Prostate cancer is among one of the major life-threatening cancers and is the second most frequently diagnosed cancer after lung cancer in men. Even with advances in early detection and conventional treatment strategies, prostate cancer incidence has increased worldwide, and it has become more resistant to available treatments. Generally, tumour metastasis confers poor prognosis and remains a major obstacle in the inability to improve patient outcome, where at least 50% of prostate cancer patients are diagnosed with pathologic or clinical evidence of bone metastasis [
During the tumour progression, cancer cells acquire several distinct characteristic alterations from normal cells which include the capacities to proliferate independently of growth-promoting or growth-inhibitory signals, the ability to undergo metastasis and angiogenesis, and to evade apoptosis [
Cellular signalling pathways are always interconnected to form complex signalling networks. These interactions are important for a cell to regulate vital and diverse processes such as protein synthesis, cell growth, immune responses, differentiation, and homeostasis and cell death [
The actual molecular events leading to prostate metastasis remain elusive. The mitogen-activated protein kinase family members (MAPKs) are believed to play a part in mediating metastasis by inducing proteolytic enzymes that lead to degradation of basement membrane and those that promote cell migration and activate pro-survival genes and cell growth [
During tumour metastasis, the hypoxia-inducible factors (HIFs) which regulate oxygen delivery and consumption are highly expressed. 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 [
In this study, we attempted to investigate the underlying molecular mechanism of
We have previously reported four species of
Four different species of
Human prostate adenocarcinoma cancer cell, PC-3, was obtained from American Type Culture Collection (Rockville, MD). Cells were cultured with RPMI-1640 (Roswell Park Memorial Institute) and supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco). Cells were maintained in culture at 37°C in a humidified atmosphere of 5% CO2 and 95% humidity.
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. Optimization of the conditions, including amount and incubation time of plasmid construct of transcription factor-responsive reporter of each pathway into the cells, was performed to ensure high transfection efficiency and inhibit transformation. Reverse transfection protocol was implemented for this assay. Prior to experiment, PC-3 cells were seeded into a 96-well white plate and incubated overnight at 37°C. One hundred nanograms of plasmid construct of transcription factor-responsive reporter of each pathway and controls were added to cells and incubated overnight in a 37°C incubator with 5% CO2. After 24 h of transfection, the old media was discarded and the cells were treated with
Treatment of PC-3 cells at different IC50 values of
|
Extracts |
|
---|---|---|
PC-3 | ||
|
Aqueous (H2O) | 178.3 ± 2.8 |
Methanolic (MeOH) | 84.3 ± 1.1 | |
|
Aqueous (H20) | 155.0 ± 1.2 |
Methanolic (MeOH) | 117.7 ± 2.1 | |
|
Aqueous (H2O) | 155.7 ± 2.1 |
Methanolic (MeOH) | 54.2 ± 2.1 | |
|
Aqueous (H2O) | 156.7 ± 2.4 |
Methanolic (MeOH) | 100.5 ± 1.2 |
Each of the pathways/reporters consists of an inducible transcription factor-responsive firefly luciferase reporter and the constitutively expressing
The intracellular signalling molecules in MAPK (pan-Ras, c-Raf, Elk, Rsk, c-Jun, JNK, Akt, and p38 MAPK), Wnt (GSK3
A total of 500 mg total protein was subjected to 2D gel electrophoresis according to the manufacturer’s instructions (GE Healthcare). Briefly, total proteins were extracted from untreated and treated groups by incubation with lysis buffer on ice for 30 minutes. The protein pellets were resolubilized 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 500 mg of proteins were rehydrated into 13 cm 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 500 V for 1 h; step 2 at 500–1000 V for 1 h; step 3 at 1000–8000 V for 2.5 h; and step 4 at 8000 V for 0.5 h. Upon completion of first-dimensional separation, the strip was equilibrated as following: first reduction with 64.8 mM of dithiothreitol (DTT)-SDS equilibration buffer (50 mMTris-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 (IAA)-SDS equilibration buffer for another 15 minutes. The second-dimension electrophoresis was performed by electrophoresing the samples in 12.5% SDS acrylamide gels by using the SE600 Ruby system (GE Healthcare) at 25°C in an electrode buffer (25 mMTris, 192 mM glycine, and 0.1% [wt/vol] SDS) with the following settings: step 1 at 100 V/gel for 45 minutes; and step 2 at 300 V/gel until the run is completed. After electrophoresis, the gels were fixed with destaining solution for 30 minutes, followed by staining with hot Coomassie blue for 30 minutes. Lastly, the gels were scanned using Ettan DIGE Imager (GE Healthcare). Gel images were analysed using PDQuest 2-D Analysis Software (Bio-Rad, USA), and only protein spots which showed significant differences (more than 1.0 fold) were selected for mass spectrometry analysis.
The significant protein spots were manually excised from the polyacrylamide gels and kept in sterile 1.5 mL Eppendorf tubes. Excised spots (gel plugs) were washed with destaining solution (50 mM NH4HCO3) until the gel plugs were clear. The gel plugs were then incubated with reducing solution (100 mM NH4HCO3 containing 10 mM DTT) for 30 min at 60°C. Then, the plugs were alkylated with 100 mM NH4HCO3 containing 55 mM IAA for 20 min in the dark and followed with three times of 50% acetone in 100 mM NH4HCO3 for 20 min each. The gel plugs were then rehydrated with 100% ACN. In-gel digestion using trypsin gold (Promega, Mass Spectrometry Grade) was added into gel plug and incubated overnight at 37°C. Proteins were extracted from gel plugs and were purified using Ziptip (Ziptip C18, Millipore, Bedford, MA, USA). The eluted proteins were mixed with MATRIX solution and spotted on MALDI plate using dry droplet method and analysed using AbSciexTof/Tof instruments. The generated peptides were blasted with MASCOT search algorithm (version 2.1.0) to identify the possible proteins.
For all experiments, results were expressed as the mean ± standard error (SEM) of data obtained from triplicate experiments using SPSS software. The Student’s
Ten different cancer-related pathways were investigated including Wnt, Notch, p53, TGF-
The expression of transcription activities in ten-cancer related pathways of untreated and treated PC-3. Only six different pathways (Wnt, NF
One of the hallmarks of cancer is the inhibition of apoptosis. This can be achieved by suppressing the expression of proapoptotic protein, Bax, and stimulating the expression of antiapoptotic protein, Bcl-2, as shown in Figure
Disruption of
Two upstream activators, pan-Ras and c-Raf, were highly expressed in untreated PC-3 to ensure constitutive activation of MAPK pathways (Figure
Inhibition of cellular signalling pathways in PC-3 cells by
The expression of Wnt signalling pathway was detected at increased levels in PC-3 cells (Figure
Two members of NF
Differentially expressed proteins were statistically defined based on two criteria: (1) degree of intensity
Fold changes of differential proteins in aqueous- and methanolic-treated PC-3 cells. Symbols “+” indicate upregulation and “−” indicate downregulation. PA:
No. | UniProtKB/Swiss-Prot |
Protein |
|
|||||||
---|---|---|---|---|---|---|---|---|---|---|
Aqueous extract | Methanolic extract | |||||||||
PA | PN | PU | PW | PA | PN | PU | PW | |||
I | Cell adhesion, migration, invasion, and metastasis and angiogenesis | |||||||||
| ||||||||||
1 | P98172 | Ephrin-B1 | −1.53 | −1.45 | −1.21 | −1.41 | −2.14 | −2.21 | −2.01 | −2.31 |
2 | P05787 | Keratin, type II cytoskeletal 8 | 1.45 | 1.32 | 1.38 | 1.21 | 1.86 | 1.74 | 1.92 | 1.62 |
3 | P63261 | Actin, cytoplasmic 2 | −1.43 | −1.53 | −1.21 | −1.53 | −1.74 | −1.79 | −1.72 | −1.53 |
4 | Q8NDI1 | EH domain-binding protein 1 | −1.32 | −1.32 | −1.21 | −1.13 | −1.42 | −1.32 | −1.43 | −1.43 |
5 | P04792 | Heat shock protein beta-1 | −1.43 | −1.32 | −1.54 | −1.54 | −1.42 | −1.43 | −1.32 | −1.25 |
6 | P35527 | Keratin, type I cytoskeletal 9 | 1.38 | 1.32 | 1.53 | 1.42 | 1.64 | 1.34 | 1.35 | 1.37 |
7 | Q9BYR8 | Keratin-associated protein 3-1 | 1.53 | 1.32 | 1.47 | 1.64 | 1.47 | 1.53 | 1.42 | 1.46 |
8 | P08670 | Vimentin | −1.43 | −1.32 | −1.32 | −1.11 | −1.25 | −1.43 | −1.32 | −1.42 |
9 | Q9NY65 | Tubulin alpha-8 chain | −1.53 | −1.43 | −1.48 | −1.35 | −1.40 | −1.43 | −1.42 | −1.73 |
10 | Q9Y316 | Protein MEMO1 | −1.43 | −1.42 | −1.24 | −1.32 | −1.63 | −1.32 | −1.42 | −1.43 |
| ||||||||||
II | Proliferation, cell cycle, and apoptosis | |||||||||
| ||||||||||
11 | Q92466 | DNA damage-binding protein 2 | −1.42 | −1.32 | −1.43 | −1.41 | −1.42 | −1.37 | −1.43 | −1.32 |
12 | Q7RTU1 | Transcription factor 23 | −1.39 | −1.33 | −1.42 | −1.43 | −1.53 | −1.63 | −1.58 | −1.42 |
13 | Q9UQ80 | Proliferation-associated protein 2G4 | −1.37 | −1.57 | −1.42 | −1.47 | −1.63 | −1.45 | −1.53 | −1.43 |
14 | P62993 | Growth factor receptor-bound protein 2 | −1.42 | −1.52 | −1.43 | −1.32 | −1.42 | −1.42 | −1.43 | −1.64 |
15 | O60565 | Gremlin-1 | −1.43 | −1.43 | −1.33 | −1.43 | −1.56 | −1.43 | −1.54 | −1.44 |
16 | P27348 | 14-3-3 protein theta | −1.32 | −1.43 | −1.53 | −1.42 | −1.42 | −1.21 | −1.32 | −1.42 |
17 | P61981 | 14-3-3 protein gamma | −1.32 | −1.32 | −1.43 | −1.35 | −1.32 | −1.42 | −1.22 | −1.32 |
18 | P04083 | Annexin A1 | −1.52 | −1.54 | −1.42 | −1.43 | −1.64 | −1.43 | −1.43 | −1.43 |
19 | Q96LY2 | Coiled-coil domain-containing protein 74B | −1.47 | −1.34 | −1.47 | −1.46 | −1.33 | −1.33 | −1.36 | −1.57 |
20 | P31943 | Heterogeneous nuclear ribonucleoprotein H | −1.47 | −1.33 | −1.35 | −1.21 | −1.44 | −1.56 | −1.43 | −1.53 |
21 | P09211 | Glutathione S-transferase P | −1.96 | −1.84 | −1.77 | −2.05 | −1.89 | −1.87 | −2.01 | −1.97 |
22 | P41221 | Protein Wnt-5a | −1.75 | −1.87 | −1.87 | −1.92 | −1.74 | −1.84 | −1.85 | −2.01 |
23 | Q9H0R3 | Transmembrane protein 222 | −1.57 | −1.52 | −1.57 | −1.42 | −1.47 | −1.62 | −1.57 | −1.46 |
24 | Q5MJ09 | Sperm protein associated with the nucleus on the X chromosome N3 | −1.45 | −1.57 | −1.57 | −1.57 | −1.34 | −1.45 | −1.32 | −1.46 |
25 | P46063 | ATP-dependent DNA helicase Q1 | −1.57 | −1.57 | −1.36 | −1.46 | −1.56 | −1.47 | −1.46 | −1.57 |
26 | P09382 | Galectin-1 | −1.25 | −1.46 | −1.57 | −1.47 | −1.46 | −1.33 | −1.46 | −1.46 |
27 | P04792 | Heat shock protein beta-1 | −1.37 | −1.47 | −1.47 | −1.46 | −1.56 | −1.46 | −1.34 | −1.47 |
28 | P78417 | Glutathione transferase omega-1 | −1.46 | −1.46 | −1.47 | −1.47 | −1.57 | −1.54 | −1.68 | −1.57 |
29 | Q06830 | Peroxiredoxin-1 | −1.57 | −1.42 | −1.29 | −1.33 | −1.48 | −1.62 | −1.54 | −1.67 |
30 | P30048 | Thioredoxin-dependent peroxide reductase, mitochondrial | −1.45 | −1.52 | −1.12 | −1.64 | −1.58 | −1.44 | −1.57 | −1.57 |
31 | Q9Y230 | RuvB-like 2 | −1.46 | −1.47 | −1.38 | −1.32 | −1.75 | −1.57 | −1.64 | −1.57 |
32 | P50453 | Serpin B9 | −1.46 | −1.50 | −1.36 | −1.38 | −1.58 | −1.67 | −1.76 | −1.63 |
33 | Q8ND25 | E3 ubiquitin-protein ligase ZNRF1 | −1.56 | −1.43 | −1.47 | −1.47 | −1.58 | −1.67 | −1.82 | −1.54 |
34 | Q6PRD1 | Probable G-protein-coupled receptor 179 | −1.45 | −1.43 | −1.47 | −1.56 | −1.36 | −1.37 | −1.54 | −1.58 |
35 | O43521 | Bcl-2-like protein 11 | −1.66 | −1.52 | −1.42 | −1.56 | −1.57 | −1.73 | −1.65 | 1.56 |
36 | P56703 | Proto-oncogene Wnt-3 precursor | −2.18 | −1.92 | −2.06 | −2.34 | −1.79 | −1.97 | 1.83 | −2.11 |
37 | Q8N4Z0 | Putative Ras-related protein Rab-42 | −2.11 | −2.03 | −1.79 | −1.98 | −1.96 | −2.1 | −2.15 | −1.94 |
38 | P01112 | GTPase HRas precursor | −2.12 | −1.99 | −1.83 | −1.74 | −1.85 | −2.03 | −2.05 | −1.92 |
| ||||||||||
III | Glycogenesis and glycolysis | |||||||||
| ||||||||||
39 | Q969E3 | Urocortin-3 | −1.37 | −1.42 | −1.37 | −1.46 | −1.65 | −1.73 | 1.67 | −1.47 |
40 | P00558 | Phosphoglycerate kinase 1 | −2.01 | −2.12 | −2.22 | −1.93 | −1.56 | −1.37 | −1.57 | −1.36 |
41 | P06733 | Alpha-enolase | −1.42 | −1.38 | −1.37 | −1.36 | −1.58 | −1.57 | −1.65 | −1.57 |
42 | P04406 | Glyceraldehyde-3-phosphate dehydrogenase | −1.92 | −1.88 | −1.92 | −1.83 | −1.78 | −1.36 | −1.36 | −1.56 |
43 | P04075 | Fructose-bisphosphate aldolase A | −1.63 | −1.42 | −1.39 | −1.36 | −1.48 | −1.53 | −1.52 | −1.57 |
43 | P60174 | Triosephospate isomerase | −1.47 | −1.46 | −1.47 | −1.76 | −1.48 | −1.53 | −1.49 | −1.49 |
44 | Q9NPG2 | Neuroglobin | −1.47 | −1.47 | −1.58 | −1.47 | −1.56 | −1.63 | −1.74 | −1.74 |
| ||||||||||
IV | Protein synthesis and energy metabolism | |||||||||
| ||||||||||
45 | Q4U2R6 | 39S ribosomal protein L51 | −1.37 | −1.47 | −1.36 | −1.53 | −1.64 | −1.74 | −1.65 | −1.67 |
46 | Q93088 | Betaine-homocysteine S-methyltransferase 1 | −1.46 | −1.47 | −1.46 | −1.48 | −1.67 | −1.48 | −1.58 | −1.57 |
47 | P50583 | Bis(5′-nucleosyl)-tetraphosphatase [asymmetrical] | −1.45 | −1.57 | −1.30 | −1.42 | −1.39 | −1.47 | −1.67 | −1.87 |
48 | Q96A11 | Galactose-3-O-sulfotransferase 3 | −1.69 | −1.74 | −1.67 | −1.85 | −1.93 | −1.74 | −1.82 | −1.89 |
49 | Q9Y274 | Type 2 lactosamine alpha-2,3-sialyltransferase | −1.28 | −1.52 | −1.58 | −1.44 | −1.68 | −1.64 | −1.58 | −1.57 |
50 | O43852 | Calumenin | −1.53 | −1.47 | −1.47 | −1.42 | −1.57 | −1.65 | −1.56 | −1.53 |
51 | P13667 | Protein disulfide-isomerase A4 | −1.47 | −1.41 | −1.44 | −1.45 | −1.58 | −1.64 | −1.45 | −1.57 |
52 | P27797 | Calreticulin | −1.54 | −1.32 | −1.47 | −1.48 | −1.45 | −1.53 | −1.57 | −1.64 |
53 | P07900 | Heat shock protein HSP 90-alpha | −1.64 | −1.32 | −1.34 | −1.43 | −1.55 | −1.42 | −1.32 | −1.53 |
54 | P12235 | ADP/ATP translocase 1 | −1.46 | −1.38 | −1.45 | −1.35 | −1.58 | −1.68 | −1.67 | −1.47 |
55 | P52209 | 6-phosphogluconate dehydrogenase, decarboxylating | −1.46 | −1.49 | −1.54 | −1.45 | −1.57 | −1.52 | −1.48 | −1.56 |
56 | Q9Y478 | 5′-AMP-activated protein kinase subunit beta-1 | −1.38 | −1.46 | −1.47 | −1.47 | −1.67 | −1.75 | −1.47 | −1.57 |
57 | P11021 | 78 kDa glucose-regulated protein | −1.46 | −1.46 | −1.47 | −1.57 | −1.57 | −1.54 | −1.57 | −1.84 |
58 | Q9UI09 | NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 | −1.42 | −1.36 | −1.33 | −1.33 | −1.57 | −1.73 | −1.57 | −1.46 |
59 | P30101 | Protein disulfide-isomerase A3 | −1.20 | −1.47 | −1.37 | −1.21 | −1.57 | −1.63 | −1.56 | −1.57 |
60 | P49411 | Elongation factor Tu, mitochondrial | −1.48 | −1.45 | −1.57 | −1.33 | −1.38 | −1.54 | −1.68 | −1.47 |
61 | Q5TGZ0 | Mitochondrial inner membrane organizing system protein 1 | −1.47 | −1.46 | −1.47 | −1.49 | −1.57 | −1.47 | −1.67 | −1.65 |
62 | P19404 | NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial | −1.21 | −1.32 | −1.33 | −1.43 | −1.57 | −1.57 | −1.57 | −1.74 |
63 | Q9H0F7 | ADP-ribosylation factor-like protein 6 | −1.76 | −1.50 | −1.57 | −1.54 | −1.56 | −1.54 | −1.49 | −1.70 |
64 | O75436 | Vacuolar protein sorting-associated protein 26A | −1.46 | −1.54 | −1.63 | −1.76 | −1.47 | −1.38 | −1.57 | −1.57 |
65 | Q9NXV2 | BTB/POZ domain-containing protein KCTD5 | −1.47 | −1.47 | −1.33 | −1.50 | −1.58 | −1.65 | −1.67 | −1.66 |
66 | O00429 | Dynamin-1-like protein | −1.47 | −1.63 | −1.47 | −1.57 | −1.56 | −1.57 | −1.67 | −1.73 |
67 | P40261 | Nicotinamide N-methyltransferase | −1.29 | −1.22 | −1.34 | −1.28 | −1.45 | −1.48 | −1.83 | −1.42 |
68 | P49720 | Proteasome subunit beta type-3 | −1.53 | −1.22 | −1.34 | −1.47 | −1.58 | −1.53 | −1.67 | −1.57 |
69 | Q6IQ16 | Speckle-type POZ protein-like | −1.42 | −1.67 | −1.47 | −1.47 | −1.63 | −1.64 | −1.68 | −1.47 |
70 | P21796 | Voltage-dependent anion-selective channel protein 1 | 1.57 | 1.63 | 1.52 | 1.79 | 1.67 | 1.84 | 1.77 | 1.76 |
71 | Q9Y3D8 | Adenylate kinase isoenzyme 6 | −1.51 | −1.33 | −1.43 | −1.49 | −1.46 | −1.64 | −1.74 | −1.76 |
72 | Q14152 | Eukaryotic translation initiation factor 3 subunit 12 | −1.40 | −1.37 | −1.39 | −1.49 | −1.53 | −1.67 | −1.63 | −1.53 |
Proteomic profiles of
In Group I (cell adhesion, migration, invasion, and metastasis), 10 proteins were found to be differentially expressed in
In Group II (proliferation, cell cycle, and apoptosis), 28 proteins were significantly downregulated in treated PC-3 cells. Among these altered proteins, five, namely gluthathione S-transferase P, protein Wnt-5a, proto-oncogene Wnt-3, putative Ras-related protein Rab-42, and GTPAse HRas precursor, showed the greatest reduction in their expression with a range of 1.7–2.2 folds higher than untreated cells (
In Group III (glycogenesis and glycolysis), 7 downregulated proteins were identified in the treated PC-3 cells, and five of them were enzymes: phosphoglycerate kinase-1, alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase (G3PD), fructose-biphosphate aldolase, and triosephosphate isomerase.
In Group IV (protein synthesis and energy metabolisms), 27 proteins were differentially expressed with only one being significantly upregulated in PC-3 after treatment with
Presently, modern therapies for cancer treatment such as surgery, chemotherapy, and immunotherapy are deemed relatively unsuccessful due to their ineffectiveness and safety issues, as well as costliness [
We have previously shown
Cancer cells survival requires the inhibition of apoptosis, which is accomplished by suppressing the expression of proapoptotic proteins (Bax) as well as promoting the expression of antiapoptotic proteins (Bcl-2) [
Schematic diagram illustrating that
Ras proteins are membrane-bound GTPases responsible for transmitting extracellular signals into the nucleus to regulate genes-driven malignancy of cancer including increased proliferation, apoptosis evasion, metastasis, and angiogenesis [
In cancer cells, the active ERK1/2 protein will activate RSK2 and Elk1 proteins that subsequently activate c-Jun and c-Fos proteins. Both c-Jun and c-Fos will then combine to form activator protein 1 (AP-1) which is a transcription factor that regulates survival genes [
The PI3K/Akt pathway is an overactive intracellular signalling pathway in prostate cancer that is involved in the regulation of apoptosis, cell cycle progression, and cellular growth [
Glycogen synthase kinase 3-beta (GSK3
In benign and malignant human prostate tissue, NF
The rapid uncontrolled proliferation of cancer cells usually outpaces new blood vessels generation, hence resulting in insufficient blood supply/oxygen to tumour tissues. In this condition, the cancer cells are forced to upregulate the expression of genes and enzymes which are involved in anaerobic glycolytic pathway as the main route of energy production, and this phenomenon is known as the Warburg effect [
In our study,
The network of protein-protein interaction in cancer cells plays an important role in the regulation of cellular function and biological processes [
In cell cytosol, Akt protein protects vimentin from caspase-induced proteolysis, and in
Glutathione S-transferases (GSTs) are a family of enzymes that play an important role in redox homeostasis [
In the
Mitochondrion, a vital component cell, is best known for its oxidative phosphorylation ability to produce energy source, ATP [
In conclusion, this study has provided a comprehensive perspective on how
The authors declare that they have no conflict of interests.
The authors thank Anusyah Rathakrishnan, Thamil Vaani, and Rajendran Peramauyan for their critical reading and editing of the paper. This study was funded by the Postgraduate Research Grant, University of Malaya (PV053/2011B), UMRG RG391/11HTM, and Malaysian Agricultural and Research Development Institute (MARDI) (53-02-03-1002).