Mol Biol Rep (2014) 41:3475–3480 DOI 10.1007/s11033-014-3209-3
Matrine inhibits the growth of retinoblastoma cells (SO-Rb50) by decreasing proliferation and inducing apoptosis in a mitochondrial pathway Qingliang Shao • Xiaxia Zhao • Li Yao
Received: 15 April 2013 / Accepted: 27 January 2014 / Published online: 11 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Matrine, one of the main active components of extracts from the dry roots of Sophora flavescens, has potent anti-tumor activity in vitro and in vivo. Here, we investigated the apoptosis in matrine-treated retinoblastoma cells. The results showed that matrine could inhibit cell proliferation and induce apoptosis in a dose- and time-dependent manner. Further investigation revealed that a disruption of mitochondrial transmembrane potential and an up-regulation of reactive oxygen species in matrine-treated cells. By western blot analysis, we found that the up-regulation of cleaved Apaf-1, cleaved caspase-3, cleaved caspase-9, cleaved caspase-7, Bax/Bcl-2, varying with different concentration of matrine. These protein interactions may play a pivotal role in the regulation of apoptosis. Taken together, these results overall indicate that matrine could be used as an effective anti-tumor agent in therapy of retinoblastoma targets the caspase-dependent signaling pathway.
Keywords Matrine Apoptosis Mitochondrial transmembrane potential Retinoblastoma Caspase
Introduction Retinoblastoma is the most common primary malignant intraocular tumor in infants and children [1]. Slightly more
Q. Shao X. Zhao L. Yao (&) Department of Pediatrics, The Second Affiliated Hospital of Harbin Medical University, 148 Baojian Street, Nangang District, Harbin 150081, Heilongjiang, China e-mail:
[email protected] than half of the patients have the sporadic or non-inherited form of the disease, which results from the spontaneous inactivation of the retinoblastoma gene (RB1). In the heritable form, the patient inherits usually one defective gene from the parents and a subsequent ‘‘hit’’ of the uninvolved gene results in tumor formation. The heritable form is more often bilateral than the non-heritable form of the disease. Despite progress in the treatment of retinoblastoma [2], significant problems remain unsolved. Metastatic disease is often fatal [3]. Although several treatments are available for retinoblastoma, including enucleation and/or combination of chemotherapy, laser and cryotherapy, each of them has major drawbacks in pediatric patients. There is a need for alternative new treatment modalities for retinoblastoma with better safety and efficacy profile. Preliminary studies have shown that matrine has antitumor effects by inducing apoptosis and inhibiting proliferation of many different cancer cells including cervical, stomach, breast, and lung cancers, as well as hepatocellular carcinoma, leukemia, and multiple myeloma [4–8]. Additionally, it is considered a useful agent in the treatment of oesop and laryngeal cancers and murine hepatocellular carcinoma [9, 10]. The ability of matrine to inhibit tumor growth has been proposed to be through the modulation of apoptosis- and/or proliferationrelated genes and proteins, including N-ras, p53, c-myc, Bcl-2 family members [5, 6]. Moreover, a recent study reported that matrine inhibited pancreatic cancer growth through the induction of Fas/FasL [11]. Unfortunately, very few studies have been carried on inhibitory effect of matrine on retinoblastoma and the mechanisms of the anti-cancer capacity remain poorly understood. Therefore, the aim of the present study was to thoroughly research matrine-induced apoptosis and explore the potential mechanisms.
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Materials and methods Reagents and antibodies Matrine was purchased from Sigma–Aldrich, and its purity was [99 % confirmed by high-performance liquid chromatography (HPLC). The molecular formula of matrine is C15H24N2O, and its molecular weight is 248.36 (Fig. 1) [12]. It was dissolved in cell culture medium at a stock concentration of 20 mg/ml and stored at -20 °C. Matrine stock solution was freshly diluted in the medium just before the use in each experiment. Materials used included Annexin V–fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Becton–Dickinson, Franklin Lakes, NJ, USA), Hoechst–propidium iodide (PI) staining assay kit (Beyotime Institute of Biotechnology, Shanghai, China), 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology, Shanghai, China); Apaf-1, Bcl-2, Bax, anti-caspase-9, anti-caspase-3, anticaspase-7 and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cell lines and culture conditions The retinoblastoma SO-Rb50 cells were maintained in DMEM supplemented with 10 % heat inactivated fetal calf serum (FCS), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37 °C with 5 % CO2. The cells were kept in an exponential growth phase during experiments. Proliferation assay Cell growth inhibition by matrine was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, SO-Rb50 cells were seeded in 96-well plates at a density of 6 9 103 cells per well. After treatment with various concentrations of matrine (0–1.5 mg/ ml) for 24, 48, and 72 h, 20 ll MTT (5 mg/ml) was added. Fig. 1 The chemical structure of matrine. The molecular formula of matrine is C15H24N2O, and its molecular weight is 248.36
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Four hours later, 100 ll DMSO was added to each well to dissolve the resulting formazan crystals. Absorbance was read at 490 nm using an enzyme-linked immunosorbent assay reader (SpectraMax; Molecular Devices, Sunnyvale, CA, USA). Data were collected from three separate experiments and the percentage of matrine-induced cell growth inhibition was determined by comparison to control cells. Annexin V–FITC/PI double staining Annexin V–FITC/PI double staining was employed to quantify the apoptosis of retinoblastoma cells treated with matrine. Briefly, cells were seeded in 6-well plates (2 9 105 cells/ml) and exposed to matrine (0.0–1.5 mg/ml) for 24 h. The cells were then stained using Annexin V–FITC/PI double fluorescence apoptosis detection kit (Biouniquer Technology) following the manufacturer’s instruction. Samples were analyzed using a FACS Calibur Flow cytometer within 1 h after the staining. Cells were grown in 6-well plates for 12 h and treated with caspase inhibitor Z-VAD-FMK (5 mM) for 1 h before treated with matrine. After 24 h, cells were washed twice with PBS, adjusted to 100 ll of the solution and transferred to a 1-ml centrifuge tube (1 9 105 cells). 10 ll of Annexin V–FITC and 10 ll of PI were added and cells were incubated for 15 min at room temperature (RT) (25 °C) in the dark before being analyzed as described above. Measurement of mitochondrial transmembrane potential (MMP) with fluorescent dye JC-1 Cells cultured as described above were resuspended with culture medium to a concentration of 1 9 105 cells/ml and incubated with fluorescent dye JC-1 (20 nM) at 37 °C for 30 min in the dark. Then fluorescent dye JC-1 fluorescence was immediately analyzed with a flow cytometer (Becton– Dickinson). Measurement of intracellular ROS levels The intracellular accumulation of ROS in the cells was assessed using 20, 70-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology, Shanghai, China). This fluorescent compound accumulates within cells, and on deacetylation, DCFH-DA then reacts with ROS to form fluorescent dichlorofluorescein (DCF). Briefly, cells were seeded in 6-well plates (2 9 105 cells/ml) and exposed to matrine (0.0–1.5 mg/ml) for 24 h were washed twice with PBS and incubated with DCFH-DA (20 lM) in PBS for 1 h at 37 °C in the dark. Cells were then washed three times with PBS and finally examined in PBS supplemented with 10 % FCS with a flow cytometer (Becton–Dickinson).
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Immunoblotting Cells were separately washed, collected and homogenized in a lysis buffer (10 mM Tris–HCl, pH 8, 0.32 mM sucrose, 5 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethyl sulfonylfluoride, and 1 % Triton X-100), and centrifuged (13,0009g, 10 min, 4 °C). To ensure that an equal amount of protein was loaded in each case, western blots were also carried out, using the Bradford protein assay. Equal amounts of proteins (50 lg) were subjected to electrophoresis in a sodium dodecyl sulfate– polyacrylamide gel (10 %). The gel-separated proteins were transferred to Nitropure nitrocellulose membranes (Santa Cruz Biotechnology) and the membranes were blocked with 10 % bovine serum albumin in TBST [10 mM Tris–HCl (pH 8.0), 137 mM NaCl, and 0.05 % Tween-20 by vol] overnight at 4 °C and probed with primary antibodies at 37 °C for 2 h. Each of the targeted proteins was immunostained by specific antibodies. The antibodies used were anti-cleaved caspase-9 (1:500 dilution), anti-cleaved caspase-3 (1:500 dilution), anticleaved caspase-7 (1:500 dilution), anti-Apaf-1, (1:500 dilution), anti-Bcl-2 (1:200 dilution), anti-Bax (1:200 dilution) and anti-b-actin (1:1,000 dilution). The membranes were washed three times with TBST and then incubated for 1 h at RT with alkaline phosphatase-conjugated secondary antibodies (Santa Cruz Biotechnology) before being visualized by using a chemiluminescence detection kit (Beyotime Institute of Biotechnology, Shanghai, China). Statistical analysis Data are presented as the mean ± standard deviation (SD). Differences between groups were analyzed using Student’s t test for continuous variables. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS, version 17.0; SPSS, Inc.) and P \ 0.05 was considered statistically significant.
Results Matrine inhibit the proliferation of retinoblastoma cells in a dose- and time-dependent manner To investigate the effects of matrine on the proliferation of retinoblastoma cells, we measured the growth of retinoblastoma cell lines using the MTT incorporation assay. The treatment of SO-RB50 cells with 0.0–1.5 mg/ml of matrine resulted in a dose and time-dependent inhibition of cell growth (Fig. 2). Matrine induce the apoptosis of retinoblastoma cells Flow cytometry assays showed marked changes in cell profiles after treatment with 0–1.5 mg/ml matrine, which strongly
Fig. 2 SO-Rb50 cell proliferation was inhibited by Matrine. SORb50 cells were treated with or without different concentrations of matrine for 24, 48, or 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out as described. Data are presented as mean ± SD from three independent experiments. Each experiment was conducted in triplicate. *P \ 0.05; **P \ 0.01
indicated that matrine could induce apoptosis (Fig. 3a). All matrine treatment groups showed significant increases in apoptosis compared with control groups (P \ 0.01). Apoptotic rates ranged from 0.73 ± 0.1 to 39.42 ± 1.35 % (Fig. 3b). The 1.5 mg/ml matrine group was the highest in all experiments (compared with control group, P \ 0.01). Additionally, when added caspase inhabitor Z-VAD-FMK (5 mM) before exposure to 1.5 mg/ml matrine for 24 h, apoptotic rates decreased to 23.32 ± 0.55 % (P \ 0.01) (Fig. 3b). Measurement of MMP with fluorescent dye JC-1 MMP decreased in response to matrine was in a dosedependent manner (Fig. 3c). Cells treated with 0.5 mg/ml matrine showed a 16.3 ± 2.79 % decrease in MMP (P \ 0.01). Cells treated with 1.0 mg/ml matrine showed a 20.7 ± 1.45 % decrease in MMP (P \ 0.01). Finally, cells treated with 1.5 mg/ml matrine showed a 54.3 ± 1.29 % decrease in MMP (P \ 0.01). Additionally, when added caspase inhibitor Z-VAD-FMK (5 mM) before exposure to 1.5 mg/ml matrine for 24 h, MMP rates decreased to 33.12 ± 0.29 % (P \ 0.01) (Fig. 3d). Matrine regulate the expression of apoptosis-related protein in retinoblastoma cell lines To determine the mechanism responsible for matrinemediated apoptosis, the apoptotic protein expressions were evaluated by western blot analyses. Because Apaf-1 is considered to be characteristic of apoptosis, the Apaf-1 and
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Fig. 3 Changes were induced in SO-Rb50 cells by matrine. a Representative photomicrographs (Olympus, Tokyo, Japan; magnification 9200) of SO-Rb50 cells stained with Annexin V–FITC/PI after exposure of the cells to 0–1.5 mg/ml matrine for 24 h. b Flow cytometric analysis of SO-Rb50 apoptotic cells stained for Annexin V ? PI after treatment with 0–1.5 mg/ml matrine, 1.5 mg/ml matrine ? Z-VAD-FMK. c Mitochondrial membrane hyperpolarization
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induced by matrine in SO-Rb50 cells. The mean fluorescence intensity was detected using a flow cytometer. The differences in the MMP levels between each group were expressed as a percentage of the control. d ROS production in the SO-Rb50 cells treated with various concentrations of matrine was significantly increased compared with control. Data are presented as mean ± SD of three independent experiments, and each experiment was carried out in triplicate (*P \ 0.01)
capase-3, cleaved capase-7 (Fig. 4) and Bax proteins, which are all involved in apoptosis. In contrast, there was a decrease in the expression of Bcl-2 protein in cultures exposed to different concentrations of matrine (Fig. 4).
Discussion
Fig. 4 Western blot analysis of SO-Rb50 cells after being exposed to 0–1.5 mg/ml matrine for 24 h. Martine caused an up-regulation in the levels of Apaf-1, cleaved caspase-9, cleaved caspase-3, cleaved caspase-7 and a decrease expression of Bcl-2 and an increase expression of Bax were observed in SO-Rb50 cells after treatment with different concentrations of matrine. The results are expressed as mean ± SEM, and each experiment was carried out in triplicate
caspases was evaluated. Figure 4 shows the results of western blot analysis for Apaf-1, cleaved capase-9, cleaved capase-3, cleaved capase-7, Bcl-2 and Bax proteins relative to b-actin in control untreated cultures and cultures exposed to 0–1.5 mg/ml matrine. It can be seen that exposure of cultures to matrine resulted in the up-regulation of Apaf-1, cleaved PARP, cleaved capase-9, cleaved
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Retinoblastoma is a malignant tumor of the retina of the eye and generally affects children under the age of 6 years. Retinoblastoma affects approximately 1 in 15,000 live births. Worldwide, approximately 5,000 new cases occur per year [13–15]. It is the most common eye cancer in children and is caused by mutation on chromosome 13, called the RB1 gene. The defective RB1 gene can be inherited from either of the parents in some children; however, the mutation occurs in the early stages of fetal development. Characterized by the typical cat’s eye or the white pupil reflex (leukocoria) noted by parents, approximately 63 % of all retinoblastomas arise in the first 2 years of life. In some cases, retinoblastoma metastasizes to extraocular organs including bone, lung and brain. Although non-metastatic tumors can be treated by enucleation (removal of the eye), currently, there is no treatment for metastatic retinoblastoma. Currently, the standard
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treatment for RB is neoadjuvant chemotherapy, however, the effectiveness of cytotoxic drugs often declines, due to acquired chemoresistance. Finding new therapeutic agents to target the malignant behavior of retinoblastoma cells is, therefore, important for improving the prognosis. The results of the present study showed that SO-RB50 cells growth inhibited by matrine at different concentrations, ranging from 0.0–1.5 mg/ml, for 24, 48, and 72 h. Matrine significantly decreased the proliferation of SORb50 cells in a concentration and time-dependent manner (Fig. 2). The apoptosis induced by matrine was found in a concentration-dependent manner through Annexin V– FITC–PI staining fluorescence imaging (Fig. 3a) and flow cytometry (Fig. 3b). Apoptosis plays a crucial role in protecting organisms against tumorigenesis. Many anti-cancer drugs act to induce apoptosis, eliminating cells that harbor genetic damage or divide inappropriately [16]. In mammalian cells, apoptosis has been divided into two major pathways: the extrinsic pathway, activated by pro-apoptotic receptor signals at the cellular surface; and the intrinsic pathway, regulates apoptotic cascades by the signaling convergence in the mitochondrion, which results in the alteration of the MMP, the release of cytochrome C into the cytosol, and the activation of caspase-9 [17]. Independently from cell type and apoptosis inducer that MMP disruption is a constant feature of the apoptotic effector phase [18]. Disruption of MMP and the subsequent release of apoptosis-promoting factors are considered key cellular events that trigger the intrinsic apoptotic pathway. Results from the present study clearly demonstrated that SO-Rb50 cells treated with matrine exhibited an early reduction of MMP, suggesting that MMP might play critical roles in matrine induced apoptosis (Fig. 3c). ROS are generated in and around mitochondria and are regarded as the byproducts of normal cellular oxidative processes. It has been indicated that they can regulate initiation of apoptotic signaling. High levels of ROS produced by the SO-Rb50 cells after treated with matrine were observed (Fig. 3d). These results confirmed that ROS were crucial in the induction of apoptosis and acted as upstream signaling molecules to initiate cell death. Similar to the role of mitochondria in the control of cell death, Bcl-2 and Bax, survival or apoptotic factors can also prevent or facilitate the release of apoptogenic factors such as cytochrome C [19–21]. The loss of the Bcl-2 protein promotes the opening of the mitochondrial permeability transition pore. A decrease expression of Bcl-2 and an increase expression of Bax were observed in SO-Rb50 cells after treatment with different concentrations of matrine. This change in Bcl-2 and Bax expression may be enough to facilitate pore opening. Disruption of MMP is an early event in mitochondrialmediated apoptosis [22]. After the reduction of membrane
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potential, a critical step is the formation of apoptosomes, which ultimately cleave pro-caspase-3 to form active caspase-3. Caspases play critical roles in the execution of apoptosis [23]. The results of the present study demonstrated that matrine-induced apoptosis in SO-Rb50 cells was mediated by both caspase-9 and caspase-3 following Apaf-1 cleavage. Furthermore, the role that the caspases played in matrine-induced apoptosis was confirmed by the attenuation of apoptosis in cells that were pretreated with Z-VAD-FMK (Fig. 4). In conclusion, matrine could induce SO-Rb50 cells apoptosis through perturb mitochondrial permeability transition pore and caspase activation. Moreover, down regulation of Bcl-2/Bax sustain caspase in the active state and trigger the mitochondrial pathway. Taken together, these findings provide a basis for further analysis of matrine as a promising candidate for the treatment of retinoblastoma. Acknowledgments The authors are grateful to Dr. Zhang (Harbin Medical University) for his technical help.
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