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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 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.
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pone-0112220-g001: 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.

Mentions: Anticancer activity of NPRL-Z-1 was assessed in human colorectal cancer HCT 116 cells, human non-small cell lung carcinoma A549 cells, human renal carcinoma ACHN, and A498 cells. Cells were treated at various NPRL-Z-1 concentrations (0.3, 1, 3, 10, and 15 µM) for 48 h, and cell viability was determined using the MTT assay (Fig. 1A). NPRL-Z-1 inhibited cell viability in HCT116, ACHN, and A498 in a concentration-dependent manner, with IC50 values of 9.73 µM, 7.25 µM, and 2.38 µM, respectively. Therefore, the most potent cytotoxic effect of NPRL-Z-1 was observed in A498 cells. However, there was no significant decrease in cell viability among NPRL-Z-1-treated A549 cells even at a high concentration (15 µM). In addition, etoposide inhibited A498 cell viability in a concentration-dependent manner with an IC50 value of 39.52 µM (Fig. 1B). Moreover, NPRL-Z-1 significantly induced nuclear DNA fragmentation as determined by the TUNEL assay (Fig. 1C). In light field pictures, A498 cells appeared spindle-shaped, adhered to the surface of the culture plate and were growth confluent after 24 h incubation. After treatment with 10 µM NPRL-Z-1, prominent blebs on cell surface could be observed and the cells had started shrinking and rounding up, thus gradually detaching from the culture plate. Thus, these results demonstrated that NPRL-Z-1 induced cancer cell death and was 16-fold more potent than etoposide in A498 cells.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112220-g001: 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.
Mentions: Anticancer activity of NPRL-Z-1 was assessed in human colorectal cancer HCT 116 cells, human non-small cell lung carcinoma A549 cells, human renal carcinoma ACHN, and A498 cells. Cells were treated at various NPRL-Z-1 concentrations (0.3, 1, 3, 10, and 15 µM) for 48 h, and cell viability was determined using the MTT assay (Fig. 1A). NPRL-Z-1 inhibited cell viability in HCT116, ACHN, and A498 in a concentration-dependent manner, with IC50 values of 9.73 µM, 7.25 µM, and 2.38 µM, respectively. Therefore, the most potent cytotoxic effect of NPRL-Z-1 was observed in A498 cells. However, there was no significant decrease in cell viability among NPRL-Z-1-treated A549 cells even at a high concentration (15 µM). In addition, etoposide inhibited A498 cell viability in a concentration-dependent manner with an IC50 value of 39.52 µM (Fig. 1B). Moreover, NPRL-Z-1 significantly induced nuclear DNA fragmentation as determined by the TUNEL assay (Fig. 1C). In light field pictures, A498 cells appeared spindle-shaped, adhered to the surface of the culture plate and were growth confluent after 24 h incubation. After treatment with 10 µM NPRL-Z-1, prominent blebs on cell surface could be observed and the cells had started shrinking and rounding up, thus gradually detaching from the culture plate. Thus, these results demonstrated that NPRL-Z-1 induced cancer cell death and was 16-fold more potent than etoposide in A498 cells.

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