Inhibition of Raf-MEK-ERK and Hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line

Background Lung cancer constitutes one of the malignancies with the greatest incidence and mortality rates with 1.6 million new cases and 1.4 million deaths each year. Prognosis remains poor due to deleterious development of multidrug resistance resulting in less than 15% lung cancer patients reaching five years survival. We have previously shown that Phyllanthus induced apoptosis in conjunction with its antimetastastic action. In the current study, we aimed to determine the signaling pathways utilized by Phyllanthus to exert its antimetastatic activities. Methods Cancer 10-pathway reporter array was performed to screen the pathways affected by Phyllanthus in lung carcinoma cell line (A549) to exert its antimetastatic effects. Results from this array were then confirmed with western blotting, cell cycle analysis, zymography technique, and cell based ELISA assay for human total iNOS. Two-dimensional gel electrophoresis was subsequently carried out to study the differential protein expressions in A549 after treatment with Phyllanthus. Results Phyllanthus was observed to cause antimetastatic activities by inhibiting ERK1/2 pathway via suppression of Raf protein. Inhibition of this pathway resulted in the suppression of MMP2, MMP7, and MMP9 expression to stop A549 metastasis. Phyllanthus also inhibits hypoxia pathway via inhibition of HIF-1α that led to reduced VEGF and iNOS expressions. Proteomic analysis revealed a number of proteins downregulated by Phyllanthus that were involved in metastatic processes, including invasion and mobility proteins (cytoskeletal proteins), transcriptional proteins (proliferating cell nuclear antigen; zinc finger protein), antiapoptotic protein (Bcl2) and various glycolytic enzymes. Among the four Phyllanthus species tested, P. urinaria showed the greatest antimetastatic activity. Conclusions Phyllanthus inhibits A549 metastasis by suppressing ERK1/2 and hypoxia pathways that led to suppression of various critical proteins for A549 invasion and migration.


Background
Lung cancer constitutes one of the malignancies with the greatest incidence and mortality rates with 1.6 million new cases and 1.4 million deaths each year [1,2]. Although initial use of platin-based cytotoxic chemotherapy improved the overall survival rate and life quality of the patients, the outcome remains poor due to deleterious development of drug resistance resulting in less than 15% lung cancer patients surviving for at least five years [2,3]. Approximately 80 -85% of the lung cancer patients are diagnosed with an advanced stage of non-small cell lung cancer (NSCLC) that has limited therapeutic options due to metastasis [1,4,5] and among the three types of NSCLC, patients identified with squamous carcinoma which involves lymph node metastasis accounts for 60% [5,6]. In the advance stage of NSCLC, surgery is not possible to remove all apparent lesions hence leading to the high rate of cancer recurrence [7]. Therefore, lethality of lung cancer is often attributed to late diagnosis, metastasis, and the occurrence of drug resistance [8]. Thus, the development of an efficient cancer diagnostic method is crucial since an early detection of cancer while it is still localized and curable is one of the most promising approaches to reduce increasing cancer burden [9]. This requires an understanding of the pathophysiology of the disease [10,11] which can be made possible via proteomic studies. Cancer proteomics allow the screening of early diagnostic markers or potential drug targets [6] by comparing the proteomes of the diseased and diseased-treated samples that allows identification of aberrantly expressed proteins [5,10]. These proteins could be biomarker candidates to expedite non-invasive diagnosis of early-stage malignant tumor, as well as to aid the monitoring of tumor progression and therapy effectiveness [12]. Similar to other malignant pathologies, tumor markers for NSCLC remained inadequate. Serum biomarkers employed in the current clinical setting such as ENO (enolase alpha), CEA (carcinoembryonic antigen), SCC (squamous cell carcinoma), CA-125 (cancer antigen 125) or TPA (tissue polypeptide antigen) are not satisfactory due to their low sensitivity and specificity [13,14]. In the quest for novel diagnostic or prognostic biomarkers, the proteomic technique is ideal since it permits qualitative and quantitative analysis of numerous proteins simultaneously [9,13]. Moreover, the search for biomarkers at the protein level is more dependable than at the transcriptional level as protein expression may not necessarily associate with mRNA expression [14].
Natural-product based drugs are gaining their popularity as preventive medicines or for health management, and hence this has spurred an intensive search for bioactive plant-derived anticancer compounds [15]. The genus Phyllanthus is one of the most widely distributed plants throughout the Amazon rainforests as well as other tropical and subtropical regions. Abundant studies on Phyllanthus spp. started in the late 1980′s when the clinical efficacy of Phyllanthus niruri against viral Hepatitis B was observed [16]. Various therapeutic actions of this genus have been reported, including being antihepatotoxic, antilithic, antihypertensive, anticarcinogenic, and most recently anti-HIV as well [16][17][18][19]. We have demonstrated antimetastatic and antiproliferative activities of Phyllanthus against several metastatic cell lines such as lung carcinoma (A549), breast (MCF-7) carcinomas [17], melanoma (MeWo), as well as prostate (PC-3) carcinoma [20].
Nevertheless, the exact mechanisms for the antimetastatic activities of Phyllanthus are still uncertain. MAP kinase (MAPK) is one of the signaling pathways known to mediate metastasis via transmission of extracellular stimuli into the nucleus that activate the serine/threonine kinases which belong to the MAPK superfamily [21]. MAPK serine/ threonine superfamily is made up of three well-described subgroups, including ERK1/2 or p44/42 MAPK, JNK/ SAPK, and p38 MAPK. These diverse pathways with distinct downstream targets are activated by various stimuli to regulate cell proliferation, apoptosis, and metastasis [21,22]. Regulation of metastasis by MAPK is mainly via controlling the cells' expression of matrix metalloproteinases (MMPs) enzymes by decreasing the nuclear levels of NFκB, c-Fos, or c-Jun [21,22]. The MMPs are a family of at least 20 members of highly homologous, zinc-and calcium-dependent endopeptidases which can degrade virtually all extracellular matrix (ECM) components [23]. Among these MMPs, MMP2 and MMP9 are abundantly expressed, secreted, and activated in various malignant tumors [22,24]. Recent studies also shown that MMP7 plays an essential role in ectodomain shedding of cell-surface molecules such as EGFR, HB-EGF, Fas ligand, and E-cadherin in addition to its ability to degrade ECM components. Therefore, these enzymes are the most vital ones implicated in cancer invasion and metastasis [24,25].
Hypoxia pathway is another chief regulator of metastatic process. Hypoxia-inducible factor-1 (HIF-1) is the determining factor for oxygen-dependent gene regulation and is a dimer of HIF-1α and HIF-1β. Its regulation is mainly governed by the transactivation and stabilization of HIF-1α protein resided in the cytoplasm [26]. Under normoxia condition, HIF-1α instantaneously interacts with von Hipple-Lindau (pVHL) ubiquitin E3 ligase complex and is targeted for proteasomal degradation due to hydroxylation of two specific proline residues within the oxygen-dependent degradation domain by prolyl hydroxylases [7,26]. Contrarily, HIF-1α will be stabilized with increased expression during hypoxic condition, allowing it to translocate into the nucleus and dimerize with HIF-1β. HIF-1 will subsequently bind to the hypoxia response elements to regulate transcription of over 100 target genes which favors tumor growth and metastasis, including vascular endothelial growth factor (VEGF) as well as genes that regulate cellular processes such as energy metabolism, cell proliferation, vascular development and remodelling, as well as vasotone [2,7,26].
In our previous study, Phyllanthus extracts were shown to inhibit A549 (lung carcinoma) cells growth with IC 50 values ranging from 60-130 μg/ml and 200-470 μg/ml for methanolic and aqueous extracts respectively [17]. We also demonstrated that they effectively reduced invasion, migration, and adhesion activities of A549 cells in a dosedependent manner and was capable of inducing apoptosis along with its antimetastastic action [17]. Therefore, the main objective of this study was to determine the protein expression profile of Phyllanthus-treated A549 cells and how the signaling pathways were utilized by Phyllanthus in order to exert its antiproliferative and antimetastatic activities on this cell line.

Plant extracts and standard drugs
The crude extracts (aqueous and methanolic) of each Phyllanthus spp., namely P. niruri, P. urinaria, P. watsonii and P. amarus, were obtained from the Malaysian Agriculture and Research Development Institute (MARDI), Malaysia. The aqueous extracts were prepared by dissolving 10 mg in 1ml of sterile PBS (Final concentration 10 mg/ml), whereas, the methanolic extracts were prepared by dissolving 40 mg in 1ml of DMSO (Final concentration 40 mg/ml). The tubes containing the extracts were wrapped with aluminium foil and stored at -20°C until use. A single batch of extracts was used for all the experiments. The IC 50 concentrations that were used as one of the treatment conditions in most of the experimental assays (Table 1) as well as the polyphenol contents of these extracts have been determined and published in the previous study [17]. Cisplatin and Doxorubicin (MERCK -1 mg/ml) as standard anticancer drugs for lung and breast carcinomas respectively were included as the controls.

Cell culture
Human lung carcinoma (A549) cell line was purchased from American Type Culture Collection (ATCC, USA) and was grown in RPMI-1640 (Roswell Park Memorial Institute). To ensure growth and viability of the cells, the mediums were supplemented with 10% FBS (Gibco) and incubated in a humidified atmosphere with 5% CO 2 at 37°C.

Transient transfection and cancer 10-pathway reporter array
Cancer is a group of diseases strongly correlated with defects in signal transduction proteins. Various key signaling pathways have been implicated in human tumorigenesis. Analysis of these signaling pathways was performed using Cignal Finder Cancer 10-pathway Reporter Array kit (SABiosciences, QIAGEN, USA). Transient transfection was performed using TransIT-LT1 (Mirus Bio, USA). Both plasmid DNAs for the respective signaling pathways provided in the kit as well as TransIT-LT1 were diluted using Opti-MEM I reduced serum medium (Invitrogen, USA). After that, plasmid DNAs and the TransIT-LT1 was mixed to allow TransIT-LT1/DNA complex formation. Subsequently, 90 μl (approximately 10000 cells) of the cell suspension were mixed with 10 μl of the complex and added into the designated wells of a 96-well cell culture white microplate (Nunc, Thermo Fisher Scientific, USA). The culture plate was rocked for 5 minutes on a rocker before it was incubated in a 5% CO 2 incubator at 37ºC overnight. After the transfection of cells with various plasmid DNAs for the respective signaling pathways, the cells were incubated with different Phyllanthus extracts for another 24 hours. Dual-Glo Luciferase reagent was then added into each well and incubated at room temperature for 10 minutes before reading the firefly luminescence generated using the GloMax Multi Detection System (Promega, USA). This was followed by the addition of Dual-Glo Stop & Glo reagent to all wells. Similarly, another renilla luminescence reading was obtained after the plate was incubated for another 10 minutes. The firefly constructs monitor changes in the activity of a key transcription factor which is a downstream target of a particular signaling pathway. Meanwhile, renilla construct acts as an internal control for transfection efficiencies normalization as well as to monitor cell viability. Luminescence for each wells were determined by calculating the ratio of its firefly to renilla luminescence.

Cell cycle analysis
DNA content analysis is performed by Propidium iodide (PI) staining which binds to the distinct amount of DNA content in the cells at different phases of the cell cycle. Briefly, cells were seeded at 10 5 cells/well, treated with extracts at their IC 50 values, and incubated at various time periods from 0 to 72 hours. At the end of each incubation period, cells treated with or without Phyllanthus extracts were harvested and fixed with ice-cold 70% ethanol for at least 1 hour at -20ºC. Cells were then pelleted, washed once with PBS, resuspended in the PI solution [10μg/ml PI (Sigma) and 1mg/ml RNase A in PBS], and incubated in a 37°C water bath for 30 minutes. Data acquisition was performed using a Becton Dickinson FACSCalibur flow cytometer and CellQuest software and subsequently analysed using WinMDI 2.9 software. The distribution of cell percentages in each cell cycle phase is determined by setting gates based on their amount of DNA content.

Preparation of cytoplasmic protein lysate
Protein lysates were prepared for western blotting and proteomic analysis for both Phyllanthus-treated and untreated samples. Cells treated with Phyllanthus extracts were detached from the culture plates using 0.5% trypsin-EDTA and washed twice with phosphate buffered saline (PBS), centrifuging at 1500 rpm for 5 minutes. Two hundred microliters of lysis buffer EDTA, 0.02% bromophenol blue) before they were loaded on a 12.5% of SDS-polyacrylamide gel. The proteins were separated at 100V for approximately 1 hour. Alternatively, one hundred and fifty micrograms for each protein samples were separated by two-dimensional (2D) gel electrophoresis method using 7 cm IPG gel strips with pH 3-11 NL range (GE Healthcare, USA). After electrophoresis, the stack or sandwich was assembled and the proteins were transferred onto a nitrocellulose membrane (GE Healthcare, USA) at 250 mA for 1 hour. After the transfer, the membrane was blocked with Tris buffered saline buffer consisting 0.1% Tween 20 (TBST) and 5% dry milk. Subsequently, membranes were incubated with various primary antibodies and secondary antibodies diluted using blocking solution. The immune-reacted proteins were detected via chromogenic method by addition of DAB substrate onto the membrane to form a protein band. Anti-pan-Ras, anti-c-Raf, anti-c-Myc, anti-Bcl-2, anti-Hif-1α, anti-c-Jun/AP-1, anti-p53, anti-Elk1, anti-JNK1/2, anti-VEGF, goat anti-mouse IgG peroxidase conjugate, and goat anti-rabbit IgG peroxidase conjugate antibodies were purchased from Merck Millipore, Germany while anti-RSK antibody was purchased from Thermo Fischer Scientific, USA. These antibodies were chosen based on their essential roles in the pathways modulated by Phyllanthus as detected using the Cignal Finder Cancer 10-pathway Reporter Array. A p53 antibody was included to determine any involvement of p53 pathway in response to Phyllanthus treatment.

Zymography assay
Zymography is a simple and sensitive technique employed to study extracellular matrix-degrading proteases such as MMPs based on their substrate specificity and molecular weight. Briefly, cells were seeded at 1 × 10 5 cells/well in a 24-well microtiter plate and treated with increasing concentrations of aqueous (50 μg/ml, IC 50 , 500 μg/ml) and methanolic (20 μg/ml, IC 50 , 200 μg/ml) extracts. After 72 hours incubation, supernatants were collected and stored at -20°C to be used as conditioned media. The conditioned media was mixed with 2× sample buffer (0.5M Tris-HCl [pH6.8], 87% glycerol, 10% SDS, 0.1% bromophenol blue) and subsequently loaded onto a 12.5% SDS-polyacrylamide gels that had been copolymerized with 0.1% gelatin or 0.2% casein. The gel was then run at approximately 125V for about 60 minutes. When the proteins were completely resolved, the gel was washed twice with renaturing buffer (2.5% Triton X-100) on a shaker at room temperature, 1 hour for each washing. Next, the gel was incubated with developing buffer (12.1 g Tris, 63 g Tris-HCl, 117 g NaCl, 7.4 g CaCl 2 , 0.2% Brij35, 1L distilled water) overnight at 37°C before it was stained with 0.1% Coomasie blue for 1 hour. Finally, the gel was destained with destaining solution and the presence of matrix metalloproteinase enzyme was indicated as an opaque, unstained band against a dark blue background.

Human total iNOS and GADPH immunoassay
Inducible Nitric Oxide Synthase (iNOS) is often upregulated in tumor cells and is an important regulator for vascularization and angiogenesis. In order to measure the total iNOS in whole cell, a cell-based ELISA, Human Total iNOS Immunoassay (R&D Systems, USA) was performed according to the manufacturer's instructions.

2-Dimensional gel electrophoresis
Five hundred microgram proteins for A549 cells (treated and untreated samples) were rehydrated overnight on 13 cm IPG gel strips with pH 3-11 NL range (GE Healthcare, USA). Strips rehydrated with proteins were then transferred into IPG chambers and focused using Ettan IPGphor Isoelectric Focusing unit (GE Healthcare, USA). Before proceeding with SDS-PAGE, the strips were first subjected to a two-step equilibration procedure for 15 minutes each. Firstly, they were equilibrated with an SDS-PAGE equilibration base buffer added with 2% (w/v) Dithiothreitol (DTT), followed by equilibration with 2.5% (w/v) Iodoacetamide (IAA). The strips were then placed onto a 12.5% SDS-PAGE gel and sealed with agarose sealing solution. Second dimensional separation was carried out using Ettan Dalttwelve Separation Unit (GE Healthcare, USA). The gels were then fixed using gel fixative solution and stained with Coomasie dye. Finally, the gel was destained with destaining solution and imaged using Ettan DIGE Imager (GE Healthcare, USA). Three independent gels were run for each treatment (n = 3). Gel images were analyzed using PDQuest 2-D Analysis Software (Bio-Rad, USA) which performed background removal, normalization, and automatic matching of the detected protein spots. Protein spots with more than 2-fold differential expression that showed significant difference (p < 0.05) were selected and excised for mass spectrometry analysis.

In-gel enzyme digestion
Protein spots excised from gels were first destained using 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate and then incubated with 10mM DTT for 30 minutes at 60°C followed by incubation with 55 mM iodoacetamide for 20 minutes in the dark. The gel plugs were then washed with 50% ACN in 100 mM ammonium bicarbonate before incubation with 100% ACN for 15 min on a shaker and dried using SpeedVac. The dried gel plugs were subsequently incubated with 6 ng/μl trypsin in 50 mM ammonium bicarbonate overnight at 37°C, vortexed briefly and spun down before 50% and 100% ACN were added and shaken for 15 minutes each. The supernatant of each round of extraction was transferred into a fresh tube before the digested samples were completely dried using SpeedVac. The samples can be kept at -20°C until further use. Otherwise, extracted peptides were concentrated or desalted using ZipTip C18 microcolumns (Merck Millipore, Germany).

MALDI-TOF/TOF mass spectrometry and database searching
Prior to MALDI-TOF/TOF analysis, 3 μl of each extracted peptide sample solution was mixed with 3 μl of alpha-cyano-4-hydroxycinnamic acid (Sigma-Aldrich, USA) matrix solution dissolved in 50% aqueous ACN containing 0.1% trifluoroacetic acid (Sigma-Aldrich, USA). A volume of 0.7 μl of each sample was applied onto a MALDI plate and was allowed to air dry at room temperature. Analysis was performed with ABSCIEX 4800 MALDI-TOF/TOF (AB SCIEX, USA) operated in the reflector for MALDI-TOF/TOF with fully automated mode using the 4800 Series Explorer software at an accelerating voltage of 20 kV. Calibration was performed using Mass Standards Kit for Calibration of AB SCIEX TOF/ TOF™ Instruments (AB SCIEX, USA). Data collected from the MALDI-TOF/TOF were submitted to the SwissProt database using the MASCOT search algorithm (version 2.1.0, Matrix Science, London, UK). Typical search parameters for both search engines were defined as follows: trypsin digestion allowing up to two tryptic-mass cleavages, variable modifications of oxidation and carbamidomethyl, maximal mass tolerance of 0.1Da, precursor tolerance of 100 ppm, and taxonomy Homo sapiens. Protein scores greater than 55 were considered significant (p < 0.05). The protein with the highest number of peptides was considered as those corresponding to the spot if multiple proteins were identified in a single spot. The proteins identified were then compared with Uniprot KB/Swiss-Prot database and grouped according to the Eukaryotic Orthologous Group of Classifications (COGs).

Data analysis
Results were expressed as the mean ± Standard Error Mean (SEM) of data obtained from three independent experiments. All data were analyzed using one way ANOVA, followed by Dunnett's test for pairwise comparison. P < 0.05 was considered statistically significant for all tests.

Determination of signaling pathways affected by Phyllanthus
The Cignal Finder Cancer 10-pathway reporter array was used to simultaneous screen 10 main cellular pathways that are targeted by Phyllanthus in its anticancer activities.
The pathways included in this array include Wnt, Notch, p53/DNA damage, TGFβ, Cell cycle/pRb-E2F, NFκB, Myc/Max, Hypoxia, MAPK/ERK, and MAPK/JNK, with GFP construct plasmid DNA as the positive control for this array. As shown in Figure 1A (aqueous extractstreated A549) and 1B (methanolic extracts-treated A549), the expression of GFP construct was consistent in both the aqueous and methanolic extracts-treated and untreatedcontrol A549 cells, hence the results obtained were deemed valid. Upon treatment with various aqueous and methanolic extracts, most of these pathways' expression [Myc/Max, Hypoxia, and MAPK (ERK and JNK)] decreased significantly (p < 0.05) except for NFκB which indicate Phyllanthus probably did not modulate this pathway. The aqueous Phyllanthus extracts showed better inhibitory activity on the expression of both MAP kinase pathways, while the methanolic extracts showed enhanced inhibition on the expression of Hypoxia and Myc/Max pathways. Among the four plant species, P. watsonii exhibited greatest suppression on Hypoxia (aqueous -60% and methanolic -86%), ERK (aqueous -47% and methanolic -27%), and JNK (aqueous -50% and methanolic -26%) pathways, followed by P. urinaria, P. amarus, and P. niruri. From the flow cytomery-based cell cycle analysis, we did not observe cell cycle phase arrest in the A549 cells treated with Phyllanthus extracts (Figure 2) since the percentage of gated cells for each cell cycle phases (G 0 /G 1 , S, and G 2 /M) did not change significantly (p > 0.05) between the untreated and extracts-treated cells. Thus, this further explained the low expression level of cell cycle/pRb-E2F pathway in both the untreated and treated A549 cells. Nevertheless, the percentages decreased with a concurrent increase in the percentage of apoptotic cells (increase in Sub G 1 phase) at increasing incubation time points (24,48, and 72 hours). Conversely, there was an accumulation of cells at G 2 /M phase for A549 cells treated with Cisplatin and Doxorubicin.

Modulation of intracellular signaling molecules expression by Phyllanthus
In order to confirm the effects of Phyllanthus on MAPK and Hypoxia pathways, western blots were performed with available antibodies (Anti-pan-Ras, anti-c-Raf, anti-c-Myc, anti-Bcl-2, anti-Hif-1α, anti-c-Jun/AP-1, anti-p53, anti-Elk1, anti-JNK1/2, anti-VEGF, and anti-RSK) to determine the specific targets of Phyllanthus, whether it affects during the early or late stages of the signaling cascade. Figure 3A to I showed the blots for untreated A549 control as well as the A549 treated with various aqueous and methanolic extracts while Figure 3K depicts the expression level of each protein. In untreated A549, the proteins detected were Pan-Ras, c-Raf, c-Jun/AP-1, Elk-1, c-Myc, and HIF-1α. The presence of these proteins reflects the specific involvement of MAPK/ERK and Hypoxia pathways in regulating the A549 cells' growth and survival. In addition, Bcl-2 protein was also detected in untreated A549 which explains its role as an antiapoptotic agent to ensure cell's survival [27]. Besides that, FUSE-binding proteins were also detected although antibody specific to this protein was not included in this experiment. This might probably be attributed to its role for proper regulation of the c-Myc protooncogene [28], as it has a certain percentage of similarity with c-Myc protein and was therefore detected when c-Myc antibody was used. As expected, most of these proteins' expression decreased when A549 was treated with various Phyllanthus extracts. Among the four species, P. urinaria showed better inhibition on those proteins, followed by P. amarus, P. watsonii, and P. niruri for both the aqueous and methanolic extracts. The expression of Pan-Ras and Elk-1 proteins were only mildly affected as compared to the other proteins and p53 expression was hardly seen in both the treated and untreated A549 cells via western blot technique, thus confirming the findings obtained from the previous cancer 10-pathway array which demonstrated low p53 expression in A549.

Inhibition of matrix metalloproteinases (MMPs) expression by Phyllanthus
MMPs play an important role during tumor metastasis and angiogenesis since its expression level is often correlated with the tumor invasiveness [29]. Among the variety of MMPs, MMP2, MMP7, and MMP9 were more commonly associated with cancer metastasis as they have the ability to degrade collagen type IV which is the major component of basement membrane [23,25]. MMP7 probably plays a greater role in A549 metastasis since its expression was higher with brighter and clearer bands as compared to MMP2 and MMP9. Nonetheless, their expressions decreased in a dose-dependent manner as shown by the untreated-control and Phyllanthus-treated bands' intensity in Figure 4. However, the bands for proMMPs with their respective MMPs were not well separated in the zymogram, hence leading to the inability to distinguish between the active and latent MMPs. Methanolic Phyllanthus extracts demonstrated greater inhibition on the MMPs' expression than aqueous extracts, with P. urinaria showing the greatest inhibitory activity compared to the other Phyllanthus species.

Inhibition of iNOS and VEGF expression by Phyllanthus
Angiogenesis or blood vessel formation is the key process in the survival and metastasis of tumors in a hypoxic environment. During hypoxia, cytoplasmic HIF-1α subunit stabilization will lead to the activation of various genes including vascular endothelial growth factor (VEGF) and inducible nitric oxide synthases (iNOS) [30]. This was verified with the high expression of both VEGF and iNOS detected in the untreated-control A549 cells ( Figure 5) using a cell-based ELISA assay. Upon treatment with various Phyllanthus, both the aqueous and methanolic extracts for all four species were observed to demonstrate inhibition on iNOS, whereby most of their expressions dropped markedly to approximately 20% except for methanolic P. urinaria which retained 40% of iNOS expression. Generally, P. urinaria (78% reduction) scored highest iNOS inhibition among aqueous extracts while P. watsonii (82% reduction) exhibited strongest activity among methanolic extracts. This suppression ability was comparable to Cisplatin and Doxorubicin with 15% and 10% iNOS expression that remained after treatment respectively.
On the other hand, methanolic Phyllanthus extracts showed better suppression of VEGF expression with 60 -80% reduction compared to aqueous extracts which caused 20 -50% reduction. In addition to this ELISA assay, expression of VEGF was also confirmed by western blot assay when VEGF expression dropped to 30 -80% after A549 cells were treated with aqueous and methanolic Phyllanthus extracts ( Figure 3J and K). Among the four species, methanolic P. amarus displayed better VEGF repression with only 20% VEGF expression. Meanwhile, P. urinaria exhibited slightly weaker capability to inhibit VEGF expression with 25% (aqueous) and 65% (methanolic) reduction respectively.
Differentially expressed proteins in Phyllanthus-treated A549 Figure 6 showed representative 2D-PAGE gels for untreatedcontrol, aqueous P. watsonii-treated, and methanolic P. watsonii-treated A549 samples. Two-dimensional gel electrophoresis proteomic analysis picked out 68 and 79 protein spots differentially expressed in aqueous and methanolic extracts-treated groups respectively. Subsequent mass spectrometry analysis and database examination using MASCOT identified 52 protein spots significantly downregulated by aqueous Phyllanthus extracts as listed in Table 2. These protein spots were further categorized according to Clusters of Orthologous Groups (COGs) classification and majority of them fell into the category of post-translational modification, protein turnover, and chaperones, followed by intracellular trafficking, secretion, and vesicular transport, cytoskeleton, as well as energy production and conversion ( Figure 7A). On the other hand, methanolic Phyllanthus extracts significantly suppressed 64 proteins as tabulated in Table 3. Most of these proteins fell into the category of signal transduction mechanisms, transcription, defense mechanisms, amino acid transport and metabolism, as well as secondary metabolites biosynthesis, transport, and catabolism ( Figure 7B). The negative (-) symbol in both Tables 2 and 3 signified suppression of the proteins and a value of 1.00 indicates the complete absence of this protein in the Phyllanthus-treated sample as compared to the untreated-control sample and the degree of suppression reduces as the value increases.

Discussion
Numerous reports describe poor therapeutic efficacy and prognostic survival for lung malignancy which is largely due to its capability to invade, metastasize, as well as to induce angiogenesis. Therefore, it is crucial to develop novel antimetastatic drugs with low toxicity and high efficacy [17,29]. In our previous study, aqueous and methanolic extracts of Phyllanthus was shown to possess the potential to inhibit growth of A549 in a time-and dose-dependent manner with minimal toxicity to the normal lung epithelial (NL20) cells. Besides that, Phyllanthus suppressed the invasion, migration, and reattachment of A549 in a dose-dependent manner and was capable of inducing apoptosis in conjunction with its antimetastastic action [17]. However, the underlying mechanisms that confer their antimetastatic and apoptosis-inducing abilities were uncertain.
In the current study, numerous differentially expressed proteins were being identified in A549 cells in response to Phyllanthus treatments using 2DE-based proteomic approach. Both aqueous and methanolic Phyllanthus extracts modulate expression of different set of proteins [17]. Among the four Phyllanthus species, P. urinaria generally demonstrated the greatest activity on A549, closely followed by P. watsonii, P. amarus, and P. niruri. This could be delineated by the higher number of polyphenol compounds present in both the aqueous (9 out of 10 polyphenols) and methanolic (3 out of 4 polyphenols) P. urinaria extracts [17,20], hence having a higher capability to cause antimetastatic activities on A549. Some of those proteins were represented by more Figure 5 Expression level of (a) iNOS and (b) VEGF in untreated and Phyllanthus-treated A549 cells. APN -aqueous P. niruri, APU -aqueous P. urinaria, APW -aqueous P. watsonii, APA -aqueous P. amarus, MPN -methanolic P. niruri, MPU -methanolic P. urinaria, MPW -methanolic P. watsonii, MPA -methanolic P. amarus, CIS -Cisplatin, DOX -Doxorubicin. Error bar indicates the standard error of the mean of three independent experiments. P < 0.05 for all extracts-treated A549 compared to untreated-control A549. than one spot, which represents different splicing forms of the same protein as a result of post-translational modification [14].
Therefore, protein turnover, chaperones, and posttranslational modification are indeed crucial to produce many variants of the common amino acid that possess distinctive structures and functions essential for tumor growth [31,32]. In order to ensure uninterrupted cell growth, continuous protein synthesis is necessary. Therefore, 40S ribosomal protein is normally upregulated in the tumor cell as it is an essential component of the higher eukaryotic ribosome that is necessary for proper protein translational function [33]. Eukaryotic translation initiation factor 3 subunit 12 is another important component as it binds to poly(A)-binding protein to initiate translation process [34]. Protein folding is the subsequent critical process upon successful protein translation to ensure formation of functional proteins and this step usually requires the presence of chaperone proteins. One of these proteins is Hsp60 whose primary role is to guide the folding of mitochondrial proteins while facilitating proteolytic degradation of denatured or misfolded proteins in an ATP-dependent manner [35]. Other chaperones include peptidyl-prolyl cis-trans isomerase A and T-complex protein 1 subunit epsilon which also assists the proper folding of proteins [36,37].
Besides facilitating protein folding, chaperones also guide the proper assembly of other proteins for them to carry out their activities. An important example is stressinduced phosphoprotein 1 that facilitates association of molecular chaperones Hsp70 and Hsp 90 which have been implicated in MMP2 activity that lead to increased invasiveness [38]. Downregulation of these proteins by Phyllanthus is therefore one of the key processes for A549 growth and metastasis inhibition by reducing functional protein synthesis and suppression of MMP expression. In addition to MMP2, suppression of other MMPs in response to Phyllanthus treatment was also observed including MMP7 and MMP9. Tumor invasion and metastasis depends largely on the integrity of the basement membrane which is frequently destroyed by a number of proteolytic enzymes such as MMPs in order to access the vasculature to develop distant metastases [22]. Among the MMPs family, MMP2 and MMP9 were more commonly associated with cancer invasion and metastasis since they had been known to be able to degrade type IV collagen-rich basement membrane of vessel wall [23]. MMP7 (28 kDa) is another member of the MMP family with broad substrate specificity against ECM components such as elastin, type IV collagen, fibronectin, vitronectin, aggrecan, and proteoglycans [25]. Reduced expression of these MMP enzymes explains the decreased aggressiveness of A549 cells' invasion upon treatment with Phyllanthus.
A constant activation of various growth-promoting signaling pathways is also required to ensure continuous cell growth and survival. This activation often involves numerous proteins which require some form of modifications such as phosphorylation for their biological role. Thus, post-translational modification process that alters the proteins properties is important [39,40]. The first step for pathway activation often requires the presence of receptor molecules on the cell surface for ligand Figure 6 Representative 2D-PAGE gels for (a and b) untreated-control A549 and its aqueous P. watsonii-treated A549, as well as (c and d) untreated-control A549 and its methanolic P. watsonii-treated A549.  The negative (-) symbol signified suppression of the proteins and a value of 1.00 indicates the complete absence of this protein in the aqueous Phyllanthus-treated sample as compared to the untreated-control sample and the degree of suppression reduces as the value increases. APN -Aqueous P. niruri, APU -Aqueous P. urinaria, APW -Aqueous P. watsonii, APA -Aqueous P. amarus, N/A -Not Affected.
binding. So, guanine nucleotide-binding protein, putative Ras-related protein Rab-42, and GTPase HRas precursor protein which can be broadly grouped as G-proteins, as well as probable G-protein coupled receptor 179 precursor expressions are usually elevated in the tumor cells to accommodate the large amount of extracellular signals to be transduced into the cells [41]. Both G-protein coupled receptor and G-proteins form the G-protein mediated signaling cascade whereby binding of ligands to receptors will lead to activation of G-proteins by promoting GDP/GTP exchange, which in turn regulates many effector molecules such as proteins kinases [41]. Therefore, the subsequent upregulated protein in the tumor cell is serine/threonine-protein kinase 6 which is a family of kinases including MAP kinase [42], which functions to turn on the downstream kinases via serine/ threonine phosphorylation [43]. Suppression of these cell signaling proteins by Phyllanthus could therefore decelerate or stop the constitutive activation of the growth-promoting pathways such as MAP kinase and hypoxia. ERK1/2 pathway is one of the MAP kinase subgroup which was frequently found to be inhibited in A549 to repress cells' continuous growth and metastasis. In a study by Shih et al. [21], α-tomatine found in tomatoes was shown to be capable of inactivating extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway to inhibit metastasis    occurrence in A549 cell line. Similarly, A549 metastasis was also inhibited by Silibinin isolated from Silybum marianum which suppresses ERK1/2 pathway that led to a reduced expression of MMP2 and u-PA concomitantly with a significant inhibition on cell invasion [22]. Indeed, our study also showed that Phyllanthus inhibit A549 metastasis via targeting specifically ERK1/2 pathway. ERK1/2 module is often thought as a linear pathway since ERK is the effector of an evolutionarily conserved signaling component that is triggered exclusively by the Raf serine/threonine kinases [44,45]. Therefore, Phyllanthus might possibly downregulate Raf protein at the early stage of the pathway, resulting in the subsequent suppression of other proteins' expression down the pathway. As a result, various biological activities controlled by ERK1/2 pathway to increase cell growth and malignancy are repressed, including regulation of transcriptional, cell cycle, apoptosis, and metastasis [46]. Transcription is an important biological activity in a cell as it is the first step for the transmission of genetic information from DNA into RNA to be translated into proteins [47]. With the suppression of the essential transcriptional proteins for eukaryotic chromosomal DNA replication such as DNA-directed RNA polymerase II 16kDa polypeptide, transcription elongation factor B polypeptide 1, and proliferating cell nuclear antigen, transcription initiation and elongation process becomes inefficient [48,49]. Besides that, zinc finger protein 174 is a DNA binding protein that acts as a cofactor for transcription factor. Its downregulation will lead to increased transcription of proteins such as E-cadherin which in turn represses cell invasion [50]. Also, cell cycle disorder plays a critical role in cancer progression. So, modulation of cell cycle by phytochemicals from natural product sources is gaining worldwide attention to control carcinogenesis [51]. However, our findings showed that cell cycle pathway was not modulated with the flow cytometric data showing insignificant shifts in each cell cycle phases for the cells treated with Phyllanthus extracts. Hence, cell cycle arrest was ruled out as one of the mechanism of actions of the extracts. Since Phyllanthus do not inflict cell cycle arrest on A549, the only approach to inhibit A549′s continuous growth is by causing toxicity. As showed in the previous study, Phyllanthus does induce apoptosis in A549 with more than three folds increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells [17]. In order to determine whether Phyllanthus induces extrinsic or intrinsic apoptotic pathway, Bcl-2 expression was examined using immunoblot analysis since it is one of the main regulators of the mitochondrial outer membrane permeabilization which initiates intrinsic apoptotic cell death [52,53]. The data obtained agrees with our previous hypothesis that Phyllanthus probably activates the intrinsic pathway of apoptosis by inhibiting antiapoptotic Bcl-2 protein to release cytochrome c for caspases activation. In addition to this, proteomic analysis also observed downregulation of Bcl-2-like protein 11 which is similar to Bcl-2 protein that has a role as an antiapoptotic protein [52]. Apart from that, suppressing MAP kinases and its downstream factors such as AP-1 have been shown to decrease MMPs expression and subsequently inhibit various pathological processes such as tumor invasion, adhesion, metastasis, and angiogenesis [22,24]. This is in accordance with results from the current study that showed decreased ERK1/2 pathway and MMPs activity by Phyllanthus which led to inhibition of A549 metastasis. Moreover, Phyllanthus extracts also suppressed cytoskeletal proteins such as actin, vimentin, tubulin alpha chain, actin-related protein, and cofilin-1/2. Besides being the components of the cytoskeleton, both actin and tubulin-binding proteins are also mediators of motility [8]. Structure, conformational dynamics, and mechanical properties of actin filaments are mainly controlled by cofilin [54]. Meanwhile, vimentin constitutes the intermediate filaments of the cytoskeleton which stabilizes cytoskeletal interactions as well as affecting cell motility and movement. Elevated expression of vimentin in several invasive cell lines suggests the possibility of it being a representative marker for epithelial to mesenchymal transition [38]. Therefore, the significant inhibitory effects of Phyllanthus on the A549 cell's cytoskeleton most probably involve the alteration of the microfilament organization and function, therefore suppressing motility and metastasis [55].
Hypoxia is largely perceived as another major obstacle to cancer therapy as increasing evidence in the cancer therapy-related literatures suggests the involvement of proangiogenic factors in the progression of lung tumorigenesis [7]. Angiogenesis is essential for tumor growth and survival due to the imbalance of nutrient and oxygen supplies to solid tumors larger than 1 mm 3 , resulting in tumor hypoxia [56]. One of the major regulatory component which responds to hypoxia to ensure cell survival and to promote angiogenesis is HIF-1α [2,7]. Therefore, it could possibly be the target for the development of novel anticancer agents. Lin et al. [7] demonstrated suppression of lung tumor angiogenesis and metastasis by andrographolide isolated from Andrographis paniculata which downregulates HIF-1α. In addition, inhibition of HIF-1α pathway by HIF-1α-siRNA displayed a direct correlation with A549 cellular proliferation and angiogenesis, a prerequisite for metastasis [26]. In agreement to these studies, our results obtained also showed the reduction of HIF-1α expression in A549 cells treated with Phyllanthus extracts. This in turn led to the suppression of various target genes controlled by HIF-1α via hypoxia-responsive-element (HRE) [26], including angiogenesis, metabolism, cell growth, and death [2]. VEGF is another crucial angiogenic growth factor which induces endothelial cell proliferation from the pre-existing capillary bed for wound healing, tumor growth, and metastasis. Its expression is therefore increased prior to an invasive and metastatic phenotype [56]. Also iNOS, one of the three distinct isoforms of NOS which is widely expressed and often upregulated in multiple tumor tissues [57] is expressed in tumor cells associated with vascularization and hence, is probably another important regulator of angiogenesis [56,57]. Nitric oxide (NO) produced by NOS have been shown to affect vascular permeability, induce extracellular matrix degradation, trigger VEGF production, as well as stimulate endothelial cell proliferation and migration [30]. Hence, inhibition of VEGF and iNOS by Phyllanthus can greatly reduce A549 angiogenesis, resulting in tumor cells malnutrition and hypoxia thereby preventing tumor growth, survival, and metastasis. Adenosine 5′-triphosphate, a major source of energy for cells and its involvement in a variety of cellular activities which are ATP-dependent is often increased in tumor cells [58]. Downregulation of these enzymes activities by Phyllanthus causes cellular energy deficit that can result in cancer cell death. One example is enolase which catalyzes conversion of 2-phosphoglyceric acid (PGA) to phosphoenolpyruvate (PEP) in the anabolic pathway during gluconeogenesis to enhance aerobic glycolysis in cancer cells [59]. Other enzymes suppressed include glyceraldehyde 3 phosphate dehydrogenase, fructose-biphosphate aldolase A, phosphoglycerate mutase 1, and triosephosphate isomerase that are also involved in energy metabolism [55,60].
Besides exploiting cellular signaling pathways for their growth and metastasis, tumor cells possess efficient drug detoxification system to remove compounds that may be fatal to them. This includes upregulation of glutathione transferase omega-1 that catalyzes binding of glutathione to various anticancer compounds such as cisplatin, thereby decreasing production of platinum-DNA adducts and rendering them useless while glutathione synthetase catalyzes production of glutathione substrate for the detoxifying activity [61,62]. Similarly, metallothionein also plays a role in chemotherapy binding and detoxification since its elevated expression was noticed in several cisplatin-resistant lung cancer cell lines [61]. Meanwhile, Annexin A4 is normally associated with chemoresistance in part by enhancing drug efflux [63]. Inhibition of these detoxification enzymes expressions in A549 after treatment with Phyllanthus as demonstrated from the proteomic analysis therefore advocates the reduced A549 drug-resistance capability resulting in their susceptibility to death-inducing compounds.

Conclusions
All the findings obtained in this study point to the involvement of ERK1/2 and hypoxia pathways which were suppressed by Phyllanthus to inhibit A549 proliferation, angiogenesis, invasion, and metastasis. Inhibition of ERK1/2 pathway led to downregulation of invasion and mobility proteins (MMP2; MMP7; MMP9; cytoskeletal proteins), transcriptional proteins (proliferating cell nuclear antigen; zinc finger protein), and antiapoptotic protein (Bcl2) while inhibition of hypoxia pathway causes repression of angiogenic proteins (VEGF; iNOS) and various glycolytic enzymes. Suppression of drug detoxification enzymes such as gluthathione transferase and metallothionein also increases sensitivity of A549 to Phyllanthus treatment. Among the four Phyllanthus species tested in this study, P. urinaria was found to be the most effective to inhibit A549 growth and metastasis, closely followed by P. watsonii. Thus, Phyllanthus could be a valuable candidate in the treatment of metastatic cancers. However, the main concern before application of Phyllanthus as an antimetastatic or antiproliferative agent is its in vivo effect. Thus, further testing of the extracts activity in vivo is necessary to exploit it as a chemotherapeutic agent. Preliminary work has identified low toxicity of Phyllanthus in an animal model as dosage greater than 50 g/kg is needed to cause 100% acute death in the mice tested (unpublished data) and subsequent efficacy testing in a tumor-bearing mice model needs to be carried out.