Introduction
Berberine is the major active component of the Chinese herbal medicine Huang Lian (1), which has been widely used clinically as a natural microbial agent in the treatment of diseases like dysentery and gastroenteritis (2,3). In recent years, a number of studies have reported that berberine has antitumor effects on numerous cancer cell line types, including leukocyte, glioblastoma, esophageal, breast and osteosarcoma (4–8). The anticancer mechanisms of berberine appear to be complex: Certain studies have suggested that berberine may interfere with DNA replication, acting as an inhibitor of replication and repair enzymes (6), while others have implied it may induce DNA damage directly (9). In addition, it has been reported that berberine is capable of inducing apoptosis through DNA damage-independent signaling pathways, including fatty acid synthase (Fas) receptor and estrogen receptor (10,11).
As high doses of berberine provoke a wide variety of cellular responses, the mechanisms that explain its genotoxicity remain difficult to define. Rev3, a catalytic subunit of DNA polymerases ζ (Polζ), participates in translesion DNA synthesis (TLS) and has been identified as an important component in the maintenance of chromosomal DNA, in its ability to enhance tolerance to DNA damage (12). Previously, we generated Rev3 deficient DNA repair deficient chicken B lymphocyte (DT40) cells (Rev3−/−) and identified they were hypersensitive to various DNA damaging agents, including IR, cisplatin and UV (13,14). Besides their specific sensitivity, Rev3−/− cells also possess a number of advantages as DT40 clones. Firstly, the proliferation of DT40 clones with a cell cycle time of 8 h is more rapid than that of the majority of mammalian cell lines. Secondly, during logarithmic growth, 70% of DT40 cells are in S phase, in contrast to the 50% for mammalian cells, so that exogenous DNA damage may target DNA replication directly. Finally, due to the absence of functional p53, DT40 cells lack the G1/S checkpoint. Therefore, DNA repair activity contributes a greater extent to cell survival, which aids our observations of the chromosomal breaks in mitotic cells, as cells carrying double-strand breaks (DSBs) can enter the M phase without stimulating the apoptosis pathway (15). As a result, Rev3−/− cells have been characterized as an excellent tool for studying the DNA damage and repair mechanisms of different chemicals, including tamoxifen and nitric oxide (NO) (11,16).
In the present study, we used Rev3−/− and other DNA repair gene deficient cells to study the mechanisms of berberine. We identified that Rev3−/− and several other DNA repair gene deficient clones including Rev1−/−, Parp1−/−, Rad18−/−, Xrcc2−/−, were hypersensitive to the naturally occurring alkaloid. Cell cycle and chromosomal break analysis demonstrated that berberine induced a significant increase in G2/M phase cells and chromosomal breaks in Rev3−/−, compared with the wild-type (WT) cells. These results suggest that berberine is able to directly induce DNA damage, and that Rev3 participates in the processes that aid its repair.
Materials and methods
Chemicals
The berberine chloride and alachlor was obtained from Sigma-Aldrich (St. Louis, MO, USA). Stock solution of berberine (20 mM) was prepared in DMSO and stored at −20°C in aliquots until use.
Cell line and cell culture
The cells were cultured as described previously (17,18). The cell lines used in this study are listed in Table I. The phenotypes of these cells have been previously described (12,19–25). The WT cell line and the DNA repair-deficient cell lines differ only in the presence/absence of an endogenous DNA repair gene, other than this difference, their genetic backgrounds were identical (15). All cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% chicken serum, 0.1% β mercaptoethanol and 1% penicillin. The cell lines were maintained at 39°C under a humidified atmosphere and 5% CO2. The study was approved by the Ethics Committee of Sichuan University (Chengdu, China).
Measurement of cell proliferation by ATP assay
To assess cell numbers following treatment with berberine, we measured the amount of ATP in the whole cell lysate (26). Cells (1.5×103–1.5×104) were incubated in 1 ml culture medium per well, containing various concentrations of berberine. At 72 h, the ATP in the cellular lysates was measured to assess the number of live cells. At least three independent experiments were conducted. Sensitivity was calculated by dividing the number of drug-treated cells, by the number of untreated cells (13).
Cell-cycle analysis
Following treatment with 5 μM berberine for 16 h, cells were labeled for 10 min with 20 μM Bromodeoxyuridine (BrdU) and subsequently harvested. Harvested cells were fixed and analyzed as previously described (19): (i) In 4N HCl, 0.5% Triton X-100 for 30 min at room temperature; (ii) in FITC-conjugated anti-BrdU antibody for 1 h at room temperature; (iii) in 5 μg/ml propidium iodide (PI) in PBS. Following incubation, cells were washed with PBS containing 2% FCS and 0.1% sodium azide. Subsequent flow cytometric analysis was performed on a FACScan. Fluorescence data were displayed as dot plots using the Cell Quests software.
Chromosomal aberration (CA) analysis
Measurement of CA was performed as previously described (12). The chicken karyotype consisted of 80 chromosomes, including 11 major autosomal macrochromosomes, the ZW sex chromosomes, and 67 microchromosomes (15). In the present study, only the 11 major autosomal macrochromosomes were measured. For CA analysis, cells (3.0×106/3 ml) were treated with the test doses of 2.5 and 5 μM for berberine and 5 μM for alachlor for 16 h. Then cells were treated for 30 min with 0.1 μg/ml colcemid and went on harvesting for 2.5 h. We then suspended the cells with 75 mM KCL and incubated them for 15 min at room temperature, adding 5 ml of Carnoy’s solution (mixture of acetic acid and methanol, 1:3) and left for at least 30 min at room temperature. The cells were then dropped onto ethanol-cleaned slides and dried by a flame. The dried slide was then dipped into 5% Giemsa solution, left for 8 min at room temperature and rinsed carefully with water and finally, they were air-dried. The cells were observed under a light microscope (magnification, ×1,000).
Statistical analysis
Survival data were log-transformed giving approximate normality. Analysis of covariance (ANCOVA) was used to test for differences in the linear dose-response curves between WT and a series of mutant cells. Viability of the DT40 cells was estimated using regression curves. Regression-curve equations were used to calculate LC50 (lethal concentration, 50%) values. Relative LC50 values were normalized according to the LC50 value of the parental WT cells.
Results
Rev3<sup>−/−</sup> cells are sensitive to berberine
As Rev3−/− are specifically sensitive to a variety of DNA damaging agents, in the present study we assessed whether Rev3−/− cells exhibit a higher sensitivity to berberine. We treated WT and Rev3−/− cells with varying concentrations of berberine for 72 h, and measured the viability of cells using an ATP assay. As summarized in Fig. 1, Rev3−/− cells exhibited a significant increase in sensitivity to berberine when compared with WT cells. This observation indicated that berberine may induce DNA damage, and that Rev3 associated DNA repair is an important regulatory process in cellular tolerance to berberine-induced genotoxicity. To study whether other DNA repair pathways were also involved in tolerance to berberine, we assessed the sensitivity of berberine in a panel of DT40 clones, which were separately deficient in different DNA repair pathways (Table I). The DNA repair pathway mutants demonstrated sensitivity to berberine in the following order: Rev3 > Rev1 > Parp1 = Xrcc2 = Rad18 > WT (parental DT40 cells)
Berberine induces enhanced arrest of cell cycle in G2/M phase
As DT40 cells are deficient in p53, and agents causing DNA damage would lead to cell arrest in G2/M phase, we assessed if the increased sensitivity of Rev3−/− to berberine was also correlated with G2/M arrest. Data from the pulse labeled with BrdU indicated that berberine induced an increase in the accumulation of Rev3−/− cells in the G2/M phase compared with that in the WT cells (Fig. 2).
Berberine induces an increase of chromosome breaks in Rev3<sup>−/−</sup> cells
To examine if the higher sensitivity of Rev3−/− to berberine was specifically associated with DNA damage, we measured the number of chromosome breaks in mitotic cells. Fifteen CAs were detectable in 50 mitotic Rev3−/− cells when the concentration of berberine was 2.5 μM, while only 4 CAs were identified in WT cells. As the concentration elevated to 5 μM, the CAs in Rev3−/− cells markedly increased to 23, by contrast, WT cells showed fewer aberrations of only 7 (Table II and Fig. 3). This result was consistent with the positive control, alachlor. The data suggest that the hypersensitivity of Rev3−/− to berberine is correlated with a significant increase of berberine-induced CAs.
Discussion
In the present study, we utilized a panel of DNA repair deficient DT40 cells to study the genotoxicity mechanisms of the natural alkaloid, berberine. Our results demonstrated that cells deficient in Rev3 exhibited the highest sensitivity to berberine. The cell cycle and chromosome breaks assay provided evidence that more G2/M arrest and chromosome breaks occurred in Rev3−/− cells than in WT cells. While the Rev−/− cells were the most sensitive, the other DNA repair gene deficient cells, including Rev1−/−, Parp1−/−, Rad18−/− and Xrcc2−/− cells were also sensitive to berberine, but polβ−/−, XPA−/−, DNA-PK−/− cells were not. Two important homologous recombination (HR) enzymes, Brca1 and Brca2, were slightly resistant to berberine. These results provided clear evidence that berberine is able to induce DNA damages, and that Rev3 as well as Rev1, Rad18, Parp1 and Xrcc2 are important in processes that repair these damages.
Rev3 is a catalytic subunit of Polζ. Studies in yeast have revealed that Polζ cells are able to efficiently bypass abasic sites, through extending from nucleotides inserted opposite the lesion, by other TLS DNA polymerases. These data suggest Polζ may be involved in the extension process of TLS in higher eukaryotes (27). One reverse genetics study in DT40 cells identified that Rev3 participated in not only TLS but also HR (12). Rev1 and Rad18 appear to have a similar function to Rev3 in maintaining genomic stability by TLS and HR (19). The high sensitivity of Rev3−/−, Rev1−/− and Rad18−/− to berberine implies that TLS or HR may participate in berberine-induced DNA damage. TLS and HR are two major pathways that promote survival following post-replication DNA damage (28,29). HR accurately repairs DSBs that arise during the mitotic cell cycle or those induced by radiotherapy (30,31). This process also releases replication forks that stall at damaged template DNA strands, by using intact sister chromatids as a template (32,33). The function of TLS is to release stalled replication forks by filling gaps on daughter strands that remain, following DNA replication caused by damage on the mother strand (34). Although cells deficient in Brca1 or Brca2, were not sensitive but slightly resistant to berberine, cells deficient in Xrcc2, a RAD51 paralog in HR (35), did exhibit sensitivity to berberine. Parp1 regulates several processes of DNA repair and also participates in HR. The sensitivity of Parp1−/− and Xrcc2−/− cells to berberine suggested that HR may participate in berberine-induced DNA repair. The importance of HR in repairing berberine-induced DNA damage has also been proved in yeast (9). Earlier studies have demonstrated that Brca1−/− and Brca2−/− cells were highly sensitive to several chemotherapeutic agents including cisplatin, camptothecin and olaparib, which separately stalled DNA replicative polymerases, via the formation of intra- and inter-strand crosslinks, inhibition of topoisomerase I and inhibition of Parp1 (36). In addition, previous studies have shown that Rad18−/− rather than Rev3−/− was more sensitive to camptothecin (37). These differences imply that the anticancer mechanisms of berberine are different from the aforementioned chemotherapeutic agents, although all of them are able to induce DSBs and HR repair. These results present that there are different DNA damage mechanisms between berberine and these agents, although certain studies considered that berberine may function as a topoisomerase I inhibitor like camptothecin (38).