pp. 122-134.

[18]   N. Balakrishnan and P. Yang, “De-Aliasing Effects Using Semifoldover Techniques,” Journal of Statistical Planning and Inference, Vol. 139, No. 9, 2009, pp. 3102-3111. doi:10.1016/j.jspi.2009.02.010

[19]   O. Tajima, et al., “Statistically Designed Experiments in a Tiered Approach to Screen Mixtures of Fusarium Mycotoxins for Possible Interactions,” Food and Chemical Toxicology, Vol. 40, No. 5, 2002, pp. 685-695. doi:10.1016/S0278-6915(01)00124-7

[20]   V. Cortin, et al., “Efficient in Vitro Megakaryocyte Maturation Using Cytokine Cocktails Optimized by Statistical Experimental Design,” Experimental Hematology, Vol. 33, No. 10, 2005, pp. 1182-1191. doi:10.1016/j.exphem.2005.06.020

[21]   M. Lim, et al., “Optimization of in Vitro Erythropoiesis from CD34+ Cord Blood Cells Using Design of Experiments (DOE),” Biochemical Engineering Journal, Vol. 55, No. 3, 2011, pp. 154-161. doi:10.1016/j.bej.2011.03.014

[22]   C. S. Yang, et al., “Cancer Prevention by Tea: Evidence from Laboratory Studies,” Pharmacological Research, Vol. 64, No. 2, 2011, pp. 113-122. doi:10.1016/j.phrs.2011.03.001

[23]   K.-S. Chen, et al., “Tannic Acid-Induced Apoptosis and Enhanced Sensitivity to Arsenic Trioxide in Human Leukemia HL-60 Cells,” Leukemia Research, Vol. 33, No. 2, 2009, pp. 297-307. doi:10.1016/j.leukres.2008.08.006

 
 
Top