Physalin F Induces Cell Apoptosis in Human Renal Carcinoma Cells by Targeting NF-kappaB and Generating Reactive Oxygen Species

<div><h3>Background</h3><p>The aim of this study was to determine the molecular mechanisms of physalin F, an effective purified extract of <em>Physalis angulata</em> L. (Solanacae), in renal carcinoma A498 cells.</p> <h3>Methodology/Principal Findings</h3><p>Physalin F was observed to significantly induce cytotoxicity of three human renal carcinoma A498, ACHN, and UO-31 cells in a concentration-dependent manner; this was especially potent in A498 cells. The physalin F-induced cell apoptosis of A498 cells was characterized by MTT assay, nuclear DNA fragmentation and chromatin condensation. Using flow cytometry analysis, physalin F induced A498 cell apoptosis as demonstrated by the accumulation of the sub-G1 phase in a concentration- and time-dependent manner. Moreover, physalin F-mediated accumulation of reactive oxygen species (ROS) caused Bcl-2 family proteins, Bcl-2, and Bcl-xL degradation, which led to disruption of mitochondrial membrane potential and release of cytochrome <em>c</em> from the mitochondria into the cytosol. These effects were associated with induction of caspase-3 and caspase-9 activity, which led to poly(ADP-ribose) polymerase cleavage. However, the antioxidant <em>N</em>-acetyl-_L-cysteine (NAC) and glutathione (GSH) resulted in the inhibition of these events and reversed physalin F-induced cell apoptosis. In addition, physalin F suppressed NF-κB activity and nuclear translocation of p65 and p50, which was reversed by NAC and GSH.</p> <h3>Conclusion</h3><p>Physalin F induced cell apoptosis through the ROS-mediated mitochondrial pathway and suppressed NF-κB activation in human renal cancer A498 cells. Thus, physalin F appears to be a promising anti-cancer agent worthy of further clinical development.</p> </div

Effects of NPRL-Z-1 on cell cycle distribution and expression of cell cycle-related proteins in A498 cells.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112220#s2" target="_blank">Materials and Methods</a>. (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. *^{, #}<i>p</i><0.05, and ^##<i>p</i><0.01 compared with the control group.</p

Effects of NPRL-Z-1 treatment on cell apoptosis induction and expression of apoptosis-related proteins in A498 cells.

<p>(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 ELISA^PLUS kit. Data are expressed as the mean percentage of control ± S.D. of three independent experiments. * <i>P</i><0.05, and *** <i>P</i><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.</p

NPRL-Z-1, as a New Topoisomerase II Poison, Induces Cell Apoptosis and ROS Generation in Human Renal Carcinoma Cells

<div><p>NPRL-Z-1 is a 4<i>β</i>-[(4″-benzamido)-amino]-4′-<i>O</i>-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.</p></div

Effect of physalin F on NF-κB.

<p>A498 cells were incubated in the absence or presence of physalin F (10 µg/mL) for indicated time and detection the expression of phospho-IκBα and IκBα (A). (B) The nuclear and cytosol extract were prepared as indicated in Methods and Materials and were analyzed by Western blotting for the detection of specific protein, as indicated (p-p65, p65, p50, GAPDH, and nucleolin). (C) cells were exposed to physalin F (10 µg/mL) for 6 hr and nuclear extracts were incubated with a hot NF-κB probe (lane 2–4) or cold probe (lane 1, indicate “cold”) and demonstrate the specificity of the bands obtained on EMSA. The data are representative of three independent experiments. P indicates positive nuclear extract. ** <i>P</i><0.01 compared with the control group. (D) Effect of ROS scavengers on physalin F-inhibited NF-κB nuclear translocation in A498 cells. Cells were pretreated with NAC (10 mM) and GSH (3 mM) for 0.5 hr and then treated with 10 µg/mL of physalin F for 6 hr. The nuclear extract were prepared as indicated in Methods and Materials. The levels of p65 and p50 were detected by Western blotting.</p

Effects of NPRL-Z-1 on cell viability in four human cancer cell lines.

<p>(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. * <i>p</i><0.05,** <i>p</i><0.01, and *** <i>p</i><0.001 compared with the control group.</p

Effects of NPRL-Z-1 on TOP2cc formation in A498 cells.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112220#s2" target="_blank">Materials and Methods</a>. TOP2-free form was partitioned into fractions between 1 and 4. TOP2αcc (A) and TOP2βcc (B) were trapped between fractions 5 and 8, respectively.</p

Effect of physalin F on reduction of Δψ_m and release of cytochrome <i>c</i>.

<p>A498 cells were incubated in the absence or presence of physalin F (10 µg/mL) for indicated time, and cells were harvested and prepared for detection (A) mitochondria membrane potential by using FACScan analysis, (B) release of cytochrome <i>c</i> in cytosol (The fractions were collected by using the protocol of preparation of cytosolic and mitochondrial fractions as mentioned in Materials and Methods.), and (C) Bcl-2, Bcl-xL and Bax expression by using Western blotting analysis. * <i>P</i><0.05 compared with the 12 hr-time point control group. ^#<i>P</i><0.05 compared with the 18 hr-time point control group. ^&&<i>P</i><0.01 compared with the 24 hr-time point control group.</p

Effects of NPRL-Z-1 on TOP2 expression.

<p>(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.</p

Effects of NPRL-Z-1 on DNA DSBs and DNA checkpoint pathway.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112220#s2" target="_blank">Materials and Methods</a>. (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.</p