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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.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.

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 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.
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pone-0112220-g002: 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.

Mentions: The effects of NPRL-Z-1 on cell apoptosis induction were studied using a cell death detection ELISA kit. NPRL-Z-1 administration significantly triggered A498 cell apoptosis in a concentration-dependent manner (Fig. 2A). Upon apoptosis induction, the caspase cascade was also activated [28]. NPRL-Z-1 decreased pro-caspase-3, -8, and -9 expression and induced PARP and caspase-3, -8, and -9 cleavage (Fig. 2B). Furthermore, NPRL-Z-1 displayed a strong downregulatory effect on the Akt pathway. The phosphorylation of Akt, 4EBP1 and p70S6K were downregulated after NPRL-Z-1 treatment (Fig. 2C and 2D).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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pone-0112220-g002: 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.
Mentions: The effects of NPRL-Z-1 on cell apoptosis induction were studied using a cell death detection ELISA kit. NPRL-Z-1 administration significantly triggered A498 cell apoptosis in a concentration-dependent manner (Fig. 2A). Upon apoptosis induction, the caspase cascade was also activated [28]. NPRL-Z-1 decreased pro-caspase-3, -8, and -9 expression and induced PARP and caspase-3, -8, and -9 cleavage (Fig. 2B). Furthermore, NPRL-Z-1 displayed a strong downregulatory effect on the Akt pathway. The phosphorylation of Akt, 4EBP1 and p70S6K were downregulated after NPRL-Z-1 treatment (Fig. 2C and 2D).

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.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.

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