Inhibitory Effect of Tellimagrandin I on Chemically Induced Differentiation of Human Leukemia K562 Cells
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中国科技论文在线 http://www.paper.edu.cn Toxicology Letters 147 (2004) 109–119 Inhibitory effect of tellimagrandin I on chemically induced differentiation of human leukemia K562 cells Zongchun Yi a,b, Zhao Wang a,∗, Haixia Li a, Mingjie Liu a a Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China b Department of Biological Engineering, Beijing University of Aeronautics and Astronautics, Beijing 100083, China Received 12 September 2003; received in revised form 12 September 2003; accepted 12 October 2003 Abstract Tellimagrandin I is a hydrolysable tannin compound widely present in plants. In this study, the effect of tellimagrandin I on chemically induced erythroid and megakaryocytic differentiation was investigated using K562 cells as differentiation model. It was found that tellimagrandin I not only inhibited the hemoglobin synthesis in butyric acid (BA)- and hemin-induced K562 cells with IC50 of 3 and 40 M, respectively, but also inhibited other erythroid differentiation marker including acetylcholinesterase (AChE) and glycophorin A (GPA) in BA-induced K562 cells. Tellimagrandin I also inhibited 12-O-tetradecanoylphorbol-13- acetate (TPA)-induced expression of CD61 protein, a megakaryocytic marker. RT-PCR analysis showed that tellimagrandin I decreased the expression of erythroid genes (␥-globin and porphobilinogen deaminase (PBGD)) and related transcription factors (GATA-1 and NF-E2) in BA-induced K562 cells, whereas tellimagrandin I induced the overexpresison of GATA-2 transcription factor that played negative regulation on erythroid differentiation. These results indicated that tellimagrandin I had inhibitory effects on erythroid and megakaryocytic differentiation, which suggested that tannins like tellimagrandin I might influence the anti-tumor efficiency of some drugs and the hematopoiesis processes. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Erythroid differentiation; GATA-1; GATA-2; ␥-Globin; Megakaryocytic differentiation; NF-E2; Porphobilinogen deaminase; Tellimagrandin I 1. Introduction Tannins have been showed to possess a variety of pharmacological activities such as antiviral, antimi- Tannins are present in varied plants utilized as crobial, antioxidant, antimutagenic, and anti-tumor foods and medicinal herbs (Chung et al., 1998a). In activities (Chung et al., 1998b). The adverse effects addition, tannins have been used as food additives. of tannins, including hepatotoxic, antinutritional, and It was estimated that people in the US ingested each carcinogenic activities, have been paid attention. In day 1 g of tannic acid (a tannin) (Sanyal et al., 1997). addition, several toxicity studies had been performed to evaluate the health safety of gallic acid and propyl ∗ gallate. As early as 1948, Orten and his colleagues Corresponding author. Tel.: +86-10-62772241; fax: +86-10-62772240. observed reduced food intake, growth inhibition and E-mail address: [email protected] (Z. Wang). at dietary dose levels of 11,700 mg of propyl gallate 0378-4274/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2003.12.008 转载 中国科技论文在线 http://www.paper.edu.cn 110 Z. Yi et al. / Toxicology Letters 147 (2004) 109–119 (Orten et al., 1948). In another study, propyl gallate and doxorubicin (Jeannesson et al., 1997), while at the dose of 10,000 mg/kg feed produced severe 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anemia and growth retardation (Heijden et al., 1986). K562 cells to differentiate towards the megakary- Recently, a subchronic toxicity study of gallic acid ocytic lineage (Villeval et al., 1983). Out of these on F344 rats showed that toxic effects following ad- inducers, anthracyclines, Ara-C, and BA are widely ministration of 0.6% or more in males and 5% in used in cancer therapy through their inducing differ- females included reduction of hemoglobin concentra- entiation activities, suggesting that the inhibition of tion, hematocrit, and red blood cell counts, suggest- their inducing differentiation activities would result in ing the development of anemia (Niho et al., 2001). the loss of anti-tumor function. In this study, the effect In a subacute study, oral of gallic acid at a dose of of tellimagrandin I on chemically induced erythroid 1000 mg/kg body weight (BW) for 28 days reduced and megakaryocytic differentiation was investigated hemoglobin level in mice (Rajalakshmi et al., 2001). in K562 cells. During erythroid differentiation, tel- All these studies demonstrated that gallic acid could limagrandin I not only inhibited the hemoglobin syn- induce the development of anemia. It was proposed thesis in BA- and hemin-treated K562 cells, but also that this hydrolysate (gallic acid) of hydrolysable tan- inhibited other erythroid differentiation marker in- nins and hydrolysable tannins per se could interfere cluding acetylcholinesterase (AChE) and glycophorin erythropoiesis processes. A (GPA) in BA-induced K562 cells. When K562 cells Tellimagrandin I is a hydrolysable tannin com- were simultaneously treated with TPA and tellima- pound widely present in plants such as Punica grana- grandin I, the TPA-induced expression of megakary- tum, Myrtaceae, and Elaeagnaceae, which possesses ocytic surface marker CD61 was also inhibited by various biological activities, such as inhibitory ef- tellimagrandin I. These results showed that tellima- fect against carbonic anhydrase (Satomi et al., 1993), grandin I inhibited K562 cell differentiation, which antibacterial activity against Helicobacter pylori suggested that hydrolysable tannins such as tellima- (Yoshida et al., 2000), inhibitory activity on the syn- grandin I might influence the efficiency of some cytia formation (Kim et al., 2001), toxicities towards anti-tumor agents and the hematopoiesis processes. the nematode and the brine shrimp (Yamasaki et al., 2002), restoration of effectiveness of beta-lactams and tetracycline on methicillin-resistant Staphylococ- 2. Materials and methods cus aureus (Shiota et al., 2000), anti-tumor activities against sarcoma-180 in mice (Miyamoto et al., 1993b) 2.1. Materials and induction of IL-1 production from human peripheral macrophages (Miyamoto et al., 1993a). Tellimagrandin I (purity > 95%) was kindly pro- Nevertheless, the effect of tellimagrandin I on hema- vided by Prof. Yanze Liu at Henan College of Tradi- tological differentiation and hematopoiesis processes tional Chinese Medicine, Zhengzhou, China. has not been characterized. It has been extensively demonstrated that K562 2.2. Cell culture cells can be induced to differentiate towards erythroid and megakaryocytic lineages by various differenti- K562 cells were grown in RPMI 1640 medium ation inducers (Sutherland et al., 1986). K562 cells (GIBCO) supplemented with 10% (v/v) fetal bovine have been used as a model to screen anti-tumor serum (HyClone), 100 units/ml penicillin, and drugs inducing differentiation and to research hema- 100 g/ml streptomycin (Sigma) in 5% (v/v) CO2 tological cell differentiation (Koeffler and Golde, humidified atmosphere at 37 ◦C. For the experiment, 1980). Erythroid differentiation of K562 cells could exponentially growing K562 cells were collected and be achieved by exposure to several pharmacolog- re-suspended in fresh culture medium. After 24 h, ical agents, including hemin (Dean et al., 1981), the cells was added hemin (Sigma) at a final con- arabinofuranosyl cytosine (Ara-C) (Watanabe et al., centration of 40 M, BA (Sigma) at 0.5 mM, TPA 1985), butyric acid (BA) (Lozzio et al., 1979; Chénais (Sigma) at 50 ng/ml, and tellimagrandin I at 10 M et al., 1997), and anthracyclines such as aclarubicin, or at indicated concentrations. 中国科技论文在线 http://www.paper.edu.cn Z. Yi et al. / Toxicology Letters 147 (2004) 109–119 111 2.3. Benzidine staining and determination (Biometra). The following specific primers sets were used for PCR. ␥-Globin: sense strand primer, The percentage of cells straining for hemoglobin 5-ACAAGCCTGTGGGGCAA-3, antisense strand was estimated by staining with benzidine/H2O2 es- primer, 5 -GCCATGTGCCTTGACTTT-3 ; porpho- sentially as previously described (Ngo-Nyoung et al., bilinogen deaminase (PBGD): sense strand primer, 1994). The hemoglobin-positive cells were stained 5-GGTCCTACTATCGCCTCCCTC-3, antisense str- with blue by benzidine. The benzidine-positive cells and primer, 5-CCAGCCTCTGTCCCCTCCAGC-3; were counted in a hemacytometer on a microscope GATA-1: sense strand primer, 5-CAGTCTTTCAGG- from 500 cells for each sample. Then the percentage TGTACCC-3, antisense strand primer, 5-GAGTG- of benzidine-positive cells was calculated. ATGAAGGCAGTGCAG-3; NF-E2: sense strand primer, 5-ATTTGAGCCCCAAGCCCCAGC-3, anti- 2.4. AChE activity assays sense strand primer, 5-CCAGCCTCTGTCCCCTCC- AGC-3; GATA-2: sense strand primer, 5-ATCAAGC- The method used to determine AChE activity was CCAAGCGAAGACTG-3, antisense strand primer, modified from that of Ellman et al.’s (1961). Briefly, 5-ACATTGTGCAGCTTGTAGTAGAGGC-3; - the enzyme reaction was undergone at 37 ◦Cina actin: sense strand primer, 5-TGGACTTCGAGCAA- 1-ml reaction system containing 0.5×106 K562 cells, GAGATGG-3, antisense strand primer, 5-ATCTCCT- 0.1 M potassium phosphate buffer (pH 8.0), 0.6 mM TCTGCATCCTGTCG-3. The amplification re- 5,5-dithiobis-(2-nitrobenzoic acid) and 0.75 mM actions were initiated by a denaturation step for acetylthiocholine iodide substrate. After 20 min, each 5 min at 95 ◦C and then subjected to 30 cycles sample was centrifuged at 4 ◦C. The absorbance of of 95 ◦C for 1 min, 60 ◦C for