p38 mapk and erk1/2 pathways are involved in the pro ... · of notoginsenoside ft1 on human...
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Life Sciences 108 (2014) 63–70
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p38MAPK and ERK1/2 pathways are involved in the pro-apoptotic effectof notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells
Bo Gao a,1, Hai-Lian Shi b,1, Xiang Li b, Shui-Ping Qiu b, Hui Wu b, Bei-Bei Zhang b,Xiao-Jun Wu b,⁎, Zheng-Tao Wang a,b,c,d,⁎a Department of Pharmacognosy, China Pharmaceutical University, Nanjing, Chinab Shanghai Key Laboratory of Complex Prescription, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, Chinac The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai, Chinad The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Shanghai, China
⁎ Corresponding authors at: Institute of Chinese Materof Traditional Chinese Medicine, 1200 Cailun Road, Zhan201203, China. Tel.: +86 21 5132 2578, +86 21 5132 25121 5132 2519.
E-mail addresses: [email protected] (X.-J. Wu),(Z.-T. Wang).
1 These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.lfs.2014.05.0100024-3205/© 2014 Elsevier Inc. All rights reserved.
a b s t r a c t
a r t i c l e i n f oArticle history:
Received 21 December 2013Accepted 10 May 2014Available online 23 May 2014Keywords:Notoginsenoside Ft 1ApoptosisCell cycleSH-SY5Y cells
Aims: This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compoundexclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.Main methods: CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measurethe cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morpholog-ical changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activitywas measured by colorimetric assay kit.Key findings:Among the saponins examined, Ft1 showed thebest inhibitory effect on cell proliferation of SH-SY5Ycells with IC50 of 45 μM. Ft1 not only arrested the cell cycle at S, G2/M stages, but also promoted cell apoptosis,whichwas confirmed byHoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein
expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover,Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38MAPK. However, the phosphorylation of Jak2 and p85PI3Kwas reduced by Ft1. Inhibitors of p38MAPK and ERK1/2 but not JNK abrogated the up-regulated protein ex-pressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3activity induced by Ft1.Significance: Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38MAPK andERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.© 2014 Elsevier Inc. All rights reserved.
Introduction
Radix Notoginseng (san qi), the root of Panax notoginseng (Burk.)F.H. Chen, has been used worldwide as a medicine or a dietary supple-ment for removing blood stasis, improving blood circulation and allevi-ating pain (Jia et al., 2010; Ruan et al., 2010). P. notoginseng derivedproducts such as notoginseng tea, capsules and even its extracts havealready been marketed in European countries and the United Statesas dietary supplements or functional foods (Ruan et al., 2010; Sunet al., 2010; He et al., 2012). Studies revealed that the crude extractsof P. notoginseng can inhibit the growth of many human cancer cells
ia Medica, Shanghai Universitygjiang Hi-tech Park, Shanghai7; fax: +86 21 5132 2578, +86
including hepatocarcinoma SMMC-7721, prostate cancer PC-3 andbreast cancer MCF-7 (Chuang et al., 2004; Shang et al., 2006; Ng,2006) through interfering cell cycle and eliciting apoptosis. Saponinscontaining ginsenosides and notoginsenosides are thought to be themajor components from P. notoginseng responsible for the anticancereffect (He et al., 2012).
Neuroblastoma, predominantly a peripheral nervous solid tumor,is the most common extracranial neoplasma of early childhood thataccounts for 7%–10% of all childhood cancers (Kim et al., 2007; Brardet al., 2009). Despite multimodal therapies including surgery, irradia-tion, autologous stem-cell transplantation and chemotherapy havebeen applied, however, the cure rate of neuroblastoma has not beenelevated compared with other childhood malignancies. The majorityof the neuroblastomas in children over one year of age are aggressivemetastatic tumors with poor clinical outcome (Matthay et al., 1999;Kim et al., 2007). Moreover, more than 50% of children have high recur-rence rates because of drug-resistant residual disease (Kim et al., 2007;Brard et al., 2009). Thus, new drug development targeting neuroblasto-ma with higher efficacy is still urgently needed.
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Notoginsenoside Ft1 (Ft1), one of notoginsenosides found exclu-sively in P. notoginseng, has been reported by our group to promoteblood vessel formation, thus contributing to wound healing (Shenet al., 2012) in addition to enhancing platelet aggregation (Gao et al.,2014). However, whether the natural compound possesses anti-cancer effect has not been investigated yet. In the current study, wediscovered that Ft1 can effectively prevent the proliferation of humanneuroblastoma SH-SY5Y cells and promote cell apoptosis. Thereafter,possible underlying mechanisms were investigated, which may con-tribute to the clinical application of Ft1 or its derivatives in therapy ofneuroplastomas.
Materials and methods
Materials
Saponins including notoginsenoside Ft1, notoginsenoside Fe,notoginsenoside Fc, ginsenoside Rh2, vietnamginsenoside R7,ginsenoside F2, ginsenoside Rb1, gypenoside IX, ginsenoside Rb3,ginsenoside Re, notoginsenoside R1, ginsenoside Rd, ginsenoside Rg2,pseudoginsenodide F11, and ginsenoside Rg1 were isolated, purifiedand identified using mass spectrometry by Shanghai R&D centre forstandardization of Chinese medicines (Shanghai, China). The purity ofall of the saponins was greater than 98%. Cell Counting Kit-8 (CCK-8)was from Dojindo (Kumamoto, Japan). Caspase 3/CPP 32 colorimetricassay kit was purchased from BioVision (California, USA). PD98059and SB202190 were obtained from Selleckchem (Houston, USA). BI78D3 was purchased from Tocris (Bristol, United Kingdom). PI andDMSO were purchased from Sigma-Aldrich Co. (MO, USA). Hoechst33258 was obtained from AnaSpec Inc. (CA, USA). Trypsin and annexinV were purchased from Life Technologies (NY, USA). RNase A was fromBeijing Jingkehongda Biotechology Co. (Beijing, China).
Cell culture
Human neuroblastoma SH-SY5Y, human gliomas U87-MG andU251, rat glioma C6 were obtained from Cell Bank of Type Culture Col-lection of Chinese Academy of Sciences (Shanghai, China). SH-SY5Ywas routinely maintained in a DMEM/F-12 medium, while all theother glioma cell lines were cultured in a DMEM medium (ThermoFisher, IL, USA). All the cells were grown in the medium supplementedwith 10% FBS (Gibco, NY, USA) at 37 °C with 5% CO2.
Proliferation assay
Cells were seeded in 100 μl of medium at 2.5 × 105 cells/mL in 96-well culture plates and grown overnight. After treated by drug for24 h, 10 μl of CCK-8 solution was added to each well and incubated at37 °C for another 1 h. The absorbance of the solutions was detected at450 nm by a microplate reader (ThermoSci Varioskan Flash, USA). Thecell viability rate was calculated as the percentage of CCK-8 absorptionas follows: (absorbance of drug-treated sample / absorbance of controlsample) × 100.
Cell cycle distribution analysis
Cells were seeded in 6 well plates at 2.5 × 105 cells/well in 3mLme-dium and cultured overnight. After serum starvation for 24 h, cells wereincubated with Ft1 for 24 h. The cells were harvested by trypsinization,washed twicewith phosphate buffered saline (PBS),fixedwith cold 70%ethanol overnight followed by staining with PI solution containing50 μg/mL RNase A and 0.1% Triton X-100. The distribution of cell cyclewas examined using FACScan flow cytometer (Becton Dickinson,USA), and the data were analyzed by ModFitLT V3.0 software.
Annexin V/PI double-staining
Cells were plated in 6-well culture plates at a density of 2.5 × 105
cells/mL in 3 mL of medium and allowed to adhere to the platesovernight. The cells were incubated with different concentrationsof Ft1 (45 and 67.5 μM) or vehicle solution (0.1% DMSO) in a mediumcontaining 10% FBS for another 24 h. Then, the cells were harvested bytrypsinization, washed twice with 1× annexin V binding buffer, anddouble-stained with annexin V and PI. Cell apoptosis was examinedusing the Guava flow cytometer (Millipore, USA).
Hoechst 33258 staining
Cells were seeded in 6-well culture plates at a density of 2.5 ×105 cells/mL in 3 mL of medium and allowed to adhere to the platesovernight. The cells were incubated with different concentrations ofFt1 (45 and 67.5 μM) or vehicle solution (0.1%DMSO) in amedium con-taining 10% FBS for 24 h. After the treatment, the cells were fixed with4% PFA for 10min, followed by incubation with Hoechst 33258 stainingsolution (10 μg/mL) for 5 min and finally analyzed for morphologicalcharacteristics of cell apoptosis under a fluorescence microscope(Olympus CKX41, Japan).
Western blot analysis
Cells were plated in 6-well culture plates at a density of 2.5 ×105 cells/mL in 3 mL of medium and allowed to adhere to the platesovernight. The cells were incubated with different concentrations ofFt1 (22.5, 45 and 67.5 μM) or 0.1% DMSO in a medium containing10% FBS for 48 h. For the investigation of involvement of MAPK path-ways in Ft1 induced apoptosis, the cells were pre-incubated withdifferent inhibitors for 1 h, then co-treated with Ft1 at 45 μM for24 h. Afterwards, the cells were collected, lysed with cell lysis bufferand sonicated three times each for 15 s. The cell lysates were cen-trifuged at 14,000 g for 15 min at 4 °C, and the supernatants werecollected. Protein samples were separated by SDS-PAGE (15% or8%) and transferred onto Hybond-NC membranes. Subsequently,the NC membranes were blocked with 5% non-fat milk solutionand incubated with the primary antibodies against cleaved caspase 3(Asp175), cyclin B1, Bax, Bcl-2, phospho-p53 (Ser15), p21, p-ERK1/2(Thr202/Tyr204), ERK1/2, p38 MAPK, p-p38 MAPK (Thr180/Tyr182),SAPK/JNK, p-SAPK/JNK (Thr183/Tyr185), Jak2, p-Jak2 (Tyr1007/1008),p-p85 PI3K (Tyr458), p85 PI3K (Cell Signaling Technology, MA, USA)and GAPDH (Epitomics, CA, USA) overnight at 4 °C. After thoroughlywashed with 1× TBST, the NCmembranes were incubated with respec-tive secondary antibodies. The protein bands were visualized with theECL prime kit (GE Healthcare, NA, UK) and scanned with SmartViewsoftware (Furi, China).
Caspase 3 activity analysis
The activity of caspase 3 was determined by caspase colorimetricassay kit (BioVisionm Milpitas, CA, USA). Briefly, after pre-incubationwith SB202190 (inhibitor of p38 MAPK, 20 μM), BI 78D3 (inhibitorof JNK, 2 μM) and PD98059 (inhibitor of ERK1/2, 20 μM) for 1 h, thecells were then co-treated with Ft1 at 45 μM for 24 h. Afterwards,cells were harvested and lysed with cell lysis buffer for 10 min on icefollowed by centrifugation at 10,000 g for 1min at 4 °C. The supernatantwas collected and the protein concentration was determined. Then,50 μg protein diluted in 50 μL cell lysis buffer was incubated with50 μL of 2× Reaction Buffer containing 400 μM substrate (DEVD-pNA)and 10 mM DTT at 37 °C for 2 h. The absorbance of samples was mea-sured at 405 nm by the microplate reader.
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Statistical analysis
The data were represented by the mean± standard deviation (SD).Differences among groups were analyzed by one-way ANOVA withDunnett or Bonferroni or Newman–Keuls multiple comparison testusing PrismDemo 4 software (GraphPad Software Inc., USA). P b 0.05was considered to be statistically significant.
Fig. 1. Effects of notoginsenosides and ginsenosides on cell viability of SH-SY5Y cells.A: Cell viability of SH-SY5Y cells after treated with notoginsenosides and ginsenosidesfor 24 h. The concentration of each compound was 50 μM; B: Effect of Ft1 on cell viabilityof SH-SY5Y cells after treated for 24 h; C:Morphological changes of SH-SY5Y cells inducedby Ft1 for 24 h. The cell viability was determined by CCK-8 assay as described in theMaterials and methods. Data represent mean ± SD, ⁎P b 0.05, ⁎⁎⁎P b 0.001 vs control.
Results
Ft1 showed the best antiproliferative effect on SH-SY5Y cells among fifteensaponins from P. notoginseng
Fifteen saponins isolated from P. notoginsengwere screened for theirantiproliferative effect on SH-SY5Y cells. As shown in Fig. 1A, when usedat 50 μM, both Ft1 and Fe significantly inhibited the viability of SH-SY5Ycells in 24 h. By contrast, other saponins did not affect the growth of thecells. However, Ft1 exhibited better inhibitory effect than Fe did. There-fore, further studies were focused on this compound. As displayed inFig. 1B, Ft1 dose-dependently prevented the proliferation of SH-SY5Ycells and the IC50 of it was 45 μM. Consistently, Ft1 induced remarkablemorphologic changes of the cells in a dose-dependentmanner (Fig. 1C).Generally, normal growing SH-SY5Y cells form clumps surrounded byoutspread individual cells (Fig. 1Ca). After Ft1 treatment, the size ofthe cell clumps became smaller. Cells surrounding the clumps detachedfrom the culture dishes and floated in the medium (Fig. 1Cb–c). When
Fig. 2. Effects of Ft1 on cell viability of glioma cell lines after treated for 24 h. The cellviability was determined by CCK-8 assay as described in the Materials and methods.Data represent mean ± SD, ⁎P b 0.05, ⁎⁎⁎P b 0.001 vs control.
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the concentration of Ft1 was incremental to 67.5 μM, only a few cellclumps still attached to the dishes were accompanied with more celldebris formation (Fig. 1Cd).
By contrast, as illustrated in Fig. 2, Ft1 suppressed glioma prolifer-ation only at high concentrations (50 and 100 μM). Although Ft1(100 μM) inhibited 60% of C6 proliferation, it prevented relativelylower percentage of U87-MG and U251 cells from proliferation andthe inhibitory rates of them were about 20% and 30%, respectively.
Ft1 induced S, G2/M phase arrest of SH-SY5Y cells
To examine the effect of Ft1 on cell cycle distribution, SH-SY5Y cellswere treated with Ft1 (22.5, 45 and 67.5 μM) for 24 h followed by PI
Fig. 3. Effect of Ft1 on cell cycle distribution after treated for 24 h. A: Statistical analysis of the celphase distributions induced by Ft1 for 24 h detected by cell cycle distribution analysiswith FACS⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs control.
staining. As shown in Fig. 3, flow cytometric assay exposed that Ft1 sig-nificantly arrested SH-SY5Y cells at both S and G2/M phases. However,Ft1 triggered only G2/M phase arrest when used at 67.5 μM.
Ft1 promoted apoptosis of SH-SY5Y cells
To analyze the effect of Ft1 on cell apoptosis, SH-SY5Y cells weresubjected to Hoechst 33258 and Annexin/PI staining, respectively,after Ft1 treatment. As displayed in Fig. 4A, the fluorescence of Hoechst33258 was dim in control cells. However, the fluorescence became bril-liant and aggregative in Ft1-treated cells accompanied with chromatincondensation and nuclear shrinkage or fragmentation, indicating theinitiation of apoptosis.
l cycle phase distributions after treated for 24 h; B: Flow cytometric analysis of the cell cyclecan flow cytometer as described in theMaterials andmethods. Data representmean± SD,
Fig. 4. Effect of Ft1 on cell apoptosis of SH-SY5Y cells. A: Hoechst 33258 staining of SH-SY5Y cells treatedwith Ft1 for 24 h observed and photographed under a fluorescencemicroscope asdescribed in the Materials and methods. B: Flow cytometric analysis of SH-SY5Y cells double-stained with Annexin V and PI after Ft1 treated for 24 h detected by Annexin V/PI double-staining with Guava flow cytometer as described in the Materials and methods. The scale in the pictures displays 25 μm. Data represent mean ± SD, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs control.
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After treated for 24 h, Ft1 induced significant cell apoptosis as theearly cell apoptosis rate was increased from 13.64% (in control cells)to 48.85% (in 67.5 μM Ft1-treated cells) (Fig. 4B).
MAPK pathway was involved in the anti-proliferative and pro-apoptoticeffects of Ft1 on SH-SY5Y cells
As revealed in Fig. 5, Ft1 treatment for 48 h led to prominent eleva-tion of cyclin B1. Similarly, the cleaved caspase 3, phosphorylatedp53 and p21 were increased significantly by Ft1. By contrast, Bcl-2, theanti-apoptotic protein, was decreased. However, Ft1 did not affect theexpression of Bax protein.
When treated for 48 h, Ft1 dose-dependently activated ERK1/2,SAPK/JNK and p38 MAPK. As demonstrated in Fig. 6A, phosphorylatedERK1/2, SAPK/JNK and p38 MAPK were up-regulated dramatically by
Ft1 treatment, while the total protein levels of ERK1/2, SAPK/JNK andp38 MAPK were not influenced. However, activation of both Jak2and PI3K was suppressed. As shown in Fig. 6B, Ft1 dose-dependentlycounteracted the phosphorylation of p85 PI3K and Jak2 withoutinfluencing the total p85 PI3K and Jak2.
In order to further clarify the role ofMAPKson cell apoptosis inducedby Ft1, the inhibitors of MAPKs were used. As shown in Fig. 7A, theinhibitors of MAPKs attenuated the antiproliferative effect of Ft1 onSH-SY5Y cells. After treated with Ft1 at 45 μM (Fig. 7B), the caspase 3activity in SH-SY5Y cells was significantly increased. However, the ele-vated caspase 3 activity was suppressed remarkably by the inhibitorsof p38 MAPK (SB202190) and ERK1/2 (PD98059). BI 78D3 only had atendency to inhibit the elevated caspase 3 activity induced by Ft1 butwithout significant difference. The results from western blot analysisshowed that the inhibitors of p38 MAPK and ERK1/2 could decrease
Fig. 5. Effects of Ft1 on cell cycle-related and apoptosis-related proteins. Data shownwerefrom three independent experiments determined bywestern blot analysis as described inthe Materials and methods.
Fig. 6. Effects of Ft1 on activation of MAPK, JAK2 and PI3K pathways. A: Effects of Ft1 onprotein expression of MAPK pathways; B: Effects of Ft1 on protein expressions of JAK2and PI3K pathways. Data shown were from three independent experiments determinedby western blot analysis as described in the Materials and methods.
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the effect of Ft1 on the protein expression of p21, cleaved caspase 3 andBcl-2. However, BI 78D3 had no obvious effect on the protein expressionof p21 and cleaved caspase 3 induced by Ft1, but could significantlyinhibit the effect of Ft1 on protein expression of Bcl-2 (Fig. 7C).
Discussion
In the present study, we evaluated the effects of fifteen types of sa-ponins found in P. notoginseng on the proliferation of SH-SY5Y cells. Pre-vious researches have already shown the bioactivities of P. notoginsengsaponins (PNS) on the nervous system. For instances, PNS promote pro-liferation and differentiation of neural stem cells (Si et al., 2008; Zhanget al., 2010), attenuate hypoxia/reoxygenation induced oxidative stressin cortical neurons (Yan et al., 2012) and relieve Pertussis bacilli-inducedbrain edema (Li et al., 2003). And PNS have displayed anti-tumor activ-ities in many cancer cells (He et al., 2012). However, whether PNS sup-press the proliferation of neuroblastoma has not been reported yet.Our study found that both Ft1 and Fe could prevent the proliferationof SH-SY5Y cells when used at 50 μM. As Ft1 showed better inhibitoryeffect than Fe, therefore, further studies were focused on Ft1 to uncoverthe possible underlying mechanisms of it in anti-neuroblastoma.
The imbalance between cell proliferation and apoptosis contributesto the progression of tumor cell growth. Thus, to redress the disturbedbalance of cell proliferation and apoptosis is an important therapeuticstrategy for treating human cancers. In our investigation, Ft1 dose-dependently inhibited cell proliferation, induced cell cycle arrest on Sand/or G2/M phase, and enhanced cell apoptosis of SH-SY5Y, whichindicated therapeutic effect of Ft1 on neuroblastoma.
In normal cells, cyclin B1 regulates the transition of cell cycles fromthe G2 phase to the mitotic M phase. When cyclin B1 is inhibited, cellswill be arrested in the G2 phase. On the contrary, cells will enterand proceed through mitosis, if the activity of cyclin B1 is increased.However, in tumor cells, the up-regulation of cyclin B1mediates the in-duction of mitotic prometaphase arrest (Morgan, 1995; Choi and Zhu,2012). In our study, the increased expression of cyclin B1 concomitantwith the S, G2/M arrest was elicited by Ft1. This result implicated thatFt1 induced the prometaphase arrest. JNK has been suggested to be ac-tively involved in the up-regulation of cyclin B1 (Choi and Zhu, 2012).Consistent with it, the elevated phosphorylation of JNK was observedin SH-SY5Y cells after Ft1 treatment. Butwhether it directs the elevationof cyclin B1 has not been elucidated yet.
Cell apoptosis is mediated by caspase-dependent and caspase-independent pathways. Caspase-3 is one of the key players in cell
apoptosis. At least 42 of the 58 known caspase substrates are specificallycleaved by caspase 3. Once caspase 3 is activated, it will increase cyto-chrome c release from mitochondria by cleaving Bcl-2 and convertingit from an anti-apoptotic to a pro-apoptotic protein (Cheng et al.,1997; Nicholson and Thornberry, 1997; Porter and Jänicke, 1999).Moreover, it can process pro-caspases 2, 6, 7 and 9, therefore, speedup the cascade of apoptosis. Mitochondria-dependent pathway isthe classical apoptotic pathway. Bax and Bcl-2 are the predominantregulators in controlling the release of cytochrome c in mitochondria-dependent apoptosis.When Bax inserts into the outermembrane ofmi-tochondria, cytochrome c will be released from the organelle. However,when Bcl-2 instead of Bax binds to the outer mitochondrial membrane,release of cytochrome c will be blocked (Ow et al., 2008). Many anti-cancer agents can induce the release of cytochrome c through eitherup-regulating Bax expression and/or down-regulating Bcl-2 expression(EI-Mahdy et al., 2005; Das et al., 2006; Reyes-Zurita et al., 2009). In ourstudy, Ft1 dose-dependently elicited the generation of cleaved caspase-3 but suppressed that of Bcl-2without interfering the expression of Bax.Therefore, both mitochondria-dependent and caspase-3-dependentpathways were involved in inducing cell apoptosis triggered by Ft1.
Fig. 7.MAPK pathways involved in Ft1 induced apoptosis in SH-SY5Y cells. A: Effects of MAPK inhibitors on Ft1 (45 μM) induced anti-proliferation in SH-SY5Y cells. B: Effects of MAPKinhibitors on Ft1 (45 μM) activated caspase 3 in SH-SY5Y cells. C: Effects of MAPK inhibitors on Ft1 (45 μM) regulated apoptosis-related proteins in SH-SY5Y cells. After pre-incubationwith SB (20 μM), BI (2 μM) and PD (20 μM) for 1 h, the cells were co-treated with or without Ft1 (45 μM) for 24 h, followed by CCK-8 assay, caspase 3 activity assay and western blotanalysis as described in the Materials and methods. Data represent mean ± SD, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs Ft1, #P b 0.05, ##P b 0.01, ###P b 0.001 vs control. SB: SB202190; BI: BI78D3; PD: PD98059. Data shown were from three independent experiments.
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Many anticancer compounds induce apoptosis via up-regulation ofp53 protein expression (Choi et al., 2008). However, curcumin inducesapoptosis in vascular smooth muscle cells with a tendency to decreasegene expression of p53 (Chen andHuang, 1998). p21 is the downstreammolecule of p53 which is activated by phosphorylated p53 (Personset al., 2000; Yeh et al., 2001; Lu and Xu, 2006). In this study, thephosphorylated p53 in SH-SY5Y cells was up-regulated by Ft1 dose-dependently and accompanied by the elevation of p21. Therefore, Ft1induced the apoptosis of SH-SY5Y in a p53 dependent pathway.
Mitogen-activated protein kinase (MAPK) pathway is actively in-volved in drug-induced cell apoptosis of tremendous cancer cells in-cluding SH-SY5Y (Frasca et al., 2004; Kim et al., 2005; Uehara et al.,2012). When the activation of MAPKs is inhibited, the apoptosis andcaspase-3 cleavage in tumor cells are abrogated (Jo et al., 2005; Kimet al., 2005; Uehara et al., 2012). In our study, Ft1 could enhance thephosphorylation of all of the three major MAPKs in SH-SY5Y. Activationof MAPKs results in inactivation of Bcl-2, enhanced phosphorylation ofp53 and thus activation of downstream p21 (Persons et al., 2000; Yehet al., 2001; Shimada et al., 2003; Lu and Xu, 2006). In agreement withthe reports, we also observed increased cleavage of caspase 3, enhancedphosphorylation of p53 and p21 but reduced Bcl-2 in the study, whichprovided strong evidence for the involvement of MAPK pathways inthe apoptosis induced by Ft1.
Inhibition of janus kinase 2 (Jak2) promotes cell apoptosis (Chenet al., 2010; Parada et al., 2010;Will et al., 2010). Activation of the phos-phatidylinositol 3-kinase (PI3K) pathway shows pro-survival effect. InSH-SY5Y cells, activation of the PI3K/Akt pathway has already beenfound to prevent the apoptosis of cells (Pettifer et al., 2004; Shi et al.,2010). In the current study, Ft1 could inhibit the activation of bothJak2 and p85 PI3K shown by reduced phosphorylation of Jak2 and p85PI3K, which indicated that these two pathways also were involved inFt1 elicited apoptosis.
Because of the critical role of the MAPK pathway in cell survival andapoptosis, in our further investigation, the inhibitors of p38 MAPK, JNKand ERK1/2were used to confirm the effect of theMAPKpathwayon Ft1induced apoptosis. The results showed that the increased caspase 3, p21
but decreased Bcl-2 induced by Ft1 could be blocked by p38 MAPK andERK1/2 pathway inhibitors, which indicated the involvement of p38MAPK and ERK1/2 pathways in Ft1 induced cell apoptosis.
Conclusion
Among the fifteen saponins screened, Ft1 showed the best anti-proliferative effect on SH-SY5Y cells. Ft1 treatment resulted in S and/or G2/Mphase arrest through up-regulatingprotein expression of cyclinB1. Further studies disclosed that Ft1 might act through activation ofp38 MAPK and ERK1/2 but suppression of Jak2 and PI3K pathways,therefore, regulate down-stream protein expressions of caspase 3,p53, p21 and Bcl-2 to induce the apoptosis of the neuroblastomaSH-SY5Y cells. This study provides evidences in vitro for supportingthe potential clinical usage of Ft1 in therapy of human neuroblastoma.
Conflict of interest statement
The authors declare that they have no competing interests.
Acknowledgments
This project was supported by the National Natural Science Foun-dation of China (U1032604), the Key Research Innovation Project(13ZZ099), the Key Project from Department of Education of China(20123107130002), the Shanghai Eastern Scholar Program (2013-59),and the Program of Shanghai Pujiang Talent Plan (11PJ1408800).
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