Oxidative stress in NSC-741909-induced apoptosis of cancer cells
© Wei et al. 2010
Received: 23 December 2009
Accepted: 16 April 2010
Published: 16 April 2010
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© Wei et al. 2010
Received: 23 December 2009
Accepted: 16 April 2010
Published: 16 April 2010
NSC-741909 is a novel anticancer agent that can effectively suppress the growth of several cell lines derived from lung, colon, breast, ovarian, and kidney cancers. We recently showed that NSC-741909-induced antitumor activity is associated with sustained Jun N-terminal kinase (JNK) activation, resulting from suppression of JNK dephosphorylation associated with decreased protein levels of MAPK phosphatase-1. However, the mechanisms of NSC-741909-induced antitumor activity remain unclear. Because JNK is frequently activated by oxidative stress in cells, we hypothesized that reactive oxygen species (ROS) may be involved in the suppression of JNK dephosphorylation and the cytotoxicity of NSC-741909.
The generation of ROS was measured by using the cell-permeable nonfluorescent compound H2DCF-DA and flow cytometry analysis. Cell viability was determined by sulforhodamine B assay. Western blot analysis, immunofluorescent staining and flow cytometry assays were used to determine apoptosis and molecular changes induced by NSC-741909.
Treatment with NSC-741909 induced robust ROS generation and marked MAPK phosphatase-1 and -7 clustering in NSC-741909-sensitive, but not resistant cell lines, in a dose- and time-dependent manner. The generation of ROS was detectable as early as 30 min and ROS levels were as high as 6- to 8-fold above basal levels after treatment. Moreover, the NSC-741909-induced ROS generation could be blocked by pretreatment with antioxidants, such as nordihydroguaiaretic acid, aesculetin, baicalein, and caffeic acid, which in turn, inhibited the NSC-741909-induced JNK activation and apoptosis.
Our results demonstrate that the increased ROS production was associated with NSC-741909-induced antitumor activity and that ROS generation and subsequent JNK activation is one of the primary mechanisms of NSC-741909-mediated antitumor cell activity.
We recently identified a small molecule (oncrasin-1) through cell-based synthetic lethality screening that can effectively kill several lung cancer cell lines harboring mutant K-Ras genes . Subsequent analyses of oncrasin-1 analogues led us to identify several active compounds with similar chemical structures. NSC-741909 is one of the oncrasin-1 analogues that was highly active against several cell lines derived from lung, colon, breast, ovarian, and kidney cancers when tested in NCI-60 cancer cell lines by the Developmental Therapeutics Program at the National Cancer Institute. Using a reverse-phase protein microarray assay, we determined molecular changes in 77 protein biomarkers in an oncrasin-sensitive lung cancer cell line after treatment with NSC-741909 . These results showed that treatment with NSC-741909 induced persistent activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK, Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and that persistent JNK activation is associated with apoptosis induction by this compound . Further studies revealed that treatment with NSC-741909 suppressed MAPK phosphatase-1 expression and JNK dephosphorylation, in a dose-dependent manner . Those results suggest that inhibition of JNK dephosphorylation is one of the molecular mechanisms critical for the NSC-741909-induced sustained activation of JNK and cell death.
JNKs are activated by dual phosphorylation on the Thr-Pro-Tyr motif in the activation loop through mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7) and inactivated by dephosphorylation through a group of MAP kinase phosphatases . MAP kinase phosphatases (MKPs) are a group of dual-specificity phosphatases that inactivate MAPKs by dephosphorylating their threonine and tyrosine residues. At least 16 mammalian dual-specificity phosphatases that can dephosphorylate MAPKs have been identified . Their tissue-specific transcriptional regulation, expression patterns, substrate specificities, and subcellular localization play critical roles in controlling MAPK activity and signal transduction in each cell type . Accumulating evidence has demonstrated that, like other protein tyrosine phosphatases, the conserved catalytic cysteine residue in the active motif of MKPs is highly susceptible to reversible oxidation by local reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) [5, 6], which leads to inactivation of MKPs and activation of MAPKs [7–9]. ROS-mediated inhibition of MKPs is critical for TNFα-induced sustained activation of JNK and subsequent apoptosis . Interestingly, ROS were recently identified as common mediators of antibiotic-induced cell death in bacteria . Moreover, many anticancer drugs act as prooxidants, which may trigger the generation of free radicals, such as ROS or reactive nitrogen species [11, 12], and promote apoptosis. In fact, ROS-induced oxidative stress and cell death play important roles in the efficacy of many antineoplastic agents [13, 14].
To investigate whether oxidative stress is involved in the cytotoxicity of oncrasin compounds, we examined the production of ROS and its effects on JNK activation and cell death after treatment of oncrasin-sensitive and -resistant cells with NSC-741909. We found that ROS formation is an important component of NSC-741909-induced apoptosis. Furthermore, the NSC-741909-induced generation of ROS, cytotoxicity, and JNK activation, could be dramatically attenuated by some antioxidants, such as nordihydroguaiaretic acid, aesculetin, baicalein, and caffeic acid.
The human non-small cell lung carcinoma cell lines H460, H157, H322, and H1299 were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 mg/mL penicillin-streptomycin (all from Life Technologies, Gaithersburg, MD, USA). Normal bronchial epithelial cells (HBEC) were kindly provided by Dr. John Minna (Southwest Medical School, Dallas, TX) and were cultured in serum-free keratinocyte medium (Invitrogen Corporation, Carlsbad, CA). Cells were cultured at 37°C in a humidified incubator containing 5% CO2.
NSC-741909 (the structure was shown in additional file 1) was synthesized by Zhejiang Yuancheng MST Inc. (Hangzhou, China). This compound was 98.5% pure, as determined by high-performance liquid chromatography--mass spectrometry (LC/MS) analysis. The chemical structure was confirmed by nuclear magnetic resonance. N-acetylcysteine (NAC), rotenone, Nω-nitro-L-arginine methyl ester (L-NAME), diallyl sulfide (DSE), naproxen, oxypurinol, nordihydroguaiaretic acid (NDGA), baicalein, caffeic acid, MK886, and zileuton were purchased from Calbiochem (San Diego, CA, USA). Antibodies to the following proteins were used for Western blot analysis: JNK, phospho-JNK, phospho-c-Jun (Cell Signaling Technology, Danvers, MA, USA), poly-(ADP-ribose) polymerase (BD Biosciences Pharmingen, San Diego, CA, USA), MKP1 (Santa Crutz, CA, USA), MKP7 (Sigma-Aldrich, St. Louis, MO, USA), caspase-8 (Alexis Biochemicals, Farmingdale, NY, USA), β-actin, and hemagglutinin (HA) (Sigma-Aldrich, St. Louis, MO, USA). 2',7'-Dichlorofluorescein diacetate (H2DCF-DA) was purchased from Invitrogen Molecular Probes (Carlsbad, CA, USA).
The cell-permeable nonfluorescent compound H2DCF-DA was used for measuring intracellular ROS. Inside cells, H2DCF-DA is de-esterified to 2', 7'-dichlorofluorescein (H2DCF), which is further oxidized by ROS to fluorescent dichlorofluorescein (DCF) that remains inside the cells and can be quantified by flow cytometry, as described in the manufacturer's instructions. H2DCF-DA was dissolved in dimethylsulfoxide and diluted with phosphate-buffered saline (PBS) to a final concentration of 5 μmol/L. Cells were seeded at a density of 2.5 × 105 cells/well in six-well plates and allowed to grow overnight. The cells were treated either with different concentrations of NSC-741909 for 6 h or with 1 μM NSC-741909 for different time periods (0.5, 2, 4, 6 h). Subsequently, 5 μmol/L H2DCF-DA was added, and cells were incubated for 40 min at 37°C; cells were then returned to a prewarmed growth medium and incubated for 10 min at 37°C. Cells were harvested with trypsin and washed once with PBS, and the fluorescence intensity was determined using flow cytometry, with excitation and emission settings of 488 nm and 530 nm, respectively. The mean fluorescence peak was analyzed from the gated cell population of 10,000 cells. For the NSC-741909-antioxidant combination test, the antioxidants were added 30 min before NSC-741909. All experiments were performed three times. The flow cytometry assays were performed at the Flow Cytometry and Cellular Imaging Facility at The University of Texas M. D. Anderson Cancer Center.
Cells were seeded at a density of 1 × 104 cells/well in 96-well plates. After overnight incubation, the cells were treated with NSC-741909 (0.03 - 10 μM), either alone or in combination with different antioxidant compounds for 24 h. The antioxidants were added 30 min before NSC-741909 was added, and the inhibitory effects of NSC-741909, alone or in combination with the antioxidants, on cell growth were determined using the sulforhodamine B (SRB) assay, as described previously . We determined the relative cell viability by normalizing the cells to the dimethylsulfoxide-treated control cells, which was set at 100%. Each experiment was performed in quadruplicate and repeated for a total of at least three times.
The flow cytometry assay was performed as described previously . In brief, cells were seeded at a density of 2.5 × 105 cells/well in six-well plates and allowed to grow overnight. The cells were treated with NSC-741909 (1 μM) alone or in combination with different antioxidants for 24 h. The antioxidants were added to the cells 30 min before NSC-741909 was added. After treatment, the cells were harvested with trypsin, washed once with PBS, and fixed by incubation with 70% ethanol overnight at 4°C. Before flow cytometry analysis, cells were stained with propidium iodide (PI; 1 ml PI, 10 μl RNase, 9 ml PBS; final PI concentration of 50 μg/ml) for 30 min. A flow cytometry assay was used to measure the sub-G0/G1 cellular DNA content using Cell Quest software (Becton-Dickinson (Franklin Lakes, NJ, USA). All experiments were performed three times. The flow cytometry assays were performed in the Flow Cytometry and Cellular Imaging Facility at M. D. Anderson Cancer Center.
Cells were washed with cold PBS and subjected to lysis in Laemmli's lysis buffer. The protein concentration was determined using the Bradford method. Equal amounts of lysate (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Hybond-enhanced chemiluminescence membranes (GE Healthcare Life Sciences, Piscataway, NJ, USA). Membranes were then blocked with PBS containing 5% low-fat milk and 0.05% Tween (PBST) for 1 h and then incubated with primary antibodies overnight at 4°C. After being washed three times with PBST, membranes were incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were washed with PBST again and developed with a chemiluminescence detection kit (ECL kit; GE Healthcare Life Sciences). β-Actin was used as a loading control.
Cells were seeded at a density of 1 × 105 cells per well in 6-well plates containing a 1% gelatin-treated cover slide. Cells were allowed to grow overnight. Cells were treated with 1 μM NSC-741909 for different time periods as indicated. After the treatment, cells were washed with PBS twice, then fixed with 2% paraformaldehyde for 20 min, permeablized with 0.1% Triton-100 for 20 min, and blocked with 5% normal goat serum for 1 h. The slides were incubated with primary antibodies followed by FITC or Rhodamine-linked secondary antibodies. After washed with PBS thrice, the slides were taken out and mounted with Prolong Gold antifade reagent (Molecular Probes, Carsbad, CA, USA). The slides were read under Olympus fluorescence microscope (Olympus, Melville, NY, USA).
Statistical differences between treatment groups were assessed by analysis of variance (ANOVA) using StatSoft software (Tulsa, OK, USA). P values of < 0.05 were regarded as significant.
To determine whether treatment with NSC-741909 would generate oxidative stress in sensitive cells, we treated two sensitive lung cancer cell lines, H460 and H157, with 1 μM NSC-741909. Cells were stained with H2DCF-DA, and were examined for the production of ROS by measuring the cell population with positive DCF-derived fluorescence at various time points after the NSC-741909 treatment. Cells treated with solvent alone (dimethylsulfoxide) and stained with H2DCF-DA were used as controls. We found that treatment with NSC-741909 stimulated ROS generation in a time-dependent manner in both cell lines, in contrast to the control cells (Fig. 1C). An increase in the amount of ROS generated occurred as early as 30 min to 1 h after treatment and was as high as 6- to 8-fold above baseline levels after 6 h. Similar results were obtained with cells that were treated with the lead compound, oncrasin-1 (data not shown). We then evaluated the generation of ROS as a function of NSC-741909 concentration 6 h after treatment with NSC-741909. The result showed that the generation of ROS by NSC-741909 was dose-dependent and detectable at a dose of 50 nM in both cell lines (Fig. 1D).
Our previous studies have demonstrated that the reduction in cell viability caused by oncrasin compounds is mainly caused by apoptosis induction . To further evaluate whether NDGA blocks NSC-741909-mediated cell killing, we tested the effects of NDGA and NAC on apoptosis induction by NSC-741909. H460 cells were treated with 1 μM NSC-741909 for 24 h, with or without the prior addition of NDGA or NAC, and the percentage of apoptotic cells was determined by flow cytometry analysis.
The effect of NDGA on NSC-741909-induced apoptosis was further verified by Western blot analysis. Pretreatment of cells with NDGA (20 μM) markedly blocked the NSC-741909-induced activation of caspase-8 and cleavage of poly-(ADP-ribose) polymerase (Fig. 4C). However, pretreatment with NAC did not have a similar effect. Together, these results indicate that NDGA inhibits NSC-741909-mediated apoptosis.
Our results demonstrate that NSC-741909 can effectively induce ROS generation in oncrasin-sensitive, but not in the resistant human lung cancer cells. Blocking NSC-741909-induced ROS generation with some antioxidants, such as NDGA, aesculetin, baicalein, and caffeic acid, effectively blocked NSC-741909-induced cell death. Furthermore, these antioxidants also blocked JNK activation, demonstrating that ROS generation is one of the primary mechanisms by which NSC-741909 induces the sustained activation of JNK and apoptosis.
ROS are constantly generated and eliminated inside a cell. The balance of ROS can be dramatically affected by many environmental stimuli, including cytokines, growth factors, ultraviolet radiation, radiotherapy, and chemotherapeutic agents. ROS generation and subsequent oxidative damage to the cell membrane is one of the major mechanisms of radiotherapy-mediated apoptotic cell death . Similarly, many chemotherapeutic agents, including cisplatin , paclitaxel , doxorubicin [31, 32], and the histone deacetylase inhibitor suberoylanilide hydroxamic acid, induce ROS generation in target cells . Moreover, scavenging of ROS with antioxidants causes cells to resist apoptosis induced by gamma-irradiation and various chemotherapeutic agents .
Interestingly, ROS production is often elevated in oncogene-transformed cells. For example, transformation of cells with oncogenic Ras leads to increased production of O2-, which can be suppressed by the expression of dominant-negative isoforms of Ras or Rac1 [35, 36]. Similarly, increased Akt activity sensitizes cells to ROS-mediated apoptosis by increasing the intracellular concentration of ROS through increased oxygen consumption and inhibition of the expression of ROS scavengers downstream of FoxO, particularly manganese superoxide dismutase  and sestrin 3 . In addition, overexpression of growth factor receptors, such as insulin growth factor receptor , epidermal growth factor receptor , and vascular endothelial growth factor receptor , leads to increased generation of ROS. Those changes were frequently found in malignant cells.
Elevated levels of ROS in oncogene-transformed or tumor cells potentiate the oxidative stress-mediated activation of MAP kinases, particularly the JNK and p38 kinases, which sensitizes those cells to chemotherapeutic drug- and radiation-induced cell death . Thus, oxidative stress associated with increased activities of oncogenes and growth factor receptors represents a specific vulnerability of malignant cancer cells that can be selectively targeted by novel oxidative stress-inducing anticancer agents such as NSC-741909. It has been well documented that MAPKs, such as JNK, are redox sensitive and involved in apoptosis signaling [12, 43, 44]. There are two mechanisms of JNK activation: the earlier and transient activation occurs through the pro-inflammatory cytokine signaling cascade, and the delayed and sustained activation is mediated by ROS , which inactivate MAP kinase phosphatases by reacting with catalytic cysteine and causing their aggregation. Our results also showed that treatment with NSC-741909 induced clustering of MKP1 and MKP7 in sensitive cells, suggesting that the ROS-mediated inactivation of MKPs is a primary mechanism by which NSC-741909 activates JNK signaling pathway and exerts its antitumor cell effect. However, cellular levels of MKPs are likely not the critical factors for the sensitivity to NSC-741909 in lung cancer cells, because levels of MKPs in microarrays for lung cancer cell lines described here and that in the NCI's 60 cell line panel did not reveal obvious association between IC50s and MKP expression levels (Additional file 3). This may be explained by the fact that MKPs are down stream of ROS in inactivating JNK. Factors that directly contribute to ROS inductions might be more important for apoptosis induction by NSC-741909. Nevertheless, the underlying mechanisms or the sources of NSC-741909 induced ROS remain to be characterized.
Our results showed several antioxidants, including NDGA, aesculetin, baicalein, and caffeic acid, can block NSC-741909-induced ROS generation, JNK activation, and apoptosis, whereas the ROS generation was not affected by other antioxidants, such as NAC, rotenone, L-NAME, DSE, naproxen, and oxypurinol. Interestingly, NDGA, aesculetin, baicalein, and caffeic acid are all reported to inhibit LOXs through their antioxidant activity. Nevertheless, those antioxidants mediated antagonist effect could be LOX independent because LOX inhibitors MK886 and zileuton, which do not have any intrinsic antioxidant activity, were not effective in blocking the NSC-741909-mediated ROS generation, nor did LOX specific siRNAs block NSC-741909-induced ROS generation and cell killing (Additional file 2). In addition, NAC, which acts as a precursor of GSH synthesis, did not attenuate the NSC-741909-mediated ROS generation, which suggests that the cellular reduction and oxidation regulated by intracellular GSH may not be very important for the NSC-741909-induced ROS production and cell death effects.
Taken together, our results demonstrate that NSC-741909-induced apoptosis in human lung cancer cells is mediated by the generation of ROS. Blocking the formation of ROS could sufficiently inhibit the effects of NSC-741909, including JNK activation, cell growth suppression, and apoptosis. These results indicate that the oxidative stress-mediated sustained activation of JNK and subsequent induction of apoptosis is likely the primary mechanism by which NSC-741909 exerts its antitumor cell activity.
We thank Kate Newberry for editorial review of the manuscript and the Developmental Therapeutics Program of the National Cancer Institute for testing NSC-741909 on the NCI-60 cancer cell panels. This work was supported by National Cancer Institute grant R01 CA 092487 and RO1 CA 124951 (to B. Fang), Lockton Grant matching funds, National Cancer Institute Cancer Center Support Grant CA 16672 (to M. D. Anderson Cancer Center), and National Natural Science Foundation of China No.30973563 (to X Wei).
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