Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

Abstract

Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34.

Keywords: GRP78; VPS34; autophagy; berberine; cancer cell death.

Figure 1. Berberine treatment induced autophagic cancer cells death

A. HCT-116, DLD1, HepG2 and HL-7702 cells were treated with different concentrations of berberine for 24 h. Cell viability was detected using the MTT assay and plotted against berberine concentrations, n=3. The cell viability curve was fitted using the Hill equation. IC₅₀ indicated the concentration at which 50% of the cells survived. B. Viability of HCT-116 cells after treatment with berberine plus or minus 3-MA (10mM) was measured by MTT. C. Expression of ATG5 in HepG2 cells transfected with control or ATG5 siRNA was detected by western blot. D. Expression of Beclin1 in HepG2 cells transfected with control or Beclin1 siRNA by western blot are shown. E. The cytotoxicity of berberine can be attenuated by introducing siRNA against ATG5 and Beclin1 into HepG2 cells. All experiments were performed in triplicate and the results were analyzed for statistical significance (*p<0.05, **p<0.01).

Figure 2. Berberine-activated autophagy in HCT-116 cells

A and B. Western blots of LC3, p62, Beclin1 and GAPDH were performed on HCT-116 cell lysates treated with berberine at the indicated concentrations for 24 h. The relative protein expression was calculated by Image J. C-D. HCT-116 cells were treated with the indicated concentrations of berberine for 24 h with or without CQ (50 μM). The levels of LC3, p62 and Beclin1 were monitored by western blot of the cell lysates (C) and the relative protein expression was calculated by Image J (D). E-F. Western blotting analysis of LC3-II/LC3-I, p62 and Beclin1 levels (E) and the relative protein expression were quantified by Image J (F) in HCT-116 cells treated with berberine at the indicated concentrations for 24 h, in the absence or presence of 37.5 μM BAF (treated in combination with berberine). G. Protein expression levels of LC3, p62 and Beclin1 were analyzed by western blot in HCT-116 cells after treatement with berberine at the indicated concentrations for 24 h, in the absence or presence of 3-MA at different concentrations (treated in combination with berberine). Three independent experiments were performed, and the data were expressed as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001 were compared to the untreated group.

Figure 3. Berberine activated autophagy in cancer cell lines of HCT-116, DLD1, and HepG2

A. Quantification of the relative protein expression is shown in Figure 2G of three independent experiments using Image J software. B and C. Western blot analysis of LC3, Beclin1 and p62 expression (B) and the relative protein expression were evaluated by Image J (C) in HL-7702 cells treated with various concentrations of berberine. D. HCT-116 cells incubated with 0.05 mM monodansylcadaverine (MDC) for 10 min after treatment with the indicated concentration of berberine 24 h (left panel) or transfected with mCherry-hLC3B treated with 120 μM berberine for 24 h (right panel). The cells were then analyzed using fluorescence microscopy. Scale bar, 5 μm. E-F. HepG2 and DLD1 cells were treated with the indicated concentrations of berberine for 24 h with or without BAF co-incubation. LC3, Beclin1 and p62 were analyzed by western blotting analyses (E) and relative protein expression levels were calculated by Image J (F). Three independent experiments were performed, and the data were expressed as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001 compared to the untreated group.

Figure 4. GRP78 was elevated in berberine treated cancer cell lines

A. Electron microscopic images of HepG2 control and berberine-treated cells (120 μM) for 24 h. Lipid droplets (yellow arrow); autophagosomes and autolysosome of double-membrane, single-membrane, and multivesicular body-like vesicles (blue arrows); mitochondria (red arrow). Scale bars are indicated at the bottom. B and C. HCT-116 cells were exposed to 20, 40, 80,100,120,140 and 160 μM berberine for 24 h. GRP78 was detected by qRT-PCR (B) and western blotting analyses (C). D. HCT-116 cells were exposed to 120 μM berberine for 24 h, and the cells were then fixed and stained with GRP78-specific antibody and examined using fluorescence microscopy. E and F. HepG2 and DLD1 cells were exposed to the indicated concentrations of berberine for 24 h, and GRP78 was detected by qRT-PCR (E) and western blotting analyses (F). G and H. HL-7702 cells were exposed to 100 μM berberine for 24 h, and GRP78 was detected by qRT-PCR (G) and western blotting analyses (H).

Figure 5. Knockdown of GRP78 abolished the autophagy effect of berberine in HCT-116 and DLD1 cells

A-F. HCT-116 and DLD1 cells were transfected with control siRNA or siRNA targeting GRP78, respectively. After 48 h, the cells were treated with indicated concentrations of berberine for 24 h, then GRP78 was measured by qRT-PCR (A and C) and GRP78, LC3 were examined by western blotting analyses (B and E). The LC3-II/LC3-I relative protein expression levels were calculated by Image J (C and F). Data were expressed as the mean ± SEM of three different experiments. *p<0.05 and **p<0.01 vs. respective control.

Figure 6. GRP78 played a dominant role in the modulation of autophagy in HepG2 and DLD1 cells

A-C. HepG2 cells were transfected with control siRNA or siRNA targeting GRP78, respectively. After 48 h, the cells were treated with indicated concentrations of berberine for 24 h, then GRP78 was measured by qRT-PCR (A) and GRP78, LC3 were examined by western blotting analyses (B). The LC3-II/LC3-I relative protein expression levels were calculated by Image J (C). D. The cytotoxicity of berberine can be attenuated by introducing siRNA against GRP78 into DLD1 cells. Data were expressed as the mean ± SEM of three different experiments. *p<0.05 vs. respective control.

Figure 7. Berberine induced GRP78 expression by increased ATF6 expression and GRP78 stability

A. HCT-116 and HepG2 cells were treated with berberine for 24 h. ATF6 mRNA was measured by qRT-PCR. B. HCT-116 and HepG2 cells were treated with berberine for 24 h. YY1 mRNA was measured by qRT-PCR. C. Western bloting analyses were performed to examine ATF6 expression levels in HCT-116 and HepG2 cells. D. HCT-116 cells were transfected with control siRNA or siRNA targeting ATF6. After 48 h, the cells were treated with the indicated concentrations of berberine for 24 h, and GRP78 was measured by western blot. E. In the presence of berberine or proteasome inhibitor MG132 (5 μM), HCT-116 cells were treated with cycloheximide (CHX, 0.1 mg/ml) for the indicated times and GRP78 expression was then analyzed by western blot. F. Quantification of GRP78 expression was performed on three independent experiments using Image J software and normalized against vehicle control (Figure 6E). G. HCT-116 cells were treated with MG132 in the presence or absence of berberine for 24 h. GRP78 was immunoprecipitated, and GRP78 ubiquitination was examined. Three independent experiments were performed, and the data were expresses as the mean ± SD. *p<0.05, **p<0.01 were compared to the untreated group.

Figure 8. Berberine can interact with GRP78 and induce autophagy by facilitating the ability of GRP78 to bind to VPS34

A. Effect of berberine on fluorescence spectra of GRP78. λex = 295 nm, [GRP78] = 1.0×10⁻⁶ M, curves 1–12 [berberine]/[GRP78] = 0, 0.8, 1.2, 1.6, 2.4, 2.8, 3.2, 3.6, 4.8, 6.4, 8.0, 9.6, pH 7.4, at 42°C. B. The dots represent [berberine]/[GRP78] = 0, 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, 5.6, 6.4, pH 7.4, at 42 °C. The fluorescence intensity of GRP78 declined gradually, and the blue-shift occurred for the emission maximum (from 331.5 to 320.5 nm). C. The values of Ksv, Kq and R² were determined with Stern–Volmer equation by linear regression of plots of F₀/F vs [Q] at different temperature. The Stern–Volmer equation: F₀/F=1+Ksv[Q]=1+Kqτ₀[Q], where F₀ and F were fluorescence intensities of GRP78 before and after the addition of berberine, Kq was the quenching rate constant, and τ₀ was the average lifetime of biomolecule without quencher that was valued at approximately 10⁻⁸s. [Q] was the concentration of berberine as a quencher, and Ksv was the Stern–Volmer quenching constant. D. HCT-116 cells were treated with berberine for 24 h. GRP78 was immunoprecipitated and IgG served as a negative control, VPS34 was then examined. E. Quantification of GRP78-VPS34 levels in the immunoprecipitated complex are shown in Figure 7D of three independent experiments using Image J software and normalized against the vehicle control. F. Confocal microscopy detection of co-localization of endogenous GRP78 with VPS34 in HCT-116 cells treated with berberine for 24 h. Permeabilized cells were stained with anti-GRP78 and anti-VPS34 antibody to detect GRP78 (green) (left) and VPS34 (red) (right). The merged images showed co-localizations (yellow) of GRP78 and VPS34 detected at multiple corresponding sites. Scale bars represent 6 μm.