Semi-purified extracts of Commelina benghalensis (Commelinaceae) induce apoptosis and cell cycle arrest in Jurkat-T cells

BMC Complementary and Alternative Medicine

Leseilane Mampuru²  Leshwene Jeremiah Shai¹  Vusi Mbazima²  Thabe Moses Matsebatlela²  Victor Patrick Bagla²  Matlou Phineas Mokgotho²  Kgomotso Welheminah Lebogo² 

[1]Department of Biomedical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria 0001, Gauteng Province, Republic of South Africa

[2]Department of Biochemistry, Microbiology and Biotechnology, Faculty of Science and Agriculture, University of Limpopo (Turfloop Campus), Private Bag X1106, Sovenga 0727, Limpopo Province, Republic of South Africa

关键词: Jurkat-T cells;    Commelina benghalensis;    Cell cycle;    Apoptosis;   

Others  :  1220256
DOI  :  10.1186/1472-6882-14-65

 received in 2013-09-12, accepted in 2014-02-05,  发布年份 2014

【 摘 要 】

Background

Commelina benghalensis (CB) is a small plant whose fleshy stems are used in South Africa to treat skin conditions (e.g., cancerous skin outgrowths). This study was aimed at evaluating the effect of sub-fractions of acetone extracts of CB stems on growth-associated molecular events of apoptosis and cell division cycle of Jurkat-T (JT) cells.

Methods

Acetone extract of CB stems were subfractioned into n-hexane (F1) and dichloromethane (F2) fractions. After treatment of JT cells with these subfractions, cell proliferation, viability and apoptosis were determined using a haemocytometer, the trypan blue dye exclusion assay, and Hoechst 33258 staining, respectively. Cell division cycle distribution profiles were analysed using an Epics Alba Flow Cytometer and the expression of cell division cycle regulatory genes was analysed using RT-PCR, while immunoreactive proteins were detected on western blots.

Results

The F1 and F2 fractions inhibited the proliferation and viability of JT cells in a concentration- and time-dependent manner, with IC₅₀ values of 32.5 μg/mℓ and 56 μg/mℓ, respectively. The observed cytotoxicity was established to be a consequence of apoptosis. as verified using Hoechst staining method. Both fractions induced a G₁/S interphase arrest of the cell division cycle of JT cells.

RT-PCR analyses showed an up-regulatory effect by the F1 fraction in the expression of cyclin B1, cdc2 and bax, with a down-regulatory effect in the expression levels of bcl-2. Fraction F1 also increased the protein expression levels of p53 and its downstream regulators, p21 and Cdc2. However, protein Bax and p21 and p53 transcripts were undetectable under the same experimental conditions. On the other hand, fraction F2 increased the mRNA expression levels of bax, bcl-2, cyclin B1 and cdc2. Concomitantly, fraction F2 showed an up-regulation in the protein expression levels of Cdc2, Bcl-2, Cyclin B1 and p21. Despite the up-regulation in protein expression levels by fraction F2, there was no observable expression levels of the p53 protein and p21 and p53 mRNAs under similar experimental conditions.

Conclusion

These findings suggest that the F1 and F2 fractions of CB may provide a valuable lead for the development of novel and effective anti-neoplastic drug(s).

【 授权许可】

   
2014 Lebogo et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Jansen O, Akhmedjanova V, Angenot L, Balansard G, Chariot A, Ollivier E, Tits M, Frederich M: Screening of 14 alkaloids isolated from haplophyllum A. Juss. for their cytotoxic properties. J Ethnopharmacol 2005, 105:241-245.
• [2]Shi M, Cai Q, Yao L, Mao Y, Ming Y, Ouyang G: Antiproliferation and apoptosis induced by curcumin in human ovarian cancer cells. Cell Bio 2006, 30:221-226.
• [3]Mathur R, Gupta S, Singh N, Mathur S, Kochupillai V, Velpandian T: Evaluation of the effect of Withania somnifera root extracts on cell cycle and angiogenesis. J Ethnopharmacol 2006, 105:336-341.
• [4]Farombi EO: African indigenous plants with chemotherapeutic potentials. Afr J Biotechnol 2003, 2:662-671.
• [5]Taylor JLS, Rabe LJ, McGaw AK, Jansen AK, Van Staden J: Towards the scientific validation of traditional medicinal plants. Plant Growth Regul 2001, 34:23-37.
• [6]Wu CC, Lin JP, Yang JS, Chou ST, Chen SC, Lin YT, Lin HL, Chung JG: Capsaicin induced cell cycle arrest and apoptosis in human esophagus epidermoid carcinoma CE 81T/VGH cells through the elevation of intracellular reactive oxygen species and Ca2+ productions and caspase -3 activation. Mutat Res 2006, 601:71-82.
• [7]Tani D, Monteilhi-Zoller M, Fleig A, Penner R: Cell cycle– dependent regulation of store–operated ICRAC and Mg2 + - nucleotide regulated MagNuM (TRPM7) currents. Cell Calcium 2006, 41:249-260.
• [8]Gui H, Li S, Matise MP: A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord. Dev Biol 2007, 301:14-26.
• [9]Zhang C, Ferreira TB, Cruz PE, Alves PM, Harry M, Carrondo MJT: The importance of 293 cell cycle phase on adenovirus vector production. Enzyme and Microb Technol 2006, 39:1328-1332.
• [10]Sadeghi H, Yazdanparast R: Anti-tumour activity and cell cycle arrest of a new diterpene ester from Daphne mucronata using K562 cells. Iranian Biomed J 2003, 7:127-131.
• [11]Bicknell K, Coxon CHM, Brooks G: Can the cardiomyocyte cell cycle be reprogrammed? Rev J Mol and Cell Cardiol 2007, 42:706-721.
• [12]Clark JM, Gabrielli BG: Production of a soluble Cyclin B1/Cdc2 substrate for cdc25 phosphatase. Analy Biochem 1997, 254:231-235.
• [13]Yoon DK, Jeong CH, Jun HO, Chun KH, Cha JH, Seo JH, Lee HY, Choi YK, Ahn BJ, Lee SK, Kim KW: AKAP12 induces apoptotic cell death in human fibrosarcoma cells by regulating CDK1-cyclin D1 and caspase-3 activity. Cancer Lett 2007, 254:111-118.
• [14]Hsieh WT, Huang KY, Lin HY, Chung JG: Physalis angulata induced G2/M phase arrest in human breast cancer cells. Food Chem Toxicol 2006, 44:974-983.
• [15]Herr I, Debatin KM: Cellular stress response and apoptosis in cancer therapy. Blood 2001, 98:2603-2614.
• [16]Chaudhary MI, He Q, Cheng YY, Xiao PG: Ethnobotany of medicinal plants from Tian Mu Shan biosphere reserve, Zhejiang – Province, China. Asian J Plant Sci 2006, 5:646-653.
• [17]Ibrahim J, Ajaegbu VC, Egharevba HO: Pharmacognostic and phytochemical analysis of commelina benghalensis L. Ethnobot Leaflets 2010, 14:610-615.
• [18]Mbazima VG, Mokgotho MP, February F, Rees DJG, Mampuru LJ: Alteration of Bax-to-Bcl-2 ratio modulates the anticancer activity of methanolic extract of commelina benghalensis (commelinaceae) in jurkat T cells. Afr J Biotechnol 2008, 7:3569-3576.
• [19]Huang ST, Yang RC, Yang LJ, Lee PN, Pang JH: Phyllanthus uranaria triggers apoptosis and Bcl-2 down-regulation on Lewis lung carcinoma cells. Life Sci 2003, 72(15):1705-1716.
• [20]Choi HJ, Yee SB, Park SE, Im E, Jung JH, Chung HY, Choi YH, Kim ND: Petrotetranydiol A induces cell cycle arrest and apoptosis in SK-MEL-2 human melanoma cells through cytochrome c-mediated activation of caspases. Cancer Lett 2006, 232(2):214-225.
• [21]Cho NH, Kang S, Hong S, An HJ, Choi YH, Jeong GB, Choi HK: Elevation of cyclin B1, active Cdc2, and HuR in cervical neoplasia with human papillomavirus type 18 infection. Cancer Lett 2006, 232(2):170-178.
• [22]Wang JL, Liu D, Zhang ZJL, Shan S, Han X, Srinivasula SM, Croce CM, Alnemri ES, Huang Z: Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumour cells. Proc. Natl. Acad. Sci., USA 2000, 97(13):7124-7129.
• [23]Lee YS, Wan J, Kim BJ, Bae MA, Bong BJ: Ubiquitin-dependent degradation of p53 despite phosphorylation at its N-terminus by Acetaminophen. J Pharm Exp Therap 2006, 317:202-208.
• [24]Chang YC, Lee YS, Tejima T, Tanaka K, Omura S, Heintz NH, Mitsui Y, Magae J: Mdm2 and bax, downstream mediators of p53 response, are degraded by the ubiquitin-proteasome pathway. Cell Growth Diff 1998, 9:79-84.
• [25]Collins K, Jacks T, Pavletich NP: The cell cycle and cancer. Proc Natl Acad Sci USA 1997, 94:2776-2778.
• [26]Wang W, Hasham MG, Isordia-Salas I, Tsygankov AY, Colman RW, Guo YL: Up-regulation of Cdc2 and cyclin a during apoptosis of endothelial cells induced by cleaved high-molecular–weight kininogen. Am J Physiol Heart Circ Physiol 2003, 284:1886-1891.
• [27]Mogk A, Schmidt R, Bukan B: The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies. Trends In Cell Biol 2007, 17:165-172.
• [28]Fu N, Drinnenberg I, Kels R, Paabo S, Zengo WJ, Khaitovich P: Comparison of protein and mRNA expression evolution in humans and chimpanzees. PLoS ONE 2007, 2:e216.
• [29]Chen F, Chang D, Goli M, Klibanov SA, Ljungman M: Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors. Cell Growth Diff 2000, 11:239-246.
• [30]Li B, Dou PQ: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumour survival and progression. Proc Natl Acad Sci USA 2000, 8:3850-3855.