Preparation of cell-free EBV virions and generation of EBV-transformed B cells were carried out as described previously (19). Human Burkitt's lymphoma Raji and Daudi cells and multiple myeloma IM-9 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone) and antibiotics under a humidified atmosphere with 5% CO₂. This study was approved by the Institutional Bioethics Review Board at the Medical College of Inje University, and all donors gave informed consent for the study.

The percentage of cells undergoing apoptosis in human EBV-transformed B cells (4 weeks, 2.5×10⁵ cells/ml) and normal PBMCs was determined via flow cytometry using FITC-conjugated Annexin V (BD Biosciences, San Diego, CA, USA) and 7-AAD (BD Biosciences). To determine optimal conditions, experiments were performed using different concentrations (0, 10, 25, 50, 75 and 100 μM) of berberine (Sigma-Aldrich, St. Louis, MO, USA) and different periods of incubation (2, 4, 8, 16 and 24 h). DMSO was used as a vehicle control. To examine the role of caspases, cells were pretreated with the caspase-3 inhibitor z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, 20 μM in DMSO; Calbiochem, San Diego, CA, USA), the caspase-9 inhibitor z-LEHD-fmk (z-Leu-Glu(OMe)-His-Asp-(OMe)-fluoremethylketone, 20 μM; Calbiochem), or the broad spectrum caspase inhibitor z-VAD (N-benzyloxycarbonyl-Val-Ala-Aspfluoromethylketone, 20 μM in DMSO; Calbiochem) for 2 h before treatment with berberine. To inhibit the production of ROS, cells were pretreated with the antioxidant NAC (10 mM; Sigma-Aldrich) for 1 h. To block activation of p38-MAPK or JNK, cells were pretreated with SB203580 (10 μM, Calbiochem) or SP600125 (25 μM, Calbiochem) respectively, for 2 h. To block activation of p53, cells were pretreated with PFTα (50 μM, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. Cells were harvested, washed in PBS, and incubated with Annexin V and 7-AAD in Annexin V binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at room temperature for 15 min in the dark. The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest Pro software (BD Biosciences).

Changes in mitochondrial membrane potential were measured using DiOC₆ (3, 3′-dihexyloxacarboxyanine iodide; Molecular Probes, Eugene, OR, USA). Cells were treated with berberine or DMSO for 24 h, harvested, washed twice in PBS, resuspended in PBS supplemented with DiOC₆ (20 nM), incubated at 37°C for 15 min in the dark, and immediately analyzed with flow cytometry. Intracellular accumulation of ROS was monitored via flow cytometry after staining with the fluorescent probe, DCFH-DA (10 μM, 2′, 7′-dichlorodihydro-fluorescein diacetate; Molecular Probes). DCFH-DA is deacetylated in cells by esterase to a non-fluorescent compound, DCFH, which remains trapped within the cell and is cleaved and oxidized by ROS in the presence of endogenous peroxidase to a highly fluorescent compound, DCF (2′, 7′-dichlorofluorescein). Briefly, cells were preincubated with 10 μM DCFH-DA for 30 min at 37°C. The cells were seeded in 6-well plates (5×10⁵ cells/ml) and treated with or without berberine. Cells were washed, resuspended in PBS, and ROS levels were determined using a FACSCalibur flow cytometer.

After drug treatment, cells were harvested and lysed in NP-40 buffer (Elpis Biotech, Daejeon, Korea) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). To address phosphorylation events, a set of phosphatase inhibitors (Cocktail II, Sigma-Aldrich) was added to the NP-40 buffer. Protein concentration was determined using a Pierce BCA assay kit (Thermo Scientific, Rockford, IL, USA). An equal volume of 2× Laemmli sample buffer (Elpis Biotech) was added to each sample of lysate containing 10 μg protein and immediately boiled for 5 min at 100°C. The insoluble material was spun down at 13,000 rpm. Total cell lysates (~5×10⁶ cells/sample) were subjected to SDS-PAGE on 10–15% (w/v) acrylamide gels under reducing conditions. Separated proteins were transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA, USA), the membranes were blocked with 5% skim milk and western blot analysis was performed using a commercial kit. Chemiluminescence was detected using an ECL kit (Advansta Corp., Menlo Park, CA, USA) and the multiple Gel DOC system (Fujifilm, Tokyo, Japan). Primary antibodies against the following proteins were used: caspase-3, caspase-9, PARP, β-actin, Bcl-2, Bax, phospho-Bim (Ser⁶⁹), PUMA, XIAP, phospho-p53 (Ser¹⁵), p53, phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵), JNK, phospho-p38-MAPK (Thr¹⁸⁰/Tyr¹⁸²), p38-MAPK, phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), ERK1/2, phospho-Akt (Ser⁴⁷³), and Akt (Cell Signaling Technology, Beverly, MA, USA); XAF1 (Abcam, Cambridge, UK); GADD45α, Ref-1, and COX-IV (Santa Cruz Biotechnology); phospho-Bax (Ser¹⁸⁴) (Bioss Inc., Woburn, MA, USA); Bim and β-tubulin (BD Biosciences).

Mitochondrial and cytosol cellular fractions were prepared using a Cytosol/Mitochondria Fractionation kit (Calbiochem). Untreated or berberine-treated cells (1×10⁷ cells) were harvested by centrifugation at 600 × g for 5 min at 4°C, washed twice with cold PBS, and resuspended in 250 μl Cytosol Extraction buffer containing a protease inhibitor cocktail and 1 mM dithiothreitol (DTT). After incubation on ice for 10 min, the cells were homogenized on ice using a Dounce tissue homogenizer and centrifuged at 700 × g for 10 min at 4°C. The supernatants were collected and centrifuged again at 10,000 × g for 30 min at 4°C. The resulting supernatants were harvested as cytosolic fractions and the pellets were resuspended in 50 μl Mitochondria Extraction buffer containing a protease inhibitor cocktail and 1 mM DTT and designated the mitochondrial fractions. The nuclear fraction was prepared using a Nuclear/Cytosol Fractionation kit (Biovision, Mountain View, CA, USA). Briefly, 2×10⁶ cells were harvested and resuspended in 200 μl cytosol extraction buffer A. After incubation on ice for 10 min, the cell suspension was added to cytosol extraction buffer B and incubated on ice for 1 min. After centrifugation the pellets were resuspended in 100 μl nuclear extraction buffer mix and designated the nuclear fraction.

For the protein binding assay, cells were treated with berberine for 24 h. Cells were harvested (5×10⁶ cells/sample) and lysed in NP-40 buffer containing a protease inhibitor cocktail. To reduce non-specific binding of protein, we performed pre-clearing on equal amounts of cell lysates by incubating the samples with washed protein G PLUS-agarose beads (Santa Cruz Biotechnology). For IP, pre-cleared lysate was incubated with the optimal amount of antibody against XAF1, Puma, GADD45α, or p53 antibody at 4°C for 2 h on a rotator. The immunoprecipitates were harvested using protein G PLUS-agarose beads and incubated at 4°C for 2 h under rotary agitation. The supernatant was removed and the beads were washed four times in lysis buffer. Finally, the immunoprecipitates were eluted by boiling the beads in Laemmli sample buffer for 10 min and characterized by western blotting with the appropriate antibodies.

The viability of EBV-transformed B cells was significantly decreased in a dose-dependent manner after treatment with different concentrations of berberine. However, inhibition of proliferation and induction of apoptosis was not observed at the same doses in normal PBMCs (data not shown). To confirm that berberine induced apoptosis in EBV-transformed B cells we used a 7-AAD and Annexin V staining method. Treatment with different concentrations of berberine resulted in time- and dose-dependent induction of apoptosis and altered mitochondria membrane potential in EBV-transformed B cells (Fig. 1A and B). Treatment with berberine induced activation of capase-3 and -9, which was detected by generation of the cleaved products. Appearance of cleaved PARP, a target of caspase-3, was also observed 12 h after treatment of EBV-transformed B cells or cancerous B cells with berberine (Fig. 1C and E). EBV-transformed B cells were pretreated with several caspase inhibitors including z-VAD (pan-caspase inhibitor), z-DEVD (caspase-3 inhibitor), and z-LEHD (caspase-9 inhibitor) 2 h before treatment with berberine. These inhibitors reduced caspase activity and effectively blocked berberine-induced apoptosis in EBV-transformed B cells (Fig. 1D). These data suggest that berberine induces apoptosis in EBV-transformed B cells or cancerous B cells, but in not normal PBMCs, through a mitochondria-dependent pathway.

As berberine-induced apoptosis is associated with activation of p53 in breast cancer (MCF-7) cells (4,20), we next examined whether XAF1 (an antagonist of XIAP) and GADD45α, both downstream targets of p53, and the Bcl-2 protein family are associated with mitochondrial apoptosis signaling after treatment with berberine in EBV-transformed B cells or cancerous B cells. Berberine increased the level of phosphorylated p53 in a time-dependent manner (Fig. 2A). Expression of XAF1 and GADD45α were induced in EBV-transformed B cells or cancerous B cells. However, expression of XIAP and the antiapoptotic protein Bcl-2 significantly decreased to almost undetectable levels 24 h post-treatment in EBV-transformed B cells. Similar to GADD45α and XAF1, we observed induction of PUMA, phosphorylated Bax, and phosphorylated Bim after berberine treatment in EBV-transformed B cells and cancerous B cells (Fig. 2B and C). These data suggest that berberine-induced apoptosis in EBV-transformed B cells or cancerous B cells is not only related to phosphorylated p53 and its downstream targets, XAF1 and GADD45, but also involves apoptotic members of the Bcl-2 protein family disrupting the mitochondria membrane.

Next, we further examined the interaction between XAF1 and apoptosis-related proteins to elucidate the role of XAF1 in regulating the apoptotic signaling induced by berberine. XAF1 was not detected in cytosol before treatment with berberine, whereas, XAF1 in the nuclear fraction disappeared after treatment with berberine. However, GADD45α was observed in both nuclear and cytosolic fractions of EBV-transformed B cells treated with berberine (Fig. 3A). Interestingly, XAF1 in the mitochondrial fraction were increased in berberine-treated EBV-transformed B cells. Bax and Bim disappeared from the cytosol and were accumulated in mitochondria after treatment with berberine; furthermore pro-apoptotic protein, PUMA was detected in the mitochondria (Fig. 3B). To further characterize the relationship between p53 and XAF1 and/or GADD45α, we investigated the potential interactions of these proteins using immunoprecipitation and immunoblotting. After treatment of EBV-transformed B cells with berberine, XAF1 translocated from the nucleus and interacted with p53 and GADD45α in the cytosol in addition relocated XAF bound to PUMA, Bax, and Bim in mitochondria (Fig. 3C–F). These data suggest that re-localization of XAF1 from the nucleus to the cytoplasm might be one of the key processes in the induction of apoptosis through mitochondrial signaling in cancerous B cells after treatment with berberine.

Death receptors and the mitogen-activated protein kinase (MAPK) pathway promote apoptosis in HeLa cells after treatment with 300 μM berberine (20,21). GADD family proteins are known to be upstream activators of p38/JNK MAPK (15,22). Next, we examined whether berberine-induced apoptosis involves activation of these signaling pathways. Levels of the phosphorylated forms of JNK and p38 increased in a time-dependent manner after treatment of EBV-transformed B cells and cancerous B cells with berberine, whereas expression of the phosphorylated forms of ERK1/2 and Akt decreased (Figs. 4A and 6A). PFT-α inhibits p53 apoptotic signaling by interfering with its nuclear translocation and the transcriptional activation of p53 inducible genes (23,24). PFT-α blocked the berberine-induced upregulation of phosphorylated p53, PUMA, GADD45α, and XAF1 in EBV-transformed B cells (Fig. 4B). PFT-α also abrogated the cleavage and activation of caspase-3 and -9, but did not prevent the phosphorylation of JNK and p38-MAPK (Fig. 4C). To investigate the role of p38/JNK MAPK in p53-mediated apoptosis after treatment with berberine, EBV-transformed B cells were pretreated with SB203580 (p38-MAPK inhibitor) or SP600125 (JNK MAPK inhibitor) 2 h before treatment with berberine. These inhibitors effectively prevented the upregulation of phosphorylated p53, XAF1, GADD45α, and proapoptotic Bcl-1 family proteins including PUMA, phosphorylated Bim, and phosphorylated Bax (Fig. 4D). These data suggest that functional p53 is not only required for berberine-induced apoptosis through regulation of XAF1-GADD45α, but is also regulated by the p38/JNK MAPK signaling pathway.

ROS are associated with the activation of MAPKs and caspases in apoptosis (25). To investigate whether ROS were involved in the MAPK/p53/XAF1-mediated apoptosis induced by treatment with berberine, EBV-transformed B cells were treated with berberine for the indicated times and the intracellular ROS level was measured by addition of DCFH-DA. The ROS level was significantly higher in the berberine-treated EBV-transformed B cells than in the control group at 2 h after treatment and peaked at 8 h after treatment, after which ROS levels were downregulated. N-acetylcysteine (NAC), a ROS quencher, effectively blocked berberine-induced apoptosis (Fig. 5A). NAC also suppressed ROS production by berberine and the berberine-induced mitochondria disruption (Fig. 5B). Pre-incubation with NAC almost completely blocked the activation of p38/JNK MAPK and the change in mitochondria-related apoptotic proteins (Bax, Bim, and PUMA). The phosphorylation of p53 and expression levels of its downstream targets (XAF1, GADD45α) were restored to control levels after preincubation of EBV-transformed B cells with NAC (Fig. 5C). ROS level was also significantly higher in the berberine-treated EBV-positive cancerous B cells (Raji, IM-9, Daudi cells) than in the DMSO-treated control cells at even 1 h after treatment and was maintained up at 24 h after treatment (Fig. 6B). These data suggest that ROS generation might be one of the predisposing events promoting mitochondria-mediated apoptosis through MAPK/p53 signaling after treatment of EBV-transformed B cells with berberine.