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. 2014 Nov 5;9(11):e112220.
doi: 10.1371/journal.pone.0112220. eCollection 2014.

NPRL-Z-1, as a new topoisomerase II poison, induces cell apoptosis and ROS generation in human renal carcinoma cells

Szu-Ying Wu  1 , Shiow-Lin Pan  2 , Zhi-Yan Xiao  3 , Jui-Ling Hsu  4 , Mei-Chuan Chen  5 , Kuo-Hsiung Lee  6 , Che-Ming Teng  1
Affiliations

NPRL-Z-1, as a new topoisomerase II poison, induces cell apoptosis and ROS generation in human renal carcinoma cells

Szu-Ying Wu et al. PLoS One. .

Abstract

NPRL-Z-1 is a 4β-[(4"-benzamido)-amino]-4'-O-demethyl-epipodophyllotoxin derivative. Previous reports have shown that NPRL-Z-1 possesses anticancer activity. Here NPRL-Z-1 displayed cytotoxic effects against four human cancer cell lines (HCT 116, A549, ACHN, and A498) and exhibited potent activity in A498 human renal carcinoma cells, with an IC50 value of 2.38 µM via the MTT assay. We also found that NPRL-Z-1 induced cell cycle arrest in G1-phase and detected DNA double-strand breaks in A498 cells. NPRL-Z-1 induced ataxia telangiectasia-mutated (ATM) protein kinase phosphorylation at serine 1981, leading to the activation of DNA damage signaling pathways, including Chk2, histone H2AX, and p53/p21. By ICE assay, the data suggested that NPRL-Z-1 acted on and stabilized the topoisomerase II (TOP2)-DNA complex, leading to TOP2cc formation. NPRL-Z-1-induced DNA damage signaling and apoptotic death was also reversed by TOP2α or TOP2β knockdown. In addition, NPRL-Z-1 inhibited the Akt signaling pathway and induced reactive oxygen species (ROS) generation. These results demonstrated that NPRL-Z-1 appeared to be a novel TOP2 poison and ROS generator. Thus, NPRL-Z-1 may present a significant potential anticancer candidate against renal carcinoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effects of NPRL-Z-1 on cell viability in four human cancer cell lines.
(A) HCT 116, A549, ACHN, and A498 were treated with NRRL-Z-1 at various concentrations for 48 h and analyzed using the MTT assay. (B) A498 cells were treated with vehicle or etoposide at various concentrations for 48 h and analyzed using the MTT assay. (C) Fluorescence microscopy of untreated or NPRL-Z-1-treated A498 cells for 24 h followed by TUNEL staining (at 20× magnification). Data are expressed as the mean percentage of control ± S.D. of three independent experiments. * p<0.05,** p<0.01, and *** p<0.001 compared with the control group.
Figure 2
Figure 2. Effects of NPRL-Z-1 treatment on cell apoptosis induction and expression of apoptosis-related proteins in A498 cells.
(A) Cells were treated with DMSO or NPRL-Z-1 at various concentrations (1, 3, 5, and 10 µM) for 24 h. Formation of cytoplasmic DNA was quantitatively measured by cell death ELISAPLUS kit. Data are expressed as the mean percentage of control ± S.D. of three independent experiments. * P<0.05, and *** P<0.001 compared with the control group. A498 cells were incubated in the absence or presence of NPRL-Z-1 at various concentrations (0.3, 1, 3, and 10 µM) for 24 h (B) and 6 h (C), and cells were harvested and prepared for detection by Western blotting. (D) Cells were treated for indicated times, and the cell lysates were subjected to immunoblotting by using indicated antibodies.
Figure 3
Figure 3. Effects of NPRL-Z-1 on cell cycle distribution and expression of cell cycle-related proteins in A498 cells.
Cells were incubated with (A) DMSO or various concentrations of NPRL-Z-1 for 24 h and (B) DMSO or 10 µM NPRL-Z-1 for the indicated time periods. Cell cycle phase and cell apoptosis were determined by FACS as described in Materials and Methods. (C) A498 cells were incubated with DMSO or 10 µM NPRL-Z-1 for the indicated time periods. After treatment, cells were harvested and lysed for detection of the indicated proteins via western blotting. Data are expressed as the mean percentage of control ± S.D. of three independent experiments. *, # p<0.05, and ## p<0.01 compared with the control group.
Figure 4
Figure 4. Effects of NPRL-Z-1 on DNA DSBs and DNA checkpoint pathway.
(A) A498 cells were seeded and treated with NPRL-Z-1 or etoposide for 30 min and processed for the comet assay as detailed in Materials and Methods. (B) A498 cells were incubated with DMSO or 3 or 10 µM NPRL-Z-1 for the indicated time periods. After treatment, cells were harvested and lysed for detection of the expression of indicated protein via western blotting. N3, N10 and E25 indicated as NPRL-Z-1 3 µM, 10 µM and etoposide 25 µM, respectively.
Figure 5
Figure 5. Effects of NPRL-Z-1 on TOP2 expression.
(A) DNA relaxation assay. Lane 1: 0.3 pmol of negatively supercoiled DNA substrate and no protein; lane 2: DNA, TOP2, and DMSO; lanes 3–4: DNA, TOP2, and NPRL-Z-1; and lane 5: DNA, TOP2 and etoposide. (B) A498 cells were treated with NPRL-Z-1 or etoposide for 1 h to detect the depletion of free enzymes, TOP2α and TOP2β, using the band depletion assay. (C) A498 cells were treated with NPRL-Z-1 or camptothecin for 1 h to detect the depletion of free enzymes, TOP1, using the band depletion assay. (D) Restoration of depleted TOP2 expression. After treatment with NPRL-Z-1 or etoposide for 1 h, the medium was replaced with fresh growth medium and cells were incubated for another hour (R). Cells were then harvested and prepared for TOP2α and TOP2β detection via western blotting. N10 and E25 indicated as NPRL-Z-1 10 µM and etoposide 25 µM, respectively.
Figure 6
Figure 6. Effects of NPRL-Z-1 on TOP2cc formation in A498 cells.
A498 cells were treated with NPRL-Z-1 or etoposide for 30 min and the ICE assay was performed to detect TOP2–DNA adduct formation as described in Materials and Methods. TOP2-free form was partitioned into fractions between 1 and 4. TOP2αcc (A) and TOP2βcc (B) were trapped between fractions 5 and 8, respectively.
Figure 7
Figure 7. Effects of TOP2 in NPRL-Z-1-induced cell death and DNA damage signaling.
TOP2α or TOP2β siRNA was transfected to evaluate expression of TOP2α (A) and TOP2β (B), cell viability using the MTT assay (C and D), expression of apoptosis-related proteins (PARP and pro-caspase-3) (E and F) for 24 h, and DNA damage-related proteins (p-ATM, p53, and p-histone H2AX) (G and H) for 1 h in A498 cells. Data are expressed as the mean percentage of control ± S.D. of three independent experiments. *, & p<0.05, and **, ##, && p<0.01 compared with the treatment group. N3, N10 and E25 indicated as NPRL-Z-1 3 µM, 10 µM and etoposide 25 µM, respectively.
Figure 8
Figure 8. Effect of NPRL-Z-1 on cellular ROS accumulation in A498 cells.
A498 cells were incubated in the absence or presence of NPRL-Z-1 (10 µM) for indicated time. The fluorescent intensity of DCFH-DA was detected by flow cytometric analysis. ** P<0.01 compared with the 0.5 h-time point control group. ## P<0.05 compared with the 1 h-time point control group. && P<0.01 compared with the 3 hr-time point control group. (B) Different ROS scavengers were preincubated for 30 min and cell viability was determined by MTT assay. *** P<0.001 compared with the control group. # P<0.05 compared with the NPRL-Z-1-treated group. Treatment of NAC inhibited NPRL-Z-1-induced ROS generation (C, ** P<0.01 and ## P<0.01 compared with the NPRL-Z-1-treated group.). (D) NAC (10 mM) reduced the apoptosis-related results in NPRL-Z-1-treated cells for 24 h. E25 indicated as etoposide 25 µM.
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