Figure 1.
Berberine inhibits colon tumor colony growth in soft agar.
IMCEras cells were cultured in 0.35% agar on the basal 0.5% agar’s layer in IMCE cell culture medium in the presence or absence of the indicated concentrations of berberine at 33°C for 14 days. Prior to counting colonies, cells were stained using the CellTiter®120 AQueousOne Solution Cell Proliferation Assay. Colonies of >50 µm were counted. The data are shown as fold increase in comparison with the colony cultured without berberine (control). Data represent at least 3 independent experiments, each performed in triplicate. *p<0.01 compared to the control group, #p<0.01 compared to the 50 µM of the berberine treated group.
Figure 2.
Berberine induces cell death and LDH release in the colon cancer cell line, IMCE cells.
IMCE and YAMC cells were cultured in serum-starved RPMI 1640 medium at 37°C (non-permissive condition) for 24 h with or without 50 µM berberine treatment for indicated times from the end of experiment (A and C) or berberine treatment at indicated concentrations for 18 h (B and D). Cell viability was assessed using the CellTiter®729 AQueousOne Solution Cell Proliferation Assay (A–B). % of cell number change was calculated as: [(cell number at the end of treatment-cell number before treatment)/(cell number before treatment)]×100. Cell culture supernatants were collected for detecting LDH release using the CytoTox 96® 159 Non-Radioactive Cytotoxicity Assay (C–D). LDH release is expressed as a percentage of control in each experiment. Data represent at least 3 independent experiments, each performed in triplicate. *p<0.01 compared to the control group.
Figure 3.
Berberine induces cell death through a caspase-independent mechanism in IMCE cells.
Cells were treated with berberine at 50 µM for the indicated times or TNF (100 ng/ml) and cycloheximide (1 µg/ml) for 6 h in serum-starved RPMI 1640 medium at 37°C, as described in Figure 2. Cellular lysates were collected for Western blot analysis using antibodies against cleavage (active) forms of caspases and an anti-PARP antibody (which identifies both intact and cleavage forms) (A). The actin blot was used as a protein loading control. Cells were treated with berberine at 50 µM for 18 h or TNF and cycloheximide for 6 h in the presence or absence of a caspase inhibitor, zVAD-fmk (25 µM). Cell number change (B) and LDH production (C) were detected as described in Figure 2. *p<0.01 compared to the control group. #p<0.01 compared to the TNF/cycloheximide treated group.
Figure 4.
Berberine-induced ROS production is required for cell death in IMCE.
Cells were treated with berberine at indicated concentrations for 18 h (A and B), as described in Figure 2. Intracellular ROS levels were detected using DHE probe and the amount of fluorescence was measured using flow cytometry. The fold change of the amount of fluorescence was calculated by comparing that in the treated groups to the control group (A). Cells were treated with the JC-1 probe and then analyzed by flow cytometry to measures the fraction of cells with polarized or depolarized mitochondrial membrane (B). Cells were treated with berberine at 50 µM for 18 h in the presence or absence of 5 µM of Tiron, a ROS scavenger. Cell number change (C) and LDH release (D) were detected as described in Figure 2. *p<0.01 compared to the control group, #p<0.01 compared to the berberine treated group.
Figure 5.
Berberine stimulates AIF release from the mitochondria to the cytoplasm and nuclear translocation in IMCE, which is required for berberine-induced cell death.
Cells were treated with berberine at 50 µM for the indicated times, as described in Figure 2. Cytoplasmic, nuclear, and mitochondrial fractions were prepared for Western blot analysis using anti-AIF, anti-GAPDH (cytosolic marker), anti-COX IV (mitochondrial marker) and anti-Ki-67 (nuclear marker) antibodies (A). Immunostaining of cells with 50 µM berberine treatment for 18 h was performed to localize AIF using an anti-AIF antibody and a FITC-conjugated secondary antibody (green) and nuclei using DAPI staining (blue). Arrows indicate nuclei with AIF translocation (B). IMCE cells transfected with AIF siRNA or non-targeting siRNA for 24 h were treated with berberine at 50 µM for 18 h to detect number change (D) and LDH release (E) as described in Figure 2. AIF expression levels were detected by Western blot analysis using an anti-AIF antibody (C). Actin blot was used as a protein loading control. *p<0.01 compared to the control group.
Figure 6.
Berberine-stimulated cathepsin B release through ROS production is required for AIF activation in IMCE.
Cells were treated with berberine at 50 µM for 18 h in the presence or absence of a ROS scavenger, Tiron (5 µM) or a cathepsin B inhibitor, CA-074 Me (100 nM). Immunostaining was performed to localize AIF using an anti-cathepsin B antibody and a FITC-conjugated secondary antibody (green) and nuclei using DAPI staining (blue) (A). Cathepsin B staining shows granular lysosomal pattern in control cells and berberine treated YAMC cells and a diffuse cytosolic pattern with more nuclear distribution after berberine treatment in IMCE cells. Cytoplasmic fractions were prepared for Western blot analysis using anti-cathepsin B, anti-AIF, anti-GAPDH antibodies (B). Cell number change (C) and LDH release (D) were detected as described in Figure 2. *p<0.01 compared to the control group, #p<0.01 compared to the berberine treated group.
Figure 7.
Berberine activates PARP in IMCE, which mediates berberine-mediated AIF activation and cell death.
Cells were treated with berberine at 50 µM for the indicated times (A) or at the indicated concentrations for 18 h (B). PARP activity was measured using the HT 167 Universal Colorimetric PARP Assay Kit. PARP activation is expressed as a percentage of control in each experiment. Cells were treated with berberine at 50 µM for 18 h in the presence or absence of DPG (25 µM), a PARP activation inhibitor. Cell number change (C) and LDH release (D) were detected as described in Figure 2. The cytosolic fractions were prepared for Western blot analysis of AIF, as described in Figure 3 (E). *p<0.01 compared to the control group, #p<0.01 compared to the berberine treated group.