Plumbagin from Plumbago Zeylanica L Induces Apoptosis in Human Non-small Cell Lung Cancer Cell Lines through NF- kB Inactivation

Lung cancer is currently the commonest malignancy and the leading cause of cancer-related death in the world (Jemal et al., 2009). Non-small cell lung cancer (NSCLC) represents about 80% of primary lung cancer cases and approximately two thirds of these patients are diagnosed at an advanced stage (Jemal et al., 2009). Although several effective chemotherapeutic agents are administrated, the platinum-based regimen is the standard initial treatment for NSCLC patients (Pujol et al., 2006). However, the efficacy of the regimen for NSCLC has been reported to be only 30-40% based on NSCLC trials involving unselected patients (Ohe et al., 2007). Identifying novel agents with fewer side effects and more effective therapy is of significance for prognosis of patients with NSCLC. Plumbagin (5-hydroxy-2methyl-1, 4-naphthoquinone), a Chinese traditional herb extracted from the root of Plumbago zeylanica L has been reported in the literature to have various pharmacological


Introduction
Lung cancer is currently the commonest malignancy and the leading cause of cancer-related death in the world (Jemal et al., 2009). Non-small cell lung cancer (NSCLC) represents about 80% of primary lung cancer cases and approximately two thirds of these patients are diagnosed at an advanced stage (Jemal et al., 2009). Although several effective chemotherapeutic agents are administrated, the platinum-based regimen is the standard initial treatment for NSCLC patients (Pujol et al., 2006). However, the efficacy of the regimen for NSCLC has been reported to be only 30-40% based on NSCLC trials involving unselected patients (Ohe et al., 2007).
Identifying novel agents with fewer side effects and more effective therapy is of significance for prognosis of patients with NSCLC. Plumbagin (5-hydroxy-2methyl-1, 4-naphthoquinone), a Chinese traditional herb extracted from the root of Plumbago zeylanica L has been reported in the literature to have various pharmacological
Reports in relation to the anticancer potential of plumbagin toward lung cancer have been described previously. It was found to induce apoptosis and inhibition of metastasis in A549 lung cancer cell line Shieh et al., 2010). However, the antitumor mechanisms of plumbagin remain inconclusive. The aim of present research is to investigate the anticarcinogenic activity of plumbagin toward three non-small cell lung cancer (NSCLC) cell lines and to detect the potential mechanism.

Clonogenicity assay
To determine long-term effects, cells seeded in sixwell plates (10 3 cells/well) were treated with various concentrations of plumbagin for 6 h. Then the medium was discarded, and fresh medium was added to the wells, after which cells were allowed to grow for 14 days to form colonies, which were stained with crystal violet (0.4 g/l; Sigma). The images were collected, and the number of colonies in each well was counted.

Annexin V-FITC/PI staining assay
The extent of apoptosis was evaluated by Annexin V-FITC/PI kit (BD Pharmingen USA) and flow cytometer. The concentration of the plumbagin stock solution was adjusted so that the final concentration of DMSO in the culture medium 0.1%. In control cultures, DMSO 0.1% was added as a vehicle control. After incubation, the plumbagin-treated A549, H292, and H460 cells were harvested, washed with PBS (pH 7.4), centrifuged, and stained with Annexin V-FITC and PI in binding buffer for 15 min at 37 °C in the dark. The samples were analyzed using FACSCalibur flow cytometer (Becton, Dickinson and Co, San Jose, CA, USA). Data analysis was performed using FACSD iva software (BD, USA).

TUNEL assay
TdT-UTP nick end labeling (TUNEL) assays were performed with the ApopTag ® Peroxidase TUNEL kit (Chemicon Company, USA& Canada) according to the manufacturer's instructions. Cells grown in 96 well culture plates were treated with plumbagin. After incubating for 24 hours, cells were stained by TUNEL kit. The TUNELpositive cells were imaged under a light microscope. Cells with brown precipitate were defined as early stage of apoptotic cells.

Detection of reactive oxygen species (ROS)
H460 cells (0.5×10 6 ) were labeled for 1h with 5 μM carboxy-H2DCFDA (Molecular Probes, Eugene, OR). Next, excess carboxy-H2DCFDA was removed by washing the cells and then suspending them in serumfree, phenol red-free RPMI. The labeled cells were then exposed to vehicle control (DMSO) or various concentrations of plumbagin for 1h and the levels of ROS were determined by flow cytometric analysis using a FACSAria II flow cytometer (Becton, Dickinson and Co, San Jose, CA, USA). In all experiments, 10,000 viable cells were analyzed using forward/side-scatter gating.

Electrophoretic mobility shift assay (EMSA)
To detect NF-κB DNA-binding activity, we implemented electrophoretic mobility shift assay (EMSA) using the Viagene EMSA Kit following the manufacturer's protocol. Briefly, nuclear extracts (5 μg) from plumbagintreated and NAC plus plumbagin-treated cells were incubated with biotin-labeled NF-κB oligonucleotide probes, 5'AGTT GAGG GGAC TTTC CCAGGC 3' and 3'TCAA CTCC CCTG AAAG GGTC CG5' in reaction buffers, for 30 min at 37 °C. Specific competition binding assays were performed using unlabeled probe as a specific competitor. DNA-protein complex was separated from free oligonucleotide on 6.5% native polyacrylamide gels, and then transferred to positively charged nitrocellulose membranes (Milipore, Bedford, MA) by a transfer blotting apparatus. Gel shifts were visualized with streptavidinhorseradish peroxidase followed by chemiluminescent detection and quantitated using labworks4.6 software.

NF-κB luciferase reporter assay
H460 cells were plated in 96-well plates with 1 × 10 4 cells per well in 10% FBS-supplemented RPMI medium. After overnight incubation, the cells were transfected with the NF-κB reporter plasmid linked to a luciferase gene or with the dominant-negative IκBa (IκBa-DN) plasmid. NF-κB luciferase plasmid was obtained from Stratagene (La Jolla, CA). Transfections were done according to the Plumbagin Induces Apoptosis in Human NSCLC Cell Lines through NF-kB Inactivation Cells were incubated with 0, 5, 10, and 15 μmol/L plumbagin for 12 h. The apoptosis was analyzed by Annexin V-FITC/PI double-staining assay manufacturer's protocols using FuGENE-6 (Roche). At 24h post-transfection, cells were treated with indicated concentrations of plumbagin for 2 h and then washed and lysed in luciferase lysis buffer (Promega), and the luciferase activity was measured with a luminometer using a luciferase assay kit (Promega) and was normalized to β-galactosidase activity. All the experiments were done in triplicates and repeated two or more times.

Western blot analysis
Cells seeded in 10 cm dishes were treated with 6 μmol/L or 3 μmol/L of plumbagin for indicated time. Total cell extracts were prepared using M-PER mammalian protein extraction reagent and protease inhibitors (Pierce, Rorkford, IL, USA). The nuclear extracts were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China).Protein concentrations were determined using the BCA protein assay (Pierce, Rorkford, IL, USA) following the manufacturer's protocol. Equivalent amounts of protein (30 μg) were loaded per lane, resolved by SDS-polyacrylamide gel electrophoresis (8%-12%), and transferred to PVDF membranes. After being blocked with a 5% skim milk solution for 1 h, the membranes were incubated with their respective primary antibodies overnight at 4 °C, followed by secondary antibody incubations for 1 h at room temperature. The proteins were visualized using an enhanced chemiluminescence system (ECL, Beyotime Institute of Biotechnology, Jiangsu, China).

Statistical analysis
Data are expressed as mean ± standard deviation. Statistical comparisons were performed using a one-way analysis of variance followed by the Fisher test. Significant differences between the groups were determined using an unpaired Student t-test.

Plumbagin decreased viability of NSCLC cells
Cell viability was assayed by treating NSCLC cell lines, including A549, H292, and H460 cells, with various concentrations of plumbagin followed the CCK-8 viability assay. We observed that cellular proliferation was inhibited by plumbagin for 24 hours in a dose-dependent manner in all three of the NSCLC cell lines ( Figure 1B). The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 μmol/L, 7.3μmol/L, and 6.1 μmol/L, respectively. The inhibition of growth of the three cell lines by plumbagin was also time-dependent (data not shown). While, there was no significant alteration in the viability of normal lung epithelial cells (BEAS-2B) on exposure (up to 6 μM concentration) to plumbagin (data not shown).
To examine the long-term effects of plumbagin, clonogenic assay was performed. The clonogenicity of the three cell lines in the plumbagin groups was decreased in a concentration-dependent manner. At a concentration of 6μM, plumbagin inhibited about 55%, 65% and 70% of clone formation in A549, H292, and H460, respectively ( Figure 1C and 1D). The in vitro clonogenic assay has been reported to correlate well to in vivo assays of tumorigenicity in nude mice (Freedman and Shin, 1974).

Exposure to plumbagin led to ROS-mediated apoptosis in H460 lung cancer cells
Plumbagin has been reported to correlate to increased levels of ROS in divergent carcinomas including prostate, melanoma, cervical, and promyelocytic leukemia (Powolny et al., 2008;Xu et al., 2010). In the present study, we detected the influence of plumbagin treatment on H460 cells ROS levels. To this end, H460 cells were prelabeled with 5 μM carboxy-H2DCFDA and then exposed to diverse concentrations of plumbagin for 1h. Exposure of H460 cells to various concentration of plumbagin led to an increase level of ROS-associated mean fluorescence intensity (MFI) compared with vehicle treated cells. The level of ROS correspondingly increased from 12% to 40%, 72% and 78%, respectively ( Figure 3A). The implicated of the plumbagin-induced increase in ROS in H460 cells apoptosis was confirmed using NAC. More specifically, H460 cells were exposed for 24h to plumbagin ± NAC (10 mM) and then analyzed for viable cell number and induction of apoptosis using the Annexin V-FITC/PI double-labeled assay. The results demonstrated that treatment of H460 cells with NAC led to a significant reduction in plumbagin-induced cell killing and apoptosis ( Figure 3B and 3C). Together, these results suggest that plumbagin-induced increase in ROS levels plays a crucial role in mediating apoptosis of H460 cells.

Plumbagin suppressed activation of NF-κB in H460 cells and the activation of ROS was involved
NF-κB played a pivotal role in regulating cell apoptosis is relatively well documented (Karin et al., 2006). To identify whether plumbagin induced cell apoptosis is mediated by NF-κB and whether ROS is involved in the process, we investigated NF-κB activation using EMSA assay and luciferase reporter assay. Firstly, using EMSA assay, we demonstrated that plumbagin alone could suppress endogenous NF-κB DNA binding activity in a dose-dependent manner and ROS scavenger NAC could highly abrogate plumbagin induced NF-κB inactivation ( Figure 4A). However, DNA binding alone is not always The role of ROS in plumbagin-mediated proliferative inhibition was assessed using ROS scavenger NAC. The influence of NAC on plumbagininduced cytotoxicity was determined by CCK-8 assay. C) The effect of NAC on plumbagin-induced apoptosis was detected by Annexin V-FITC/PI double-staining assay. The data represent the mean ± SD. Asterisk denoted statistically significant difference (p<0.05) between vehicle (0 μM Plumbagin) and plumbagin treated cells. Double asterisk indicated statistically significant difference (p<0.05) between Plumbagin and Plumbagin + NACtreated cells

Figure 4. Plumbagin Inhibits NF-κB Activity in H460 Lung Cancer Cells. A) Plumbagin suppressed endogenous
NF-κB DNA binding activity in a concentration-dependent manner and ROS scavenger NAC largely retrieved NF-κB activity.H460 lung cancer cells were pretreated with indicated concentrations of plumbagin for 6 h, incubated with or without 10 mM NAC for 30 minutes, and then subjected to EMSA to examine NF-κB activation. B) H460 cells were transiently transfected with an NF-κB-luciferase plasmid and then treated with the indicated concentrations of plumbagin for 2 h, presence or absence of 10 mM NAC for 30 minutes. Cell supernatants were thereafter collected and assayed for luciferase activity as described in Methods. Representative results of three independent experiments are shown. Results are expressed as fold activity over the activity of the vector control. Bars indicate standard deviation. Asterisk denoted statistically significant difference (p<0.05) between vehicle (0 μM Plumbagin) and plumbagin treated cells. Double asterisk indicated statistically significant difference (p<0.05) between Plumbagin and Plumbagin + NACtreated cells. C) plumbagin inhibited p65 nuclear translocation and IκBα degradation by Western blot, while NAC partly retrieved the effect of plumbagin. H460 lung cancer cells were incubated with indicated concentrations of plumbagin for 6 h, incubated with or without 10 mM NAC for 30 minutes, and then nuclear extracts were analyzed for p65 by Western blot and equal amounts of cytoplasmic proteins for IκBα. C23 and GAPDH antibodies were used as controls. Results from three repeated and separated experiments were similar associated with NF-κB-dependent gene transcription (Nasuhara et al., 1999;Manu et al., 2011), suggesting that additional regulatory steps are involved. So we further performed NF-κB-luciferase reporter assay to confirm the inhibition effect of plumbagin on NF-κB. Secondly, similar results were also observed by NF-κB-luciferase reporter gene assay. As shown in Figure 4B, Plumbagin significantly inhibited NF-κB reporter gene activity in a dose-dependent manner and NAC partly retrieved NF-κB activity. Together, our results suggested that plumbagin significantly inhibited NF-κB activity in lung cancer cells and the activation of ROS may be implicated in the mechanism.

Plumbagin inhibited p65 nuclear translocation and IκBα degradation implicated in ROS
The effects of plumbagin on p65 nuclear translocation and the influence of ROS were examined in lung cancer cells. To confirm the data, we performed western blot analysis of p65 in nuclear extracts. As showed in Figure  4C, treatment of plumbagin inhibited p65 nuclear translocation and ROS scavenger NAC could highly reverse its nuclear translocation activity. To understand whether treatment of plumbagin affects upstream of NF-κB/p65 pathway, we examined IκBα degradation. Treatment of plumbagin markedly inhibited degradation of IκBα in lung cancer cells and NAC partly reversed the effect ( Figure 4C).

Effect of plumbagin on expression of NF-κB-regulated apoptotic-related proteins in lung cancer cells
NF-κB is known to regulate a variety of cell functions, including apoptosis, proliferation, invasion and angiogenesis through regulating target gene expression (Karin et al., 2006). Using western blot analysis, we examined the effects of plumbagin on apoptotic gene expression which is regulated by NF-κB activation. As shown in Figure 5A, levels of the proapoptotic and antiapoptotic proteins were altered after treatment of plumbagin. Western blot analysis showed an increase in the levels of Bax, CytoC and Bak proteins in a timedependent manner in H460 lung cancer cells, and a decrease of Bcl-2 protein. The involvement of caspase activation in plumbagin mediated cell apoptosis in lung cancer cell line was also determined with the use of western blot. The activation of caspase-9 and caspase-3 but not caspase-8 was observed ( Figure 5B).

Discussion
Lung cancer has long been the leading mortality in both developed and developing countries. Unfortunately, there is limited therapy available for this malignant disease due to fewer efficacies of chemotherapy and radiotherapy. Non-small cell lung cancer (NSCLC) is the predominant subtype and has unfavorable outcome. In our study, we have found that plumbagin effectively inhibited three kinds of NSCLC cell lines including A549, H262, and H460 growth in vitro, concomitant with induction of apoptosis.
Emerging research has demonstrated that plumbagin exerts anti-carcinogenic effects. Previous studies examining the effect of plumbagin on lung cancer A549 cell line showed the inhibition was dose and timedependent and with the IC50 value of 11.69μM for 48h treatment . In the present research, we also observed the concentration and time dependent anti-

Figure 5. Effect of Plumbagin on Expression of NF-κB-regulated Apoptotic-related Proteins in H460 Cells.
A) Western blot for protein expression of Bak, Bax, Bcl-2, and cytoC. Cells were treated with plumbagin (3 μM) for 6h, 12h and 24h. The expression of proteins was assessed by western blotting, as described under Methods. The blot is representative of three separate experiments. B) H460 cells were treated with plumbagin (3 μM) for 6h, 12 h and 24 h. Protein lysates were prepared and subjected to Western blot analysis as described in Methods using antibodies against caspase-8, caspase-3, and caspase-9. Equal protein loading was ensured by demonstrating uniform GAPDH expression. The blot is representative of three separate experiments Figure 6. Schematic Diagram of Plumbagin Induced Apoptotic Pathway in Lung Cancer Cells proliferatition of plumbagin in A549, H292, and H460 cells with the IC50 values 10.3 μM, 7.3 μM, and 6.1 μM for 12h, respectively ( Figure 1B).
The occurrence and development of carcinoma is closely related to cell apoptosis (Krysko et al., 2008). Mitochondrial apoptotic pathway has been described as an important signaling of apoptotic cell death for mammalian cells (Hengartner et al., 2000). It is well known that Bcl-2 family and cytochrome C (cytoC) of proteins play a central role in the regulation of the intrinsic (mitochondrial) apoptotic pathway. Many reports have shown that plumbagin induced apoptosis through the mitochondrialmediated pathway in breast cancer and lung cancer Ahmad et al., 2008;Kawiak et al., 2012). Following the treatment of H460 cells with plumbagin, we observed that plumbagin treatment resulted in a significant increase of Bax, Bak and cytoC expression, and a decrease of Bcl-2, which was consistent with Hsu et al. Caspases play a central role in apoptosis execution. Their activation is essential for the occurrence of the typical hallmarks of apoptosis such as chromatin condensation and DNA fragmentation. As shown in Figure 5B, treatment with plumbagin increased caspase-9 activity in H460 cells. This is consistent with the release of cytochrome c into the cytosol from mitochondria, potentially activating caspase-9. The activated upstream caspase-9 acts on the downstream target of caspase-3 enzymes, subsequently, the activated caspase-3 acts on the target cells as an effector molecule, damaging the cell structure and causing functional disorder by proteolysis, ultimately inducing apoptosis through the caspase-dependent mitochondria pathway (Deng et al., 2008) .
The function of NF-κB for regulating target gene expression, including apoptosis-related and proliferationrelated is also well documented (Mocellin et al., 2005;Karin, 2006). We determined that plumbagin was able to inhibit the expression of proteins involved in antiapoptosis (ie, Bcl-2) and enhance the expression in pro-apoptosis (ie, Bax, Bak, and cytoC), all of which are linked to, or regulated by NF-κB. Based on our findings, we hypothesized that plumbagin may have an effect on the NF-κB pathway in lung cancer cells. In current study, we found that plumbagin suppressed activation of NF-κB and inhibited NF-κB-p65 nuclear translocation and IκBα degradation. Our result was consistent with many reports which have investigated the effect of down-regulating NF-κB activation by plumbagin in various tumor cells (Sandur et al., 2006;Shieh et al., 2010;Manu et al., 2011;Li et al., 2012). Together, those results showed that inhibition of NF-κB activation by plumbagin was not cell-type specific.
Plumbagin is a naturally occurring naphthoquinone, its cytotoxic properties is related to its quinone core and contribute to its therapeutic activity. There are two main mechanisms with the therapeutic activity of quinones including the production of the semiquinone radical under aerobic conditions which participates in redox cycling to generate reactive oxygen species (ROS) like superoxide anion (O 2· ) and hydrogen peroxide (H 2 O 2 ) and as a potent electrophile reacting with the thiol groups of proteins and glutathione (GSH) (Castro et al., 2008). The production of ROS has been suggested to play a pivotal role in the DNA damage, and may exert the cytotoxic effects of menadione in cultured human cells (Noto et al., 1989;Ngo et al., 1991). Previous research found that plumbagininduced apoptosis was associated with generation of ROS in various cancer cell lines, including leukemia, cervical, prostate, and melanoma (Srinivas et al., 2004;Powolny and Singh, 2008;Wang et al., 2008;Xu and Lu, 2010). We postulated that plumbagin induced apoptosis also through an increase in ROS in H460 cells. This was directly confirmed by our studies that plumbagin exposure led to increase ROS levels in H460 cells and indirectly in experiments that treatment with free radical scavenger NAC prevented plumbagin-mediated apoptosis. To our knowledge, we have demonstrated for the first time that plumbagin can generate ROS in non-small lung cancer cells. We propose that this effect may be important for the induction of apoptosis after treatment with plumbagin. However, the mechanism by which plumbagin generates ROS remains elusive. The quinone structure of plumbagin allows it to conduct electrons from two-electron donors to single-electron acceptors in the respiratory pathways of both prokaryotic and eukaryotic cells thus forming as a superoxide generator (Imlay and Fridovich, 1992). Thus, we hypothesized that the target of plumbagin induced ROS may be mitochondrial respiratory chain; however, further elaborately experiments should be conducted before reaching to such a conclusion.
In the present study, we investigated that plumbagin could both increase ROS level and inhibit activation of NF-κB. Which is the upstream mediator, ROS or NF-κB? ROS is well known to be involved in diverse physiological and pathological processes, and oxidative stress can activate the NF-κB pathway (Barchowsky et al., 1996;Qian et al., 2012). To explore whether or not ROS is the upstream mediator of NF-κB, ROS scavenger NAC was added to plumbagin treated H460 cells. We observed that NAC could partly abrogate the effect of plumbagin on inhibition of NF-κB activity. These results indicated that excess ROS generation may be an upstream signal of NF-κB inactivation in this process. However, whether there is a feedback loop between ROS and NF-κB, and the precise regulation mechanism should be further identified.
It can be concluded from the above results that plumbagin inhibited the proliferation of A549, H292, and H460 cells in a dose and time-dependent manner. The cytotoxic effect of plumbagin-induced cell death is through the induction of apoptosis, as indicated by the current data. Plumbagin exposure led to generation of ROS in H460 cells. Subsequently, excess ROS generation inhibited the activation of NF-κB, which down-regulated the expression of anti-apoptotic proteins and up-regulated pro-apoptotic proteins. Thereafter, caspase-dependent mitochondrial pathway was activated, as shown by the plumbagin-mediated activation of caspases-3 and -9 ( Figure 6). Plumbagin therefore have the potential to be a potent agent for non-small lung cancer treatment.