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

Wu SY, Pan SL, Xiao ZY, Hsu JL, Chen MC, Lee KH, Teng CM - PLoS ONE (2014)

Bottom Line: We also found that NPRL-Z-1 induced cell cycle arrest in G1-phase and detected DNA double-strand breaks in A498 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan.

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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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.
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pone-0112220-g005: 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.

Mentions: An in vitro DNA relaxation assay was performed to investigate whether TOP2 was involved in the NPRL-Z-1-induced DNA damage pathway. NPRL-Z-1 inhibited the ability of TOP2 to convert supercoiled DNA to relaxed DNA as well as etoposide (Fig. 5A). Furthermore, the band depletion assay was performed to detect free intracellular topoisomerases that were not covalently bound to cellular DNA. NPRL-Z-1 or etoposide treatment of A498 cells caused depletion of TOP2α and TOP2β, suggesting that NPRL-Z-1 increased intracellular accumulation of TOP2α- and TOP2β-mediated cleavable complexes (Fig. 5B). However, NPRL-Z-1 had no effect on TOP1–DNA complexes in A498 cells (Fig. 5C). The TOP1 poison camptothecin was used as a positive control. Moreover, after replacement of medium containing NPRL-Z-1 or etoposide with fresh growth medium, TOP2α and TOP2β expression was restored (Fig. 5D). These data clearly showed that NPRL-Z-1 dose-dependently stabilized TOP2 in A498 cells and the cleavable complexes were reversible.


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

Wu SY, Pan SL, Xiao ZY, Hsu JL, Chen MC, Lee KH, Teng CM - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221609&req=5

pone-0112220-g005: 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.
Mentions: An in vitro DNA relaxation assay was performed to investigate whether TOP2 was involved in the NPRL-Z-1-induced DNA damage pathway. NPRL-Z-1 inhibited the ability of TOP2 to convert supercoiled DNA to relaxed DNA as well as etoposide (Fig. 5A). Furthermore, the band depletion assay was performed to detect free intracellular topoisomerases that were not covalently bound to cellular DNA. NPRL-Z-1 or etoposide treatment of A498 cells caused depletion of TOP2α and TOP2β, suggesting that NPRL-Z-1 increased intracellular accumulation of TOP2α- and TOP2β-mediated cleavable complexes (Fig. 5B). However, NPRL-Z-1 had no effect on TOP1–DNA complexes in A498 cells (Fig. 5C). The TOP1 poison camptothecin was used as a positive control. Moreover, after replacement of medium containing NPRL-Z-1 or etoposide with fresh growth medium, TOP2α and TOP2β expression was restored (Fig. 5D). These data clearly showed that NPRL-Z-1 dose-dependently stabilized TOP2 in A498 cells and the cleavable complexes were reversible.

Bottom Line: We also found that NPRL-Z-1 induced cell cycle arrest in G1-phase and detected DNA double-strand breaks in A498 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan.

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.

Show MeSH
Related in: MedlinePlus