CLL samples were obtained from the Affiliated Hospital of Xuzhou Medical College according to the diagnostic criteria for CLL between September 2013 and October 2015, while 6 healthy volunteers served as a normal control group. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood obtained from 35 CLL patients.

All procedures performed in the study involving human participants were in accordance with the ethical standards of the Institutional and/or National Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

CLL cell lines, HG3 and MEC-1, a kind gift from Anders Rosén at Linköping University, were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA). The Bcl-2 and Bax antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and anti-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit IgG and rabbit anti-mouse IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Xiongben, Japan). The Cell-Light EdU Apollo^® 567 In Vitro Imaging kit was from Ruibo Biotechnology (Guangzhou, China). Annexin V and 7-AAD double-labeled apoptosis detection kit and propidium iodide (PI) were obtained from eBiosience (San Diego, CA, USA). Plumbagin was purchased from Sigma-Aldrich.

Extracted RNA from the CLL samples was reverse-transcripted to cDNA which was used for quantification of mRNA expression of Bcl-2 and Bax by real-time PCR with GADPH as internal control. Amplification was performed in triplicate on LightCycler^® R480 II (Roche Life Science) in a total volume of 20 μl. The primers for Bcl-2, Bax and GADPH were as follows: Bcl-2 (F), 5′-ACGACTTCTCCCGCCGCTA-3′ and (R), 5′-ACCCCACCGAACTCAAAGAAG-3′; Bax (F), 5′-AGAGGATGATTGCCGCCGT-3′ and (R), 5′-CAACCACCCTGGTCTTGGATC-3′; GAPDH (F), 5′-TGAAGGTCGGAGTCAACGGATT-3 and (R), 5′-CCTGGAAGATGGTGATGGGATT-3′. qPCR reaction was performed according to the SYBR-Green qPCR SuperMix manuals. The relative mRNA expression of target genes was calculated by the comparative Ct method which was performed using the following formula: Relative expression = 2^−ΔΔCt.

Three thousand cells in 100 μl of medium were seeded into 96-well plates with three replicates. The cells were incubutated with 0.625, 1.25, 2.5, 5, 10 and 20 μM of plumbagin or fludarabine for 48 h, or incubated with 10 μM of plumbagin or fludarabine for 0, 20, 40, 60 and 80 h, and then CCK-8 reagent (5 μl) was added into each well to incubate for an additional 4 h. The cells were exposed to measure the absorbance at 450 nm by a microplate reader (Wellscan MK-3; Labsystems, Dargon, Finland).

EdU-incorporation is a method for labeling DNA in vivo during DNA replication. The HG3 and MEC-1 cells were pretreated with 0, 2.5, 5 and 10 μM of plumbagin for 48 h, and then incubated with EdU for an additional 3 h. After fixation, permeabilization and staining according to the kit manual, the cells were observed and the number of EdU-positive cells was calculated under a microscope.

The HG3 and MEC-1 cells following the same treatments with plumbagin were collected and fixed in 70% ethanol on ice for 10 min, rinsed with phosphate-buffered saline (PBS) and incubated with 100 mg/ml RNase A (0.25 mg/ml) for 15 min. After washing with PBS for two times, the cells were further incubated with 50 μg/ml PI at room temperature for 10 min, and then the cells were subjected to cell cycle analysis.

The HG3 and MEC-1 cells following the same treatments with plumbagin were collected and then resuspended in 1× binding buffer at 1–5×10⁶/ml, and incubated with Annexin V-APC and 7-AAD for 10 min at room temperature for analysis of apoptosis by flow cytometry. Early apoptotic cells were labeled with Annexin V and late apoptotic cells were double labeled with Annexin V and 7-AAD.

The HG3 and MEC-1 cells following the same treatments with plumbagin were collected and proteins were extracted for western blotting. Equal amount of protein lysates were subjected to 10–12% SDS-PAGE, and then transferred to a 0.45-μm pore size polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membrane was probed with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 h. Bound antibodies were detected using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) and visualized by ImageQuant LAS 4000 (GE Healthcare, Fairfield, CT, USA).

The results are representative of experiments repeated at least three times and quantitative data are expressed as means ± SEM. Student's t-test and ANOVA test were used to analyze the difference between groups. P<0.05 was considered statistically significant, and P<0.01 as very significant. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).

The mRNA expression of Bcl-2 and Bax was determined in 35 CLL patients and 6 healthy donors. Bcl-2 exhibited an increasing trend in Rai II–IV patients compared with the control group, particularly in Rai III/IV patients (P<0.05; Fig. 1A). By contrast, Bax showed a decreasing trend in the CLL patients, but without statistical significance (Fig. 1B). However, the expression ratio of Bcl-2/Bax was found to be significantly increased (3.78-fold) in the Rai III/IV patients compared with this ratio in the control group (P<0.05; Fig. 1C).

To investigate the potential growth inhibition of plumbagin in CLL cells, the HG3 and MEC-1 cell lines were cultured with 0.625, 1.25, 2.5, 5, 10 and 20 μM of plumbagin for 48 h, and the cell viability was determined by CCK-8 assay. As shown in Fig. 2A and E, HG3 cells treated with plumbagin and fludarabine presented decreased cell viability in a dose-dependent manner, while the cells treated with plumbagin showed a lower IC₅₀ value (8.5±0.5 μM) compared with fludarabine (11.5±0.7 μM) (P<0.05). Similarly, under the same conditions, MEC-1 cells were more susceptible to plumbagin (IC₅₀=7.5±0.8 μM) compared to fludarabine (IC₅₀=10.2±0.5 μM) (Fig. 2B and E). Next, HG3 and MEC-1 cells were incubated with 10 μM of plumbagin or fludarabine for 0, 20, 40, 60 and 80 h. Almost all CLL cells showed growth retardation at 72 h when treated with plumbagin, while 34.5±0.7% (P<0.05) and 28±0.3% (P<0.05) of HG3 and MEC-1 cells presented strong cell viability after the same treatment with fludarabine (Fig. 2C and D).

To assess the effect of plumbagin on cell proliferation, we performed EdU incorporation assay with HG3 and MEC-1 cells in the presence or absence of plumbagin. As expected, the numbers of EdU-positive cells distributed as 68, 62 and 30% in HG3 cells vs. 58, 54 and 28% in MEC-1 cells at 2.5, 5 and 10 μM of plumbagin, were significantly decreased in a dose-dependent manner (P<0.05, P<0.05, P<0.01; Fig. 3).

To investigate the role of plumbagin in cell cycle progression, HG3 and MEC-1 cells treated with plumbagin were collected and stained with PI for cell cycle analysis via flow cytometry. The distribution of the cell cycle in the HG3 cells exhibited no obverse changes (38–41% in G1, 19–27% in S and 35–40% in G2/M phases) when treated with 0, 2.5 and 5 μM of plumbagin. However, ~60% of the HG3 cells were arrested in G0/G1 phase following treatment with 10 μM of plumbagin (P<0.01; Fig. 4A and B). Notably, MEC-1 cells were blocked at the S phase following the same treatment, and showed an increase from 33 to 53% compared with the control when treated with 10 μM of plumbagin (P<0.01; Fig. 4A and C).

To assess whether plumbagin induced these growth-suppressive effects on CLL cells by apoptosis, we collected HG3 and MEC-1 cells treated with plumbagin for double-staining using Annexin V-APC and 7-AAD. As expected, plumbagin significantly induced cell apoptosis in a dose-dependent manner, both in the HG3 and MEC-1 cell lines. In addition, statistical analysis showed that the number of apoptotic cells treated with 5 or 10 μM of plumbagin was 18±0.7 or 50±0.5% in the HG3 cells, and 19±0.5 or 58±0.2% in the MEC-1 cells (P<0.05; P<0.001; Fig. 5A and B). Furthermore, western blotting showed that plumbagin treatment notably decreased the expression of Bcl-2 and increased Bax in the HG3 cells. Similarly, the expression of Bcl-2 was slightly decreased, while the Bax level was strongly elevated even following treatment with a dose of 2.5 μM of plumbagin in the MEC-1 cells (Fig. 5C).