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. 2014 Jul;5(7-8):285-92.
doi: 10.18632/genesandcancer.26.

Impact of DNA Repair Pathways on the Cytotoxicity of Piperlongumine in Chicken DT40 Cell-Lines

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Free PMC article

Impact of DNA Repair Pathways on the Cytotoxicity of Piperlongumine in Chicken DT40 Cell-Lines

Saki Okamoto et al. Genes Cancer. .
Free PMC article

Abstract

Piperlongumine is a naturally-occurring small molecule with various biological activities. Recent studies demonstrate that piperlongumine selectively kills various types of transformed cells with minimal toxicity to non-transformed cells by inducing a high level of reactive oxygen species (ROS). ROS generates various types of DNA lesions, including base modifications and single strand breaks. In order to examine the contribution of ROS-induced DNA damage to the cytotoxicity by piperlongumine, various DNA repair-deficient chicken DT40 cell-lines with a single DNA repair gene deletion were tested for cellular sensitivity to piperlongumine. The results showed that cell lines defective in homologous recombination (HR) display hyper-sensitivity to piperlongumine, while other cell lines with a deficiency in non-homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), Fanconi anemia (FA) pathway, or translesion DNA synthesis (TLS) polymerases, show no sensitivity to piperlongumine. The results strongly implicate that double strand breaks (DSBs) generated by piperlongumine are major cytotoxic DNA lesions. Furthermore, a deletion of 53BP1 or Ku70 in the BRCA1-deficient cell line restored cellular resistance to piperlongumine. This strongly supports the idea that piperlongumine induces DSB- mediated cell death. Interestingly, piperlongumine makes the wild type DT40 cell line hypersensitive to a PARP-inhibitor, Olaparib. The results implicate that piperlongumine inhibits HR. Further analysis with cell-based HR assay and the kinetic study of Rad51 foci formation confirmed that piperlongumine suppresses HR activity. Altogether, we revealed novel mechanisms of piperlongumine-induced cytotoxicity.

Keywords: BRCA1; BRCA2; chemotherapy; homologous recombination; oxidative stress; piperlongumine.

Figures

Figure 1
Figure 1. The cellular sensitivities of DNA repair deficient DT40 mutants to piperlongumine
a. Obtaining IC50 values of piperlongumine in each DNA repair –deficient cell-line. In each experiment, the relative viabilities are measured as N/N0, where N is the number of colonies at each dose of piperlongumine-treated cells and N0 is the mean colony number of non-treated controls. The surviving fractions are marked with red symbols and the mean value at each dose is represented by a black open circle. b. Piperlongumine specifically sensitizes HR-deficient DT 40 cells. DNA repair deficient cells were treated with various doses of piperlongumine for 24 h and grew in the methylcellulose- containing medium for 7 days at 39°C. After the Giemsa staining, the numbers of colonies formed were counted and IC50 was determined. The representative dose response curves used to determine IC50 were shown in Supplementary Figure S1. All experiments were performed in triplicate. The IC50 values were plotted as dots and the SEMs were shown as error bars; and the dotted vertical line represents the IC50 of the wild type (2.0 μM). The order of DT40 mutants in the graph is based on their IC50 values.
Figure 2
Figure 2. A deletion of 53BP1 or Ku70 abrogates the piperlongumine-induced cytotoxicity in brca1−/−. DT40 cell-line
Each cell-line was treated with the indicated doses of piperlongumine (PL) and the surviving fractions were obtained. The error bars represent SEM obtained from three independent experiments. While a single deletion mutant of 53BP1 or Ku70 did not show sensitivity to PL, a deletion of 53BP1 or Ku70 in the brca1−/− cell-line abolished the cellular sensitivity of PL in this mutant cell-line. The two graphs were generated from the same set of experiments. In order to better demonstrate the results, the experiments with 53BP1 and the ones with Ku70 were graphed separately.
Figure 3
Figure 3. Evidence for piperlongumine-induced DNA damage
a. Induction of Rad51 foci by piperlongumine. Wild type DT40 cells were stained with anti-RAD51 antibody after a 24 h exposure to 2 μM piperlongumine. Cells with more than three bright Rad51 foci were counted as positive. b. Piperlongumine induces chromosomal aberrations. Wild type DT40 cells were incubated for 24 h with indicated doses of piperlongumine. Number of chromosomal aberrations per 50 metaphase nuclei from the indicated cells was counted. Data are presented as mean ± SEM. * p-value<0.05, ** p-value<0.01.
Figure 4
Figure 4. Effect of PARP-inhibitor olaparib on the cellular sensitivity to piperlongumine in brca1−/− and brca2tr/− cell-lines
Cellular sensitivity of piperlongumine was investigated in the presence and absence of olaparib. Cells were treated with the indicated combinations and doses of chemicals for 24 hr and, after washing the drugs, the cells were grown for 7 days. a. brca1−/−., b. brca2tr/− and c. wild type. Piperlongumine was added at 1 μM and olaparib was at 25 nM in a and b, while piperlongumine was at 2 μM and olaparib was at 5 μM in c. Data were presented as mean ± SEM. * p-value<0.05, ** p-value<0.01.
Figure 5
Figure 5. Suppression of homologous recombination by piperlongumine
a. Cell-based homologous recombination (HR) assay in DT40. The expression vector encoding I-SceI is transfected to cells with SCneo (3′neo and S2neo) in the Ovalbumin locus. Black and white box represent 5′-untranslated region and coding regions of the neoR gene, respectively. The figure is not drawn to the scale. b. Pieprlongumine suppresses HR in DT40. Immediately after wild type DT40 cells were transfected with I- SceI expression vector, the cells were grown for 48 h in the RPMI medium in the presence and absence of 1 μM of piperlongumine. Then, the cells were diluted appropriately, seeded, and grown in the presence of 2 mg/ml G418 in 96-well plates. The recombination frequency was calculated by dividing the number of neomycin-resistant colonies by the number of the total colonies. Data are presented as mean ±SEM. * p-value<0.05. c. Impact of piperlongumine on the γ-ray-induced Rad51 foci formation. Wild type DT40 cells were treated with piperlongumine (PL) at 1 μM for 1 h. After removing piperlongumine, the cells were irradiated with γ-ray at 2 Gy. Foci-formations of Rad51 were examined at the indicated time points after the irradiation. Irradiated wild type cells without treatment with piperlongumine were used as a control. A cell containing more than three distinct foci was scored as positive. Each bar represents the results of scoring at least 50 cells. Data are presented as mean ±SEM.

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