Preparation Of Phyllanthus Extracts Biology Essay


Methodology/Principal Findings: The cancer ten-pathway reporter array was performed to profile the changes in the activities of ten different signalling pathways relevant to cancer influenced by Phyllanthus. Expression of five pathway reporters were significantly decreased (Wnt, NFκB, Myc/Max, Hypoxia, MAPK/ERK and MAPK/JNK) in PC-3 cells after treated with Phyllanthus. Western blot was conducted to identify signalling molecules in the identified pathways. To get insight into PC-3 cell response to Phyllanthus, proteomics-based approach was performed to identify possible proteins that involved in anti-cancer effects of Phyllanthus Proteins up- and down-regulated in response to Phyllanthus were identified by MS and mapped into specific metabolic and cellular processes, including tumour cell adhesion, migration, apoptosis, glycogenesis and glycolysis, invasion, metastasis angiogenesis and protein synthesis and energy metabolism.

Conclusions/ Significance: This study provides insight into the understanding of Phyllanthus regulation on major cancer pathways in prostate cancer and might be offered further application as anti-cancer agent.


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Although there are tremendous advances in anticancer research and treatment, the morbidity and mortality rate caused by cancer are still in dilemma. Cancer is one of the top leading causes of death in the worldwide. Most of these deaths are mainly due to tumour metastasis, which is the ability of cancer cells to metastasize from their primary tumour to distant size in human body. During the tumour progression, cancer cells are acquires several distinct characteristic alterations from normal cells. These include the capacities to proliferate independently of growth-promoting or growth-inhibitory signals, metastasis and angiogenesis, and evade from apoptosis.

Cellular signalling pathways are always interconnected each other to form complex signalling networks. This interaction is important for a cell to regulate vital and diverse processes such as protein synthesis, cell growth, immune responses, differentiation, and homeostasis and cell death. These interactions can be achieved either directly by cell-cell contact or indirectly by signalling molecules that are communicated between cells. However, when this intercellular communication disrupted either by external (UV radiation and chemicals) or internal factors, a number of diseases can be result includes cancer, diabetes and defect in developmental abnormalities. In anticancer research, most of the signalling proteins have become targets to halt progression of cancer by induction of apoptosis and inhibits tumour metastasis and angiogenesis. Thus, understanding these underlying (molecular, biochemical, cellular and physiological) mechanisms in cancer is of paramount importance.

Prostate cancer is one of the major life-threatening cancers and second most frequently diagnosed cancers after lung cancer in men. The incidence of diagnosed prostate cancer in men health has increased worldwide over the past decades. Despite advances in early detection and conventional treatment strategies, prostate cancer progress and becomes resistant to treatment. In addition, metastasis has been a major prevailing in cancer treatment to improve patient outcome and confers poor prognosis. About 50% of prostate cancer patients are diagnosed with pathologic or clinical evidence of bone metastasis (Seo, et al., 2008). Bones metastases can cause a wide range of symptoms that can impaired health-related quality of life or reduce life expectancy. Complications of this bone metastasis include severe pain, pathologic fractures, and epidural spinal cord compression. In addition, osteolytic metastases can result in life-threatening hypercalcemia (Coleman, 2001). The urge for development of new anticancer agents to combat both locally advanced and metastatic prostate cancer is necessity due to limitations of current treatment modalities.

The actual molecular events leading to the acquisition of the prostate metastasis is remains unclear. The mitogen activated protein kinase family member (MAPK) is believed to plays a part to mediate metastasis by inducing proteolytic enzymes that leads degradation of basement membrane, cell migration, activates pro-survival genes and cell growth. Highly expressed of MAPK pathway has been detected in various human cancers including breast, colon, kidney, melanoma, prostate and lung suggesting the possibility that MAPK may play a major role in tumour progression and metastasis. There are three major mammalian MAP kinase have been widely described; ERK1/2, c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK), and p38 MAPK. These MAP kinase members are activated by different extracellular stimuli and have distinct downstream targets, thus serving different roles in cellular response. Inhibitions of the MAPK pathway might have been the potential to target angiogenesis, proliferation, invasion and metastasis for a wide range of tumours. In addition, the PI3K/Akt, NFκB and Wnt signal transduction pathways involved in regulation of prostate cancer and are closely associated with the development and progress of other tumours.

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In normal physiology, human cells require adequate oxygen from respiration to produce energy source, ATP, used in most biochemical reactions. These oxygen delivery and consumption are highly regulated by the hypoxia-inducible factors (HIFs) (Semenza, 2011). HIFs are highly expressed during tumour metastasis as surrounding blood vessels of solid tumour are often structurally and functionally abnormal, resulting in severe hypoxia in cancer cells. The activated HIFs will induce transcription of survival-related genes to produce vascular endothelial growth factor (VEGF) and stimulate tumour angiogenesis and thereby increase oxygen transport. These genetic changes can result the clinical lethal phenotype of cancer leads hypoxic cancer cells to acquire invasive and metastatic properties as well as develops resistance to chemotherapy.

The plant of the genus Phyllanthus belongs to the family Euphorbiaceae. It widely distributed in tropical regions such as China, India and Malaysia. The pharmacological effects of Phyllanthus such as antiviral [1-4], anti-bacteria [5, 6], anti-hepatotoxic [7-11], and hypoglycaemia properties [13, 14]. Phyllanthus also exhibits anticancer effects on several cancers (Pinmai et al., 2008; Rajkapoor et al., 2007). We have reported anti-proliferation, apoptosis induction, anti-metastasis, anti-angiogenesis properties of Phyllanthus on human prostate carcinoma cell line, PC-3.

A better comprehension of these prostate cancer-related mechanisms is an essential step to explore deeper in the function and regulation in prostate cancer cells. In this present work, to gain a better understanding of the molecular mechanism changes in prostate cancer cell during Phyllanthus, a proteomic approach was performed to identify sets of up- or down-regulated proteins. These can give complimentary results from proteome-based approach and provided a more global view of the molecular and cellular changes elicited by Phyllanthus in prostate cancer cell, PC-3. Besides, these changes can offers new opportunity for the identification of new targets for therapeutic intervention and pharmacological manipulation in cancer research field.

Materials and Methods

Preparation of Phyllanthus Extracts

Four different species of Phyllanthus (P.amarus, P.niruri, P.urinaria, and P.watsonii) were used in this study. Each of these species, two extracts were prepared; aqueous and methanolic. Briefly, plant samples were harvested freshly, washed, and freeze dried. Ultra-pure water was used to soak the dried plant samples for aqueous extract preparation. While absolute methanol was used to prepare methanolic extract. Then, the samples were subjected for homogenization with extraction buffer and the supernatant was obtained after three rounds of extraction. Lyophilized forms of aqueous and methanolic extracts were collected after evaporation and stored at -20°C prior of experiment.

Cell culture

Human prostate adenocarcinoma cancer cell, PC-3 was obtained from American Type Culture Collection (Rockville, MD). Cells were cultured with RPMI-1640 1640 (Roswell Park Memorial Institute) and supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco). Cells were maintained in culture at 37°C in a humidified atmosphere of 5% CO2 and 95% humidity.

Dual luciferase pathway reporter transient transfection

Ten different cancer-related pathways analysis was performed using the Cignal Finder 10-Pathway Reporter Arrays (SA Biosciences, Fredrick, MD) according to the manufacturer's instructions. Reverse transfection protocol was implemented for this assay. Prior of experiment, PC-3 cells were seeded into 96-well white plate and were incubated overnight at 37°C. An l00ng from dual luciferase Cignal transcription factor-responsive reporter of each pathway, negative and positive control constructs were added to cells and was incubated overnight in a 37°C incubator with 5% CO2. After 24 hr of transfection, old media was discarded and replaced with complete growth medium (MEM supplemented with 10% FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin) and was further incubated for another 48 h. Each transfection condition was carried in triplicate.

Luciferase assay

After 72 h, the activity of each pathway were determined by measured generated luminescent signals using the Dual-Glo Luciferase Assay system (Promega, Madison, WI) on Glomax machine (Promega, USA). The relative luciferase units were determined by divided the firefly/Renilla luciferase activity ratio generated from the transcription factor-responsive reporter transfections with the firefly/Renilla activity ratio generated from the negative control transfections. The fold changes of each reporter's expression between Phyllanthus and negative control was calculated from the relative luciferase units.

Western Blot analysis

To analyse the pathways-related signalling protein, western blot was performed as follows. The samples were resolved on 12% SDS-PAGE gels. Proteins were transferred onto nitrocellulose membranes. Nonspecific bindings of the membrane was blocked with Tris-buffered saline (TBS) containing 1% (w/v) nonfat dry milk and 0.1% (v/v) Tween-20 (TBST) for more than 2 hr. Membranes were washed with TBST three times for 10 min and incubated with appropriate dilution of specific primary antibodies in TBST overnight at 4°C. Subsequently, the membranes were washed with TBST and incubated with appropriate secondary antibodies (horseradish-conjugated goat anti-mouse or anti-goat IgG) for 1 hr. after washing the membrane three times for 10 min in TBST, the band detection was revealed by substrate A and B.

Two-Dimensional Gel Electrophoresis

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Five hundred grams of total protein were subjected to 2D gel electrophoresis according to the manufacturer's instructions (GE Healthcare). Briefly, total proteins were extracted from control and experimental samples by incubation with lysis buffer on ice for 30 minutes. The protein pellets were re-solubilized in rehydration solution (8 M urea, 2% CHAPS, 40 mM DTT, 0.5% IPG buffer pH3-11NL, bromophenol blue) and kept at -80°C until further analysis. Total amount of proteins was determined using 2D Quant kit (GE Healthcare) and 250µl of proteins were rehydrated into 13cm immobilized pH gradient (IPG) strips (pH 3-11 nonlinear) (GE Healthcare). The first dimension was run on the IPGphor III machine (GE Healthcare) at 20°C with the following settings: step 1 at 500V for 1 hour; step 2 at 500-1000V for 1 hour; step 3 at 1000-8000V for 2.5 hour, and step 4 at 8000V for 0.5 h. Once first dimensional separation was completed, the gel was equilibrated as follows; first reduction with 64.8 mM of dithiothreitol-SDS equilibration buffer (50 mM Tris-HCl [pH 8.8], 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) for 15 minutes, followed by alkylation with 135.2 mM of iodoacetamide-SDS equilibration buffer for another 15 minutes. The second dimension was carried out using the SE600 Ruby system (GE Healthcare) at 25°C in an electrode buffer (25 mM Tris, 192 mM glycine, and 0.1% [wt/vol] SDS) with the following settings: step 1 at 100V/gel for 45 minutes; step 2 at 300V/gel until the run is completed. The gels used in the second dimension were 12.5% homogenous acrylamide gels casted in the laboratory. After electrophoresis, the gels were fixed with destaining solution for 30 minutes, followed by staining with hot Coomasie blue for 10 minutes. The gels were scanned using Ettan DIGE Imager (GE Healthcare). Gel images were analyzed using PDQuest 2-D Analysis Software (Bio-Rad, USA) and only protein spots which showed significant differences (more than 1.5 fold) were selected for mass spectrometry analysis.

Protein Digestion, Desalting and MALDI-TOF/TOF Analysis

The interested protein spots were manually excised from polyacrylamide gels were kept in sterile 1.5ml eppendorf tubes. Excised spots (gel plugs) were washed several times with destaining solution (50mM NH4HCO3) until the gel plugs are clear. The gel plugs were incubated with reducing solution (100mM NH4HCO3 containing 10mM DTT) for 30 min at 60°C. Subsequently, gel plugs were first alkylated with 100mM NH4HCO3 containing 55mM for 20 min in the dark and follow with three times of 50% acetone in 100mM NH4HCO3 for 20 min each. The gel plugs were rehydrated with 100% ACN. In-gel digestion using trypsin gold (Promega, Mass Spectrometry Grade) was added into gel plug and incubated overnight in 37°C.

Proteins were extracted from gel plugs and were purified by Ziptip (Ziptip C18, Millipore, Bedford, MA, USA). The eluted proteins were mixed with MATRIX solution and spotted on MALDI plate using dry droplet method.

Statistical Analysis

For all experiments, results were expressed as the mean ± standard error (SE) of data obtained from triplicate experiments using SPSS software. The Student's t-test was used where values of p < 0.05 considered significant.


Phyllanthus alters expression of several cancer pathways

To determine the signalling pathways altered upon Phyllanthus treatment in PC-3, a transcription factors array consisting of 10 dual-luciferase reporters' assays were used. Each of the pathways/reporters consist an inducible transcription factor responsive firefly luciferase reporter and constitutively expressing Renilla construct act as positive control in this assay. Thus, Phyllanthus-treated cells (experimental reporter) were represented by firefly luciferase, while untreated cells (normalization reporter) were represented by Renilla luciferase. As summarized in Figure 1, the expressions of six pathway reporters (Wnt, NFκB, Myc/Max, Hypoxia, MAPK/ERK and MAPK/JNK) were significantly decreased and Notch, p53, TGF-beta and cell cycle/pRB-E2F and pathways were not significantly affected by Phyllanthus. The fold changes in the transcription factor activities of these pathways in treated cells are displayed in Table 1. The transcription factor activities of the Wnt, NFκB, Myc/Max, Hypoxia, MAPK/ERK and MAPK/JNK pathway were one fold lower from untreated PC-3 cells.

Phyllanthus disrupts anti-apoptotic/pro-apoptotic balance

One of the hallmarks of cancer is inhibition of apoptosis. This can be achieved by suppressing the expression of pro-apoptotic factors (Bax) as well as stimulaing the expression of antiapoptotic factors (BCL-2). As shown in Figure 1, Bax and Bcl-2 proteins were detected at 23 kDa and 26 kDA, respectively. Figure 2B shows the percentage of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) of untreated and treated PC-3 based on their intensity. The percentage of the pro-apoptotic (Bax) of treated PC-3 was significantly one fold higher than untreated cells. The percentage of anti-apoptotic (Bcl-2) of treated PC-3 cells was significantly decreased compared to untreated PC-3. In addition, there is no expression of p53 proteins were detected in both untreated and treated PC-3 cells.

Inhibition of MAPK pathways in PC-3 cells

High expressions in MAPK signalling in prostate cancer impinge on most signalling pathways and play a critical role in the progression of cancer such as tumour metastasis and angiogenesis. In cancerous cells, two proteins are frequently tightly regulated; Ras and Raf to ensure constitutive activation of MAPK pathways. In addition, several studies have indicated that transcription factors in MAPK pathways such as JNK1/2, ERK1/2, p38 MAPK and Akt are highly expressed in prostate cancer cells. As shown in Figure 3, the expression of these proteins and their downstream targets (RSK, Elk and c-Jun) were significantly down-regulated in treated PC-3 after compared to untreated cells.

Phyllanthus alters activities of cell signalling molecules in other cancer pathways

An aberrant activation of Wnt signalling was detected at has been reported to be involved in cancers including prostate cancer. As shown in Figure 4, the expression of the DSH, Gsk3ß and ß-catenin were detected at 95kDA, 47kDA and 65 kDA, respectively. The expression of DSH and ß-catenin were significantly down-regulated in treated PC-3 by Phyllanthus extracts. However, the expression of Gsk3ß was significantly up-regulated in PC-3 cells after treated with Phyllanthus treatment. Activated Gsk3ß proteins will degrade ß-catenin and thus, suppressed activity of c-myc in treated PC-3 (Figure 5).

As shown in Figure 5, the expression of the c-myc was detected at 67kDA. The downstream targets of c-myc were HIF-α and VEGF and both were detected at 120kDa and 50kD, respectively. Tumour growth and angiogenesis are correlated with the expression of HIF-α and VEGF. The high level expressions of these three proteins were detected in highly metastatic PC-3 cells. However, their levels of expression were significantly decreased in treated PC-3 compared to untreated cells.

Inhibitions of NFκB pathway by Phyllanthus extract

The constitutively activated NFκB signalling pathway leads to activation of multiple genes associated with cancerous cell proliferation, metastasis, angiogenesis and suppression of apoptosis. The NFκB protein or known as p50/p65 heterodimer was highly detected in untreated PC-3 cells (Figure 6). However, their expressions were significantly reduced after treated with Phyllanthus extracts.

Proteomic profiling of the differentially expressed proteins in Phyllanthus treated PC-3

Differentially expressed proteins were defined as statistically based on two criteria: 1) degree of intensity >1.0 fold (Protein scores greater than 70 are significan, p<0.05) and 2) reoccurrence more than two times in the three repeated experiments. According to these criteria, a total of 63 proteins were identified by MS/MS and grouped in four biological processes as listed in Table2. Only XXXX were up-regulated and the others were down-regulated in PC-3 after treated with Phyllanthus.


Nowadays, modern therapies for cancer are unsuccessful due to their ineffectiveness, lack of safety and costly. Although chemotherapy was advocated at one time, recent studies shows these agents are no longer effective as it can evoke other pathways in cancerous cell to survive and cause toxic to normal cells as well. Therefore, agents that could target multiple pathways can become more promising potential in cancer treatment.

In our previous studies, Phyllanthus extracts exhibits selective cytotoxicity on cancer cells and leads to apoptosis induction. This mainly attributed to the presence of different bioactive compounds within the Phyllanthus plant such as gallic acid and geraniin.. Kandil et al. (2002) has reported that no any individual class of components could be fully responsible for activity produced by whole extracts. Therefore, it is meaningful to assess the activity of Phyllanthus extract as a whole mixture of bioactive compounds rather than as their individual compounds.

Cancer cells survival requires the inhibition of apoptosis, which accomplished by suppressing the expression of pro-apoptotic proteins (Bax) as well as promoting the expression of anti-apoptotic proteins (Bcl-2). However, the expression of Bcl-2 was greatly enhanced with accompanied by reduction of Bax expression in Phyllanthus-treated PC-3 cells. Other pro-apoptotic protein, including also found has been to be activated by Phyllanthus.

The PI3K/AK pathway is an overactive intracellular signalling pathway in prostate cancer that involved in the regulation of apoptosis, cell cycle progression and cellular growth. However, the suppression on Akt protein would induce induction of apoptosis through activation of the proapoptotic factors such as Bad, GSK-3ß, procaspase-9 and TRAIL/APO-2L (TNF-Related Apoptosis-Inducing Ligand). Induction of apoptosis by Phyllanthus will be further implemented when the transcription factor cyclic AMP response element-binding protein (CREB), and the IκB kinase (IKK), a positive regulator of NF-κB, were dephosphorylated by Akt protein, thus leads reduction in expression of genes with anti-apoptotic activity.

Glycogen synthase kinase-3 (GSK-3ß), cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1, and Raf, are downstream targets for Akt proteins to regulate protein synthesis, glycogen metabolism and cell cycle regulation of cancer. In cancer cell, active Akt will inhibits GSK-3ß function in degradation of ß-catenin and c-myc, thus allowing expression of several genes including cell cycle, such as Cyclin D1. Therefore, down-regulation of Akt protein by Phyllanthus would leads to activation of GSK-3ß and then causes degradation of ß-catenin and c-myc and cell cycle arrest at G1-phase in Phyllanthus-treated PC-3. The cell cycle arrest of PC-3 by Phyllanthus was further implemented by antiproliferative effects p21CIP1/WAF1 and p27KIP1 due to both dephosphorlation of Akt and degradation of c-myc.

Ras protein is membrane bound GTPases and responsible to transmit extracellular signals into nucleus to regulate genes drive malignancy of cancer including increased proliferation, evasion of apoptosis, metastasis and angiogenesis. The down-regulation of Ras proteins by Phyllanthus was observed and this will leads to suppression of its downstream targets; Raf and Akt. The major downstream targets of Ras are three mitogen-activated protein kinase (MAPK) pathways; ERK 1/2, JNK1/2 and p38 MAPK.

In cancer cells, the active ERK1/2 wills activates RSK2 and Elk1, and subsequently activate C-Jun and C-Fos proteins. Both C-Jun and C-Fos will combine to form activator protein 1 (AP-1) which is a transcription factor that regulates genes in DNA. Besides, JNK1/2 and p38 MAPK will enhance AP-1 formation by further produce C-Jun and C-Fos. However, all these proteins involved in MAPK signalling were down-regulated in PC-3 after treated with Phyllanthus extracts. This indicates Phyllanthus extracts disrupts Ras/MAPK signalling to inhibit proliferation, metastasis and angiogenesis and induce apoptosis. However, the actual mechanisms of ERK signalling to induce apoptosis in cancer cells still remain unknown.

In benign and malignant human prostate tissue, NFkB is constrictively active. The effect of Phyllanthus extract on NFkB as measured by p65 and p50 nuclear translocation. There are several studies to support anti-apoptotic role of NF-κB in cancer cells. NFκB induces the expression of the Inhibitors of Apoptosis (IAPs) and some anti-apoptotic proteins. The IAPs will inhibit apoptosis in both extrinsic and intrinsic pathways through direct suppression of effector caspases (caspases-3, -6, -7, and 9), whereas anti-apoptotic proteins will antagonize the function of the pro-apoptotic proteins. However, the expression of these proteins was decreased after treated with Phyllanthus in PC-3. The secretion and activation of matrix metalloprotenaises (MMP) by active NFkB are well documented. However, the down-regulation of NFkB expression and their signalling molecules in treated PC-3 cells was detected and hence leads to inhibit proliferation, metastasis and angiogenesis and induce programmed cell death.

The uncontrolled proliferation of cancer cells and rapid growth of the tumour mass usually outpace new blood vessels generation, resulting in an insufficient blood supply/oxygen to the tumour tissues. The insufficient of oxygen supply for use in mitochondrial respiration to synthesis of ATP. Thus, this condition will force the cancer cells to up-regulate the glycolytic pathway as the main route of energy production, and known as Warbury effect. This metabolic adaptation in response to these alterations is always associated with resistance to therapeutic agents.

From our results, Phyllanthus extracts were inhibits the glycolytic pathway and ATP productions in prostate cancer cells by down-regulated HIF-α and eIF-4e proteins. The deactivated HIF-α and eIF-4e proteins will reduce production vascular endothelial growth factor (VEGF) and thus, inhibits tumour angiogenesis and thereby decrease oxygen transport. There are several signalling pathway involved in Warburg effect such as PI3K/Akt and Ras. However, suppression of Akt by Phyllanthus has leads inhibition on glycolytic pathway through deactivation of phosphoglycerate kinase enzymes. Other low levels of glycolytic enzymes were detected including urocortin-3, alpha-enolase, GAPDH, fructose-bisphosphate aldolase, triosephosphate isomerase and neuroglobin. In addition, Phyllanthus can also deactivate glycolysis-apoptosis integrating molecule, Bax through dephosphorylation by Akt and makes cancer cells are more susceptible to apoptosis. Thus, inhibition of glycolysis by Phyllanthus could effectively kills cancer cells in a hypoxic environment in which the cancer cells exhibit high glycolytic activity and induce apoptosis.

The networks of protein-protein interaction are important and play important roles in cellular function and biological processes. Dysfunction of these interactions may cause many diseases, including cancer. The cancer-related proteins are tightly regulated to ensure immortality of cancer cells. Disruption of these cancer-related proteins could be potential in cancer treatment. There are various types of proteins in PC-3 cells have been detected and involved involve in proliferation, protein synthesis, energy metabolism, adhesion, metastasis and angiogenesis. However, their expressions have been disrupted by Phyllanthus in PC-3 cells. .

Vimentin is one of intermediate filament family of proteins and found overexpressed in various types of cancers. Its overexpression always correlates with tumour growth, and invasion; however, the exact role of this protein in cancer progression still remains unclear. Vimentin has used as a marker for epithelial-mesenchymal transition (EMT) due to its expression is associated with ECM transition. Epithelial-mesenchymal transition (EMT) is a series of rapid changes in cancer cell including a loss of cell-cell adhesion, enhanced migratory capacity, invasiveness, and resistance to apoptosis. In cell cytosol, vimentin is protected by AKT from caspase-induced proteolysis. Vimentin stabilizes ERK protein and allows ERK translocate into nucleus. In addition, vimentin also binds to 14-3-3 proteins to prevent assembly of Raf-14-3-3 and control various intracellular signalling and cell cycle control pathways. The down-regulation of vimentin and 14-3-3 proteins, together with AKT and ERK proteins were detected in Phyllanthus-treated PC-3 cells, thus leads to decrease prostate tumour growth, adhesion, invasion and sensitive to apoptosis.

ECM transition also involved expression of differentiation-specific keratin. There are several studies proved correlation of keratin down-regulation with increased tumour aggressiveness. The exact mechanism of this regulation is still unclear till present. Paccione et al (2008) suggested that down-regulation of keratin is could be due to expression of a factor(s) that induced by vimentin. Our results revealed that few keratin types were increased with decreased of vimentin in PC-3 after treated with Phyllanthus.

One of the endocytic proteins, sorting nexin 3 (SNX3), is significantly up-regulated in after treated with Phyllanthus. The actual function of the sorting nexins in cancer in not entirely clear, but SNX may involve in degradation of cell surface receptors. Thus, Phyllanthus may restore SNX3 function in promoting degradation of cell surface receptor in prostate cancer cells, thus cause the cells insensitive to growth factors and halts their growth.

Protein MEMO1 (mediator of ErbB2-driven cell motility 1) is involved in ErbB2 expression in tumour cells. Active ErbB2 protein will suppress GSK-3ß activity, mediated by MEMO protein. The expression of ErB2 is always positively correlated to have more aggressive and metastatic tumour in cancer patient. However, MEMO proteins were decreased in PC-3 with Phyllanthus treatment. The down-regulated protein MEMO1 was in conjuction with down-regulated of MAPK and PI3K pathways and up-regulated GSK-3ß thus could reduce metastatic properties of prostate cancer cells and leads to apoptosis.

Alteration in DNA repair mechanism in cancer cell could leads to active cell cycle and give rise to characteristic of uncontrolled proliferation and inhibition of apoptosis. However, there are several proteins involved in DNA repair were suppressed by Phyllanthus extract in prostate cancer cells such as DNA damage-binding protein 2, transcription factor 23, gremlin-1, proliferation-associated protein 2G4, growth factor receptor-bound protein 2, 14-3-3 proteins, annexin A1, glutathione, galectin-1, heat shock proteins, peroxiredoxin, RuvB-like 2 and Serpin B9.

Glutathione S-transferases (GSTs) are a family of enzymes that play an important role in redox homeostasis and play an essential role in several vital signalling pathways involved in proliferation, apoptosis and inflammatory responses with reduction of glutathione. The catalytic activity of c-Jun N-Terminal kinase (JNK) pathway is regulated by glutathione S-transferase P (GSTP1-1) (Wang et al. 2001). The high levels of GSTP1-1 in treated PC-3 cells will leads to assembly of JNK-GSTP1-1 complex, and resulting in decreased JNK catalytic activity. The active GSTP1-1 can act as a thiol donor and has inhibition impact on several proteins, which shown decrease in their activity after treated with Phyllanthus; includes enzymes involved with protein folding and stability (peroxiredoxin I, protein disulphide isomerase, alpha enolase, phosphoglycerate kinase), cytoskeletal proteins (vimentin, actin, tubulin, Annexin), signalling proteins (ERK, caspase 3), transcription factors (c-jun, NFκB subunit p65 and p50), ras proteins, heat shock proteins and energy metabolism and glycolysis (GAPDH and NADH ubiquinone).

Galectin-1 is a multifunctional protein, which can act as apoptosis inhibitor (Yang et al., 1996), mRNA splicing promoter (Dagher et al., 1996), adhesion molecule (Glinsky et al., 2001) and promotes cancer progression and metastasis (Liu et al., 2005; Takenaka et al., 2004). High levels of this galectin-3 are seen in patients with melanoma (Vereecken et al., 2006), breast, colorectal, lung (Lurisci et al., 2000) and head and neck (Saussez et al., 2008) cancers. The inhibition of this galectin-3 by Phyllanthus in PC-3 could explicit apoptosis, G1 cell cycle arrest and growth inhibition (Fischer et al., 2005), disrupts cell-cell interaction and inhibits metastasis of cancer cells.

Cancer cells generally have a high affinity to glucose for protein synthesis and energy production for rapid growth of cancer cells. Calumenin, Calreticulin, 39S ribosomal protein L51, betaine--homocysteine S-methyltransferase 1, protein disulfide-isomerase A4, heat shock proteins, 78 kDa glucose-regulated protein, dynamin-1-like protein and proteasome subunit beta type-3 are some of the proteins involved in protein synthesis and energy production in PC-3, were down-regulated after Phyllanthus treatment in PC-3 cells.

Heat shock proteins (Hsps) are molecular chaperones that regulate integrity and stability of proteins. The overexpression of Hsp found in a wide range of human cancers and is implicated cancer cell proliferation, differentiation, invasion and metastasis. The down-regulation of Hsps could explicit inhibition on proliferation, invasion and metastasis in PC-3 after treated with Phyllanthus.

In summary, our study provided new insight into the understanding of Phyllanthus regulation on major cancer pathways and protein regulation in prostate cancer and might be offered further application as anti-cancer agent. In the future, the other anticancer effects in term of anti-metastasis and anti-angiogenesis of Phyllanthus are in need to be examined.