Tumor Biol. DOI 10.1007/s13277-014-2754-7
RESEARCH ARTICLE
Berberine-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species generation and mitochondrial-related apoptotic pathway Juan Xie & Yinyan Xu & Xinyan Huang & Yanni Chen & Jing Fu & Mingming Xi & Li Wang
Received: 23 July 2014 / Accepted: 20 October 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Berberine has drawn extensive attention toward their wide range of biochemical and pharmacological effects, including antineoplastic effect in recent years, but the precise mechanisms remain unclear. Treatment of human breast cancer cells (MCF-7 and MDA-MB-231 cells) with berberine induced inhibition of cell viability in concentration- and time-dependent manner irrespective of their estrogen receptor (ER) expression. Hoechst33342 staining confirmed berberine induced breast cancer cell apoptosis in time-dependent manner. Because apoptosis induction is considered to be a crucial strategy for cancer prevention and therapy, berberine may be an effective chemotherapeutic agent against breast cancer. To explore the precise mechanism, berberine-induced oxidative stress and mitochondrial-related apoptotic pathway in human breast cancer cells were investigated in this study. In both MCF-7 and MDA-MB-231 cells, berberine increased the production of reactive oxygen species (ROS), which activated the pro-apoptotic JNK signaling. Phosphorylated JNK triggered mitochondria membrane potential (ΔΨm) depolarization and downregulation expression of anti-apoptotic protein Bcl-2 concomitant with the upregulation expression of proapoptotic protein Bax. Downregulation of anti-apoptotic Bcl2 family protein in parallel with loss of ΔΨm, leading to increased the release of cytochrome c and apoptosisinducing factor (AIF) from mitochondria, and eventually triggered the caspase-dependent and caspase-independent
Juan Xie and Yinyan Xu contributed equally to this work. J. Xie (*) : Y. Xu : X. Huang : Y. Chen : J. Fu : M. Xi : L. Wang (*) State key Laboratory of Reproductive Medicine, Department of Pharmacy, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China e-mail:
[email protected] e-mail:
[email protected] apoptosis. Taken together, our study reveled that berberine exerted an antitumor activity in breast cancer cells by reactive oxygen species generation and mitochondrial-related apoptotic pathway. These finding provide an insight into the potential of berberine for breast cancer therapy. Keywords Berberine . Apoptosis . Reactive oxygen species . Mitochondria
Instruction Breast cancer is one of the most common malignancies in women and represents a major issue of public health with 1,380,000 million new cases and 458,000 annual deaths worldwide [1, 2]. In recent years, the incidence of breast cancer has become a major threat to the lives of women in some big cities in China [3]. Currently, patients can be cured by surgery and radiotherapy, and frequently supported by adjuvant chemo- or hormone-therapy. However, breast cancer is highly resistant to chemotherapy, and there is still no effective cure for patients with advanced stages of the disease [4]. Therefore, research is urgently needed to identify effective therapeutic drugs for breast cancer. Berberine is an isoquinoline quaternary alkaloid isolated from many kinds of medicinal plants such as Hydrastis canadensis, Berberis aristata, Coptis chinensis, Coptis rhizome, Coptis japonica, Phellondendron amurense, and Phellondendron chinense schneid [5]. Berberine has been found to possess a wide variety of pharmacological and biological activities such as antimicrobial, antihelmintic, and antiinflammatory [6–8]. In recent years, researchers also focus on the anticancer activity of berberine because of its antineoplastic evidence. Researchers have demonstrated its anticancer effects against a variety of human cancer cells both in vitro
Tumor Biol.
and in vivo, through suppression of tumor cell proliferation and induction of tumor cell apoptosis [9]. However, the precise mechanism and the molecular involved in berberineinduced apoptosis remain to be elucidated. In the present study, we focused on the effects of berberine on human breast cancer. Apoptosis is a process that plays an important role in the control of the growth and development of organisms, and apoptosis induction is considered to be a crucial strategy for cancer prevention and treatment [10, 11]. The successful eradication of cancer cells through apoptosis is the ultimate aim of chemotherapy. Reactive oxygen species (ROS) are produced by all aerobic cells to regulate cell development, growth, survival, and death. ROS normally exist in balance with biochemical antioxidants in all aerobic cells [12, 13]. When this critical balance is disrupted by excess ROS production and/or antioxidant depletion, oxidative stress may come to exist [14]. ROS exert their damaging effects by modification of intracellular or extracellular macromolecules, hyper- or hypo-functionality of the signaling pathways, and finally leading to exert pathological effects or alter their physiological action [14]. It was reported that many chemotherapeutic drugs may be selectively toxic to cancer cells by induced oxidant stress [15]. Therefore, ROS has been an important target for developing antitumor drugs. In the present study, we first detected that berberine inhibited the cell viability and induced cell apoptosis in two cell lines, estrogen-dependent ER (+) MCF-7 human cancer cell line and the estrogen-independent ER (−) MDA-MB231cells. To further establish the anticancer mechanism of berberine, we assayed the levels of intracellular ROS and mitochondrial membrane potential, which are strongly associated with the apoptosis signal transduction pathway and affect the chemosensitivity of tumor cells to anticancer agents. Our findings suggest that berberine is a promising candidate for clinical use in human breast cancer chemotherapy.
Chemical reagents and antibodies Berberine was purchased from Wuhan Fortuna Chemical CO. Ltd. (China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RIPA lysis buffer, Hoechst33342, and 2′,7′dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was purchased from Life Technologies Inc. (Carlsbad, CA, USA). Caspase-3 Colorimetric Assay Kit was purchased from Nanjing Keygen Biotech CO. Ltd. (China). High Pure Mitochondria Isolation Kit was purchased from Shanghai Genmed Biotech CO. Ltd. (China). Antibodies against Bax, Bcl-2, phosphorylated JNK, JNK, cytochrome c, apoptosis-inducing factor (AIF), CoxIV, and β-actin were from Cell Signaling Technology, Inc. (Danvers, MA, USA). The secondary antibodies, namely, horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody and HRP-labeled anti-rabbit IgG antibody were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Cell viability assay Cell viability was measured with MTT (5 mg/ml). The cells were cultured in 96-well plates at a density of 4×104 cells per well. After 24 h incubation, cells were treated with various concentrations of berberine (10, 25, 50, 75, 100 μM) and cultured for 48 or 72 h. After incubation, 20 μl of MTT reagent was added to each well and incubated for 2.5 h at 37 °C in the dark. After the culture medium containing MTT aspirated, DMSO was then added into each well. The absorbance of each well was recorded with a Varioskan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at a test wavelength of 490 nm. The cell viability was calculated by dividing the absorbance of treated cells in each well to the mean absorbance of control cells. Hoechst staining
Materials and methods Cell culture and reagent treatment The MCF-7 and MDA-MB-231 breast cancer cell lines were kindly provided by Pro. Sun Y (Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing, People’s Republic of China). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (Life Technologies Inc., Carlsbad, CA, USA) containing 10 % heatinactivated fetal bovine serum (Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin, and maintained as monolayer in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C, and culture medium was replaced every 2 days.
After treated with berberine 50 μM for 24, 48, or 72 h, the changes in nuclear morphology of Hoechst33342-stained cells were examined using fluorescence microscopy Olympus BX60 (Olympus Optical Co Ltd, Tokyo, Japan). The morphological features of apoptosis (cell shrinkage, chromatin condensation, and fragmentation) were monitored. At least 400 cells from 12 randomly selected fields per well were counted, and each treatment was performed in duplicate. Measurement of intracellular ROS Formation of ROS was monitored using DCFH-DA, a membrane-permeable probe. The non-fluorescent dye freely penetrates cells and is then hydrolyzed by intracellular esterases to DCFH and trapped inside the cells. Intracellular H2O2
Tumor Biol.
or low-molecular-weight peroxides oxidize DCFH to the highly fluorescent compound DCF. Following treatment with 50-μM berberine, cells were incubated with DCFH-DA dye (50 μM, final concentration) in medium for 30 min in the dark. After rinsed twice with PBS solution, lysis buffer was added into dishes to break membrane of cells. Cell suspensions were transferred to 96-well plate, and fluorescence was read at the Ex of 490 nm and the Em of 520 nm.
nitrocellulose membrane. After blocking at room temperature in 10 % BSA with TBST buffer (10 mM Tris-HCl, 120 mM NaCl, 0.1 % Tween-20, pH 7.4) for 1 h, the membrane was incubated with various primary antibodies at 4 °C overnight. Membranes were then washed three times in TBST buffer, followed by incubation with different second antibodies for 1 h followed by four washings. Signal detection was performed with an enhanced chemiluminescence kit. Statistical analysis
Measurement of mitochondrial membrane potential The loss of mitochondrial membrane potential (ΔΨm) was monitored with the fluorescent probe JC-1. The cells was treated with berberine (50 μM) for different time and then incubated with JC-1 (10 μM, final concentration) in dark for 15 min at 37 °C. After washing with PBS, the cells were observed with Nikon Optical TE2000-S inverted fluorescence microscope at the Ex of 490 nm the Em of 520 nm. Cells with polarized mitochondria were seen with distinct mitochondria fluorescing red-orange, and in cells with depolarized mitochondria, the cytoplasm and mitochondria turned green. Caspase-3 activity assay After treated with berberine for 24 or 48 h, the enzymatic activity of caspase-3 in the cell lysate were detected by Caspase-3 Colorimetric Assay Kit as described in the manufacturer’s protocol. Briefly, 50 μl of cell lysate were incubated with 50 μl of 2× reaction buffer and 5 μl caspase-3 substrate in 37 °C for 4 h. A reading was then taken from Varioskan flash at a test wavelength of 405 nm. Isolation of mitochondria After treated with berberine for 24 or 48 h, the mitochondria of cells were isolated by the High Pure Mitochondria Isolation Kit following the manufacture’s protocol. Briefly, 107 cells were washed with ice-cold reagent A, and then the cells were lysed in mixed reagent (contains reagent B, C, and D) on ice. Following a centrifugation step at 800g at 4 °C for 10 min, the supernatant was separated from the pellet consisting of cellular debris. The mitochondrial pellet was collected by centrifugation at 13,000g at 4 °C for 10 min. Western blot assaying Cells were washed twice with PBS after treatment and solubilized in RIPA lysis buffer. Protein concentrations were determined by the Lowry method. Protein samples (40 μg) were separated by 10 % SDS/PAGE and transferred on to a
All values were expressed as mean±SEM. The significance of the difference between control and samples treated with berberine was determined by one-way ANOVA followed by the post hoc least significant difference test. Differences were considered significant at p≤0.05.
Results Effects of berberine on cell viability The effects of berberine on cell viability of MCF-7 and MDAMB-231 cells were determined by MTT assay. MCF-7 and MDA-MB-231 breast cancer cells were incubated with berberine (ranging from 10 to 100 μM) for 48 and 72 h, respectively. As shown in Fig. 1, treatment of MCF-7 and MDAMB-231 cells with berberine resulted in a significant inhibition of cell viability in concentration- and time-dependent manner. Concentration treatment of berberine induced reduction of MCF-7 cell viability ranged from 88 to 29 % after 48 h, and from 75 to 20 % after 72 h, while induced reduction of MDA-MB-231 cell viability ranged from 91 to 32 % after 48 h and from 79 to 14 % after 72 h. However, we observed no difference in growth inhibition between two cell lines. Berberine induced MCF-7 and MDA-MB-231 cell apoptosis To explore the underling mechanisms about the berberine on MCF-7 and MDA-MB-231 cells, we chose berberine at 50 μM in our further study. As illustrated in Fig. 2a, both MCF-7 and MDA-MB-231 cells showed a change in the morphology after treatment with berberine at 50 μM. As observed under a microscope, both MCF-7 and MDA-MB231 cells in control groups showed regular and round nuclei, while after cells being exposed to berberine 50 μM for 24, 48, or 72 h, condensation and fragmentation of nuclei were evident, which was the characteristic of apoptotic cells. The apoptotic rate was measured by counting at least 400 cells from 12 randomly selected fields per well. As shown in Fig. 2b, in MCF-7 cells, the apoptotic cells increased from
Tumor Biol.
Fig. 1 Berberine inhibits cell viability of MCF-7 and MDA-MB-231 cells. The effect of berberine on cell viability was measured by MTT assay. a MCF-7 and b MDA-MB-231 cells were treated with berberine for 48 and 72 h. Berberine significantly inhibited cell viability of both cell lines in a dose- and time-dependent manner. The experiments were
performed in triplicate and data presented as the mean±SEM of six separate experiments. *, #p≤0.05, berberine-treated group compared with control group. &p≤0.05, berberine-treated 48 h group compared with 72 h group
4.87 % in control cells to 22.56, 32.28, and 45.26 % after treated with berberine 50 μM for 24, 48, and 72 h, respectively, while in MDA-MB-231 cells, the apoptotic cells increased from 7.2 % in control cells to 19.74, 30.08, and 52.98 % after
treated with berberine 50 μM for 24, 48, and 72 h, respectively, suggesting the berberine induced MCF-7 and MDA-231 cell apoptosis in time-dependent manner. While after treated with different time of berberine, the apoptotic rate was similar
Fig. 2 Berberine induced MCF-7 and MDA-MB-231 cell apoptosis. a Fluorescence microscopy images of Hochest33342-stained cells showing the appearance of apoptotic morphology in berberine-treated breast cancer cells. In contrast to the control group, the group treated with berberine showed characteristic apoptotic phenotypes including cell shrinkage,
chromatin condensation, and fragmentation. Scale bar 100 μm. b The apoptotic rate was measured by counting at least 400 cells from 12 randomly selected fields per well. The data presented as mean±SEM of four individual experiments. *p≤0.05, berberine-treated group compared with control group
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in MCF-7 and MDA-MB-231 cells as compared to untreated control cells. Berberine affected the levels of ROS and mitochondrial membrane potential (ΔΨm) in cells To determine whether treatment of cells with berberine is associated with the generation of reactive oxygen species (ROS), MCF-7 and MDA-MB-231 cells were incubated with DCFH-DA. As shown in Fig. 3, compared with control cells, both MCF-7 and MDA-MB-231 cells treated with berberine 50 μM for 6 h markedly increased ROS generation to 221.0 and 227.9 %. The vital mitochondrial dye JC-1 is a useful tool for investigating mitochondrial function. The dye undergoes a reversible change in fluorescence emission from red to green as ΔΨm decreases. Cells with high membrane potential promote the formation of dye aggregates (red fluorescence), and cells with low membrane potential contain monomeric JC-1 (green fluorescence) [16]. To investigate the loss of ΔΨm during the early phase of apoptosis induced by berberine, cells were stained with JC-1 and monitored through a fluorescent microscope. As shown in Fig. 4, JC-1 was accumulated in untreated control cells, indicating a high membrane potential, while JC-1 was poorly accumulated in berberine-treated cells, indicating the disruption of ΔΨm. Berberine induced activation of JNK in MCF-7 and MDA-MB-231 cells The activation of the redox sensitive JNK signaling pathway is followed by an increase in ROS production [17–19]. To investigate whether berberine-induced ROS leads to the activation of JNK in MCF-7 and MDA-MB-231 cells, we determined the phosphorylation state of JNK in MCF-7 and MDAMB-231 cells treated with berberine. The results were shown in Fig. 5; berberine 50-μM treatment for 3 h and 6 h significantly increased the phosphorylation of JNK both in MCF-7 and MDA-MB-231 cells. Fig. 3 Berberine induced generation of ROS in MCF-7 and MDA-MB-231 cells. Compared with control group, treatment with berberine 50 μM for 6 h markedly increased ROS generation. The data presented as mean±SEM of four individual experiments. *p≤ 0.05, berberine-treated group compared with control group
Berberine induced change of anti-apoptotic and pro-apoptotic protein expression level in MCF-7 and MDA-MB-231 cells To investigate the mitochondrial apoptotic events involved in apoptosis induced by berberine, the levels of the antiapoptotic protein Bcl-2 and the pro-apoptotic protein Bax were analyzed. Western blot analysis showed (Fig. 6) that treatment of MCF-7 and MDA-MB-231 cells with berberine resulted in a markedly reduction in the expression of Bcl-2 and an increase in the levels of Bax. These results suggest that berberine alters the levels of pro- and anti-apoptotic proteins of the Bcl-2 family in a manner that contributes to the susceptibility of MCF-7 and MDA-MB-231 cells to berberineinduced apoptosis. Berberine induced releasing of pro-apoptotic factors from mitochondria of MCF-7 and MDA-MB-231 cells ROS can initiate apoptosis via the mitochondrial pathway by inducing loss of the ΔΨm and release of mitochondrial proapoptotic proteins such as cytochrome c and apoptosisinducing factor (AIF) [20]. To determine whether the mitochondrial pathway was involved in induction of cell death by berberine, we examined the AIF and cytochrome c level in mitochondria of MCF-7 and MDA-MB-231 cells. As shown in Fig. 7, the mitochondrial levels of cytochrome c and AIF were decreased in the MCF-7 and MDA-MB-231 cells treated with berberine 50 μM for 24 and 48 h. These results indicated that berberine increased the release of cytochrome c and AIF from mitochondria and subsequently increased the cytosolic cytochrome c levels and AIF levels, and eventually triggered the caspase-dependent and caspase-independent apoptosis pathway, respectively. Berberine induced activation of caspase-3 in both MCF-7 and MDA-MB-231 cells Caspase-3 activation represents the irreversible or execution stage of apoptosis. To further examine the involvement of caspase-3 in apoptosis induction of berberine, we examined the activities of caspase-3 as shown in Fig. 8. It was found that
Tumor Biol.
Fig. 4 Berberine induced loss of mitochondrial membrane potential in MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were stained with a mitochondria-specific dye, JC-1, and its florescence was monitored using fluorescence microscopy. JC-1 was accumulated in
control cells, where it displayed a bright-red fluorescence indicating a high potential. In contrast, JC-1 was poorly accumulated in berberinetreated cells, which displayed green fluorescence, indicating the loss of the mitochondrial membrane potential. Scale bar 50 μm
caspase-3 was significantly activated after berberine treatment for 24 and 48 h in both MCF-7 and MDA-MB-231 cells. In MCF-7 cells, incubated with berberine 50 μM for 24 and 48 h, caspase-3 activity was markedly increased by 148.8 and 197.8 %, respectively, and in MDA-MB-231 cells, incubated with berberine 50 μM for 24 and 48 h, caspase-3 activity was further enhanced by 173.2 and 249.0 % above control cells, respectively.
property for candidate anticancer drug, the morphological observation was conducted to explore whether the cytotoxic effect was related with the apoptotic process. It was found that the cell death induced by berberine exhibited a clear morphological sign of apoptosis. After being treated with berberine, cell shrinkage, chromatin condensation, and fragmentation were observed. These observations suggest that berberine may be an effective chemotherapeutic agent against breast cancer. Further studies were performed to elucidate the mechanism underlying the inhibition in cell viability through induced of apoptosis. ROS are thought to participate in a wide variety of cellular functions, including cell proliferation, differentiation, and apoptosis [22, 23]. Elevation of ROS in excess of the buffering capacity designed to modulate ROS levels result in potentially cytotoxic “oxidative stress”. ROS and oxidative stress are known as apoptosis triggers and modulators [20, 24], and they have been shown to play an important role in the therapeutic principle of many chemotherapeutics such as cisplatin, bleomycin [25], and paclitaxel [26]. Mitochondrial electron transport chain is a major endogenous source of ROS, of which NADH is the main composition. Berberine contains both a quaternary nitrogen atom and
Discussion Berberine has a definite potential as drug in a wide spectrum of clinical applications, such as gastroenteritis, abdominal pain, and diarrhea. Recent years multiple reports have indicated that berberine exerts in vitro anti-proliferative effects on different cancer cell lines [21]; however little is known about the mechanisms. The results of this study showed that berberine significantly inhibited the proliferation in a time- and dosedependent manner in both MCF-7 and MDA-MB-231 cells. Because apoptosis induction appears to be the most important Fig. 5 Berberine induced activation of JNK in MCF-7 and MDA-MB-231 cells. Cells were incubated with berberine for 3 and 6 h, and then, cell proteins were obtained and analyzed with antiphospho-JNK anti-JNK antibodies by Western blot. Berberine significantly increased the phosphorylation of JNK. *p≤ 0.05, berberine-treated group compared with control group
Tumor Biol. Fig. 6 Berberine induced change of anti-apoptotic and proapoptotic protein expression level in MCF-7 and MDA-MB-231 cells. Cells were incubated with berberine for 24 and 48 h, and then, cell proteins were obtained and analyzed with anti-Bcl-2 and anti-Bax antibodies by Western blot. Treatment of MCF-7 and MDA-MB-231 cells with berberine resulted in a marked reduction in the expression of Bcl-2 and an increase in the levels of Bax. *p≤0.05, berberinetreated group compared with control group
Fig. 7 Berberine induced releasing of cytochrome c and AIF from mitochondria in MCF-7 and MDA-MB-231 cells. Cells were incubated with berberine for 24 and 48 h, then, mitochondrial extracts were obtained and analyzed with anti-cytochrome c (a) or anti-AIF (b) antibody by Western blot. Treatment of MCF7 and MDA-MB-231 cells with berberine resulted in a marked releasing of cytochrome c and AIF from mitochondria. *p≤0.05, berberine-treated group compared with control group
Tumor Biol. Fig. 8 Berberine induced activation of caspase-3 in both MCF-7 and MDA-MB-231 cells. Cells were incubated with berberine for 24 and 48 h, then, the cell lysate were detected by Caspase-3 Colorimetric Assay Kit. Treatment of MCF-7 and MDA-MB-231 cells with berberine resulted in activation of caspase-3. *p≤0.05, berberinetreated group compared with control group
methoxy group which causes its accumulation inside the organelle and has a marked inhibitory effect on NADH [27]. The dysfunction of electron transport chain caused excessive release of ROS from mitochondria. Figure 3 clearly shown that berberine induced apoptosis concomitant with increased ROS generation in both MCF-7 and MDA-MB-231 cells, indicating that berberine may exert anticancer activity by increase the production of ROS. Mitochondria are both a major endogenous source and target of ROS; they have become target for drug discovery in recent years [28], because they have been described to play a central role in the apoptotic process. Currently, many studies have suggested that mitochondria are the main site of action for JNK in apoptosis. Intracellular ROS accumulation inactivates MAPK phosphatases (MKPs) by oxidation of their catalytic cysteine, which leads to sustained activation of JNK [17]. JNK serves as an important pro-apoptotic mechanism in mitochondrial apoptosis in cells. First, phosphorylated JNK translocates from cytosol to mitochondria, where JNK triggers ΔΨm depolarization. Second, phosphorylate JNK Fig. 9 Schematic model for the mechanisms of berberine-exerted antitumor activity. Berberineinduced ROS generation activated the pro-apoptotic stress kinase JNK, inhibited the antiapoptotic Bcl-2 family protein, and resulted in loss of ΔΨm, which subsequently increased the release of cytochrome c and AIF from mitochondria, and eventually triggered the caspasedependent and caspaseindependent apoptosis pathway
executes its pro-apoptotic activity via regulation of Bcl-2 family members. We revealed that berberine treatment resulted in a significant activation of JNK both in MCF-7 and MDA-MB-231 cells, which cause loss of ΔΨm and regulation of Bcl-2 family members. After being treated with berberine, both in MCF-7 and MDA-MB-231 cells, downregulation of Bcl-2 expression was detected, and this downregulation was concomitant with the upregulation of Bax. This result indicated that berberine may induce MCF-7 and MDA-MB-231 cell apoptosis by ROS-mediated activation of JNK. Numerous studies have demonstrated that induction of downstream caspase activity was dependent on cytochrome c released from mitochondria, which was regulated by ΔΨm and Bcl-2 family members [29]. It is assumed that disruption of ΔΨm is the onset of mitochondrial membrane transition pores (MPTP) formation. Reduction of ΔΨm and generation of MPTP must facilitate the localization of cytochrome c from mitochondria to cytoplasm to gain access to their substrates or interacting partners [30]. Recently, Bcl-2 proteins were
Tumor Biol.
thought to regulate the mitochondrial-mediated apoptotic pathway exclusively [31]. Downregulation of Bcl-2 and upregulation of Bax also stimulates the release of cytochrome c from the mitochondria into the cytosol. Then, we detected the classical mitochondrial proteins cytochrome c released into cytosol. We found that the expression of cytochrome c in cytosol was significantly increased after berberine treatment. Mitochondrial release of cytochrome c into the cytoplasm induces the formation of an oligomeric complex containing cytochrome c and Apaf-1. This complex, called the apoptosome, finally activates the effector caspase-3 (as shown in Fig. 8) resulting in the subsequent degradation of cellular death substrates [32]. We unexpectedly found that in both MCF-7 and MDA-MB-231 cells, AIF was also upregulated in the cytoplasm after berberine treatment. AIF induces caspase-independent cell death primarily [33]. Following AIF translocation from the mitochondria to the nucleus, classic apoptotic features, such as phosphatidylserine exposure, partial chromatin condensation, and nuclear condensation, occur in the absence of caspase activation. Our results indicated that both caspase-dependent and caspase-independent mechanism participate in the berberine induced MCF-7 and MDA-MB-231 cell death. In conclusion, the results of the present study indicated that berberine inhibited proliferation and induced apoptosis in both of MCF-7 and MDA-MB-231 cells have nothing to do with estrogen receptor expression. Based on our study, we proposed a model by which berberine mediated generation of ROS leading to MCF-7 and MDA-MB-231 cell apoptosis. The constructed model of action (Fig. 9) illustrated that berberine acted on mitochondria, and induced ROS generation, which activated the pro-apoptotic stress kinase JNK, inhibited the anti-apoptotic Bcl-2 family protein, and subsequently resulted in loss of ΔΨm. The loss of ΔΨm increased the release of cytochrome c and AIF from mitochondria, and eventually triggered the caspase-dependent and caspaseindependent apoptosis pathway. This study may provide an insight into the potential of berberine for breast cancer therapy. Acknowledgments This work was supported by grants from the Nanjing Medical Science and Technique Development Foundation, Nanjing, Jiangsu, China (Grant No.QYK10156), and the natural science foundation of Jiangsu Province (Grant No.BK20130073). We thank Pro. Sun Y (Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing, People’s Republic of China) for providing MCF-7 and MDA-MB-231 breast cancer cell lines.
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