The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

Abstract

DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells.

Figure 1

Screening of herbal extracts and characterization of berberine and coptisine. (a) PFGE analysis of DSB accumulation after treatment with extracts of Phellodendron Bark and Coptis Rhizome. Total broken DNA was detected by ethidium bromide (EtBr) staining, whereas DSBs at DNA replication sites were detected by immunoblotting with the anti-BrdU antibody. (b) Immunofluorescence analysis of DSB accumulation by detecting the DSB markers γ-H2AX and 53BP1 foci. (c) Molecular structures of the BIAs berberine, coptisine, palmatine, and magnoflorine. (d) PFGE analysis of DSB accumulation after treatment with berberine, coptisine, palmatine, and magnoflorine. (e) Quantification of broken DNA. The data are presented as the ratios of the amount of broken DNA per total DNA (intact DNA + broken DNA). (f) Quantification of broken DNA. The data are presented as fold inductions relative to the untreated control.

Figure 2

Analysis of the cytotoxic effects of berberine, coptisine, and palmatine. (a) PFGE analysis of DSB accumulation after treatment with aphidicolin combined with extracts of Phellodendron Bark and Coptis Rhizome. (b) PFGE analysis of DSB accumulation after treatment with aphidicolin combined with extracts of berberine and coptisine. (c) PFGE analysis of DSB accumulation after treatment with Z-VAD-FMK combined with extracts of berberine and coptisine. (d) Time-course PFGE analysis of DSB accumulation after treatment with CPT, palmatine, berberine and coptisine. (e) Quantification of broken DNA. The data are presented as fold inductions relative to the untreated control. (f) Survival curves of MRC5sv cells against berberine, coptisine, palmatine, and magnoflorine. (g) Survival curves of MRC5sv cells treated with CPT and etoposide.

Figure 3

Analysis of Topo I inhibition by BIAs in vivo. (a) Western blot analysis of Topo I after transfection with siRNA against the Top1 gene. (b) PFGE analysis of DSB accumulation after treatment with transient Topo I depletion by siRNA combined with berberine and coptisine. (c) Quantification of broken DNA per total DNA after treatment with transient Topo I depletion by siRNA combined with berberine and coptisine. The means and SEs were determined from four independent experiments. (d) Analysis of the accumulation of the Topo I-DNA complex by ICE assay. Following the manufacturer’s recommended protocol, the cells were treated with 50 μM CPT, coptisine, and berberine, and the Topo I-DNA complexes were visualized by immunoblotting using an anti-Topo I antibody.

Figure 4

Analysis of Topo I inhibition by BIAs in vitro. (a,b) Inhibition of the relaxation activity of Topo I by berberine, coptisine, and palmatine. (c) Relaxation activity of Topo I after treatment with magnoflorine. (d) Detection of nicked DNA based on the effects on the relaxation activity of Topo I by CPT, coptisine (Cop), berberine (Ber), and palmatine (Palm). (d) NMR titration curves, ¹H chemical shift changes versus the concentration of dsDNA, and dissociation constant (Kd) values for coptisine, berberine, and palmatine. The inset shows the proton in the alkaloid backbone used for the NMR analysis.

Figure 5

Analysis of the inhibitory effects of BIAs in nicking and rejoining assays. (a) Schematic representation of the nicking assay. Nicked DNA products were detected by the Cy-3 fluorescent signal. Unaffected DNA molecules were detected as 36-mer fragments, and nicked DNAs were detected as 22-mer fragments. (b) Inhibitory effects of treatment with CPT, berberine (Ber), and coptisine (Cop) on Topo I-dependent nicking activity. (c) Schematic representation of the rejoining assay. The rejoined products were detected by Cy-3 and Cy-5 fluorescent signals. Unaffected DNA molecules were detected as 18-mer fragments on a Cy-3-visualized gel, and nicked DNAs were detected as 36-mer fragments on both the Cy-3- and Cy-5-visualized gels. (d) Inhibitory effects of treatment with CPT, berberine (Ber), and coptisine (Cop) on Topo I-dependent re-joining activity.

Figure 6

Analysis of MUS81 function on BIA-induced DSB formation in vivo. (a) Western blot analysis of MUS81 after transfection with siRNA against the MSU81 gene. (b) PFGE analysis of DSB accumulation after treatment with transient MUS81 depletion by siRNA combined with berberine and coptisine. (c) Quantification of broken DNA per total DNA after treatment with transient Topo I depletion by siRNA combined with berberine and coptisine. The means and SEs were determined from four independent experiments.

Figure 7

Analysis of the inhibitory effects of BIAs against CPT-resistant Topo I proteins. (a) Schematic representation of CPT-resistant TOP1 mutations. (b) Relaxation activity of CPT-resistant Topo I proteins in the presence of CPT in vitro. (c) Inhibitory effect of coptisine, berberine, epiberberine and palmatine on CPT-resistant Topo I proteins in vitro.

Figure 8

Analysis of the inhibitory effects of BIAs against CPT-resistant cells and Topo I proteins. (a) Analysis of CPT resistance of CPT-K5 cells carrying D533G mutations in the TOP1 gene. (b,c) Survival curve of RPMI-8402 and CPT-K5 cells after treatment with berberine (b) and coptisine (c). (d) Analysis of CPT resistance of CEM/C2 cells carrying N722S mutations in the TOP1 gene. (e,f) Survival curve of CCRF-CEM and CEM/C2 cells after treatment with berberine (e) and coptisine (f).

Figure 9

Effect of treatment with CPT combined with berberine and coptisine. (a) Effect of treatment with CPT combined with berberine and coptisine on the relaxation assay in vitro. (b-e) Effect of treatment with CPT combined with berberine and coptisine on the survival of CPT-resistant cell lines, as determined through the MTT assay. (b,c) Effect of treatment with CPT combined with berberine (b) and coptisine (c) on the survival of the CPT-K5 cell line, as determined through the MTT assay. (d,e) Effect of treatment with CPT combined with berberine (d) and coptisine (e) on the survival of CEM/C2 cells, as determined through the MTT assay.

Figure 10

PFGE analysis of DSB accumulation after treatment with berberine and coptisine in various cancer cell lines. Various cancer cell lines were treated with 10 μM coptisine and berberine for 24 h, and the accumulation of DSBs was analysed by PFGE.