Basic & Clinical Pharmacology & Toxicology, 2014, 115, 488–498
Doi: 10.1111/bcpt.12267
Riccardin D, a Macrocyclic Bisbibenzy, Inhibits Human Breast Cancer Growth through the Suppression of Telomerase Activity Cui-Cui Sun1, Hui-Min Xu2, Yi Yuan1, Zu-Hua Gao3,4, Hong-Xiang Lou1 and Xian-Jun Qu1,3 2
1 Department of Pharmacology, Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan, China, Faculty of Radiologic Sciences, School of Medicine, Qingdao University, Qingdao, China, 3Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital Medical University, Beijing, China and 4Department of Pathology, McGill University, Montreal, QC, Canada
(Received 23 January 2014; Accepted 2 May 2014) Abstract: Riccardin D, a liverwort-derived naturally occurring macrocyclic bisbibenzyl, has been found to exert anticancer effects in multiple cancer cell types. In this study, we investigated the effect and mechanism of Riccardin D on human breast cancer. Experiments were performed on human breast cancer MCF-7 and MDA-MB-231 cells. The antitumour effects of Riccardin D were assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and human breast cancer xenografts mice model. TRAPezeâ XL Telomerase Detection assay was used for the detection of telomerase activity. c-H2AX foci formation was tested for the induction of DNA damage response. Cell cycle distribution was analysed by flow cytometry, and cell apoptosis was determined by annexin V-FITC/PI staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay and Western blotting. Riccardin D effectively inhibited the growth of MCF-7 and MDA-MB-231 cells in vitro. And Riccardin D also effectively delayed the growth of MCF-7 and MDA-MB-231-luc-D3H2LN xenografts without significant loss of body-weight. Further analysis suggested that Riccardin D’s effects may arise from its suppression of telomerase activity, which led to telomere dysfunction. Telomerase inhibition and telomere dysfunction could activate the canonical ataxia telangiectasia-mutated (ATM) kinase-mediated DNA damage response, as shown by elevated expression of c-H2AX, pATM and p-Chk2. This is finally followed by the induction of cell cycle arrest and apoptosis, as shown by the increase of TUNEL-stained cells, caspase activation, PARP cleavage and the increase of bax/bcl-2 ratio. Moreover, Riccardin D induced p53-proficient MCF-7 cells to arrest in G1 phase and p53-deficient MDA-MB-231 cells to arrest in G2/M phase. Overall, these results demonstrate that Riccardin D may inhibit human breast cancer growth through suppression of telomerase activity.
Breast cancer, accounting for 23% of all cancers, is the most common malignant disease and the second most frequent cause of cancer death in women [1]. About one million new cases are diagnosed, and 400,000 patients die from the disease each year [2]. Over the last 40 years, advances in the development of breast cancer drugs have led to improved treatments and outcome for patients. However, mortality has remained relatively unchanged over the same period. In addition, toxicity of many anticancer drugs impacts patients’ compliance with treatment and results in serious long-term health effects. Therefore, identification of novel agents that can suppress the growth of human breast cancers but are relatively safe is highly desirable. Telomerase is active in more than 90% of breast carcinomas, whereas in normal tissues, it is not active or detectable. Telomerase is a ribonucleoprotein reverse transcriptase that maintains telomeric ends of eukaryotic chromosomes during DNA replication [3]. It consists of a catalytic protein subunit (human telomerase reverse transcriptase, hTERT), an RNA Authors for correspondence: Xian-Jun Qu, Hong-Xiang Lou and Zu-Hua Gao, Department of Pharmacology, Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan and Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital Medical University, Beijing, China (fax 86-531-88382490, e-mails [email protected], louhongxiang@ sdu.edu.cn and [email protected]).
component (human telomerase RNA, hTR) and other associated proteins. Telomerase is highly associated with carcinogenesis. Telomerase enables cancer cells to bypass replicative senescence and to be capable of self-replicating indefinitely by compensating cell-division-dependent telomere attrition [4]. hTERT is an important catalytic protein subunit of telomerase, and its expression level is closely associated with telomerase activity [5]. hTERT is generally repressed in normal cells and up-regulated in immortal cells. Inhibition of the hTERT component of telomerase enzyme could induce telomere shortening to a critical length, called telomere ‘uncapped’, initiating end-to-end fusions and the DNA damage response [6]. This in turn leads to cell cycle arrest, DNA repair or apoptosis. Therefore, telomerase is an attractive target for the development of anticancer drugs. Riccardin D is a novel macrocyclic bisbibenzyl compound isolated from the liverwort plant Dumortiera hirsuta [7]. It has been considered as a potential candidate agent for cancer treatment. Riccardin D was found to inhibit the proliferation of human leukaemia cells HL60, K562 and its multidrug-resistant counterpart K562/A02 cells [8]. Riccardin D inhibited tumour angiogenesis in human lung carcinoma H460 xenografts mouse model [9]. Riccardin D is also a potential chemopreventive regimen for intestinal cancers derived from APC gene mutation [10]. In this study, we first investigated the effects of
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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Riccardin D on human breast cancer MCF-7 and MDA-MB231 cells. We then evaluated the efficacy of Riccardin D in nude mice bearing human breast cancer xenografts in vivo. To study the molecular mechanism of the anticancer effect of Riccardin D, we examined its effect on telomerase activity in human breast cancer cells. We further examined the effect of Riccardin D on DNA damage response using p53-proficient MCF-7 cells and p53-deficient MDA-MB-231 cells. Our study provides scientific evidence that Riccardin D could be a therapeutic agent against human breast cancer. Materials and Methods Drug. Riccardin D was isolated from the liverwort plant D. hirsuta as described previously [7]. The purity of Riccardin D was measured at 98.6% by high performance liquid chromatography (HPLC). Riccardin D was dissolved in dimethylsulphoxide (DMSO; Sigma, St. Louis, MO, USA) for in vitro assays. For the animal experiments, Riccardin D nanosuspension was prepared by the high-pressure homogenization (HPH) method, as described previously [11,12]. Cell lines and cell culture. Human breast cancer cell lines, MCF-7 (p53-proficient) and MDA-MB-231 (p53-deficient), were purchased from Cell Bank, China Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin at 37°C in a humid atmosphere (5% CO2– 95% air). MDA-MB-231-luc-D3H2LN cells, expressing stable firefly luciferase, were purchased from Caliper Life Sciences. MDA-MB-231luc-D3H2LN cells were cultured in minimum essential medium with Earl’s balanced salts solution (MEM/EBSS) supplemented with 10% foetal bovine serum, 1% non-essential amino acids, 1% L-glutamine, 1% sodium pyruvate, 100 U/mL penicillin and 100 lg/mL streptomycin at 37°C in a humid atmosphere containing 5% CO2. MTT assay for cell viability. MCF-7 and MDA-MB-231 cells seeded in 96-well plates (5 9 103 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5, 10, 20 and 40 lM) for 24, 48, 72 and 96 hr, respectively. The cells were then incubated with 20 lL MTT reagent (5 mg/mL, Sigma) for 4 hr at 37°C. The MTT reagent was removed, and 100 lL DMSO was added to each well. The spectrophotometric reading was taken at 570 nm using a microplate reader (Perkin-Elmer, Waltham, MA, USA). Triplicate experiments were performed with triplicate samples. Assessment of cancer growth in mice. Female BALB/c nude (nu/nu) mice, 6 weeks of age, were purchased from the Animal Center of the China Academy of Medical Sciences (Beijing, China). All experimental protocols were strictly approved by the Committee of Animal Care and Use of Shandong University. Mice were housed under pathogen-free conditions. MCF-7 and MDA-MB-231-lucD3H2LN cells (1 9 107 per mouse) were subcutaneously injected into the left flank [13]. Mice were then divided into three groups with seven mice in per group: vehicle (nanosuspension without drug), Riccardin D (20 mg/kg) and etoposide (20 mg/kg). Tumour growth was monitored with a calliper or by in vivo bioluminescence imaging (BLI) on the last day. In vivo BLI was performed with an IVIS-200 imaging system (Xenogen Corporation, Hopkinton, MA, USA). Mice were anaesthetized and injected i.p. with 200 mL of luciferin (16.7 mg/mL in saline). Ten minutes after the injection, mice were placed onto a warm stage. Imaging was performed for 1–3 min., depending on tumour size and time-point. Regions of interest were quantified as photon flux (p/s) using Xenogen Living Imageâ software
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[14]. Tumour volume was calculated by the formula, 1/2 9 D 9 d2, in which D is the long diameter, and d is the short diameter. All measurements were performed in a coded and blinded fashion. Telomerase activity assay. Telomerase activity was determined using the TRAPezeâ XL Telomerase Detection Kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. Briefly, MCF-7 and MDA-MB-231 cells seeded in 24-well plates (3.0 9 104 per well) were incubated with medium alone or with different concentrations of Riccardin D (5, 10, and 20 lM) for 72 hr. Cells were lysed in CHAPS lysis buffer. Cell lysates were then mixed with TRAPezeâ XL reaction mix containing Amplifuorâ primers and incubated at 30°C for 30 min. Amplified telomerase products were quantified using a fluorescence plate reader (Victor-1420 Multilabel Counter; Wallac, Turku, Finland). Telomerase activity (in TPG units) was then calculated by comparing the ratio of telomerase products to an internal standard for each lysate. Each sample was examined for three times. Reverse transcription and RT-PCR. MCF-7 and MDA-MB-231 cells seeded in 6-well plates (2.0 9 105 per well) were collected after incubation with medium alone or with different concentrations of Riccardin D (2.5, 5, and 10 lM) for 48 hr. Total RNA was extracted using RNA easy kit, according to the manufacturer’s instructions (Sangon, Shanghai, China) and reverse transcribed into cDNA using the first-strand cDNA synthesis kit (Toyobo, Osaka, Japan). Real-time quantitative RT-PCR was performed by SYBR Green Real-time PCR Master Mix (Toyobo, Japan) [15]. For semi-quantitative RT-PCR, specific primers were carried out in a Takara Bioscience PCR machine. The products were electrophoresed on 2% agarose gel followed by staining with ethidium bromide and visualized under UV light. The gene-specific oligonucleotide sequences were as follows: hTERT, forward 50 -gcggaagacagtggtgaact-30 , reverse 50 -agctggagtagtcg ctctgc-30 [16]; TRF2, forward 50 -gtacccaaaggcaagtggaa-30 , reverse 50 tgacccactcgctttcttct-30 [17]; GAPDH, forward 50 -gaggggccatccacagtctt30 , reverse 50 -ttcattgacctcaactacat-30 [9]. Each reaction was performed in triplicate. Western blotting assay. MCF-7 and MDA-MB-231 cells seeded in 6well plates (3.0 9 105 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5, 10 and 20 lM) for up to 72 hr. Cells were then harvested and lysed with RIPA buffer. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred into PVDF membranes [18]. Membranes were incubated with primary antibodies overnight at 4°C, followed by three washes and exposure to HRP-conjugated secondary antibodies for 1 hr at room temperature. The primary antibodies included those specific for hTERT (ab32020; Abcam, Cambridge, UK), bax (2772; Cell Signaling, Danvers, MA, USA), bcl-2 (2872; Cell Signaling), caspase-3 (9662; Cell Signaling), caspase-9 (9502; Cell Signaling), cleaved PARP (9541; Cell Signaling), ATM (2873; Cell Signaling), p-ATM (5883; Cell Signaling), p53 (9282; Cell Signaling), TRF2 (2645; Cell Signaling), Chk2 (3428-1; Epitomics, Burlingame, CA, USA), p-Chk2 (2661; Cell Signaling), c-H2AX (7631; Cell Signaling), p21 (2990-1; Epitomics) and b-actin (ab6276; Abcam). The membranes were developed with the ECL Western blotting detection system (Millipore) and quantified by densitometry using ChemiDoc XRS + image analyser (Bio-Rad, Hercules, CA, USA). Data were expressed as the relative density of the proteins normalized to b-actin. Triplicate experiments with triplicate samples were performed. Immunofluorescence assay. MCF-7 and MDA-MB-231 cells seeded in 24-well plates (3.0 9 104 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5 and 10 lM) for up to 72 hr. Cells grown on coverslips were fixed in cold acetone–methanol (1:1) for 10 min., washed twice with PBS,
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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permeabilized with 0.1% Triton X-100 for 20 min. and blocked with 10% bovine serum albumin for 1 hr at room temperature. Cells were then incubated with anti-c-H2AX (7631; Cell Signaling) in a humidified chamber overnight at 4°C. The slides were then washed and incubated with goat anti-rabbit IgG-FITC antibody (ZSGB-BIO, Beijing, China) in the dark for 1 hr at room temperature. DAPI (2 mg/mL in PBS; Sigma) was used to stain the nucleus. Untreated MCF-7 or MDA-MB-231 cells that were not subjected to primary antibody incubation served as the negative control; the positive control included MCF-7 or MDA-MB-231 cells treated with 1 mM hydrogen peroxide for 10 min. Images were acquired under a laser scanning microscope (Carl Zeiss, Oberkochen, Germany) [19]. Cell cycle assay. After synchronizing in 0.5% serum medium for 24 hr, MCF-7 and MDA-MB-231 cells seeded in 6-well plates (1.5 9 105 per well) were exposed to 10% serum medium alone or 10% serum medium containing different concentrations of Riccardin D (2.5, 5 and 10 lM) for another 48 hr. Cells were harvested, washed and fixed in cold 70% ethanol overnight. Cells were then collected and suspended in 1 mL propidium iodide (PI) solution (50 lg/mL, Sigma, 50 lg/mL DNase-free RNase A, 0.1% Triton X-100 in PBS) for 30 min. Data acquisition was performed on a FACScan flow cytometer (Becton Dickinson and Company, Franklin Lakes, NJ, USA). The percentages of cells in G0/G1, S and G2/M phases were determined using ModFit LT software 3.0 (Verity Software House, Topsham, ME, USA). The experiments were repeated three times. Apoptosis assay. Apoptotic cells were detected using Annexin VFITC/PI detection kit (Labtek, Dalian, China). MCF-7 and MDA-MB231 cells seeded in 6-well plates (1.5 9 105 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5 and 10 lM) for 48 hr and then washed with cold PBS. Cells were resuspended in 19 binding buffer. After adding 5 lL annexin V and 2.5 lL PI working solution to 100 lL cell suspension, the apoptotic cells were identified by a FACScan flow cytometer (Becton Dickinson and Company) [20]. The experiments were repeated three times. TUNEL assay. The apoptotic cells in both MCF-7 and MDA-MB231-luc-D3H2LN xenografts were identified using the TUNEL staining method and in situ cell death detection kit (Roche, Basel, Switzerland). Serial 4-lm sections were cut from formalin-fixed MCF7 and MDA-MB-231-luc-D3H2LN xenografts. Staining was performed according to the manufacturer’s instructions. Cancer cells with brown-stained nuclei were considered to be TUNEL positive [21]. The proportion of positive cells was scored at random under a microscope in at least three mice per group.
from 0.1% to 59.2%, for 24-hr exposure; from 7.3% to 87.9%, for 48-hr exposure; from 27.8% to 90.9%, for 72-hr exposure; and from 34.2% to 93.3%, for 96-hr exposure, respectively (fig. 1A and table 1, 40 lM, p < 0.01 versus the vehicle control). Figure 1B and table 2 show the similar profiles of growth inhibition in MDA-MB-231 cells. At concentrations ranging from 2.5 lM to 40 lM, the rates of inhibition in MDA-MB-231 cells were increased from 0.6% to 47.5%, for 24-hr exposure; from 5.9% to 49.5%, for 48-hr exposure; from 26.8% to 51.5%, for 72-hr exposure; and from 37.7% to 69.8%, for 96-hr exposure, respectively (40 lM, p < 0.01 versus the vehicle control). Inhibitory effect on the growth of human breast cancer xenografts in mice. The activity of Riccardin D was further evaluated on the MCF-7 xenografts mouse model. As shown in fig. 2A,B, Riccardin D (20 mg/kg) significantly delayed the growth of MCF-7 xenografts by 47.2% (p < 0.01 versus the vehicle control). The effect was also observed by a delayed increase in tumour volume (fig. 2C). Riccardin D also inhibited cancer growth in mice without significant body-weight loss (fig. 2D, p > 0.05 versus the vehicle control). The control drug etopoA
B
Statistical analysis. Data were expressed as mean S.D. for three different determinations. Statistical significance was analysed by oneway analysis of variance (ANOVA) followed by Dunnett’s multiple range tests. p < 0.05 was considered as statistically significant. Statistical analysis was performed using the SPSS/Win13.0 software (SPSS, Inc., Chicago, IL, USA).
Results Inhibition of cancer growth in vitro. MCF-7 or MDA-MB-231 cells were incubated with different concentrations of Riccardin D for different time periods and then subjected to MTT analysis. Riccardin D demonstrated an inhibitory effect on cancer cell growth in a time- and dosedependent manner. At concentrations ranging from 2.5 lM to 40 lM, the rates of inhibition in MCF-7 cells were increased
Fig. 1. Proliferation inhibition of MCF-7 cells (A) or MDA-MB-231 cells (B) exposed to Riccardin D. Cancer cells were exposed to different concentrations of Riccardin D for up to 96 hr. Viable cell number was evaluated by MTT assay and was denoted as a percentage of vehicle control at the concurrent time-point. The bars indicate means S.D. (n = 3). *p < 0.05, **p < 0.01 versus the vehicle control.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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Table 1. Proliferation inhibition of MCF-7 cells exposed to Riccardin D. Concentrations Time
2.5 lM, % (p)
5 lM, % (p)
10 lM, % (p)
20 lM, % (p)
40 lM, % (p)
24 48 72 96
0.1 7.3 27.8 34.2
0.2 12.0 48.9 55.7
9.0 13.1 76.5 88.4
18.6 55.4 88.7 92.0
59.2 87.9 90.9 93.3
hr hr hr hr
(>0.05) (>0.05) (
Doi: 10.1111/bcpt.12267
Riccardin D, a Macrocyclic Bisbibenzy, Inhibits Human Breast Cancer Growth through the Suppression of Telomerase Activity Cui-Cui Sun1, Hui-Min Xu2, Yi Yuan1, Zu-Hua Gao3,4, Hong-Xiang Lou1 and Xian-Jun Qu1,3 2
1 Department of Pharmacology, Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan, China, Faculty of Radiologic Sciences, School of Medicine, Qingdao University, Qingdao, China, 3Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital Medical University, Beijing, China and 4Department of Pathology, McGill University, Montreal, QC, Canada
(Received 23 January 2014; Accepted 2 May 2014) Abstract: Riccardin D, a liverwort-derived naturally occurring macrocyclic bisbibenzyl, has been found to exert anticancer effects in multiple cancer cell types. In this study, we investigated the effect and mechanism of Riccardin D on human breast cancer. Experiments were performed on human breast cancer MCF-7 and MDA-MB-231 cells. The antitumour effects of Riccardin D were assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and human breast cancer xenografts mice model. TRAPezeâ XL Telomerase Detection assay was used for the detection of telomerase activity. c-H2AX foci formation was tested for the induction of DNA damage response. Cell cycle distribution was analysed by flow cytometry, and cell apoptosis was determined by annexin V-FITC/PI staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay and Western blotting. Riccardin D effectively inhibited the growth of MCF-7 and MDA-MB-231 cells in vitro. And Riccardin D also effectively delayed the growth of MCF-7 and MDA-MB-231-luc-D3H2LN xenografts without significant loss of body-weight. Further analysis suggested that Riccardin D’s effects may arise from its suppression of telomerase activity, which led to telomere dysfunction. Telomerase inhibition and telomere dysfunction could activate the canonical ataxia telangiectasia-mutated (ATM) kinase-mediated DNA damage response, as shown by elevated expression of c-H2AX, pATM and p-Chk2. This is finally followed by the induction of cell cycle arrest and apoptosis, as shown by the increase of TUNEL-stained cells, caspase activation, PARP cleavage and the increase of bax/bcl-2 ratio. Moreover, Riccardin D induced p53-proficient MCF-7 cells to arrest in G1 phase and p53-deficient MDA-MB-231 cells to arrest in G2/M phase. Overall, these results demonstrate that Riccardin D may inhibit human breast cancer growth through suppression of telomerase activity.
Breast cancer, accounting for 23% of all cancers, is the most common malignant disease and the second most frequent cause of cancer death in women [1]. About one million new cases are diagnosed, and 400,000 patients die from the disease each year [2]. Over the last 40 years, advances in the development of breast cancer drugs have led to improved treatments and outcome for patients. However, mortality has remained relatively unchanged over the same period. In addition, toxicity of many anticancer drugs impacts patients’ compliance with treatment and results in serious long-term health effects. Therefore, identification of novel agents that can suppress the growth of human breast cancers but are relatively safe is highly desirable. Telomerase is active in more than 90% of breast carcinomas, whereas in normal tissues, it is not active or detectable. Telomerase is a ribonucleoprotein reverse transcriptase that maintains telomeric ends of eukaryotic chromosomes during DNA replication [3]. It consists of a catalytic protein subunit (human telomerase reverse transcriptase, hTERT), an RNA Authors for correspondence: Xian-Jun Qu, Hong-Xiang Lou and Zu-Hua Gao, Department of Pharmacology, Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan and Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital Medical University, Beijing, China (fax 86-531-88382490, e-mails [email protected], louhongxiang@ sdu.edu.cn and [email protected]).
component (human telomerase RNA, hTR) and other associated proteins. Telomerase is highly associated with carcinogenesis. Telomerase enables cancer cells to bypass replicative senescence and to be capable of self-replicating indefinitely by compensating cell-division-dependent telomere attrition [4]. hTERT is an important catalytic protein subunit of telomerase, and its expression level is closely associated with telomerase activity [5]. hTERT is generally repressed in normal cells and up-regulated in immortal cells. Inhibition of the hTERT component of telomerase enzyme could induce telomere shortening to a critical length, called telomere ‘uncapped’, initiating end-to-end fusions and the DNA damage response [6]. This in turn leads to cell cycle arrest, DNA repair or apoptosis. Therefore, telomerase is an attractive target for the development of anticancer drugs. Riccardin D is a novel macrocyclic bisbibenzyl compound isolated from the liverwort plant Dumortiera hirsuta [7]. It has been considered as a potential candidate agent for cancer treatment. Riccardin D was found to inhibit the proliferation of human leukaemia cells HL60, K562 and its multidrug-resistant counterpart K562/A02 cells [8]. Riccardin D inhibited tumour angiogenesis in human lung carcinoma H460 xenografts mouse model [9]. Riccardin D is also a potential chemopreventive regimen for intestinal cancers derived from APC gene mutation [10]. In this study, we first investigated the effects of
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
RICCARDIN D INHIBITS TELOMERASE ACTIVITY
Riccardin D on human breast cancer MCF-7 and MDA-MB231 cells. We then evaluated the efficacy of Riccardin D in nude mice bearing human breast cancer xenografts in vivo. To study the molecular mechanism of the anticancer effect of Riccardin D, we examined its effect on telomerase activity in human breast cancer cells. We further examined the effect of Riccardin D on DNA damage response using p53-proficient MCF-7 cells and p53-deficient MDA-MB-231 cells. Our study provides scientific evidence that Riccardin D could be a therapeutic agent against human breast cancer. Materials and Methods Drug. Riccardin D was isolated from the liverwort plant D. hirsuta as described previously [7]. The purity of Riccardin D was measured at 98.6% by high performance liquid chromatography (HPLC). Riccardin D was dissolved in dimethylsulphoxide (DMSO; Sigma, St. Louis, MO, USA) for in vitro assays. For the animal experiments, Riccardin D nanosuspension was prepared by the high-pressure homogenization (HPH) method, as described previously [11,12]. Cell lines and cell culture. Human breast cancer cell lines, MCF-7 (p53-proficient) and MDA-MB-231 (p53-deficient), were purchased from Cell Bank, China Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin at 37°C in a humid atmosphere (5% CO2– 95% air). MDA-MB-231-luc-D3H2LN cells, expressing stable firefly luciferase, were purchased from Caliper Life Sciences. MDA-MB-231luc-D3H2LN cells were cultured in minimum essential medium with Earl’s balanced salts solution (MEM/EBSS) supplemented with 10% foetal bovine serum, 1% non-essential amino acids, 1% L-glutamine, 1% sodium pyruvate, 100 U/mL penicillin and 100 lg/mL streptomycin at 37°C in a humid atmosphere containing 5% CO2. MTT assay for cell viability. MCF-7 and MDA-MB-231 cells seeded in 96-well plates (5 9 103 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5, 10, 20 and 40 lM) for 24, 48, 72 and 96 hr, respectively. The cells were then incubated with 20 lL MTT reagent (5 mg/mL, Sigma) for 4 hr at 37°C. The MTT reagent was removed, and 100 lL DMSO was added to each well. The spectrophotometric reading was taken at 570 nm using a microplate reader (Perkin-Elmer, Waltham, MA, USA). Triplicate experiments were performed with triplicate samples. Assessment of cancer growth in mice. Female BALB/c nude (nu/nu) mice, 6 weeks of age, were purchased from the Animal Center of the China Academy of Medical Sciences (Beijing, China). All experimental protocols were strictly approved by the Committee of Animal Care and Use of Shandong University. Mice were housed under pathogen-free conditions. MCF-7 and MDA-MB-231-lucD3H2LN cells (1 9 107 per mouse) were subcutaneously injected into the left flank [13]. Mice were then divided into three groups with seven mice in per group: vehicle (nanosuspension without drug), Riccardin D (20 mg/kg) and etoposide (20 mg/kg). Tumour growth was monitored with a calliper or by in vivo bioluminescence imaging (BLI) on the last day. In vivo BLI was performed with an IVIS-200 imaging system (Xenogen Corporation, Hopkinton, MA, USA). Mice were anaesthetized and injected i.p. with 200 mL of luciferin (16.7 mg/mL in saline). Ten minutes after the injection, mice were placed onto a warm stage. Imaging was performed for 1–3 min., depending on tumour size and time-point. Regions of interest were quantified as photon flux (p/s) using Xenogen Living Imageâ software
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[14]. Tumour volume was calculated by the formula, 1/2 9 D 9 d2, in which D is the long diameter, and d is the short diameter. All measurements were performed in a coded and blinded fashion. Telomerase activity assay. Telomerase activity was determined using the TRAPezeâ XL Telomerase Detection Kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. Briefly, MCF-7 and MDA-MB-231 cells seeded in 24-well plates (3.0 9 104 per well) were incubated with medium alone or with different concentrations of Riccardin D (5, 10, and 20 lM) for 72 hr. Cells were lysed in CHAPS lysis buffer. Cell lysates were then mixed with TRAPezeâ XL reaction mix containing Amplifuorâ primers and incubated at 30°C for 30 min. Amplified telomerase products were quantified using a fluorescence plate reader (Victor-1420 Multilabel Counter; Wallac, Turku, Finland). Telomerase activity (in TPG units) was then calculated by comparing the ratio of telomerase products to an internal standard for each lysate. Each sample was examined for three times. Reverse transcription and RT-PCR. MCF-7 and MDA-MB-231 cells seeded in 6-well plates (2.0 9 105 per well) were collected after incubation with medium alone or with different concentrations of Riccardin D (2.5, 5, and 10 lM) for 48 hr. Total RNA was extracted using RNA easy kit, according to the manufacturer’s instructions (Sangon, Shanghai, China) and reverse transcribed into cDNA using the first-strand cDNA synthesis kit (Toyobo, Osaka, Japan). Real-time quantitative RT-PCR was performed by SYBR Green Real-time PCR Master Mix (Toyobo, Japan) [15]. For semi-quantitative RT-PCR, specific primers were carried out in a Takara Bioscience PCR machine. The products were electrophoresed on 2% agarose gel followed by staining with ethidium bromide and visualized under UV light. The gene-specific oligonucleotide sequences were as follows: hTERT, forward 50 -gcggaagacagtggtgaact-30 , reverse 50 -agctggagtagtcg ctctgc-30 [16]; TRF2, forward 50 -gtacccaaaggcaagtggaa-30 , reverse 50 tgacccactcgctttcttct-30 [17]; GAPDH, forward 50 -gaggggccatccacagtctt30 , reverse 50 -ttcattgacctcaactacat-30 [9]. Each reaction was performed in triplicate. Western blotting assay. MCF-7 and MDA-MB-231 cells seeded in 6well plates (3.0 9 105 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5, 10 and 20 lM) for up to 72 hr. Cells were then harvested and lysed with RIPA buffer. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred into PVDF membranes [18]. Membranes were incubated with primary antibodies overnight at 4°C, followed by three washes and exposure to HRP-conjugated secondary antibodies for 1 hr at room temperature. The primary antibodies included those specific for hTERT (ab32020; Abcam, Cambridge, UK), bax (2772; Cell Signaling, Danvers, MA, USA), bcl-2 (2872; Cell Signaling), caspase-3 (9662; Cell Signaling), caspase-9 (9502; Cell Signaling), cleaved PARP (9541; Cell Signaling), ATM (2873; Cell Signaling), p-ATM (5883; Cell Signaling), p53 (9282; Cell Signaling), TRF2 (2645; Cell Signaling), Chk2 (3428-1; Epitomics, Burlingame, CA, USA), p-Chk2 (2661; Cell Signaling), c-H2AX (7631; Cell Signaling), p21 (2990-1; Epitomics) and b-actin (ab6276; Abcam). The membranes were developed with the ECL Western blotting detection system (Millipore) and quantified by densitometry using ChemiDoc XRS + image analyser (Bio-Rad, Hercules, CA, USA). Data were expressed as the relative density of the proteins normalized to b-actin. Triplicate experiments with triplicate samples were performed. Immunofluorescence assay. MCF-7 and MDA-MB-231 cells seeded in 24-well plates (3.0 9 104 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5 and 10 lM) for up to 72 hr. Cells grown on coverslips were fixed in cold acetone–methanol (1:1) for 10 min., washed twice with PBS,
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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permeabilized with 0.1% Triton X-100 for 20 min. and blocked with 10% bovine serum albumin for 1 hr at room temperature. Cells were then incubated with anti-c-H2AX (7631; Cell Signaling) in a humidified chamber overnight at 4°C. The slides were then washed and incubated with goat anti-rabbit IgG-FITC antibody (ZSGB-BIO, Beijing, China) in the dark for 1 hr at room temperature. DAPI (2 mg/mL in PBS; Sigma) was used to stain the nucleus. Untreated MCF-7 or MDA-MB-231 cells that were not subjected to primary antibody incubation served as the negative control; the positive control included MCF-7 or MDA-MB-231 cells treated with 1 mM hydrogen peroxide for 10 min. Images were acquired under a laser scanning microscope (Carl Zeiss, Oberkochen, Germany) [19]. Cell cycle assay. After synchronizing in 0.5% serum medium for 24 hr, MCF-7 and MDA-MB-231 cells seeded in 6-well plates (1.5 9 105 per well) were exposed to 10% serum medium alone or 10% serum medium containing different concentrations of Riccardin D (2.5, 5 and 10 lM) for another 48 hr. Cells were harvested, washed and fixed in cold 70% ethanol overnight. Cells were then collected and suspended in 1 mL propidium iodide (PI) solution (50 lg/mL, Sigma, 50 lg/mL DNase-free RNase A, 0.1% Triton X-100 in PBS) for 30 min. Data acquisition was performed on a FACScan flow cytometer (Becton Dickinson and Company, Franklin Lakes, NJ, USA). The percentages of cells in G0/G1, S and G2/M phases were determined using ModFit LT software 3.0 (Verity Software House, Topsham, ME, USA). The experiments were repeated three times. Apoptosis assay. Apoptotic cells were detected using Annexin VFITC/PI detection kit (Labtek, Dalian, China). MCF-7 and MDA-MB231 cells seeded in 6-well plates (1.5 9 105 per well) were incubated with medium alone or with different concentrations of Riccardin D (2.5, 5 and 10 lM) for 48 hr and then washed with cold PBS. Cells were resuspended in 19 binding buffer. After adding 5 lL annexin V and 2.5 lL PI working solution to 100 lL cell suspension, the apoptotic cells were identified by a FACScan flow cytometer (Becton Dickinson and Company) [20]. The experiments were repeated three times. TUNEL assay. The apoptotic cells in both MCF-7 and MDA-MB231-luc-D3H2LN xenografts were identified using the TUNEL staining method and in situ cell death detection kit (Roche, Basel, Switzerland). Serial 4-lm sections were cut from formalin-fixed MCF7 and MDA-MB-231-luc-D3H2LN xenografts. Staining was performed according to the manufacturer’s instructions. Cancer cells with brown-stained nuclei were considered to be TUNEL positive [21]. The proportion of positive cells was scored at random under a microscope in at least three mice per group.
from 0.1% to 59.2%, for 24-hr exposure; from 7.3% to 87.9%, for 48-hr exposure; from 27.8% to 90.9%, for 72-hr exposure; and from 34.2% to 93.3%, for 96-hr exposure, respectively (fig. 1A and table 1, 40 lM, p < 0.01 versus the vehicle control). Figure 1B and table 2 show the similar profiles of growth inhibition in MDA-MB-231 cells. At concentrations ranging from 2.5 lM to 40 lM, the rates of inhibition in MDA-MB-231 cells were increased from 0.6% to 47.5%, for 24-hr exposure; from 5.9% to 49.5%, for 48-hr exposure; from 26.8% to 51.5%, for 72-hr exposure; and from 37.7% to 69.8%, for 96-hr exposure, respectively (40 lM, p < 0.01 versus the vehicle control). Inhibitory effect on the growth of human breast cancer xenografts in mice. The activity of Riccardin D was further evaluated on the MCF-7 xenografts mouse model. As shown in fig. 2A,B, Riccardin D (20 mg/kg) significantly delayed the growth of MCF-7 xenografts by 47.2% (p < 0.01 versus the vehicle control). The effect was also observed by a delayed increase in tumour volume (fig. 2C). Riccardin D also inhibited cancer growth in mice without significant body-weight loss (fig. 2D, p > 0.05 versus the vehicle control). The control drug etopoA
B
Statistical analysis. Data were expressed as mean S.D. for three different determinations. Statistical significance was analysed by oneway analysis of variance (ANOVA) followed by Dunnett’s multiple range tests. p < 0.05 was considered as statistically significant. Statistical analysis was performed using the SPSS/Win13.0 software (SPSS, Inc., Chicago, IL, USA).
Results Inhibition of cancer growth in vitro. MCF-7 or MDA-MB-231 cells were incubated with different concentrations of Riccardin D for different time periods and then subjected to MTT analysis. Riccardin D demonstrated an inhibitory effect on cancer cell growth in a time- and dosedependent manner. At concentrations ranging from 2.5 lM to 40 lM, the rates of inhibition in MCF-7 cells were increased
Fig. 1. Proliferation inhibition of MCF-7 cells (A) or MDA-MB-231 cells (B) exposed to Riccardin D. Cancer cells were exposed to different concentrations of Riccardin D for up to 96 hr. Viable cell number was evaluated by MTT assay and was denoted as a percentage of vehicle control at the concurrent time-point. The bars indicate means S.D. (n = 3). *p < 0.05, **p < 0.01 versus the vehicle control.
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Table 1. Proliferation inhibition of MCF-7 cells exposed to Riccardin D. Concentrations Time
2.5 lM, % (p)
5 lM, % (p)
10 lM, % (p)
20 lM, % (p)
40 lM, % (p)
24 48 72 96
0.1 7.3 27.8 34.2
0.2 12.0 48.9 55.7
9.0 13.1 76.5 88.4
18.6 55.4 88.7 92.0
59.2 87.9 90.9 93.3
hr hr hr hr
(>0.05) (>0.05) (
