Induction of Apoptosis by Puerarin in Colon Cancer HT-29 Cells

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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].

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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. Signals were detected using the at concentrations of 50, 75, and 100 mM after puerarin ECL kit (Amersham Pharmacia Biotech). For all of treatments for 24, 48, and 72 h (P!0.05). the experiments, Ponceau staining of filters was performed to confirm loading of equal amounts of 3.2. Effect of atRA on cell viability in HT-29 cells protein. In this set of experiments, we evaluated the effects 2.7. Assay for caspase-3 activity of puerarin on cell viability in HT-29 cells. Cells were treated with different concentrations of puerarin for Cells were washed twice with cold PBS and 72 h and cell viability was determined by MTT assay. resuspended in the same lysis buffer as Western blot, A significant inhibition in cell viability was observed 中国科技论文在线 http://www.paper.edu.cn

56 Z. Yu, W. Li / Cancer Letters 238 (2006) 53–60

5 thought to be one of the characteristics of apoptosis. 0 µM As shown in Fig. 4, puerarin induced an additional 4 25 µM peak in front G0/G1 peak at concentrations of 50, 75, 50 µM cells/well) and 100 mM treatment for 72 h, respectively. The 4 3 75 µM results are consistent with the previous results of DNA 100 µM 2 fragmentation analysis by ELISA.

1 3.5. Expressions of Bcl-2, Bax and c-Myc in HT-29 Cell Number (×10 0 0 h 24 h 48 h 72 h cells treated with puerarin Time In order to understand the induction mechanism of Fig. 1. Effect of atRA on the growth of HT-29 cells assayed by apoptosis by puerarin, this study examined the trypan blue dye exclusion test. Cells were cultured for 72 h with expression levels of bcl-2, bax, and c-myc using puerarin at concentrations of 0 (control), and 25–100 mM. Puerarin inhibited the growth of HT-29 cells time- and dose-dependently. Western blot. The treatment of the HT-29 cells with Values represent meansGSD. These data are representative of four the puerarin resulted in a marked decrease of the independent experiments. c-myc and bcl-2 at protein levels in a dose-dependent manner (Fig. 5). In contrast, the bax protein upon treatments with various concentrations of expression level was slightly increased compared to puerarin (Fig. 2) and was in a dose-dependent manner. the vehicle control. Indeed, 50, 75, and 100 mM puerarin inhibited cell viable number by 31.5, 49.3, and 60.1%, respectively, compared with control cells that were treated only 3.6. The effect of puerarin on caspase-3 activity with vehicle control (indicated as 0 mM). in HT-29 cells

Apoptosis is mediated by the sequential activation 3.3. Induction of apoptosis by puerarin treatment of caspases, which are constitutively present in most cells as inactive proenzymes. These, in turn, are A cell death ELISA assay was used to determine activated by specific proteolytic cleavage. Special role whether the significant decrease in cell growth in this process seems to be played by caspase-3, a key observed after treatment with puerarin was the result of enhanced apoptosis in HT-29 cells. Fig. 3 shows the significant (P!0.01) increases in relative apoptotic 120 level at concentrations of 50, 75, and 100 mM puerarin 100 treatment for 72 h, respectively. Indeed, a 2.1, 3.9, and 80 ** 6.2-fold increase in DNA fragmentation over that of 60 control cells was observed, respectively. * * ** 40

3.4. The results assayed by ﬂow cytometry 20 0

Cell Viability (% Vehicle Control) 0 µM 25 µM 50 µM 75 µM 100 µM Flow cytometry was employed to determine the Concentrations of puerarin ratios of apoptotic cells in HT-29 cells. In the cells undergoing apoptosis, DNA was degraded to frag- Fig. 2. Effects of puerarin on cell viability in HT-29 cells. HT-29 ments of low molecular weight and subsequently cells were exposed to culture medium containing various leaked out from the cells, and DNA content was concentrations of puerarin for 72 h and cell vitality was measured by MTT assay. Results are shown as meanGSD (shown by bars); stained with a DNA-specific fluoro-chrome, propi- each bar represents average of 12 measurements in four independent dium iodide (PI), a special DNA peak (usually called experiments. ** denotes significant difference (P!0.01) when sub-G1 peak or apoptotic peak) appeared. This peak is compared with vehicle control (C). 中国科技论文在线 http://www.paper.edu.cn

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8 4. Discussion 7 * * 6 In the current study, we conducted a detailed 5 * * investigation of the effects of puerarin on colon cancer 4 HT-29 cells. Puerarin inhibited cell growth in a dose- 3

OD / mg protein ** and time-dependent manner (Fig. 1). After 72 h of 2 incubation, 50, 75, and 100 mM puerarin signiﬁcantly 1 reduced the viability of HT-29 cells to 31.5, 49.3, and 0 0 µM 25 µM 50 µM 75 µM 100 µM 60.1%, respectively, of the vehicle control. Concentrations of puerarin In further studies, we aimed to get more information about the possible stimulation of apopto- Fig. 3. DNA fragmentation in HT-29 cells treated with puerarin. sis in HT-29 cells by puerarin. Apoptosis is a major HT-29 cells were incubated in culture medium containing given form of cell death, characterized initially by a series of concentrations of puerarin. DNA fragmentation was measured by a sandwich enzyme immunoassay using anti-histone antibody and stereotypic morphological changes [22]. The most anti-DNA antibody. Data are expressed as absorbance per milligram common change is the DNA fragmentation, which is a of protein in each sample. Values are meansGSD of quadruplicate relatively late event during apoptosis. Cell Death cultures. Detection ELISA Plus kit allowed the detection of DNA fragments together with the detection of executioner of apoptosis, for its active form is necrosis. The random sized DNA fragments resulted responsible for the cleavage and breakdown of several from necrosis can be detected by analyzing the cellular components related to DNA repair and supernatant (culture medium) after treatment. For regulation. As shown in Fig. 6, caspase-3 activity apoptotic cells, DNA fragments including high was upregulated after 72 h of treatments with given molecular weight DNA fragments remain in the cell concentrations of puerarin. cytoplasm, which can only be detected after

Fig. 4. Sub-G1 analysis of puerarin-treated human colon cancer HT-29 cells. The HT-29 cells were incubated with various concentrations of puerarin for 72 h, and the cells were harvested and submitted to ﬂow cytometric analysis. The appearance of sub-G1 peak (apoptotic peak) was observed after concentrations of 75 and 100 mM puerarin treatment for 72 h, respectively. Here is the representative DNA histograms of HT-29 cells at three independent experiments. *P!0.05, **P!0.01, versus vehicle controls at the same time point. 中国科技论文在线 http://www.paper.edu.cn

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A 1 2 3 4 5 stained with a DNA-specific fluoro-chrome, propidium iodide (PI), a special DNA peak (usually called bcl-2 sub-G1 peak or apoptotic peak) appeared. This peak is thought to be one of the characteristics of apoptosis. c-myc As shown in Fig. 4, puerarin induced an additional peak in front G0/G1 peak at concentrations of 50, 75, bax and 100 mM after 72 h treatment, respectively, and the mean ratio from four independent experiments were β-actin 10.1, 23.7, and 39.6%. These results are consistent with the results of DNA fragmentation analysis by B 3 ELISA. bcl-2 During the process of apoptosis, the initiation of all 2.5 bax intracellular events seems to be connected with the 2 c-myc activation of specific proteases called caspases 1.5 (cysteinyl–aspartic-acid–proteases) [23,24]. Special 1 role in this process seems to be played by caspase-3, 0.5 a key executioner of apoptosis, for its active form is Relative Protein Level responsible for the cleavage and breakdown of several 0 0 µM 25 µM 50 µM 75 µM 100 µM cellular components related to DNA repair and Concentrations of puerarin regulation. We have noticed that the cell treatment with puerarin stimulates the caspase-3 activity about Fig. 5. Effect of puerarin on protein level of apoptosis-related 5.6-fold at concentration of 100 mM. Moreover, the molecules. (A) The cells were treated with given concentrations of puerarin. After 72 h, the cells were harvested. Total cell lysates increase in caspase-3 activation is synchronized with were prepared, and proteins were subjected to Western blotting the increase in bax expression and the decrease in bcl- analysis with polyclonal antibodies against bax, bcl-2, and c-myc, 2 [25–27]. The bcl-2 family of proteins plays an respectively. Lane 1, vehicle control; lane 2, 25 mM; lane 3, 50 mM; important role in the regulation of apoptosis in many lane 4, 75 mM; lane 5, 100 mM. (B) West bolting signals were cellular systems, by either inhibiting (Bcl-2, Bcl-XL, transferred to Adobe Photoshop software through high resolution scanning (Microtek ScanMaker 630) of the XB-1 films (Kodak) and Bcl-W, Bfl-1, and Mcl-1) or promoting apoptosis digitized with Image Quant software. From four independent (Bax, Bak, Bad, Bcl-Xs, Bid, and Hrk) [28,29]. experiments, for all different samples, the ratio between the tested Heterodimerization between pro- and anti-apoptotic target protein and internal standard b-actin band was calculated. members of this family and relative levels of both Within each separate experiment, ratios of the samples were compared to the ratio obtained from control cells, which were set at 1. Data were from four independent experiments and shown as 7 meanGSD. *, P!0.05; **, P!0.01. ** 6 ** 5 the disruption of cell membrane by cell lysis. After 4 treatment with puerarin, the supernatants and the cell 3 * lysate were analyzed separately by ELISA. As shown 2 in Fig. 4, a significant increase in DNA fragments was 1

induced by puerarin at concentrations of 50, 75, and Relative caspase-3 activity 0 100 mM, respectively, that were in a dose-dependent 0 µM 25 µM 50 µM 75 µM 100 µM manner. To conﬁrm the apoptosis induced by Concentrations of puerarin puerarin, we further employed ﬂow cytometry to determine the ratios of apoptotic cells. In the cells Fig. 6. The effect of puerarin on caspase-3 activity. Each point is undergoing apoptosis, DNA was degraded to frag- expressed as a value of caspase-3 activity by measuring the release of p-nitroaniline (pNA) at OD405 nm. Data are the results of four ments of low molecular weight and subsequently separate experiments and presented as meansGSD. *, P!0.05; **, leaked out from the cells, and DNA content was P!0.01. 中国科技论文在线 http://www.paper.edu.cn

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