Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.
Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALD1TOF-TOF MS. The down-regulation of stathmin. Ran GTPase activating protein-1 (RanBPl % and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji ceil cycle regulation showed that it induced a Gl cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin Dl expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-kB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells.
[c] 2011 Elsevier GmbH. All rights reserved.
Keywords: EB1 Maslinic acid NF-kB RanBPI Stathmin
Triterpenoids, as major constituents of plants used in Asian traditional medicine, have been reported to have anti-inflammatory, hepatoprotective, analgesic, antimicrobial, antimycotic, virostatic, immunomodulatory and tonic effects (Dzubak et al. 2006). Their biosynthesis involves cascade cyclizations and rearrangements of the acyclic precursor squalene and 2,3-oxidosqualene (Abe 2007), leading to tetra and pentacyclic triterpene skeleta. Maslinic acid is a naturally occurring pentacyclic triterpene found in medicinal plants suchasAsteryunnanens/s(Yu etal. 1995), Elaeagnus oldahmi (Tsuen-Ih et al. 1976), Eugenia gustavioides (Yazaki 1977), Centau-riurn erythraea (Bellavita et al. 1974), Coleus tuberosus (Mooi et al. 2010) and Olea europaea (Mussini et al. 1975). Maslinic acid has antioxidant (Montilla et al. 2003), anti-inflammatory (Martin et al. 2006), anti-tumour (Reyes et al. 2006; Hsum et al. 2011), antidiabetic (Chen et al. 2006) and anti-viral properties (Xu et al. 1996).
There are several mechanisms that are responsible for the anti-carcinogenic properties of chemopreventive compounds (Surh 2003). Some chemopreventive mechanisms are interconnected. Modulation of a given end-point may be the result of a specific mechanism or the consequences of upstream mechanisms (Flora and Ferguson 2005). Maslinic acid induces apoptosis in HT-29 human colon cancer cells by inhibiting expression of anti-apoptotic protein Bcl-2, increasing the ratio of pro-apoptotic protein Bax, releasing cytochrome c from the mitochondria and activating caspase-9 followed by caspase-3 (Reyes-Zurita etal. 2008). The pro-apoptotic and cytotoxic effects of maslinic acid on tumour cell lines may be mediated by the inhibition on NF-kB and AP-1 DNA binding activities (Hsum et al. 2011).
Proteomic techniques facilitate the qualitative and quantitative measurement of a broad-spectrum of proteins (Pastwa et al. 2007). Information derived from 2-DE and MALD1TOF-TOF MS allow the assessment of differentially expressed proteomes of cancer cells in response to chemopreventive drug treatment. This may serve as a model for cancer therapeutics, and is useful for the discovery of drug-specific biomarkers (Liebold et al. 2006). The study of the effects of maslinic acid at the proteomics level may help to elucidate the complex interactions of the protein network that relates signaling pathways.
Cell cycle deregulation is a common feature of human cancers. The cell cycle includes a gap period (Gl phase) which is controlled by positive (growth, survival and mitogenic) and negative (apoptotic and cytostatic) signals (Massague 2004). Another gap period (G2 phase) monitors replication errors before mitosis. Oncogenic transformation may result from the aberrancy in these Gl and G2 mechanisms. Deregulation of the cell cycle check points and overexpression of growth-promoting factors such as cyclin Dl and CDKs have been associated with tumourigenesis (Aggarwal and Shishodia 2006). Constitutive and deregulated CDK activation may contribute to unscheduled proliferation, genomic instability, and chromosomal instability (Malumbres and Barbacid 2009). Maslinic acid has been reported to induce Gl arrest in human colon cancer cells Caco-2 without affecting the cell cycle of non-tumoural intestine cell lines IEC-6 and IEC-1 (Reyes et al. 2006). However, its regulation of the human B lymphoma Raji cell cycle, cyclin and CDK inhibitor levels has not yet been investigated.
In this study, a proteomics approach was used to identify new molecular targets of maslinic acid in Raji cells. The growth inhibition effect of maslinic acid was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After determining the growth inhibition concentration, 2-DE and MALDI TOF-TOF MS were used to identify the target proteins. The expression of proteins which had been identified were evaluated by Western blotting to validate the variation in their levels in response to treatment. This study also determined a possible block in the cell cycle induced by maslinic acid in Raji cells and its effect on the Gl phase cell cycle regulatory molecules cyclin Dl and CDK inhibitor protein p21. A time course study on NF-kB DNA binding activity was undertaken to further investigate the molecular basis for the growth inhibition properties of maslinic acid.
Materials and methods
Chemical reagents and tissue culture
Maslinic acid (Fig. 1) was obtained from the tubers of Coleus tuberosus Benth following to the procedure of Mooi et ah (2010). The compound used was a chemically pure white powder (>95% pure as determined by HPLC). A stock solution of l0mg/ml in dimethyl sulphoxide (DMSO) was stored at -20[degrees]C This solution was diluted in the cell culture medium for use in treatment. FBS, RPMI and trypsin-EDTA were purchased from Gibco (NY, USA). EBl, stathmin, p21 and cyclin Dl antibodies were obtained from Cell Signaling Technology (Beverly, MA). RanBPl was purchased from Calbiochem, Merck (Germany). MTT was purchased from Sigma-Aldrich (Deutschland). Raji cells were grown in RPMI medium supplemented with 10% FBS in a 5% C[O.sub.2] humidified environment at 37[degrees]C. Maslinic acid was applied to the cell cultures dissolved in DMSO to a final concentration of 0.5% (v/v) in all the experiments. Controls were treated with the same amount of DMSO.
Cell proliferation assessment with MTT assay
Raji cells were seeded at a density of 5 x [10.sup.4]cells/weN in a 96-well plate in complete medium (100 [micro]l/well). The cells were treated with 12.5, 25, 50, 100, and 200 [micro]M of maslinic acid and were incubated in a humidified environment with 5% C[O.sub.2] at 37 C for 72 h. The ceils were centrifuged at 1500 rpm for 5 min and the medium was removed. Twenty [micro]l of 5 mg/ml MTT was added and the plates were incubated at 37[degrees]C for 3 h. Hundred micolitres of DMSO was added to each well. The plate was then gently agitated for 5 min and the absorbance at 570 nm was determined. The experiments were performed in triplicates.
Two dimensional-polyacrylamide gel electrophoresis (2D-PAGE)
Protein extraction and sample preparation for 2D-PACE
Raji cells (5 x [10.sup.5] cells/ml) were seeded in 24-well plates and incubated with 50 [micro]M of maslinic acid for 4, 8, 16, and 24 h. After incubation, the cells were lysed with lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 40 mM DTT, 0.5% pH 3-11 NL IPC buffer, protease inhibitor mix). The cell lysate was centrifuged at 14,000 x g for 10 min at 4 C. After centrifugation, pre-chilled acetone was added to the supernatant to remove contaminants. The samples were then incubated for 2h at -20[degrees]C, after which they were centrifuged at 13,000 x g for 10 min at 4[degrees]C, and the supernatant was removed. The resulting protein pellets were dissolved in a rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 0.5% pH 3-11 NL IPG buffer, 20 mM DTT, 0.02% bromophenol blue). Biorad RCDC Protein Assay reagents (Biorad, Hercules, CA) were used to determine the protein concentration. The protein samples were then stored at -80[degrees]C.
Protein separation and protein profile analysis were carried out as described previously (Wang et al. 2009), with minor modifications. One hundred and fifty micrograms of proteins was loaded to the non-linear pH 3-11 IPC strips (13cm; GE Healthcare) which were used for SDS-PAGE gels for silver staining while 1 mg of proteins was loaded to the IPG strips which were eventually used for SDS-PAGE gels for Coomassie staining. The IPG strips were rehy-drated with protein samples for 12 h at room temperature. After rehydration, proteins on the strips were focused at 20,000 Vh at 20[degrees]C using a Ettan[TM] IPGphore system (GE Healthcare). After focusing, the strips were reduced using 1% DTT and alkylated with 2% iodoacetamide (GE Healthcare) in equilibration buffer (6M urea, 50 mM Tris-Cl, 30% glycerol, 2% SDS, 0.02% bromophenol blue) for 15 min at room temperature with gentle agitation. For the second dimension of electrophoresis, the equilibrated strips were loaded on 12.5% polyacrylamide gels. 0.5% agarose was used to fix the strip. Electrophoresis was carried out at 20 mA for 2.5 h.
Image analysis and spot selection
ProteoSilver [TM] Plus Silver Stain kit (Sigma, USA) was used for silver staining and colloidal blue staining kit (Invitrogen Life Technologies) for Coomassie-blue staining of SDS-PAGE gels. Gels were scanned to produce the proteome maps of the control Raji cells and maslinic acid-treated Raji cells. Silver-stained gels (in triplicates) of untreated Raji cells and maslinic acid-treated cells were compared using Image-Master 2D Platium 7 software (GE Healthcare). Protein spots with [less than or equal to] 2-fold changes and p < 0.05 were selected for in-gel digestion.
Selected protein spots were excised from Colloidal Blue-stained gels. These spots were destained for 10min, thrice, with 25 mM ammonium bicarbonate. The destained gel spots were washed thrice with 10% acetic acid/50% methanol for 1 h each time and then washed twice with deionized water for 30 min each time. The gel pieces were immersed in 100% ACN until they turned opaque and were dried in a vacuum centrifuge. The dried gel particles were rehydrated with l0ng/nl trypsin in 50 mM ammonium bicarbonate (pH 8.0) and were incubated at 37[degrees]C for 4h. The digests were dried by vacuum centrifugation for about 15 min and subjected to MALDITOF-TOF MS analysis.
Database searching and protein identification
The digested peptide samples were analyzed by MALDI TOF-TOF MS using a 4800 Proteomics Analyzer (TOF/TOF) (Applied Biosystems, USA). The instrument was operated in a positive ion reflection mode of 20 kV accelerating voltage. Combined MS-MS/MS searches were conducted with Data Processing Software GPS Explorer [TM] software v 3.6 (Applied Biosystems). The spectra were processed and analyzed with the MASCOT v 2.1 software (Matrix Science Ltd., London, UK) to search for the peptide mass fingerprints and MS/MS data in the NCBInr database using the parent ion mass. The error tolerance was l00ppm and the MS/MS fragment mass tolerance was 0.2 Da. Carbamidomethylation of cysteine (fixed modification) and methionine oxidation (variable modification) were taken into consideration. A protein score greater than 82 was considered to be significant (p < 0.05).
Cell cycle analysis
Raji cells (5 x [10.sup.5] cells/ml) were seeded in 24-well plates and incubated with 50 [micro]M of maslinic acid for 4, 8, 16, and 24 h. The cells were then fixed in 70% (v/v) ethanol and stored overnight at -20 [degrees]C. The ethanol-suspended cells were centrifuged for 5 min at 200 x g. The ethanol was discarded. The cell pellet was rinsed with PBS and centrifuged. The supernatant was removed and the pellet was resuspended in 500 [micro]l PBS together with 4[micro]l of propidium iodide (PI) staining solution (2.5mg/ml) and 1 [micro]l RNase (l0mg/ml). Cells in the Pi-containing solution were incubated for 10 min at 37[degrees]C after which they were analyzed by flow cytometry. The flow cytometer was equipped with a 488 nm laser and PI flow emission was detected on the FL2 channel. Ten thousand cells were recorded for each data point and the results analyzed on CellQuest [R] software. Boundary markers were manually positioned on the histogram plots to determine the percentage of cell population at different stages of the cell cycle.
Nuclear protein extraction and electrophoretic mobility shift assay (EMSA)
Raji cells were plated in 24-well plates and treated with 50 [micro]M maslinic acid for 1, 2, 4, and 8ht respectively. Nuclear extracts were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (PIERCE, Rockford). NF-kB DNA binding reactions were carried out using NoShift Transcription Factor Assay kit and NoShift NF-kB (p65) reagents (Merck, Germany). The nuclear extract was mixed with the biotinylated double stranded NF-kB oligonucleotides (wild type sequence), NoShift Bind Buffer, poly(dI/dC) and salmon sperm DNA to a final volume of 20 [micro]l and incubated on ice for 30min. The reaction mixture was then transferred to a streptavidin-coated plate where the protein-DNA complexes were bound to the plate. The bound transcription factor (NF-kB) was then detected with a specific primary antibody (p65) followed by the secondary HRP-conjugated antibody. After reaction with the tetramethylbenzidine (TMB) substrate, the absorbance was read at 450 nm. Several controls were included in the experiments to establish the specificity of NF-kB binding activity. These include reactions performed in the absence of DNA-binding protein extract (blank), reactions with non-specific competitor DNA and reactions with specific competitor DNA. A 10-fold molar excess of specific and non-specific competitor DNA were added for each competition analysis.
Raji cells (5 x [10.sup.5] cells/ml) were seeded in 24-well plates and incubated with 50 [micro]M of maslinic acid for 4, 8, 16, and 24 h. The protein expression of EB1, stathmin, RanBPl, cyclin Dl, and p21 were determined by Western blotting. SDS-PAGE and Western blotting were done as previously described (Hsum et al. 2011). Total protein was extracted using M-PER Mammalian Protein Extraction Reagent from PIERCE (Rockford, USA). Protein concentration was determined using Bio-Rad DC protein assay. Samples containing 50 [micro]g of protein were fractionated by SDS-PAGE in 12% gels with constant current of 20 mA for about 2 h. The gel was then carefully removed and electroblotted to a 0.45 [micro]m PVDF membrane (PIERCE, Rockford, USA) using a standard tank transfer apparatus for mini gels. Immunodetection was carried out using respective primary antibodies (1:1000 in 3% BSA in PBST buffer) and HRP-conjugated secondary antibodies (1:10,000 in 3% BSA in PBST buffer).
Statistical analyses were performed using SPSS 15.0 software. Treatment effects were analyzed using one-way ANOVA. p < 0.05 was used to indicate significance.
Inhibition of Raji cell growth
In order to analyze the anti-proliferative effect of maslinic acid, Raji cells were treated with 12.5,25,50,100 and 200 [micro]M of maslinic acid for 24,48, and 72 h. As shown in Fig. 2, maslinic acid inhibited cell proliferation in a concentration- and time-dependent manner. Treatment with 50 [micro]M maslinic acid resulted in 46% inhibition of cell proliferation at 24 h, 76% inhibition at 48 h, and 97% inhibition at 72 h. The percentage of cell viability was less than 5% when treated with 100 and 200 [micro]M of maslinic acid for 24, 48, and 72 h.
Differential proteomic changes in maslinic acid-treated and untreated Raji cells
The protein expression profile of Raji cells in response to maslinic acid treatment at a sub-cytotoxic level (50 [micro]M) for 4, 8, 16, and 24 h was investigated. Fig. 3 show silver-stained 2D gels. About 1000 spots were detected and around 40 spots showed [greater than or equal to] 2-fold changes in spot intensity compared to the untreated control. Tryptic digests from 17 spots were analyzed by MALDI TOF-TOF MS. Table 1 lists the proteins that were identified by the database. The functions of the differentially regulated proteins were obtained using their protein accession numbers from the NCBI protein function summary (http://www.ncbi.nlm.nih.gov/protein). The 17 proteins identified in this study were assigned into several functional groups, namely cell proliferation, cell cycle, cytoskeleton organization, energy metabolism, cell signaling, and apoptosis. Among the proteins that were down-regulated after treatment with maslinic acid, dUTPase and inorganic pyrophosphatase are involved in cell proliferation. Proteins participating in energy metabolism, e.g. isocitrate dehydrogenase and enoyl-coA hydratase were highly expressed in maslinic acid-treated Raji cells. Voltage-dependent anion-selective channel protein 1 (VDAC1) which participates in the formation of the permeability transition pore complex (PTPC) responsible for releasing mitochondrial products that trigger apoptosis was up-regulated while prohibitin which regulates mitochondrial respiration and aging was down-regutated. VDAC1 and prohibitin have been implicated in apoptosis. Expression of transgelin-2 and tumour protein D52, which are involved in tumourigenesis were decreased.
Table 1 Proteins differentially expressed by Raji cells after treatment with 50 [micro] M maslinic acid. Spot Protein Swiss-prot MW/pl Sequence Mowse Fold number identity score change database coverage entries (%) 1 dUTPase P33316 17,908/6.15 62 553 -5.3 2 Inorganic Q15181 33,095/5.54 33 369 -1.8 pyrophos phatase 3 Stathmm 1 PI 6949 17,292/6.02 43 407 -5.2 isoform 4 Micratubule Ql5691 30,151/5.02 46 452 -2.7 associated protein RP/EB family member 1 5 F-actin P47756 30,852/5.52 21 348 -2.1 capping protein subun itbeta 6 Actin O15511 16,367/5.47 58 447 -3.4 related protein 2/3 complex subunit 5 7 lsocitrate P50213 34,967/6.02 39 356 +3.7 dehydr ogenase [NAD] subunit alpha, mitoch ondrial 8 Enoyl-coA P30084 31,361/6.09 53 542 +2.6 hydratase mitocho ndrial 9 14-3-3 P63104 29,413/4.97 53 391 -3.2 zeta/delta 10 Nucleoside P22392 17,270/8.55 33 260 -2.7 diphosphate kinase B 11 Ran P434B7 23,310/5.21 21 82 -5.3 GTPase- activating protein RANBP1 12 Transgelin-2 P378D2 24,609/8.41 86 709 -3.5 13 Tumour P55327 24,369/4.79 30 336 -7.3 protein D52 14 Voltage- P21796 30,737/8.63 57 414 +2.6 dependent anion- selective channel protein 1 (VDAC1) 15 Prohibitin P35232 29,859/5.57 73 726 -1.9 16 ESI protein P30042 24,999/8.29 16 202 +2.4 homo log, mitocho ndrial 17 Purine P00491 32,382/7.09 73 354 -4.2 nucleoside Spot Protein Biological number identity function 1 dUTPase Cell proliferation 2 Inorganic pyrophos phatase 3 Stathmm 1 Cell cycle isoform 4 Micratubule associated protein RP/EB family member 1 5 F-actin Cytoskeleton capping protein subun itbeta 6 Actin related protein 2/3 complex subunit 5 7 lsocitrate Energy dehydr Metabolism ogenase [NAD] subunit alpha, mitoch ondrial 8 Enoyl-coA hydratase mitocho ndrial 9 14-3-3 Cell zeta/delta signaling 10 Nucleoside diphosphate kinase B 11 Ran GTPase- activating protein RANBP1 12 Transgelin-2 13 Tumour protein D52 14 Voltage- Apoptosis dependent anion- selective channel protein 1 (VDAC1) 15 Prohibitin 16 ESI protein Others homo log, mitocho ndrial 17 Purine nucleoside
Validation of time-dependent differential expression of stathmin, EBlr and RanBPl
Validation of the proteomics findings by immunoblot analysis was performed to confirm the differential expression of specific proteins. The expression of 3 proteins, i.e. stathmin, EB1 and RanBPl are shown in Fig. 4. Raji cells were treated with 50 [micro]M maslinic acid for up to 24 h. Cells treated for 4, 8, 16, and 24 h, together with the controls, were harvested and their total proteins were extracted. Maslinic acid significantly reduced the expression of stathmin (Fig. 4A and B), EB1 (Fig. 4C and D), and RanBPl (Fig. 4E and F) within 24 h of treatment. EB1 and RanBPl expression were almost undetectable after 24 h of treatment.
Induction of Gl cell cycle arrest
Based on the protein expression profile, maslinic acid appeared to regulate proteins that are involved in cell growth, proliferation and survival in Raji cells. Flow cytometry analysis of maslinic acid-treated Raji cells showed that it induced Gl cell cycle arrest. Cells in the Gl phase increased sharply from 47% (0 h) to 70% after 16 h treatment and remained at 65% up to 24 h (Fig. 5). The increase in Gl cell population was accompanied by a decrease in the number of cells in both S and G2 phase. Treatment of cells with maslinic acid decreased the proportion of S phase cells from 25% (Oh) to 11% (24 h). The percentage of cells in the G2 phase decreased from 23% (0 h) to 12% at 24 h upon treatment.
Effects on cell cycle regulatory molecules
Based on the observation that maslinic acid induced Gl arrest in Raji cells, the effect of maslinic acid on cell cycle regulatory molecules of the Gl-S phase transition were assessed by Western blot analysis. Cyclin Dl was down-regulated by maslinic acid in a time-dependent manner (Fig. 6). After 16 and 24 h of maslinic acid treatment, cyclin Dl levels was undetectable. The CDK inhibitor p21 protein expression was, however, transiently up-regulated by maslinic acid. These effects were observed as early as 4h of treatment and reached a maximum expression at 8 h.
Inhibition of NF-tcB (p65) activity
The effect of maslinic acid on NF-kB activity was studied. Raji cells were treated with 50 [micro]M of maslinic acid at early time points (1,2,4, and 8 h). As shown in Fig. 7A, the NF-kB (p65) binding activity in Raji cells was specific; presence of non-specific competitor (mutant DNA) did not affect the NF-kB binding activity in Raji cells but was significantly reduced in the presence of specific competitor DNA. Maslinic acid suppressed NF-kB (p65) binding activity in a time-dependent manner, with 8.3%, 42.9%, 63% and 70% inhibition observed at 1,2,4, and 8 h treatment (Fig. 7B).
Previous studies suggest that the growth inhibition response of maslinic acid is mediated via activation of the mitochondrial apoptotic pathway as apoptotic proteins such as Bax, caspase 3 and caspase 9 were activated upon maslinic acid treatment (Reyes-Zurita et at 2008). However, the effect of maslinic acid at the proteomic level has not been investigated. In this study, a comprehensive proteome profiling which deduced the target genes of maslinic acid as well as its effect on the cell cycle are reported. Maslinic acid was shown to affect the expression of proteins involved in nucleotide metabolism, microtubule filament assembly, cytoskeleton organization, and adaptor proteins in the signal transduction pathway. The results suggest that maslinic acid induces G1 cell cycle arrest with induction of Gl phase cell cycle regulatory proteins p21 and the degradation of cyclin Dl, and inhibits nuclear NF-kB (p65) activity.
Maslinic acid inhibited Raji cell growth in a concentration- and time-dependent manner. This confirms the anti-proliferative activity of maslinic acid as similar observations were also reported in other tumour cell lines (Kim et al. 2000). As revealed from the proteome profile, the cytotoxic and pro-apoptotic effects of maslinic acid might be resulted from interference with DNA replication (down-regulation of dUTPase and inorganic pyrophosphatase), nucleo-cytoplasmic trafficking (down-regulation of RanBPl), and microtubule filament assembly (reduction of stathmin and EB1) as well as induction of apoptosis related protein VDACl. In the case of mitochondrial-regulated apoptosis, the process is ATP dependent and energy demanding (Mayer and Oberbauer 2003). Down-regulation of VDACl expression by shRNA is associated with a decrease in energy production (Shoshan-Barmatz et al. 2008). Indeed, maslinic acid increases energy metabolism in cells by up-regulating mitochondria isocitrate dehydrogenase and enoyl-CoA hydratase. This supported the suggestion by Reyes et al. that maslinic acid induces cell death via the mitochondrial apoptotic pathway (Reyes-Zurita et al. 2008).
Maslinic acid also inhibited adaptor proteins such as 14-3-3 zeta/delta and tumour protein D52 which regulate a large spectrum of both general and specialized signaling pathways. 14-3-3 zeta/delta is an important regulatory protein in intracellular signaling pathways and is known to interact with more than 100 cellular proteins, including oncogene and protooncogene products (Xing et al. 2000). 14-3-3 zeta/delta may regulate inflammatory pathways in the tumour microenvironment (Kobayashi et al. 2009). Tumour protein D52 is a multi-functional adaptor protein that influences numerous cellular processes. Increased D52 expression may be an early event in cancer which contributes to subsequent tumour progression (Boutros et al. 2004). The expression of transgelin-2, a protein of the calponin family found in high abundance in Raji cells (Gez et al. 2007) was down-regulated by maslinic acid. Elevated levels of transgelin were observed in tumourigenic cells and it significantly promotes cell migration and invasion (Lee et al. 2010).
Maslinic acid plays a critical role in the regulation of cell cycle progression. Proteins such as stathmin, EB1 and RanBPl which are involved in microtubule dynamics and cell division were down-regulated. Elevated stathmin and RanBPl expression had been reported in a variety of human malignancies (Antonucci et al. 2003) while EBl expression was recently reported to be up-regulated in human breast cancer cell lines (Dong et al. 2010). The up-regulation of stathmin, EBl and RanBPl in neoplastic tissues may be part of the regulatory mechanism altered during tumourigenesis. Silencing or inhibition of stathmin, EBl and RanBPl causes aberrant mitotic spindle formation, inhibition of cancer cell proliferation, and apoptosis (Jeha et al. 1996; Gez et al. 2007). These observations, together with the findings of this study suggest that the inhibition of proteins regulating microtubule dynamics and cell division may suppress tumour cell growth.
Reyes et al. reported that maslinic acid inhibits cell growth and induces Gl cell cycle arrest in Caco-2 colon cancer cells (Reyes etal. 2006). This study reports a similar effect of maslinic acid on the Raji cell cycle. Maslinic acid also down-regulated proteins associated with the cell cycle arrest at S and G2 phase. Inhibition of dUTPase induces early S (Tinkelenberg et al. 2002) or G2 (Marie et al. 2006) arrest due possibly to stalled replication complexes caused by a lack of thymidine equivalents (dTTP or dUTP) available for DNA replication. Inhibition of stathmin expression leads to accumulation of cells in the G2 phase which is associated with severe mitotic spindle abnormalities and difficulty to exit from mitosis (Rubin and Atweh 2004). In contrast, there are studies reporting that levels of stathmin phosphorylation were significantly lower in cells blocked during the Gl/S phases of the cell cycle than in proliferating cells (Brattsand et al. 1994). Knockdown of Nm23-Hl (Dabernat et al. 2004), the target gene of nucleoside diphosphate kinase B (NDPKB) which was down-regulated by maslinic acid, has been shown to arrest cells in the G0/G1 phase of the cell cycle (Jin et al. 2009).
Progression through the cell division cycle is regulated by coordinated activities of cyclin/cyclin dependent kinases (CDK) complexes. Mitogenic signals that initiate DNA replication are first detected by expression of the D-type cyclins 031, D2 and D3) that preferentially bind to, and activate, CDK4 and CDK6 during the Gl phase (Malumbres and Barbacid 2001). Over-expression of cyclin Dl has been reported in several types of human cancers (Yu et al. 2001; Besson et al. 2008). Suppressing cyclin Dl over-expression may thus prevent oncogenic signaling. Another mechanism that prevents premature entry into the S phase relies on CDK inhibitors (CKIs) that bind onto cyclin-CDK complexes and disrupt their catalytic centre (Pavletich 1999). The Cip/Kip family member of [p21.sup.Cipl/Wafl/Sdil] is an important transcriptional target of p53 and mediates DNA-damage induced cell-cycle arrest (Besson et al. 2008). Suppressing cyclin Dl over-expression and inducing p21 levels by maslinic acid may thus prevent uncontrolled cell cycle progression.
Cyclin Dl is a transcriptional target of NF-kB (Hinz et at 1999) and NF-kB activation is essential for growth and survival of Epstein-Barr virus (EBV)-transformed B lymphoma Raji cells (McFarland et al. 2000; McFarland and Kieff 2002). NF-kB also regulates target genes that are activated in the early and late stages of aggressive cancers, including apoptosis suppressor proteins such as Bcl-2 and Bel [X.sub.L] and those required for metastasis and angiogenesis, such as matrix metalloproteases (MMP) and vascular endothelial growth factor (VEGF) (Aggarwal and Shishodia 2006). The results of this study showed that maslinic acid significantly reduced nuclear NF-kB activity in a time-dependent manner. A similar effect was also observed in pancreatic cancer cells Panc-28 where maslinic acid inhibits cell proliferation by suppressing tumour necrosis factor-alpha (TNF[alpha]) -induced NF-kB activation (Li et al. 2010). The induction of Gl cell cycle arrest, modulation of Gl phase cell cycle components and suppression of NF-kB activity by maslinic acid may contribute to its growth inhibitory effect.
The identification of proteins with altered expression profiles at multiple treatment time points may serve to elucidate the cellular mechanism involved in response to maslinic acid treatment in Raji cells. Proteins such as stathmin, EB1 and RanBPl which are associated with cell division and microtubule dynamics suggest that maslinic acid might have a role in regulating the cell cycle. Maslinic acid induced a Gl cell cycle arrest and modulated Gl associated cell cycle regulatory proteins, i.e. cyclin Dl and p21. Transcriptional activation of NF-kB which is known to promote cell cycle progression through up-regulation of cyclin Dl expression and essential for Raji cells growth and survival was inhibited in response to treatment. This study supports that treatment with maslinic acid activates the mitochondrial apoptotic pathway as has been reported by Reyes-Zurita et al. (2008), and suggests that the growth inhibition properties of maslinic acid might be mediated by modulation of the cell cycle.
Conflict of interest
The authors have declared no conflicts of interest.
The authors would like to express their deepest gratitude to Universiti Tunku Abdul Rahman and MOST1 e-science fund (00-02-11-SF0028) for supporting this project.
* Corresponding author. Tel: +60 3 90194722x178; fax: +60 3 90194722. E-mail address: firstname.lastname@example.org (Y.M. Lim).
0944-7113/$ - see front matter [c] 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2011.08.058
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W.H. Yap (a), K.S. Khoo (a), S.H. Lim (b), CC Yeo (c), Y.M. Limd (d), *
(a.) Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, 31900 Kampar, Perak, Malaysia
(b.) School of Biological Sciences, Faculty of Science, Monash University, Victoria 3800, Australia
(c.) Faculty of Agriculture and Biotechnology, Universiti Darul Iman Maloysiajalan Sultan Mahmud, 20400 Kuala Terengganu, Malaysia
(d.) Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Lot PT21144, Jalan Sungai Long, Bandar Sungai Long, 43000 Kajang, Malaysia