Cell-specific pattern of berberine pleiotropic effects on different human cell lines

Abstract

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.

Figure 1

Intracellular localization of berberine and effects on viability in HDF, U343 and MIA PaCa-2 cells. (a) Confocal images of berberine distribution in HDF, U343 and MIA PaCa-2 cells. Cells were photographed 1 hour after treatment with berberine (10 μM, 50 μM or 150 μM). Black arrows point out nuclei. Scale bars represent 5 μm. (b) Reduction of cell viability after 48 hours of treatments with 0.4 μM, 2 μM, 10 μM, 50 μM berberine in HDF, U343 and MIA PaCa-2 cells. Graph columns represent mean of viable cells ± S.D. normalized versus control group (DMSO). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Berberine localizes in mitochondria and affects mitochondrial function. (a) Berberine was visualized by confocal microscopy in mitochondria of HDF, U343 and MIA PaCa-2 cells after 48 hours of exposure to 10 μM or 50 μM berberine. Merge columns represent overlapping of the berberine green signal with the TMRM red signal. DMSO-treated cells, used as a control, lack green fluorescence. Differential interference contrast (DIC) highlighted the cell morphology. Scale bars indicate 5 μm. (b) Citrate synthase activity was measured in the three cell lines after treatments in the presence or absence of berberine as described in Methods. U = Units of enzymatic activity. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Berberine alters the cell cycle and the transcriptional profile of cell cycle regulators. (a) Representative images of cell cycle distribution of HDF, U343 and MIA PaCa-2 cells. Cell cycle distribution was assayed by flow cytometry, following treatment with 10 μM or 50 μM berberine for 48 hours; subG1 (yellow bars), G1 (blue bars), S (green bars), G2 (red bars). Upper right insets represent the zoomed in subG1 population relative to each graph. Cell number (count) is reported in y-axis; fluorescence intensity (DsRed/PE) is reported in x-axis. (b) Quantification of cell cycle distribution; (c) The transcriptional profile of P53, P21, P16 was analyzed by Real Time RT-PCR after 1 hour or 48 hours berberine treatments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Berberine induces cell senescence in HDF, U343 and MIA PaCa-2 cells. (a) Brightfield images of SA β-gal-positive HDF, U343 and MIA PaCa-2 cells after berberine treatments. Treatments: 10 μM or 50 μM berberine or DMSO for 48 hours. Scale bars indicate 50μm. (b) The graph shows the number of SA β-gal-positive cells normalized to the control (DMSO). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Effects of berberine on caspase-3 activity. (a) HDF, U343 and MIA PaCa-2 cells were incubated for 48 hours with 50 μM berberine and analyzed for caspase-3 activity monitored for 5 hours. Caspase-3 activity was measured as absorbance variation/hour/mg protein. Ber = berberine. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Berberine induces autophagy in U343 and MIA PaCa-2 cells, but not in HDF. (a) Relative mRNA expression is reported in y-axis. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. (b) Immunofluorescence experiments. Cells were treated with berberine or DMSO for 48 hours and immunohistochemistry was performed with LC3 antibody (red fluorescence), as described in Methods. Nuclei are visualized by Hoechst staining. (c) Cells were treated as in (b) and proteins were subjected to electrophoresis and western blotting as described in Methods. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Wound healing and cell invasion assays. (a) Representative brightfield images of the scratches (marked by white lines) in MIA PaCa-2 cells at 0, 24 and 48 hours, after treatments with berberine or DMSO. (b) Representative brightfield images of the scratches in U343 cells at 0, 24, 48 and 72 hours, after treatments with berberine or DMSO. (c,d) The values reported in the graphs represent the mean distance taken at each time from the wound edges (normalized to the DMSO group) ±S.D. Ber = berberine. (e) Impaired invasion of berberine-treated MIA PaCa-2 in transwell assay. (f) The graph reports the relative invasive score (±S.D.) corresponding to each berberine concentration, compared to DMSO. (g) The transcriptional profile of DAP1 and CXCR4 was analyzed by Real Time RT-PCR. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8

Effects of berberine on the transcriptional profile of DNMT1, DNMT3A, DNMT3B and MGMT in HDF, U343 and MIA PaCa-2 cells. Cells were treated with berberine for 1 hour or 48 hours and Real Time RT-PCR was performed as described in Methods. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.