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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 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.
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pone-0112220-g004: 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.

Mentions: The comet assay was performed to determine whether NPRL-Z-1 induced DNA damage. The results showed that NPRL-Z-1 induced chromosomal DNA strand breaks in A498 cells (Fig. 4A). ATM phosphorylation at serine (Ser) 1981 is an indicator of DNA damage [31]. A previous study indicated that DNA DSBs could induce histone H2AX phosphorylation at Ser 139 [32]. NPRL-Z-1 treatment of A498 cells increased the levels of p-ATM, p-Chk2, and p-histone H2AX (Fig. 4B). Moreover, p53 expression and phosphorylation (Ser 15 and 20) were upregulated. These results suggested that NPRL-Z-1 was a potent DNA-damaging agent that triggered activation of checkpoint signaling.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112220-g004: 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.
Mentions: The comet assay was performed to determine whether NPRL-Z-1 induced DNA damage. The results showed that NPRL-Z-1 induced chromosomal DNA strand breaks in A498 cells (Fig. 4A). ATM phosphorylation at serine (Ser) 1981 is an indicator of DNA damage [31]. A previous study indicated that DNA DSBs could induce histone H2AX phosphorylation at Ser 139 [32]. NPRL-Z-1 treatment of A498 cells increased the levels of p-ATM, p-Chk2, and p-histone H2AX (Fig. 4B). Moreover, p53 expression and phosphorylation (Ser 15 and 20) were upregulated. These results suggested that NPRL-Z-1 was a potent DNA-damaging agent that triggered activation of checkpoint signaling.

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