Agents that cause DNA damage have been widely used as anticancer drugs because DNA lesions can initiate DNA checkpoints that induce cell death. The results presented here indicate that QS-ZYX-1-61, a derivative of VP-16, was significantly more potent than VP-16 in suppressing the viability of A549 cells. Treatment of cells with QS-ZYX-1-61 led to a DNA damage response and a dramatic increase of apoptosis. Our results also suggest that QS-ZYX-1-61 may be a topoisomerase (topo) II targeting agent, as evidenced by relaxation assay and induction of reversible cleavable complexes. Moreover, blocking of p53, topo IIα, and topo IIβ greatly protected against caspase-3 activation, poly-ADP-ribose polymerase cleavage, and cell growth inhibition, indicating that QS-ZYX-1-61 acts through these proteins. These results support our conclusion that QS-ZYX-1-61 has potential as an anticancer agent because it causes accumulation of DNA cleavable complexes, with downstream consequences that include double-strand breaks and DNA damage response signaling for apoptosis. Taken together, our results indicate that QS-ZYX-1-61 is a novel DNA damaging agent and displays an outstanding activity that could be worthy of further investigation. (Cancer Sci 2012; 103: 80–87)
Many clinically approved anticancer agents target DNA.(1–4) These DNA-targeting agents can be classified as alkylating agents, antimetabolites, topoisomerase inhibitors, or radiomimetics.(1,5) Topoisomerases are essential for the modification of DNA topology and have been established molecular targets for several decades.(1,6,7) Topoisomerases relax the helical supercoiling of DNA that is generated during replication, transcription, and chromatin remodeling.(8,9)
Topoisomerase II (topo II) catalyzes an ATP-dependent reaction in which one DNA double helix is passed through another.(6) There are two topo II isozymes, topo IIα (170 kDa) and topo IIβ (180 kDa). The expression of topo IIα is high in the S phase and peaks in the late S/G2 phase of the cell cycle, whereas topo IIβ is expressed at a constant level throughout the cell cycle.(10)“Topo II poisons”, which are highly cytotoxic, stimulate and stabilize the formation of topo II–DNA cleavable complexes, leading to accumulation of these cleavable complexes.(11,12) In contrast, “topo II catalytic inhibitors” such as ICRF-187, disrupt enzyme activity without stabilizing the cleavable complex.(13,14) Etoposide (VP-16) is a topo II poison that has been used in the chemotherapy of non-small-cell lung cancer (NSCLC) and other cancers.(15–19) In previous studies it has been shown that etoposide induces double-strand breaks (DSBs) and triggers the DNA damage response (DDR).(20,21) Agents that induce DNA damage in cells can trigger a complex network known as the “checkpoint pathways”, thereby promoting cell cycle delay or arrest and allowing more time for DNA repair.(22,23) This intricate signaling network can be turned on by activation of ataxia telangiectasia mutated (ATM) protein kinase, which phosphorylates numerous downstream substrates.(24)
Lung cancer is the leading cause of cancer death worldwide. The standard dual agent chemotherapy improves survival rate.(25,26) Many clinical trials have tested the combination of cisplatin and etoposide with other drugs in treatment of NSCLC(27), and found the overall survival rate has not been improved significantly. QS-ZYX-1-61, a derivative of etoposide (VP-16), was synthesized in Dr. Kuo-Hsiung Lee’s Natural Products Research Laboratories (University of North Carolina, Chapel Hill, NC, USA). In previous studies, a series of 4′-O-demethylepipodophyllotoxins have been developed(28,29) and it was found that various C4 substitutions had important roles in the activity profiles of VP-16 analogues.(28) In the present study, we propose that QS-ZYX-1-61, a novel derivative of etoposide with a better performance, triggers a DDR followed by apoptosis in A549 cells. Our data suggest that QS-ZYX-1-61 is a novel DNA damage agent that could be applied as targeted therapeutic drug for NSCLC.
Materials and Methods
Cell line and reagents. A549 (p53 wild-type) lung adenocarcinoma cells, NCI-H226 (p53 mutant) lung squamous carcinoma cell line, and H1299 (p53 null) human NSCLC cell line were obtained from ATCC (Manassas, VA, USA). Cells were maintained in 10% FBS-supplemented RPMI-1640 medium (Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (Gibco) at 37°C in a humidified incubator containing 5% CO2. QS-ZYX-1-61, 4β-[(4″-benzylpiperdin-4-yl) amino]-4′-O-demethyl-epipodophyllotoxin (Fig. 1A), was obtained from Professor Kuo-Hsiung Lee (Natural Products Research Laboratories). Figure 1(B) shows the chemical structure of VP-16, which was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against various proteins were obtained from poly-ADP-ribose polymerase (PARP), Bcl-2, Bcl-xL, Bax, Chk2, p21, puma, and topo IIβ, and anti-mouse and anti-rabbit IgGs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). P-ATM (Ser1981), p-Chk2 (Thr68), p-p53 (Ser15), γH2AX, caspase 8, and caspase 9 were obtained from Cell Signaling Technology (Danvers, MA, USA). Topoisomerase IIα, topo I, and p53 were obtained from BD Biosciences (San Jose, CA, USA). Pan-actin was from Millipore (Billerica, MA, USA). Caspase 3 and survivin were obtained from Imgenex (San Diego, CA, USA) and Novus (Littleton, CO, USA), respectively.
Cell viability assay. Cell viability was determined using the MTT assay and carried out as described previously.(30)
Flow cytometry analysis. Cells were seeded in six-well plates (2.5 × 105/well) and treated with DMSO or QS-ZYX-1-61 at various concentrations for indicated times. Cells were washed with PBS, fixed in ice-cold 70% ethanol at −20°C overnight, and stained with propidium iodide (80 μg/mL) containing Triton X-100 (0.1%, v/v) and RNase A (100 μg/mL) in PBS. DNA content was analyzed with the FACScan and CellQuest software (Becton Dickinson, Mountain View, CA, USA).
Immunoblotting. A549 cells were seeded in dishes and allowed to attach overnight. The cells were treated with QS-ZYX-1-61 at indicated concentrations. After the indicated exposure time, cells were lysed and the immunoblotting was carried out as described previously.(30) Quantification of the density of bands was carried out using Gel-Pro Analyzer (version 3.1; Media Cybernetics, Bethesda, MD, USA).
Comet assay. Cells were treated for 30 min in 12 wells (1.5 × 105 cells/well) and the assay was carried out according to the published protocol.(30)
Topoisomerase II relaxation assay. The reaction was done according to the protocol from TopoGEN (Port Orange, FL, USA). After incubation at 37°C for 30 min, the reaction was terminated by adding 5 mL stopping buffer. The reaction products were analyzed by electrophoresis on 1% agarose gel using a Tris-borate/EDTA buffer at 1 V/cm, stained by ethidium bromide, and photographed using a short-wavelength UV lamp (ChemiImager 5500; Alpha Innotech, Santa Clara, CA, USA).
Band depletion assay. The cells were lysed with SDS sample buffer (4% SDS/2%-mercaptoethanol, 20% glycerol, 125 mM Tris HCl, pH 6.8) and were separated by 7.5% SDS/PAGE. The following procedures were the same as for the immunoblotting protocol.
Apoptosis assay. Drug-induced apoptotic cell death was assessed using the Cell Death Detection ELISA kit (Roche Diagnostics, Besel, Switzerland). Cells were treated with QS-ZYX-1-61 for 24 h. Both floating and adherent cells were collected and the assay was done according to the manufacturer’s instructions.
Stable expression of shRNA. Stable expressing topo IIα and IIβ shRNA A549 cells were kindly provided by Dr. T.K. Li (Department of Microbiology, National Taiwan University, Taipei, Taiwan). Cells were cultured in RPMI-1640 medium containing 10% FBS and 0.1 μM puromycin (Sigma-Aldrich).
Small interfering RNA transfection. The siRNA for p53, topo IIα, IIβ, and negative control were purchased from Invitrogen (Carlsbad, CA, USA). Transfection was done using Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions. Following transfection, cells were allowed to recover for 24 h then treated for another 24 h.
Statistics and data analysis. Each experiment was carried out at least three times, and representative data are shown. Data in bar graphs are given as the means ± SEM. Means were checked for statistical difference using the Student's t-test and P-values < 0.05 were considered significant (*P <0.05, **P <0.01, ***P <0.001).
Effect of QS-ZYX-1-61 on cell proliferation and cell cycle distribution. The cell growth inhibition activities of QS-ZYX-1-61 were assessed in three human NSCLC cell lines, A549, H1299, and NCI-H226. Etoposide (VP-16) was included as a positive control DNA-damaging agent. Cells were grown in the absence or presence of different concentrations of QS-ZYX-1-61 and VP-16 for 48 h, and cytotoxicity was measured by the MTT assay. Figure 2(A,B) shows that QS-ZYX-1-61 and VP-16 induced cell death in a concentration-dependent manner. Notably, cells were much more sensitive to QS-ZYX-1-61 than VP-16 (IC50 of QS-ZYX-1-61: A549, 0.71 ± 0.07 μM; H1299, 1.66 ± 0.11 μM; NCI-H226, 2.38 ± 0.95 μM; IC50 of VP-16: A549, 81.56 ± 1.84 μM; H1299, 56.95 ± 3.27 μM; NCI-H226, 49.98 ± 1.92 μM). Next, we investigated the mechanism of cell growth repression by QS-ZYX-1-61 by measuring cell cycle distribution with flow cytometry and propidium iodide staining in A549 cells. Figure 2(C,D) shows that QS-ZYX-1-61 induced significant accumulation of sub-G1 phase, suggesting that QS-ZYX-1-61 causes apoptosis in A549 cells.
QS-ZYX-1-61 induces apoptosis in A549 cells. Next, we evaluated the effect of QS-ZYX-1-61 on the induction of apoptosis by use of an enzyme immunoassay for histone-associated DNA fragments. Figure 3(A) shows that QS-ZYX-1-61 increased the amount of histone-associated DNA fragments in a concentration-dependent manner. Apoptosis may occur through caspase-dependent or caspase-independent mechanisms. Thus, we evaluated the activation of caspase-3, caspase-8, caspase-9, and PARP cleavage after treatment of cells with QS-ZYX-1-61 and VP-16. Figure 3(B,C) shows that QS-ZYX-1-61 induced caspase-3, caspase-8, caspase-9, and cleavage of PARP in a concentration- and time-dependent manner. Notably, the cells were much more sensitive to QS-ZYX-1-61 than VP-16 (Fig. 3B).
Role of P53 in QS-ZYX-1-61-induced apoptosis. Next, we evaluated the effect of this drug on p53 by treatment of A549 cells with individual agents at various concentrations for 24 h, followed by immunoblotting with various antibodies against p53 and its target proteins. Figure 4(A) shows that QS-ZYX-1-61 increased the level of p53 protein, although the p53 mRNA level remained unchanged (data not shown). Moreover, QS-ZYX-1-61 treatment increased the levels of phospho-p53 (Ser15) and p53 target genes except for Noxa. Bcl-2 family proteins regulate apoptosis and cell cycle control.(31) Thus, we assessed the effect of QS-ZYX-1-61 on expression of Bcl-2 family proteins and other anti-apoptotic proteins. QS-ZYX-1-61 had no significant effect on the expression of anti-apoptotic proteins (Bcl-2, Bcl-XL) or on the expression of a pro-apoptotic protein (Bax) (Fig. 4B). Survivin is a member of the inhibitor of apoptosis protein family and interacts with caspases to block apoptosis.(32) Our results indicate that QS-ZYX-1-61 downregulates survivin within 6 h (Fig. 4B). We further examined the effect of QS-ZYX-1-61 by studying the effect of siRNA-mediated knockdown of p53 on rescue from QS-ZYX-1-61-mediated apoptosis. Figure 4(C,D)shows that knockdown of p53 rendered A549 cells resistant to QS-ZYX-1-61-mediated apoptotic death (PARP cleavage and caspase-3 activation) and cell growth inhibition. These results suggest that p53 has an important role in QS-ZYX-1-61-induced apoptosis in A549 cells.
QS-ZYX-1-61 triggers DSBs and DNA damage checkpoints. The comet assay is a rapid, simple, and sensitive method for measuring DNA strand breaks. VP-16, a topo II-targeting anticancer drug that efficiently induces topo II-mediated DNA damage,(33) may be considered as a positive control. Figure 5(A) shows that QS-ZYX-1-61, like VP-16, induces chromosomal DNA strand breaks in a concentration-dependent manner. Activation of nuclear kinase ATM is one of the earliest signs of DNA damage(34) and activated ATM kinase is known to phosphorylate and activate numerous substrates.(24) Histone H2AX, one of its substrates, is phosporylated at Ser-139.(22) A previous study indicated that phosphorylation of histone H2AX (termed γH2AX) was a biomarker for drug-induced DNA damage at DSB sites.(35,36) Thus, we assessed the effect of QS-ZYX-1-61 on the level of γH2AX and DNA damage checkpoint kinases in A549 cells. Figure 5(B) shows that treatment of A549 cells with QS-ZYX-1-61 or VP-16 increased the levels of phospho-ATM, phospho-Chk2, and γH2AX. The results also indicate that QS-ZYX-1-61 is more potent than VP-16 (lanes 9 and 10), and that both agents induce activation of ATM kinase and its downstream protein targets. Taken together, our data indicate that QS-ZYX-1-61 is a potent DNA damaging agent that triggers the DNA damage checkpoint signaling pathway, involving ATM, Chk2, p53, and γH2AX.
QS-ZYX-1-61 inhibits topo II activity and induces reversible cleavable complexes. Next, we used a commercial assay for in vitro measurement of changes in supercoiled DNA following QS-ZYX-1-61 treatment. Figure 6(A) shows that QS-ZYX-1-61 inhibited the DNA relaxation activity of topo II. VP-16 is a known topo II inhibitor and is considered a topo II poison because it induces and stabilizes DNA cleavable complexes.(20) The results of our band-depletion assay indicated that QS-ZYX-1-61 decreased free topo IIα and IIβ expression in a concentration-dependent (Fig. 6B, left panel) and time-dependent (Fig. 6B, right panel) manner because of “trapping” of topo IIα and IIβ to DNA into protein–DNA complexes. However, QS-ZYX-1-61 had no effect on topo I–DNA complexes in A549 cells (Fig. 6C). The topo I poison camptothecin was used as the positive control.(37) Further experiments indicated that these complexes were reversible, as indicated by restoration of the original condition after replacement of the medium with fresh conditioned medium (Fig. 6D). Based on these findings, we conclude that QS-ZYX-1-61 and VP-16 reduce the intensity of the topo II protein band, and QS-ZYX-1-61 was more effective than VP-16 (Fig. 6B).
Topoisomerase IIβ is required for activation of p53 and induction of apoptosis. To validate whether topo II was required for the activation of ATM and downstream signaling, we examined the effect of shRNA-mediated knockdown of topo II isoforms (topo IIα and IIβ) on rescuing QS-ZYX-1-61-mediated apoptotic death in A549 cells. We transfected A549 cells with plasmids encoding shRNAs against individual topo II isoforms, then carried out clonal selection. This shRNA knockdown was highly specific, as indicated by the absence of cross-silencing of other topo II isoforms (α and β) (Fig. 7A). The results indicate that QS-ZYX-1-61 retains its ability to trap the topo II–DNA cleavable complex in the presence of topo IIα and IIβ deficiency (Fig. 7B). In addition, the knockdown of topo IIβ led A549 cells to reduce the activation of QS-ZYX-1-61-mediated checkpoint pathways (p-ATM, p-p53) and DNA DSBs (Fig. 7C), whereas the effect on topo IIα was negligible. As there exists a mechanistic link between checkpoint pathway activation and the topo II inhibition, we further examined whether topo II inhibition was the underlying mechanism of QS-ZYX-1-61-induced cell death. Figure 7(D) shows that topo IIα and IIβ knockdown largely abolished caspase-3 activation and PARP cleavage, but that topo IIβ had a greater effect on this protection. Moreover, blockade of topo IIα and IIβ expression also protected A549 cells from QS-ZYX-1-61-induced cell growth inhibition (Fig. 7E,F). Overall, these results suggest that topo IIβ plays a more crucial role than topo IIα in QS-ZYX-1-61-induced activation of DNA damage signaling and apoptotic cell death.
Etoposide is currently approved for treatment of a wide variety of cancers, including small-cell lung cancer,(19,38) NSCLC,(19,27) testicular cancer,(19) and lymphomas.(19,39) Despite the crucial clinical role of etoposide, there are problems in its use in the treatment for human cancers,(40,41) motivating the search for additional anticancer agents. The present study indicates that QS-ZYX-1-61, an analogue of etoposide, suppressed the proliferation of A549 cells at least in part by stimulation of checkpoint kinases, leading to p53 activation, then caspase-3-dependent apoptosis. Our results also indicate that QS-ZYX-1-61 has 100-fold greater potency than VP-16 in A549 cells. Furthermore, our lactate dehydrogenase cytotoxicity assay confirmed that QS-ZYX-1-61 inhibited cell growth and stimulated apoptosis without induction of necrosis (Fig. S1).
Previous research of multiple types of cancer indicated that the level of survivin expression correlates with poor prognosis and predicts response to diverse anticancer therapies,(42) and that reducing the expression of survivin sensitizes cells to lung cancer treatment.(43,44) Our results show that QS-ZYX-1-61 downregulates the expression of the anti-apoptotic protein, survivin, but had no effect on the protein levels of Bax, Bcl-2, and Bcl-XL. Several mechanisms have been proposed to explain the effect of DNA damaging agents on the sensitization or chemosensitization of cancer cells.(45,46) In addition, some studies have shown that p53 is stabilized in response to DNA damaging agents.(47,48) Our results indicate that QS-ZYX-1-61 causes DDR, leading to accumulation of p53 protein, increased expression of p53 target genes (p21 and puma), and induction of apoptosis signaling pathways. Moreover, we found that p53 silencing by p53 siRNA protects cells from the effect of QS-ZYX-1-61 on p21 upregulation, survivin downregulation, PARP cleavage, and caspase-3 activation (Fig. 4C). It has been reported that loss of p53 function in cancer cells might lead to induction of survivin and resistance to DNA damaging agents.(49) Based on previous findings and our current results, p53 plays a crucial role in the QS-ZYX-1-61-mediated apoptosis of A549 cells.
DNA damage responses, generated by increased levels of topo II–DNA covalent complexes, are powerful activators and further engage a signal amplification cascade.(50) Cells can undergo p53-dependent and p53-independent signaling in response to DNA damage.(51) The relationship between p53 status and response to topo II inhibitor raised many questions and led to diverse conclusions.(52) Previous studies have shown that wild-type p53 accelerates cell death induced by DNA damaging agents in both normal and cancer cells.(53) Moreover, various chemotherapeutic agents can induce cell death in tumor cells through p53-independent pathways,(54,55) suggesting that those cells do not loss their ability to undergo apoptosis completely and they can activate p53-independent apoptosis through a backup system. Our results showed that QS-ZYX-1-61 can cause cell death efficiently in wild-type p53 (A549), whereas higher IC50 levels of the drug were shown in p53-mutant (NCI-H226), and p53-null (H1299) cell lines (Fig. 2A). We also carried out siRNA-mediated knockdown of topo IIα and IIβ in H1299 and NCI-H226 cells and found either topo IIα or IIβ-knockdown cells can reduce growth inhibition induced by QS-ZYX-1-61 (Fig. S2). In addition, the differential effect of QS-ZYX-1-61 was also observed in prostate cancer cells (DU145, PC3, and LNCaP) with different p53 status (Fig. S3). Such findings suggested that the direct function of QS-ZYX-1-61 in targeting topo II seems to be the crucial role in determining cellular fate, and wild-type p53 protein acts as a downstream effector in cells to facilitate the cell death triggered by QS-ZYX-1-61.
QS-ZYX-1-61 has the ability to interfere with topo II by induction of reversible cleavage complexes in a reaction, therefore, we propose that QS-ZYX-1-61 might exert its anticancer activity through topo II. Surprisingly, we also found that QS-ZYX-1-61-induced apoptotic markers are reduced more in topo IIβ-deficient A549 cells than in topo IIα-deficient A549 cells. Previous studies have reported that topo IIβ (rather than topo IIα) cleavage complexes are rapidly converted into DNA DSBs through proteasomal degradation, suggesting a preferential role for topo IIβ in DNA DSB-mediated apoptosis.(56,57)
In conclusion, our results indicate that QS-ZYX-1-61 mediates apoptosis through topo II in A549 cells and exhibits strong antitumor activities in NSCLC cells. These antitumor activities include trapping of topo II to the topo II–DNA complex, activation of DNA damage pathways, and post-translational upregulation of the apoptotic regulator, p53. Taken together, our results provide compelling evidence that QS-ZYX-1-61 has great potential as an individual agent or in combination with other drugs in the treatment of NSCLC.
We would like to give our thanks to Ting-Hsiang Huang for technical assistance. This work was supported by the National Science Council (NSC 98-2320-B-002-009-MY3).