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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan (Y.-L.H., P.-L.K., Y.-T.H.); and Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan (C.-Y.C., C.-C.L.)
Received November 20, 2005; accepted April 20, 2006.
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). Nonsmall cell lung cancers commonly develop resistance to radiation and chemotherapy and often present at stages too late for surgical intervention. Since current treatment modalities are inadequate, novel therapies are needed to reduce the effects of the increasing incidence in pulmonary neoplasm (Raez and Lilenbaum, 2004; Kelly, 2005).
The tumor suppressor protein p53 is targeted by a wide variety of intracellular and extracellular stimuli, such as withdrawal of growth factors, hypoxia, irradiation, chemicals, and defects in nucleotide synthesis (Harris and Levine, 2005). The activation of p53 leads, primarily through its transcriptional function, to either apoptosis, eliminating those cells harboring severely damaged DNA, or growth arrest, allowing damaged DNA to be repaired and thereby suppressing tumor formation (Harris Robles et al., 2002; Levine, 2005). Stability and activity of p53 are believed to be regulated in part by posttranslational modifications, such as phosphorylation and acetylation. Phosphorylation on NH2-terminal residues, especially Ser15, Thr18, Ser20, and Ser37, is believed to affect interaction with the negative regulator MDM2 and hence contribute to the stabilization of p53. Phosphorylation on COOH-terminal Ser315 and Ser392 in particular is believed to enhance the specific DNA binding of p53 in vitro (Xu, 2003; Bode and Dong, 2004).
c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase family protein kinases, is an important member of the signal transduction pathway that transduces extracellular signals in intracellular responses and has been implicated in a wide array of physiological processes, including cell growth, differentiation, and apoptosis (Liu and Lin, 2005). JNK can induce apoptosis in response to a variety of stresses. The proposed mechanisms of this effect are the change of gene expression as well as eliciting its phosphorylation activity to several specific substrates (Liu and Lin, 2005). Activated JNK phosphorylates and stabilizes p53 by abrogating MDM2 association (Fuchs et al., 1998; Buschmann et al., 2000). Recent studies have shown that JNK activation is required for stress-induced release of mitochondrial cytochrome c and for apoptosis mediated by the mitochondrial caspase-9 pathway (Chen et al., 2003; Gao et al., 2005; Zu et al., 2005). Several proapoptotic factors have indicated that phosphorylation by JNK enhances proapoptotic activity (Liu and Lin, 2005; Yoshida et al., 2005). In addition, phosphorylation of prosurvival Bcl-2 by JNK disrupts the binding motif of Bcl-2 and makes it unable to antagonize Bax (Zu et al., 2005).
Plumbagin, a quinonoid constituent isolated from the root of Plumbago zeylanica L., has been shown to exert anticarcinogenic, antiatherosclerotic, and antimicrobial effects (Mossa et al., 2004; Srinivas et al., 2004; Ding et al., 2005; Hsieh et al., 2005). It exhibits an inhibitory effect on carcinogenesis in the intestines, causes cytogenetic and cell cycle changes in mouse Ehrlich ascites carcinoma, and possesses antiproliferation activity in human cervical cancer cells (Singh and Udupa, 1997; Sugie et al., 1998; Srinivas et al., 2004). In this study, we determined the cell growth inhibition activity of plumbagin by using in vitro and in vivo experimental models and examined its effect on cell cycle distribution and apoptosis in human nonsmall cell lung cancer cell A549. Furthermore, to establish plumbagin's anticancer mechanism, we assayed the levels of cell cycle control- and apoptosis-related molecules, which are strongly associated with the programmed cell death signal transduction pathway and affect the chemosensitivity of tumor cells to anticancer agents.
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-actin, cyclinB1, Cdc2, Cdc25C, p21, Bax, Bak, Bcl-2, phospho-Bcl-2, and Bcl-XL were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies to p53, phospho-p53, MDM2, JNK, phospho-JNK, phospho-Cdc2, phospho-Cdc25C, and cytochrome c were obtained from Cell Signaling Technology (Beverly, MA). The pCMV and pCMV-p53mt135 plasmids were supplied by CLONTECH (Palo Alto, CA). Lipofectamine 2000 reagent was obtained from Life Technologies, Inc. (Rockville, MD). COS cells plus UV irradiation cell extract was obtained from Laprice (Brussels, Belgium). MCF-7 cells plus UV irradiation cell extract was obtained from Active Motif (Carlsbad, CA). Cell Culture. A549 (American Type Culture Collection CCL185; American Type Culture Collection, Manassas, VA) was maintained in RPMI 1640 supplemented with 10% FBS, 10 U/ml penicillin, 10 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. IMR-90 (American Type Culture Collection CCL-186) fibroblast cells were cultured in minimum essential medium (Eagle) with Earle's balanced salt solution, 2 mM L-glutamine, 1.5 mg/ml sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 10 U/ml penicillin, 10 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 10% fetal calf serum. Both cell lines were cultured in monolayer culture at 37°C and 5% CO2.
Cell Proliferation and Clonogenic Assay. Inhibition of cell proliferation by plumbagin was measured by sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) assay. In brief, cells were plated in 96-well culture plates (1 x 104 cells/well). After 24-h incubation, the cells were treated with plumbagin (0, 2.5, 5, 10, and 20 µM) for 6, 12, 24, and 48 h. An amount of 50 µl of XTT test solution, which was prepared by mixing 5 ml of XTT-labeling reagent with 100 µl of electron coupling reagent, was then added to each well. After 4 h of incubation, the absorbance was measured on an enzyme-linked immunosorbent assay reader (Multiskan EX; Thermo Electron, Waltham, MA) at a test wavelength of 492 nm and a reference wavelength of 690 nm.
To determine the long-term effects, cells were treated with plumbagin at various concentrations for 1 h. After being rinsed with fresh medium, cells were allowed to grow for 14 days to form colonies that were then stained with crystal violet (0.4 g/l; Sigma). Clonogenic assay was used to elucidate the possible differences in long-term effects of plumbagin in A549 and IMR-90 cells.
Cell Cycle Analysis. To determine cell cycle distribution analysis, 5 x 105 cells were plated in 60-mm dishes and treated with plumbagin (0, 10, and 20 µM) for 6 h. After treatment, the cells were collected by trypsinization, fixed in 70% ethanol, washed in phosphate-buffered saline, resuspended in 1 ml of phosphate-buffered saline containing 1 mg/ml RNase and 50 µg/ml PI, incubated in the dark for 30 min at room temperature, and analyzed by EPICS flow cytometer. The data were analyzed using Multicycle software (Phoenix Flow Systems, San Diego, CA).
Apoptosis Assay. Cells (1 x 106) were treated with vehicle alone (0.1% DMSO) and various concentrations of plumbagin for indicated times and then collected by centrifugation. Pellets were lysed by DNA lysis buffer (10 mM Tris, pH 7.5, 400 mM EDTA, and 1% Triton X-100) and then centrifuged. The supernatant obtained was incubated overnight with proteinase K (0.1 mg/ml), then with RNase (0.2 mg/ml) for 2 h at 37°C. After extraction with phenol/chloroform (1:1), the DNA was separated in 2% agarose gel and visualized by UV after staining with ethidium bromide.
Quantitative assessment of apoptotic cells was assessed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method, which examines DNA strand breaks during apoptosis by using BD ApoAlert DNA Fragmentation Assay Kit. In brief, cells were incubated with 0, 10, and 20 µM plumbagin for the indicated times. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. After being washed, the cells were incubated with the reaction mixture for 60 min at 37°C. The stained cells were then analyzed with an EPICS flow cytometer and a fluorescence microscope at 20x magnification.
Assay for Caspase-9 Activity. The assay is based on the ability of the active enzyme to cleave the chromophore from the enzyme substrate of caspase-9, LEHD-pNA (Ac-Leu-Glu-His-Asp-pNA). Cell lysates were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM HEPES, 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, and 0.1% CHAPS, pH 7.4) for 2 h at 37°C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percentage of change in activity compared with the untreated control.
Mitochondrial Membrane Potential Assay. We used mitochondrial-specific cationic dye JC-1 (Invitrogen, Carlsbad, CA), which undergoes potential-dependent accumulation in the mitochondria. It is a monomer when the membrane potential (
) is lower than 120 mV and emits a green light (540 nm) following excitation by blue light (490 nm). At higher membrane potentials, JC-1 monomers convert to J-aggregates that emit a red light (590 nm) following excitation by green light (540 nm). Cells were seeded in a 96-well plate. Following treatment with various concentrations of plumbagin for 8 and 12 h, cells were stained with 25 µM JC-1 for 30 min at 37°C. Fluorescence was monitored with the fluorescence plate reader at wavelengths of 490 nm (excitation)/540 nm (emission) and 540 nm (excitation)/590 nm (emission) pairs. Changes in the ratio between the measurement at test wavelengths of 590 nm (red) and 540 nm (green) fluorescence intensities are indicative of changes in the mitochondrial membrane potential (Martin and Forkert, 2004).
Immunoprecipitation/Immunoblot and JNK Activity Assays. Cells were treated with 20 µM plumbagin in the absence or presence of JNK inhibitors for specified intervals of time. Mitochondrial and cytoplasmic fractions were separated using Cytochrome c Releasing Apoptosis Assay Kit (BioVision, Mountain View, CA). For immunoblotting, the cells were lysed on ice for 40 min in a solution containing 50 mM Tris, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM Na3VO4, 2 mM EGTA, 12 mM -glycerolphosphate, 10 mM NaF, 16 µg/ml benzamidine hydrochloride, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was centrifuged at 14,000g for 15 min, and the supernatant fraction was collected for immunoblotting. Equivalent amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis (10-12%) and transferred to polyvinylidene difluoride membranes. After blocking for 1 h in 5% nonfat dry milk in Tris-buffered saline, the membrane was incubated with the desired primary antibody for 1 to 16 h. The membrane was then treated with appropriate peroxidase-conjugated secondary antibody, and the immunoreactive proteins were detected using an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions.
For association of p53 and MDM2, cell lysates (300 µg) were incubated with 10 µl of anti-MDM2 for 1 h at 4°C. Immunocomplexes were resolved by 7.5% SDS-polyacrylamide gel electrophoresis. Association of MDM2 with p53 was detected by incubating the blots with anti-MDM2 and anti-p53 antibodies as described above. The JNK mitogen-activated protein kinase activities were determined using kits from Cell Signaling Technology according to the manufacturer's instructions.
Stable Transfection. Transfection of A549 cells was carried out using Lipofectamine 2000 reagent (Life Technologies). A549 cells were exposed to the mixture of Lipofectamine 2000 reagent and pCMV-p53mt135 plasmid or empty vector for 6 h. After transfection, cells resistant to neomycin were selected by incubating with medium containing 1 mg/ml G418 (Life Technologies), individual A549 clones were isolated and tested for constitutive p53 expression. The p53-positive A549 cells were selected and maintained in the presence of G418 (400 µg/ml), as were p53-negative control cells (Xu et al., 2003).
In Vivo Tumor Xenograft Study. Male nude mice [6 weeks old; BALB/cA-nu (nu/nu)] were purchased from the National Science Council Animal Center (Taipei, Taiwan) and maintained in pathogen-free conditions. A549 cells were injected s.c. into the flanks of nude mice (5 x 106 cells in 200 µl). Tumors were allowed to develop for 
20 days until they reached 75 mm3, when treatment was initiated. Thirty mice were randomly divided into two groups. The mice in the plumbagin-treated group were i.p. injected with plumbagin in 25% polyethylene glycol (2 mg/kg body weight) in a 0.2-ml volume. The control group was treated with an equal volume of vehicle. After transplantation, tumor size was measured using calipers, and tumor volume was estimated according to the formula: tumor volume (millimeters cubed) = L x W2/2, where L is the length, and W is the width.
Analysis of apoptotic cells in formalin-fixed specimens was by BD ApoAlert DNA Fragmentation Assay Kit as described above. The apoptotic cells were detected using a fluorescent microscope at 20x magnification.
Statistical Analysis. Data were expressed as means ± S.D. Statistical comparisons of the results were made using analysis of variance. Significant differences (p < 0.05) between the means of control and plumbagin-treated cells were analyzed by Dunnett's test.
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; Shin et al., 1975). Figure 1, B and C, shows the effects of plumbagin on the relative clonogenicity of the control and the plumbagin-treated A549 cells. Clonogenicity of A549 cells was reduced in a dose-dependent manner after exposure to plumbagin. To examine the selection of plumbagin-mediated cell proliferation inhibition, we also evaluated the effect of plumbagin in the normal lung cell line, IMR-90. The results showed that treatment of IMR-90 cells with plumbagin failed to affect the cell proliferation at any of the examined time points (Fig. 1D). In addition, plumbagin also failed to affect the colony formation in IMR-90 cells (Fig. 1, E and F). This result demonstrated that plumbagin possessed selectivity between normal and cancer cells.
Plumbagin Induces Cell Cycle Arrest and Apoptosis in A549 Cells. To examine the mechanism responsible for plumbagin-mediated cell proliferation inhibition, cell cycle distribution was evaluated using flow cytometric analysis. The results showed that treating cells with plumbagin caused a significant inhibition of cell cycle progression in A549 cells at 6 h (Fig. 2A), resulting in a clear increase of the percentage of cells in the G2/M phase compared with the control.
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To examine the selection of plumbagin-mediated G2/M phase arrest and apoptosis, we also evaluated the effects of plumbagin in IMR-90. The results showed that treatment of IMR-90 cells with plumbagin failed to affect the distribution of cell cycle in IMR-90 cells after 6 h of treatment (Fig. 2G). In addition, plumbagin also failed to induce apoptosis in IMR-90 cells after 24 and 48 h of treatment (Fig. 2H). This result demonstrated that plumbagin possessed selectivity between normal and cancer cells.
Plumbagin Increases the Expression of p53 and Phosphorylated p53 (Ser15 and Ser392) and Regulates the Levels of Cell Cycle-Related Molecules in A549 Cells. Because our studies have shown that plumbagin treatment of A549 cells results in G2/M phase cell cycle arrest, we examined the effect of plumbagin on cell cycle-regulatory molecules, including p53, p21, cyclinB1, Cdc25C, and Cdc2. We first assessed the status of p53 in plumbagin-treated A549 cells. Exposure of cells to 20 µM plumbagin enhanced the phosphorylation of p53 on Ser15 and Ser392 (Fig. 3A) without any phosphorylation on serine residues 6, 9, 20, and 46. Plumbagin treatment was also associated with an increase in cells' levels of both p53 and its downstream target, p21 (Fig. 3A). In addition, the association of p53 and MDM2 decreased in a time-dependent manner in plumbagin-treated A549 cells, as detected by immunoprecipitation assay (Fig. 3B).
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Plumbagin Induces the Execution of Apoptosis through Activation of the Mitochondrial Pathway. To investigate the mitochondrial apoptotic events involved in plumbagin-induced apoptosis, we first analyzed the changes in the levels of proapoptotic proteins Bax, Bak, and Bcl-Xs, and antiapoptotic proteins Bcl-2 and Bcl-XL. Western blot analysis showed that treatment of A549 cells with plumbagin increased Bax, Bak, and Bcl-Xs protein levels (Fig. 3A). In contrast, plumbagin decreased Bcl-2 and Bcl-XL levels, which led to an increase in the proapoptotic/antiapoptotic Bcl-2 ratio (Fig. 4A). In addition, plumbagin also increased phosphorylation of Bcl-2 (Ser70) in A549 cells.
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m) using the mitochondria-specific dye JC-1. We investigated mitochondrial dysfunction by measuring m in plumbagin-treated A549 cells at 8 and 12 h (Fig. 4B). Cytosolic extracts were prepared under conditions to preserve the mitochondria, and cytosolic cytochrome c protein levels were measured by immunoblotting analysis. Figure 4C shows that the cytosolic fraction from untreated A549 cells contained no detectable amounts of cytochrome c, whereas it did become detectable after 48 h of 20 µM plumbagin treatment in A549 cells (Fig. 4C).
Hallmarks of the apoptotic process include the activation of cysteine proteases, which represent both initiators and executors of cell death. Upstream caspase-9 activities increased significantly, as shown by the observation that treatment with plumbagin increased caspase-9 activity in A549 cells. This is consistent with the release of cytochrome c into the cytosol (Fig. 4D).
Plumbagin Induces the Activation of JNK. Figure 5A shows that activation (phosphorylation) of JNK was evident as early as 1 h after plumbagin treatment and persisted for the duration of the experiment. On the other hand, the expression of JNK (unphosphorylated form) was not altered by plumbagin treatment. Plumbagin-mediated activation of JNK was additionally confirmed by determining phosphorylation of one of its substrates, c-jun. As shown in Fig. 5B, in comparison with the control, the Ser63 phosphorylation of c-jun increased after A549 cells were exposed for 1 h to 20 µM plumbagin. Phosphorylation of c-jun increased relative to the control at all four time points (Fig. 5B).
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The Role of p53 in Plumbagin-Mediated Cell Cycle Arrest and Apoptosis. To further define the role of p53 in plumbagin-induced cell cycle arrest and apoptosis, we transfected pCMV-p53mt135 plasmid containing the gene encoding a dominant-negative mutation of p53 that blocks normal p53 activity (Hsu et al., 2005). Overexpression of mutant p53 protein in cells transfected with the dominant-negative p53 mutant plasmid was verified by Western blot using antibody against human p53 (recognizing both wild- and mutant-type p53) (Fig. 6A). Cells expressing p53 mutant were subsequently used to document plumbagin-mediated cell cycle arrest and apoptosis. As shown in Fig. 6B, the inhibition of p53 activity was accompanied by a reduction in the sensitivity of A549 cells to plumbagin-mediated G2/M arrest. The expression of p21 was also inhibited in pCMV-p53mt-transfected A549 cells (Fig. 6C). Furthermore, compared with vehicle-treated cells, induction of apoptosis induced by 20 µM plumbagin decreased from 36.5% in A549 cells to 13.4% in p53 mutant cells after a 24-h treatment (Fig. 6D). However, the inhibition of p53 did not completely abrogate plumbagin-mediated cell cycle arrest and apoptotic death, suggesting that plumbagin-mediated cell cycle arrest and apoptosis is carried through both p53-dependent and -independent manners.
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The Role of JNK on p53 and Bcl-2 Phosphorylation. Previous studies have indicated that p53 phosphorylation at Ser15 is a critical event for stabilizing the function of p53. Phosphorylation at this site in vivo was shown to be inhibited by JNK-specific inhibitors, suggesting that JNK may target this serine directly or indirectly (Milne et al., 1995; Fuchs et al., 1998). As shown in Fig. 8A, pretreatment of cells with SP600125 decreased the plumbagin induction of p53 protein and phosphorylation at Ser15. Moreover, a major factor that influences the stability of p53 protein is the binding of p53 with MDM2, and this interaction is inhibited when p53 phosphorylation occurred. As shown in Fig. 8B, the binding of MDM2 to p53 dramatically decreased in plumbagin-treated cells. These data suggest that plumbagin induces p53 phosphorylation through JNK signaling, which stabilizes p53 protein to induce p21 expression.
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On the other hand, we tested the involvement of the mitochondrial apoptotic pathway by examining the effect of JNK inhibitors on phospho-Bcl-2 expression. As shown in Fig. 8A, cotreatment of A549 cells with plumbagin and SP600125 completely blocked plumbagin-mediated Bcl-2 phosphorylation at Ser70.
Plumbagin Inhibits Tumor Growth in Nude Mice. To determine whether plumbagin inhibits tumor growth in vivo, equal numbers of A549 cells were injected s.c. into both flanks of the nude mice. Tumor growth inhibition was most evident in mice treated with plumbagin at 2 mg/kg/day, where 80% reductions in tumor size were observed, in contrast with mice treated with the vehicle (Fig. 9, A and B). No sign of toxicity, as judged by parallel monitoring body weight, was observed in plumbagin-treated mice.
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). In our study, we have found that plumbagin effectively inhibits tumor cell growth in vitro, concomitant with induction of cell cycle arrest and apoptosis, and inhibits tumor cell growth in nude mice. Furthermore, because plumbagin does not exhibit any significant toxicity on normal lung cancer, this suggests that plumbagin possesses selectivity between normal and cancer cells.
Tumor suppressor gene p53 is a key element in the induction of cell cycle arrest and apoptosis following DNA damage or cellular stress in human cells (Harris and Levine, 2005). Cell cycle arrest that is dependent on p53 requires transactivation of p21 or other cell cycle-related factors (Taylor and Stark, 2001). The induction of p21 causes subsequent arrest in the G1/G0 or G2/M phase of the cell cycle by binding of the cyclin-cdk complex (Taylor and Stark, 2001; Coqueret, 2003). In this study, we have shown that treatment of A549 cells with plumbagin resulted in the accumulation of p53 and phospho-p53 (Ser15 and 392) both in vivo and in vitro. Indeed, we also have found that plumbagin increases the expression of p21 and arrests the cell cycle at G2/M. The up-regulation of p21 by plumbagin was inhibited by suppression of normal p53 activity via dominant-negative p53, suggesting that p21 is regulated in a p53-dependent manner. In addition, treatment of A549 with plumbagin also decreases the expression of cyclinB1, Cdc25C, and Cdc2, whereas it increases the phosphorylation of Cdc2 and phospho-Cdc25C. Therefore, we suggest that plumbagin may prove to be a valuable tool for inhibition of Cdc2/cyclinB1 in lung cancers for the following reasons: the down-regulation of cyclinB1 by plumbagin; the induction of p21 by plumbagin in a p53-dependent manner, which may subsequently inhibit the function of Cdc2 by forming Cdc2/p21 complex; and the increase of phospho-Cdc25C followed by an increase in inactivated phospho-Cdc2, suggesting that increased phospho-Cdc25C levels may also decrease functioning phosphatase for dephosphorylating and activating Cdc2.
Increased expression of p21 is associated with cell cycle inhibition, differentiation, and cellular senescence (Chen at al., 2002). In addition, p21 can bind to proliferating cell nuclear antigen, thereby blocking DNA synthesis (Gartel and Radhakrishnan, 2005). Jaiswal et al. (2002) have indicated that plumbagin treatment caused an increase of p21 expression and a decrease of DNA repair resulting in cell death in mouse embryonic fibroblast cells. However, p21 has also been reported to influence the outcome of the p53 response to DNA damage and play a protective role on survival signal against apoptosis (Seoane et al., 2002; Kuo et al., 2004). The up-regulation of p21 by p53 induction attenuates the cell death in the quercetin-treated A549 cells (Kuo et al., 2004). These different observations may be due to the cell type and cell content specificity of apoptosis inducers and their subsequent signaling transduction pathways. Although our result indicated that plumbagin induces early G2/M phase arrest, which is followed by apoptosis in A549 cells, the actual role of p21 in the relation of G2/M arrest and apoptosis requires further investigation.
Mitochondrial apoptotic pathway has been described as an important signaling of apoptotic cell death for mammalian cells (Hengartner, 2000). Following the treatment of A549 cells with plumbagin, we observed that plumbagin treatment resulted in a significant increase of Bax and Bak, Bcl-Xs expression, and a decrease of Bcl-2 and Bcl-XL, suggesting that changes in the ratio of proapoptotic and antiapoptotic Bcl-2 family proteins might contribute to the apoptosis promotion activity of plumbagin. In addition, elevation of phospho-Bcl-2 (Ser70) by plumbagin treatment further helps reduce its ability to bind with Bax and enhance the translocation of Bax from cytosol to mitochondria, leading to an enhanced susceptibility of the cells to apoptosis (Ishikawa et al., 2003; Zu et al., 2005). However, phosphorylation of Bcl-2 at Ser70 has also been reported to be required for Bcl-2's antiapoptotic activity upon IL-3 and etoposide treatment (Ito et al., 1997; Deng et al., 2001). The influence of Bcl-2 phosphorylation thus requires further investigation. Our finding also showed a collapse of m, a substantial release of cytochrome c, and the activation of caspase-9 after A549 cells were treated with plumbagin. These occurrences of mitochondria apoptotic events are correlated with the modulation of plumbagin on Bcl-2 family proteins. These results confirm that plumbagin-induced apoptosis is associated with regulation of Bcl-2 family proteins.
Activation of the JNK pathways has long been associated with the apoptotic response induced by several DNA-damaging agents (Liu and Lin, 2005). The proapoptotic targets of the activated JNK are not clearly defined, but the phosphorxylation of transcription factors such as c-Jun and p53, as well as pro- and antiapoptotic Bcl-2 family members such as Bim and Bcl-2, has been suggested to be of importance (Fuchs et al., 1998; Buschmann et al., 2000; Liu and Lin, 2005; Yoshida et al., 2005). Phosphorylation of human p53 in the N-terminal domain results in enhancement of transcriptional activity and prolongation of p53 half-life by inhibiting p53-MDM2 complex formation (Fuchs et al., 1998; Buschmann et al., 2000). In this report, we have shown that treatment of A549 cells with plumbagin resulted in the accumulation of phospho-JNK in both in vitro and in vivo. This JNK activation correlated well with the plumbagin-induced increase of JNK activity as measured by the JNK substrate phospho-c-Jun. Furthermore, we observed that blocking the plumbagin-induced activation of JNK1/2 by SP600125 could prevent p53 phosphorylation (Ser15) and enhance p53-MDM2 interaction, suggesting that plumbagin-induced JNK activation contributes to the stabilization of p53 function by Ser15 phosphorylation, which decreases the interaction of p53 and MDM2. This suggestion was strongly supported by the inhibition of the duration and phosphorylation of p53 (Ser15) by SP600125 treatment. Our results indicated that the Ser392 and Ser15 phosphorylation of p53 was also observed in plumbagin A549 cells, but SP600125 did not affect Ser392 phosphorylation on p53. Therefore, the upstream regulators and effect of p53 Ser392 phosphorylation require further investigation. Previous study has reported that Bcl-2 is inactivated by phosphorylation on three serine residues (Thr69, Ser70, and Ser87) via JNKs (Yamamoto et al., 1999). Our results showed that exposure of A549 cells to plumbagin led to concurrent phosphorylation of Bcl-2 at Ser70 and SP600125 pretreatment inhibited Bcl-2 phosphorylation, suggesting that the activation of JNK induced by plumbagin is involved in the modulation of Bcl-2. Moreover, the JNK inhibitor SP600125 prevented plumbagin-induced G2/M arrest and apoptosis, further suggesting that the cooperation of JNK with p53 and the mitochondrial apoptotic pathway play a crucial role in plumbagin-induced G2/M arrest and apoptosis.
Although SP600125 was described as an inhibitor of the JNK pathway for the treatment of autoimmune, inflammatory, and neurodegenerative diseases and shown to be selective for JNK1/2 (Bennett et al., 2001), it has also been reported to exhibit nonspecific inhibition on several cell cycle and apoptosis-related enzymes, including serum- and glucocorticoid-induced kinase, p70 ribosomal protein S6 kinase, AMP-activated protein kinase, Cdk2, and dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (Bain et al., 2003). Therefore, the influence of nonspecific inhibition of SP600125 on plumbagin-mediated cell cycle arrest and apoptosis induction could be further investigated.
In conclusion, the present study demonstrated that human nonsmall cell lung cancer A549 cells are highly sensitive to growth inhibition by plumbagin both in in vitro and in vivo experimental models; reduced survival of A549 cells after exposure to plumbagin is associated with G2/M phase cell cycle arrest and apoptosis induction; plumbagin can inhibit cell cycle progression at the G2/M phase by increasing p21 expression in a p53-dependent manner and by decreasing the expression of Cdc2, Cdc25C, and cyclinB1; plumbagin-induced cell growth inhibition in the A549 cells is mediated by activation of JNK, which stabilizes p53 by phosphorylation of p53 at Ser15 and decreasing the interaction of p53 and MDM2; and JNK also phosphorylates Bcl-2, leading to alter function of Bcl-2 to apoptosis. These findings suggest that plumbagin may be a promising chemopreventive agent against human nonsmall cell lung cancer.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: JNK, c-Jun NH2-terminal kinase; plumbagin, 5-hydroxy-2-methyl-1,4-naphthoquinone; DMSO, dimethyl sulfoxide; PI, propidium iodide; SP600125, anthra [1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone; phospho-JNK, phosphorylated c-Jun NH2-terminal kinase; XTT, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; G418, geneticin; PARP, poly(ADP-ribose) polymerase.
1 These authors contributed equally to this work. 
Address correspondence to: Dr. Chun-Ching Lin, Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. E-mail: aalin{at}ms24.hinet.net
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