Induction of Apoptosis by Puerarin in Colon Cancer HT-29 Cells
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中国科技论文在线 http://www.paper.edu.cn Cancer Letters 238 (2006) 53–60 www.elsevier.com/locate/canlet Induction of apoptosis by puerarin in colon cancer HT-29 cells Zengli Yu*, Wenjie Li School of Public Health, Zhengzhou University, Zhengzhou 450052, China Received 13 December 2004; received in revised form 6 June 2005; accepted 13 June 2005 Abstract Puerarin was isolated from Pueraria radix and has beneficial effects on cardiovascular, neurological, and hyperglycemic disorders. The current study showed that puerarin also possessed anti-cancer properties. Methyl thiazolyl tetrazolium assay (MTT) assay revealed a dose-dependent reduction of HT-29 cellular growth in response to puerarin treatment. Apoptosis was observed following treatments ‘with R25 mM puerarin, as reflected by the appearance of the subdiploid fraction and NDA fragmentations. We then investigated effects of puerarin on expression of apoptosis-associated genes and the results revealed an increase of bax and decreases of c-myc and bcl-2. Finally, puerarin treatment significantly increased the activation of caspase-3, a key executioner of apoptosis. These findings indicate that puerarin may act as a chemopreventive and/or chemotherapeutic agent in colon cancer cells by reducing cell viability and inducing apoptosis. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Puerarin; Apoptosis; Human colon cancer HT-29 cell line; Bcl-2; c-myc; caspase-3 1. Introduction have not been identified as [5,6]. Flavonoids are a class of more than 4000 phenylbenzopyrones present Colon cancer is a serious health problem in most in many edible plants. Moreover, the flavonoids developed countries and is the third leading cause of possess a remarkable spectrum of biochemical and cancer mortality throughout the world [1]. Colon pharmacological activities suggesting that they carcinogenesis is considered to be linked with significantly affect basic cell functions such as dietary habits like high animal fat intake [2].In growth, differentiation and/or programmed cell contrast, a number of studies have suggested that death (apoptosis). Their proposed protective role in high consumption of fruit and vegetables decreases colon tumor development may prevail especially in the risk of colon cancer [3,4].Thedietary the intestinal tract due to direct exposure of the compounds responsible for this biological effect large intestinal epithelia to these dietary ingredients [7]. Flavonoids reaching the large intestine may be further metabolized to deglycosylation by the * Corresponding author. Address: Department of Nutrition and microflora [8]. Possible mechanisms for the anti- Food Hygiene, School of Public Health, Zhengzhou University, Daxue Road 40, Zhengzhou 450052, China. Tel.: C86 371 cancer property of flavonoids are mainly due to 6912323. controlling cell cycle progression and altered gene E-mail address: [email protected] (Z. Yu). expression [9–11]. 0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.06.022 转载 中国科技论文在线 http://www.paper.edu.cn 54 Z. Yu, W. Li / Cancer Letters 238 (2006) 53–60 Although the main component of Pueraria radix 2.2. Trypan blue exclusion assay (RP) is starch, it contains fairly high amounts of flavonoids, including puerarin, daidzin, genistin, After incubation with 0–100 mM puerarin for 24, 48, daidzein, and genistein. Puerarin is the most abundant and 72 h, respectively, HT-29 cell growth curve was isoflavone-C-glucoside extracted from RP and has determined by dye exclusion assay. To do this, cells been shown to have beneficial effects on cardiovas- were collected by trypsinization, washed twice with cular [12], neurological [13], and hyperglycemic PBS and suspended in FBS. 0.25% trypan blue (Sigma) disorders [14]. Recently, a finding suggests that was added to cells just prior to counting them under the Pueraria mirifica possesses estrogenic effect and light microscope (40-fold magnification). The number could inhibit MCF-7 cell growth at high concentration of blue stained (dead) and unstained (viable) cells were as other flavonoids [15]. Another two studies reported then counted in six randomly chosen fields. that pueraria had anti-inflammatory, anti-nociceptive, and anti-mutagenic activities [16,17]. However, there 2.3. MTT assay have been virtually no reports that have explored anti- cancer properties of puerarin. MTT assay is another standard method used to Human colon cancer development is often assess cell viability. Briefly, cells (5!103 cells/well) characterized in an early stage by a hyperprolifera- were seeded in 96-well microtiter plates (Nunc, tion of the epithelium leading to the formation of Denmark). After exposure to various concentrations adenomas. This is mainly a consequence of of puerarin for 72 h, 50 ml MTT (Sigma) solution deregulated cell cycle control and/or suppressed (2 mg/ml in PBS) was added to each well and the plates apoptosis as usually observed in colorectal cancers were incubated for additional 4 h at 37 8C. MTT [18,19]. The current study therefore mainly focus on solution in medium was aspirated off. To achieve whether and to what extent puerarin could reduce solubilization of the formazan crystal formed in viable cell growth and promote apoptosis in the human cells, 200 ml DMSO was added to each well. The intestinal tumor cell line HT-29. In addition, we will absorbance was read at 540 nm on a Dias automatic further explore the underlying molecular mechan- microwell plate reader with DMSO as the blank. isms for the first time. 2.4. Detection of DNA fragmentations 2. Materials and methods The Cell Death Detection ELISA (Roche) was used to evaluate the presence of apoptosis and necrosis activity in the cells after incubation with 2.1. Cell culture puerarin for a period of 72 h. After treatment, the cells were then lysed to release cytoplasmic histone- Human colon cancer cell line HT-29 was associated-DNA-fragments, an indicator of apoptosis. purchased from American Type Culture Collection. Cell lysates were prepared and placed into streptavi- 2 The cells were cultured in 75-cm flask containing din-coated microplates. These were incubated for 2 h Dulbecco’s modified Eagle’s medium (DMEM, at room temperature with anti-histone-biotin and anti- Gibco.) supplementing with 10% fetal bovine serum DNA-peroxidase antibodies. Calculation was done by (FBS; Invitrogen), 4.5 g/l glucose, 10,000 U/ml of measuring the absorbance at 405 against 490 nm. penicillin, and 10 mg/ml of streptomycin at 37 8C and Enrichment factor was calculated after normalization 5% CO2. All experiments were performed using cells of protein amount in each treatment. The enrichment from passage 20 or less. factor was calculated as follows (the enrichment factor of control was defined as 1): OD of test sampleðincubation medium=cell lysateÞ=mg protein Enrichment factor Z OD of the control=mg protein 中国科技论文在线 http://www.paper.edu.cn Z. Yu, W. Li / Cancer Letters 238 (2006) 53–60 55 2.5. Flow cytometry left on ice for 40 min, then centrifuged at 8000!g for 5 min. Caspase-3 activity was measured using fluoro- To further determine and confirm whether or not genic substrate peptides 7-amine-4methylcoumarine the growth inhibitory activity of puerarin was related (Pharmingen). The supernatant containing 100 mgof to induction of apoptosis, subdiploid (character of whole cell lysates were incubated with 100 mM apoptosis) fraction was detected by flow cytometry. substrate peptide in 100 ml lysis buffer at 37 8C for Briefly, 5!104 cells were seeded in 6-well palate and 1 h. Absorbance of the samples was read at 405 nm in a incubated with given concentrations of puerarin for microtiter plate reader [20,21] using a sample without 72 h. The adhered and floating cells were mixed, substrate peptides as a blank. Caspase-3 activity of washed twice in PBS followed by centrifugation at each sample was calculated according to the formula 300!g for 5 min and fixed in 70% cold ethanol. Four below: hours later, the cells were washed twice in PBS and Caspase-3 activity resuspended in 0.1% Triton X-100 solution in PBS containing 40 mg/ml propidium iodide (PI, Sigma K Z OD of test sample OD of blank Chemical Co.) and 0.1 mg/ml RNAse (Sigma Chemi- OD of controlKOD of blank cal Co.). Cells were analyzed by FACScan (Becton Dickson) using a ModFit software package (Becton- 2.8. Statistical analysis Dickinson). All data are presented as mean valuesGSD 2.6. Western blot analysis (standard deviation). The data were evaluated by a one-way ANOVA followed by least significant After HT-29 cells were seeded in 75 ml flasks and difference (LSD) test as a post hoc test or Dunnett’s treated with given concentrations of puerarin for 72 h, T3 test using the SPSS 10.0 (SPSS Inc., Chicago, IL, the cells were harvested and lysed in a buffer USA) program. Statistical significance was at P! containing 100 mM Hepes (pH 7.4), 10% sucrose, 0.05. 0.1% CHAPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 10 mg/ml pepstain, 10 mg/ml leupetin. 50 mg protein lysate were separated by 12.5% SDS-PAGE and transferred onto nitrocellulose. After blocking in a 3. Results 5% non-fat dry milk solution in washing buffer containing 10 mmol/l Tris (pH 7.5), 150 mmol/l 3.1. Puerarin inhibited HT-29 cell growth NaCl, and 0.05% Tween-20, membranes were incubated overnight at 4 8C with different rabbit The effects of puerarin on the growth of HT-29 polyclonal antibodies: anti-bcl-2 (Santa Cruz), anti- cells were analyzed using the trypan blue dye bax (Santa Cruz), anti-c-myc, and anti-b-actin (Santa exclusion test. As shown in Fig. 1, puerarin inhibited Cruz). After washing three times with Tween-20– the growth of HT-29 cells in a time and dose- PBS, membranes were incubated for 2 h with dependent manner (with increasing concentrations horseradish peroxidase-coupled secondary antibodies from 25 to 100 mM) and showed significant inhibition at room temperature.