Berberine was obtained commercially from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). It was dissolved in dimethyl sulfoxide (DMSO; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and then diluted in HyClone™ RPMI-1640 medium (GE Healthcare Life Sciences, Logan, UT, USA) to the desired concentrations. All concentrations contained a final DMSO concentration of <0.1%, not enough to affect cell growth. MTT was also purchased from Sigma-Aldrich (Merck KGaA). The PE Annexin V Apoptosis Detection kit was purchased from BD Biosciences (San Diego, CA, USA). Rapamycin, z-VAD-fmk and 3-methyladenine (3-MA) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA).

The human MPM NCI-H2452 cell line was purchased from the Shanghai Institute of Cell Biology, the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (HyClone Laboratories; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (FBS) (Shanghai ExCell Biology, Inc., Shanghai, China), 100 mg/ml streptomycin and 100 U/ml penicillin at 37°C in a humidified atmosphere containing 5% CO₂.

The effect of berberine on NCI-H2452 cells was assessed by an MTT assay, as previously described (15). Briefly, cells were seeded onto 96-well culture plates at a density of 5,000 cells/well. After 24 h, cells were treated with berberine at a concentration of 25, 50, 100 or 200 µM, or with DMSO alone (control group) for 24, 48 and 72 h. After the designated period, 20 µl MTT (5 mg/ml) was added to each well and the plates were cultured at 37°C for additional 4 h. Formazan crystals that formed in the wells were dissolved in 150 µl DMSO, and the absorbance value of each well was measured at 490 nm using a Spectra Max M2 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). All experiments were performed in triplicate.

To investigate whether the inhibition of cell proliferation by berberine was mediated by apoptosis, we used Hoechst 33258 staining to detect apoptosis. After treatment with berberine at a concentration of 0 (DAMO alone; control group) or 100 µM, chromatin morphologic changes were observed by fluorescence microscopy after DNA staining with 10 µg/ml Hoechst 33258 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Reduced nuclear size, chromatin condensation, intense fluorescence, and nuclear fragmentation were considered to indicate apoptotic cells. Then, we further detected the apoptosis rate using Annexin V-PE/7-AAD double staining assay by flow cytometry. The cells were treated with berberine at the concentration of 0 (DMSO alone; control group), or 100 µM; or the cells were treated with 100 µM berberine in the absence or presence of 3-MA, respectively, for 48 h and then harvested and resuspended in Annexin V binding buffer. The suspension was incubated with 5 µl of Annexin V-PE and 5 µl of 7-AAD for 15 min at room temperature in the dark followed by addition of 400 µl of binding buffer. Then the samples were detected immediately by flow cytometry (FACSAria III; BD Biosciences, San Diego, CA, USA) analysis. All experiments were carried out in triplicate.

NCI-H2452 cells were treated with berberine at a concentration of 0 (DMSO alone; control group) or 100 µM for 48 h. Then, the treated cells were fixed in 4% paraformaldehyde for 15 min. After blocking with 5% normal goat serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS), the cells were incubated with an anti-LC3B antibody (1:50; cat. no. CST-2775s; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight, followed by incubation with a TRITC-conjugated goat anti-rabbit IgG secondary antibody (1:200; cat. no. BA1090; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 1 h at room temperature. Nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Shanghai, China) for 10 min. Coverslips were mounted in antifade mounting medium and images were obtained using a Leica™ fluorescence microscope equipped with a Leica™ camera (Leica Camera AG, Wetzlar, Germany).

Expression levels of LC3, p62 and proteins associated with the mitochondrial pathway were measured in NCI-H2452 cells via immunoblot analysis. The cells were treated as described in the Figure legends. Whole cell extracts were prepared in ice-cold lysis buffer. To detect the expression of cytochrome c, cytosolic and mitochondrial fractions were prepared using a Mitochondria Extraction kit (Nanjing KeyGen Biotech., Co., Ltd., Nanjing, China). After removal of the insoluble fraction by centrifuging at 20,000 × g for 30 min at 4°C, the protein content of the supernatant was determined via the bicinchoninic acid (BCA) method with a commercial protein assay reagent (CoWin Bioscience Co., Ltd., Beijing, China). Protein samples (30 µg) were subjected to precast 8–12% SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were then incubated with primary antibodies against LC3B (1:750), p62 (1:1,000), cytochrome c (1:1,000), caspase-9 (1:1,000), caspase-3 (1:1,000), PARP [poly(ADP-ribose) polymerase] (1:1,000), and Cox 4 (1:5,000) in 5% BSA in TBS-T (Cell Signaling Technology, Inc.) and β-actin (1:1,000; Zhongshan Bio., Beijing, China) overnight at 4°C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshan Bio.) at a dilution of 1:5,000 in TBS-T for 1 h at room temperature, the immunoreactive bands were detected using electrochemiluminescence (ECL; EMD Millipore) and quantified using Multi Gauge V3.2 analysis software (Fuji Film, Tokyo Japan).

Cell death was evaluated using a trypan blue exclusion assay as described previously (13). Briefly, NCI-H2452 cells were cultured for 48 h with various concentrations of berberine, or with 200 µM berberine in the absence or presence of pan-caspase inhibitor z-VAD-fmk (20 µM), autophagy inhibitor 3-MA (5 mM), or autophagy inducer rapamycin (100 nM). Then, both adherent and non-adherent cells were collected, washed three times with PBS, and resuspended in PBS at a concentration of 1×10⁶ cells/ml. After mixing with 0.4% trypan blue (ratio of cells:trypan blue, 9:1), the cells were counted using a hemocytometer. The number of dead cells with disrupted membranes (blue cells) was counted. The cell death rate was calculated as the mean percentage of blue cells/total cells. All experiments were conducted in triplicate.

All data in this study are expressed as the means ± SD. A one-way analysis of variance (ANOVA) or t-test was performed to assess differences between groups under different conditions. All statistical analyses were performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered statistically significant.

The antiproliferation effect of berberine against MPM NCI-H2452 cells was evaluated by an MTT assay. The results showed that berberine inhibited the growth of cells in a dose- and time-dependent manner. The proliferation rate of berberine-treated NCI-H2452 cells was 87.58±3.72, 46.68±8.08 and 33.24±7.17%, respectively, following treatment with berberine at a concentration of 100 mM for 24, 48 and 72 h (Fig. 1). Based on the MTT assay results, the 100 µM berberine concentration for 48 h was used in subsequent experiments.

Previous studies have reported that berberine can induce apoptosis in some cancer cells (9,10); therefore, we investigated whether it could induce apoptosis in NCI-H2452 cells using Hoechst 33258 staining. As shown in Fig. 2A, the treatment of NCI-H2452 cells with berberine at 100 µM for 48 h resulted in apoptosis, which was characterized by unique morphological nuclear changes such as nuclear condensation and fragmentation.

We further examined the apoptosis rate by Annexin V-PE/7-AAD double staining assay by flow cytometry. The results showed that after treatment with berberine at the concentration of 100 µM for 48 h, the apoptosis rate of NCI-H2452 cells increased to 32.23±1.06%, while the apoptosis rate was 3.55±0.86% in untreated cells (P<0.001; Fig. 2B and C).

Many antitumor agents induce cancer cells apoptosis through mitochondrial apoptotic pathways (16,17); therefore, we further investigated the expression of key proteins in the mitochondrial pathway. As shown in Fig. 2D and E, treatment with berberine resulted in the release of cytochrome c into the cytoplasm, as well as a significant increase in the active forms of caspase-9 and caspase-3, and the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP).

To determine whether berberine induces autophagy in NCI-H2452 cells, we examined the intracellular distribution of LC3, an autophagy marker, upon berberine treatment under immunofluorescence. As shown in Fig. 3, the distribution of LC3 fluorescence changed from diffuse cytosolic in untreated cells to punctate upon berberine treatment.

We further analyzed the expression of LC3-II using western blotting (Fig. 4A and B). The results showed an increased conversion of LC3-I to the autophagic LC3-II isoform in NCI-H2452 cells treated with berberine, in a dose- and time-dependent manner. The increased conversion was attenuated by pre-treatment with the autophagic inhibitor 3-MA, and enhanced by the autophagic inducer rapamycin (Fig. 5A and B). In addition, treatment with berberine decreased the expression of p62 in a dose- and time-dependent manner (Fig. 4A and B). These results suggest that autophagy is induced by berberine in NCI-H2452 cells.

Cell death in response to berberine was quantified by a trypan blue exclusion assay, and the cells with disrupted membranes (blue cells) were counted as dead cells. NCI-H2452 cells were treated with berberine at concentrations of 0, 50, 100 and 200 µM for 48 h. As shown in Fig. 6, berberine killed MPM cells in a dose-dependent manner. After treatment with berberine at the concentration of 200 µM for 48 h, the death rate of NCI-H2452 cells increased to 34.66±5.32% (P<0.001).

Our western blot results showed that autophagy was induced in NCI-H2452 cells by treatment with berberine in a dose-dependent manner. Based on the western blotting results, autophagy was induced at a concentration of 200 µM. As cell death was clearly induced with a concentration of 200 µM berberine in the trypan blue exclusion assay, this concentration was used in subsequent experiments.

The results showed that MPM cells treated with berberine underwent autophagy in a dose- and time-dependent manner, which was positively correlated with the cytotoxic activity of berberine; therefore, we further assessed whether berberine-induced NCI-H2452 cell death was mediated by autophagy. In addition, both apoptosis and autophagy were induced by berberine treatment in this study. Therefore, it was necessary to further investigate the interactions between berberine-induced apoptosis and autophagy. We first explored whether the inhibition of autophagy by 3-MA or the induction of autophagy by rapamycin affected the rate of cell death induced by berberine (Fig. 7A and B). The results showed that the death rate of NCI-H2452 cells treated with 3-MA plus berberine increased significantly compared with the cells treated with berberine alone, while pre-treatment with rapamycin decreased the death rate of berberine-treated cells.

The apoptosis rate of NCI-H2452 cells was assessed using Annexin V-PE/7-AAD double staining assay by flow cytometry. Compared with the cells treated with berberine alone, 3-MA-treated cells underwent extensive apoptosis after berberine treatment (Fig. 2B and C). However, the expression level of LC3-II in NCI-H2452 cells treated with berberine in the presence of z-VAD-fmk was similar to that in the cells treated with berberine alone (Fig. 7C).

Subsequently, we further investigated if berberine-induced NCI-H2452 cell death occurs via apoptosis. Cells were pretreated with z-VAD-fmk, a pan-caspase inhibitor, which only inhibits apoptosis-mediated cell death, for 1 h, followed by berberine for 48 h. As shown in Fig. 7D, berberine-induced NCI-H2452 cell death was attenuated by z-VAD-fmk.

Collectively, the results show that apoptosis is the main route by which berberine induces NCI-H2452 cell death. Contrary to previous studies, our results supported a cytoprotective autophagic activity induced by berberine, preventing cells from undergoing apoptosis; the inhibition of autophagy may enhance the antitumor potential of berberine in MPM NCI-H2452 cells.