A novel arylbenzofuran induces cervical cancer cell apoptosis and G1/S arrest through ERK-mediated Cdk2/cyclin-A signaling pathway

7-hydroxy-5,4′-dimethoxy-2-arylbenzofuran (Ary) is purified from Livistona. It has been demonstrated to have anticancer activity to various tumors in including cervical cancer, but its mechanism is still unclear. In the present, we show that Ary induces cervical cancer cells apoptosis through mitochondria degradation and mediates cervical cancer cell arrest. Further, Ary-inducing cell cycle G1/S-phase arrest is associated with increased cyclin A2 and cyclin dependent kinase 2 (Cdk2) proteins. Knockdown of cyclin A2 using small interfering RNA (siRNA), and inhibiting Cdk2 activity with flavopiridol, strikingly reduced G1/S-phase arrest. Moreover, Ary sustainedly induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2). And ERK1/2 phosphorylation inhibition using specific inhibitor U0126 effectively suppressed cyclin A2 expression, and reduced G1/S-phase arrest induced by Ary. All the experiments in vitro and in vivo verified that Ary has an anticancer effect on cervical cancer. These data provide novel evidences that Ary induces cervical cancer cells apoptosis through mitochondria degradation and cell G1/S-phase arrest. These findings also suggest that ERK-mediated Cdk2/cyclin A signaling pathway is involved in Ary-induced G1/S-phase arrest.

a major risk factor of cervical cancer development and precancerous lesions [4][5][6], such as HPV16, 18, 31, and 33 promote in cervical cancer pathogenesis [7][8][9]. Among all these types of HPV, types 16 and 18 are the most dangerous factors which are approximately 70 percent in cervical cancer [10,11], but vaccine against HPV could not effectively prevented pre-existing HPV infections and progression of HPV-associated lesions. In current, the therapeutic regimens of cervical cancer include surgical removal, radiotherapy and chemotherapy. Women with early-stage cervical cancer can be potentially cured with radical hysterectomy or chemoradiation therapy [12]. Unfortunately, up to 17% of women develop either local

Research Paper
and/or distant disease recurrence usually within the first 2 years of completing the treatment [13]. Local recurrence of cervical cancer after primary surgical therapy is also problematic, treatment directed to the site of recurrence can be performed with curative intent. Options include radiation therapy (RT) and pelvic exenteration, both resulting in suboptimal rates of local tumor control and rates of survival [14]. Chemotherapy is an important therapeutic tool, especially for the patients in advanced and metastatic. However, in the course of chemotherapy, some cancer cells may have varying degrees of drug resistance phenomenon. Most of chemotherapy drug toxicities are large, and the patients have a lot of adverse reactions after chemotherapy. Therefore, to search for a novel effective and harmless chemotherapy drug for cervical cancer is very necessary.
Livistona, a perennial tropical and subtropical evergreen tree, is mainly distributed in southern or southeastern of China. Its seeds have been used for therapeutic esophageal cancer, nasopharyngeal carcinoma, malignant mole and leukemia [15][16][17][18]. Livistona chinensis fruit has been used for anti-hyperlipidemic and anti-ulcer [19]. 7-hydroxy-5,4′-dimethoxy-2-arylbenzofuran (Ary) is purified from Livistona, it has been found to inhibit the proliferation of tumor cells including HL-60, Mata, HepG2 and CNE-1 cells [20]. Our previous works showed that Ary (also named as HDAB) activates ATMdependent DNA repair response and suppresses PARP-1 activity through docking into PARP-1 hydrophobic pocket, resulting in cell cycle arrest [21]. In the present, we showed that Ary can inhibit cervical cancer cell proliferation through G1/S arrest and initiate cervical cancer cells programmed to death by mitochondrial apoptosis way, therefore activate extracellular signalregulated kinase (ERK) phosphorylation to elicit cell G1/S phase arrest through cyclin A2 and cyclin dependent kinase (Cdk) 2 upregulation.

Inhibitory effect of Ary on cervical cancer cell
At first, we detected the inhibitory effect of Ary on cervical cancer cells growth and proliferation. Cervical cancer cell lines, HeLa and Caski cells were treated with Ary, 3-(4,5-dimethylthiazol-2-thiazolyl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium bromide (MTT) assay and Soft agar colony formation assay were used to detect growth and proliferation of the treated cell. MTT assay results showed that both Hela and Caski cell growths were inhibited by Ary treatment ( Figure  1B Figure 1E-d, P < 0.01) colony formation in the treated groups were significantly low when compared with the control group ( Figure 1D-a; Figure 1E-a). Along with increasing Ary's concentration, its inhibitory effect was increased, and the cell colony formation was decreased ( Figure 1D-d, P < 0.01; Figure 1E-d, P < 0.01). The results suggest that Ary could effectively inhibit the growth and proliferation of cervical cancer cell.
The clearance rate of drug mostly depends on metabolic activity in vivo biotransformation process [3,22]. To further confirm Ary's anticancer effect in vivo, Hela cells were transplanted in nude mice. After 10 days, the nude mice models with cervical cancer cells were constructed, and the tumor masses of nude mice were injected with Ary at 50 μg/g (bodyweight) for 10 days, and simultaneously tumor volumes were measured [21]. After treatment 12 days, the implanted tumors were significantly small when compared with the control groups ( Figure 2A). After treatment 20 days, the mice were sacrificed, the tumor mass was peeled, its masses were weighed. The tumor weights in Ary group were significantly light when compared with the control groups ( Figure 2B-a, b, P < 0.01). The results indicated that Ary can inhibit cervical cancer growth in vivo.

Ary induces cervical cancer cells apoptosis through mitochondrial
In this step, we also observed whether Ary induces cervical cancer cells apoptosis. After Hela cells were treated with Ary, the treated cells were stained with DAPI. The changes of nuclear morphology were observed under fluorescence microscope. The results showed that after Ary treatment with 1.25 μg/mL, the cell nucleus became irregular and small, and cytoplasm was concentrated and marginalized ( Figure 3A-b), the treated cells had typical apoptotic bodies when Ary concentration increased to 5 μg/ mL ( Figure 3A-c), however, in the control group, the cell nucleus were round and color uniformity ( Figure 3A-a). The treated cells were stained with Annexin V-PI, apoptosis cells were counted using flow cytometry. The apoptosis rates increased when Ary concentration increase ( Figure 3B). Thereafter, the caspase 3 was detected in Hela cells with Ary treatment. The results showed that caspase 3 was activated when Ary treatment, and caspase 3 increased with Ary concentrations increase ( Figure 3C).
Decreased mitochondrial membrane potential (MMP) is an early sign of apoptosis event. To further probe the mechanism of Ary-inducing cervical cancer cell apoptosis, Confocal microscopy was used to observe MMP in the treated cell. The results showed that Ary treatment group at 5 μg/mL had a weak staining of J-aggregates (red fluorescent) ( Figure 4A-d) and strong staining of JC-1 monomers (green fluorescent) ( Figure 4A-e), when Ary concentration was increased to 10 μg/mL, the red fluorescence completely disappeared ( Figure 4A-g), only had a strong green fluorescence ( Figure 4A-e). However, the control group had strong staining of J-aggregates ( Figure 4A-a) and weak staining of JC-1 monomers ( Figure 4A-b). These suggest that Ary may induce cervical cancer cell MMP change. Cytochrome C is an important protein which participates in the electronic respiratory chain proteins of mitochondria, it is released into cytoplasm from mitochondria When cells apoptosis [19]. In the next step, we also observed cytochrome C changes in the cells with Ary treatment. The results showed that cytochrome C was increased in cytoplasm, but reduced in mitochondria in the treated cells, displaying a dose-dependent manner ( Figure 4B, 4C). These results showed that Ary may induce cervical cancer cell apoptosis through mitochondria degradation.

Ary arrests cervical cancer cell cycle at G1/sphase through cyclin A and cdk2
The previous results showed that Ary inhibits cell growth. We continue to verify whether Ary affects cell cycle progression. HeLa cells were treated with Ary at 1.25 μg/mL or 2.5 μg/mL for 24 h, and then their cell cycle was analyzed using flow cytometry assay. Compared with the control group (17.17%), the treated cells at both Ary 1.25 μg/mL and 2.5 μg/mL had a high percentage of G1/Sphase, 35.17% and 46.10% respectively. G1/ S-phase cell  percentage was higher in 2.5 μg/mL than that in 1.25 μg/ mL ( Figure 5A-b, c). These indicated that HeLa cells were suppressed in G1/S-phase by Ary. As we all know, cyclin A2 and Cdk2 have an important role in the G1/S-phase. And then cyclin A2, cyclin D1, cyclin E, Cdk2, and p21 expressions were detected. The results showed that after Ary treatment, cyclin A2, Cdk2 and p21 expressions were increased, and cyclin D1 decreased, displaying a dose-dependent manner ( Figure 5B). To further investigate the function of cyclin A2 in Ary-mediated G1/S arrest, siRNA-cyclinA2 (siCylinA2) was used to knockdown cyclin A2 expression. Western-blotting analysis showed that siCyclinA2 effectively inhibited Ary-mediated cyclin A2 expression ( Figure 6A-a, lane 4 vs 2 in upper panel; Figure 6A-b, lane 4 vs 2). The flow cytometry assay showed that the proportion of G1/ S-phase was significantly increased when Ary treatment ( Figure 6B-b), this increase was blocked by siCyclinA2 ( Figure 6B-d). These suggest that cyclinA2 plays an important role in Ary-induce G1/S arrest. Furthermore, to probe Cdk2's roles in Ary-mediated G1/S arrest, Cdk2 inhibitor, Flavopiridol was used to inhibit Cdk2 expression, and then cell cycle of the treated cell was analyzed. The results showed that Flavopiridol effectively inhibited Cdk2 expression ( Figure 6C-a, b), similarly, Ary-induced G1/S arrest was significantly decreased when Flavopiridol treatment ( Figure 6D-d). The results demonstrated that Cdk2 may also plays some role in Ary-induce G1/S arrest. Mitogen-ativated protein kinase kinase (MAPKK) is involved in many cellular biological functions including proliferation, differentiation, motility and death. ERKs (ERK1/2) are main members of MAPKKs pathway [23,24]. ERK1/2 is activated through phosphorylation of activation-loop residues threonine (Thr) 202/tyrosine (Tyr) 204 and Thr 185/Tyr 187, respectively. Activated ERK1/2 translocates into nucleus and participates in the regulation of G1-to S-phase transition, and the nuclear translocation The abundance ratios of C-cytochrome C or M-cytochrome C expressions to β-actin were calculated. The data were shown as the mean ± SD of three independent experiments by analysis of Student's t test. *P < 0.05, **P < 0.01, vs 0 μg/mL. of ERK1/2 is served as an anticancer target [25,26]. So, we want to clarify whether ERK-pathway was activated in G1/S-phase arrest induced by Ary. The results showed that phos-ERK1/2 was increased after Ary treatment, displaying a dose-dependent manner ( Figure 7A, upper panel), while total ERK expression did not change ( Figure  7A, meddle panel). Further, ERK nuclear translocation was observed. The immunofluorescence assay results showed that after Ary treatment, phos-ERK1/2 in the cytoplasm was decreased when compared with the control ( Figure 7B, f vs b), while it was increased in the nucleolus ( Figure 7B, g vs c). This indicated that phos-ERK1/2 was induced from cytoplasm to nucleolus by Ary treatment. To further investigate phos-ERK1/2's role in Ary-mediated G1/S-phase, an inhibitor of phos-ERK1/2, U0126 was used to inhibit phos-ERK1/2 expression. After the cells were treated with U0126, ERK phosphorylation was blocked ( Figure 7C, lane 3 in upper panel) and CyclinA2 expression was also reduced ( Figure 7C, lane 3 in middle panel), simultaneously the ratios of G1/S-phase were reduced compared with the control group without U0126 ( Figure 7D, d vs b, P < 0.05). Furthermore, we also analyzed the inhibition of Ary on cell soft agar colony formation when U0126 inhibited ERK phosphorylation. Compared with the group, the inhibition rates were reduced ( Figure 7E, lane 4 vs 2, P < 0.05 ). Over all, these results suggest that Ary may activate phosphorylation ERK1/2 pathway to arrest cervical cancer cell in G1/Sphase and block the cell proliferation.

DIscUssION
Ary is a novel natural product which is isolated from Livistona, several studies have documented the anticancer activity of Ary [2,20,27]. But it has rare reports about Ary's effect on cervical cancer and its mechanism. In this study, we showed that Ary can effectively inhibit the proliferation and colony growth of HeLa and CaSki cells in vitro, and it can also inhibit cervical cancer xenograft growth in vivo, this indicated that Ary has some therapeutic effect on cervical cancer.
To further clarify its mechanism, we detected Ary's effect on cervical cancer cell apoptosis and cell cycle. It is found that Ary can indeed induce cervical cancer apoptosis, which is supported by evidences including morphology change and caspase 3 activation in Ary treated cells. Generally, apoptosis is a type of cell self-destructive in an orderly way by a series of signal cascades which include many gene products and cytokines. Caspase-3 activation plays an important role in the development of apoptosis [28,29]. In the Ary treated cells, we observed apoptosis morphology changes, and detected the changes of caspase 3. In the further study on mechanism, we found that Ary decreased J-aggregates and increased JC-1 monomers, additionally, Ary also increased cytoplasm cytochrome C and decreased mitochondria cytochrome C. We think that Ary induces cell apoptosis through mitochondria.
The Ary-treated cells were used to analyze cell cycle, we found that Ary can arrest cervical cancer in G1/S phase. Cyclin A2 is a cell cycle protein which predominantly expresses in S phase, simultaneously activates the partner of Cdk2 to regulate the initiation and progression of DNA synthesis [30,31]. Cdk2 is a member of the Cyclin Dependent Kinase (Cdk) family, which is associated with cyclin A or cyclinE. Cdk inhibitor and p21Cip1 restricts cell to G1/S phase and apoptosis [32][33][34][35][36].This study demonstrated that Ary can restrict cervical cancer cells in G1/S phase along with cyclin A2 and Cdk2 expression increase. When cyclin A2 gene expression was blocked with siRNA and Cdk2 was inhibited with Flavopiridol, Ary-induced G1/S phase arrest was dramatically decreased. This suggests that cyclin A2/ Cdk2-associated kinase activation may be responsible for Ary-mediated G1/S phase arrest.
ERK is extracellular regulated protein kinase which involves in many cellular programs. Phosphorylation activation of ERK translocates from the cytoplasm to the nucleus, and participates in the biological response of cells [37,38]. Some studies suggest that the regulation of ERK1/2 in G1-to S-phase transition and the nuclear translocation of ERK1/2 served as an anticancer target [23,24,39]. However, in this study, we found that Ary induced ERK phosphorylation, and the phosphorylation levels were positively correlated with Ary doses, when ERK1/2 phosphorylation was inhibited, the ratio of G1/S-  Colony formation ability of the treated cells was determined using soft agar colony formation assay, and the inhibition rates of colony formation were calculated. Data were shown as the mean ± SD of three independent experiments by analysis of Student's t test. *P < 0.05, **P < 0.01. phase was reduced. Yang TY, et al. found that pemetrexed mediated S-phase arrest and apoptosis conjugated with sustained activation of ERK and Cdk2/Cyclin A, and further verified that the sustained activation of ERK results in S-phase arrest [40]. ERK is thought to be a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine/ threonine kinases, Raf activates the MAPK/ERK kinase (MEK)1/2 dual-specificity protein kinases, which then activate ERK1/2 [41]. Based on these, we speculate that MAPK/ERK kinase (MEK)1/2 phosphorylating ERK is involved in Ary anti-cervical cancer, MEK1/2 may be its upstream molecular. Ary may constantly activate the phosphorylation ERK1/2 to locate the nucleolus and to delay the cervical cancer cells in G1/S-phase to prevent them proliferation.
It is documented that HPV is associated with cervical cancer, and it play an important role in cervical cancer pathogenesis. Whether Ary's anti-cervical cancer is associated with HPV infection? Based on our previous works, Ary has effects on non-cervical cancer which is HPV negative [20,21]. We speculate that Ary has anticancer effect not only to the cervical cancer with integrated HPV genomes, but also to other cancers with HPV-negative. We think that Ary's anticancer effect should not be linked with HPV. The studies in vitro showed that Ary could significantly inhibit the growth, proliferation and colony formation of cervical cancer cells. However, most of drug's concentration and clearance rate depends on drug metabolism activity in vivo through biotransformation, the effect of drug in vivo should be exactly therapeutic effect [39,42]. In this study, to confirm Ary's effect on cervical cancer in vivo, we established a human cervical cancer xenograft models, and validate its therapeutic effect. Ary significantly inhibited the growth of cervical cancer implanted-tumor. We speculate that Ary will be a novel curative medicine, and this will provide a new therapeutic strategy for cervical cancer.
In summary, our data showed that a novel natural product, Ary can effectively treat cervical cancer in vivo and in vitro, its mechanism is that it induces cervical cancer cell apoptosis through mitochondrial membrane potential, and arrests cells at G1/S-phase through constantly activating ERK1/2 to locate the nucleolus and upregulating cyclin A2 and Cdk2 expression (Figure 8). In addition, our study also suggested that the sustained-phosphorylation ERK and upregulation cyclin A2 and Cdk2 elicit cell G1/S arrest.

MAtErIALs AND MEtHODs reagents and antibodies
Ary was extracted and isolated from the fruits of L. chinensis by Hubei University of Traditional Chinese Medicine (Wuhan, China), its purity is 92%. Its chemical structure is shown at Figure 1A. Ary was dissolved in dimethyl sulphoxide (DMSO) for storage fluid, the storage fluid was diluted using cell culture medium for the indicated concentration. Its stability is 98.3% in DMSO, and 97.4% in cell culture medium (shown at Supplementary Table S1). MTT, DAPI, Annexin V, fluorescein isothiocyanate (FITC) reagents were purchased from Invitrogen Corporation (CA, USA). Antibodies against cyclin A2, cyclin D1, cyclin E, Cdk2 or p21 were purchased from Abcam Company (MA, USA). Antibodies to ERK1/2 or phos-ERK1/2 (Thr202/ Tyr204) were obtained from Cell Signaling Technology Company (Danvers, MA, USA). Antibody against β-actin, normal mouse IgG, sodium dodecyl sulfate (SDS) lysis buffer, phenylmethane-sulfonyl fluoride (PMSF) and BCA Protein Assay kit were purchased from Santa Cruz Biotechnology (CA, USA). The inhibitors of U0126 (an inhibitor for ERK1/2), Flavopiridol (an inhibitor for Cdk2) were purchased from Sigma company (MO, USA). DMSO, Triton X-100, horseradish peroxidase-linked antimouse immunoglobulin G, anti-rabbit immunoglobulin G, antibody against cytochrome C or caspase 3, and Mitochondrial Membrane Potential Assay kit with JC-1 were purchased from Biyuntian Company (Shanghai, China). Mitochondria Isolation Kit was purchased from Pierce Biotechnology (MA, USA). Double-Stranded cDNA Synthesis Kit and Lipofectamine™2000 were purchased from Invitrogen Company.

Mtt assay
MTT assay was performed as previously described [43]. Briefly, HeLa and Caski cells were seeded in 96-well plate at a density of 5×10 3 cells/well, and incubated for 24 h. The cells were treated with Ary at 1.25, 2.5 l, 5, 10 and 20 μg/mL, the saline was served as a blank control. After being incubated for 24 h, the cells were changed its media, and incubated again with 20 μL of MTT (5 mg/ mL)/well for 4 h. The cells' supernatants were discarded, each cell well was added 150 μL DMSO, and then shocked 10 min to dissolve the crystal violet. Finally, the absorbance of cell wells was measured at 490 nm. The inhibitory rate of various concentrations was calculated, inhibition rate = 1-OD value of experimental group/ control group OD value × 100%.

soft agar colony formation assay
Cell colony formation assay was performed as previously described [44]. Briefly, the treated cells at 1 × 10 3 cells/well were suspended in 1 mL of DMEM containing 0.6% low-melting-point agarose (Amresco, USA) and 10% FBS, and plated on a bottom layer containing 0.3% agarose and 10% FBS in 6-well plate in triplicate. The 6-well cell plates were added Ary at 1.25, 2.5, 5, 10 or 20 μg/ mL. After being incubated for two weeks, the cell plates were stained with 0.1% crystal violet for 15 min, and the colonies were counted under a microscopy.

cervical cancer cell xenograft experiments
Animal experiments were performed as previously described [44]. Briefly, 20 female BALB/c nude mice (approximately four to six weeks old) were purchased from Guangdong Province Medical Animal Center (Guangzhou Guangdong), fed in a specific pathogen-free environment. After one week, the mice were injected subcutaneously with Hela cell (5 × 10 7 ) or Caski cells (5 × 10 7 ). The injected mice were randomly divided into two groups (n = 6) when the tumors reached a same palpable size. One group was injected with DMSO solvent, served as the control group. Another group received Ary treatment at a dosage of 50 μg/g (bodyweight) for 10 days [21]. The mice tumor block length diameter L and short diameter D were measured with a vernier caliper at 1 time per 2 days, and tumor volume was calculated according to the formula: V = L × D 2 /2 (18). Mice were sacrificed after 20 days or while the mice appeared moribund, tumors were excised carefully and weighed tumors weight.

Morphology detection of cell apoptosis
The cells were seeded in 6-well plate, cultured for 24 h, and treated with Ary at 1.25 and 2.5 μg/mL for 24 h. The treated cells were fixed with 4% paraformaldehyde for 15 min, incubated with 0.1% Triton X-100 for 5 min. The fixed cells were stained with DAPI staining working solution at room temperature for 10 min. After being washed with PBS, the cell nuclear morphology was observed under fluorescence microscope. www.impactjournals.com/oncotarget

Detection of mitochondrial membrane potential
The cells were incubated in a 6-well plate for 24 h, and then treated with Ary at 5 and 10 μg/mL. After being treated for 24 h, the cell medium was removed. After being washed with phosphate-buffered saline (PBS), the cells were added Mito-Tracker Green staining fluid, and then incubated in 37°C for 30 min. After being removed the staining fluid, the cells were washed with PBS, added pre-warmed and fresh cell culture medium. The cells were observed under fluorescence microscopy or Confocal microscope.

Mitochondria isolation
The treated cells (5 × 10 7 ) were collected, and washed with PBS. After being washed, the cells were added 800 μL Mitochondria Isolation Reagent A, and vortexed at medium speed for 5 s, and then incubated tube on ice for 2 min. After ice incubation, the cell suspensions were added 10 μL reagent B, incubated tube on ice for 5 min, and vortexed at maximum speed every minute. After being vortexed, the cell suspensions were added 800 µL reagent C, inverted tube several times to mix, and centrifuged at 700 × g for 10 min at 4°C. The supernatants were collected, centrifuged at 3000 × g for 15 min. After being centrifuged, 500 µL reagent C was added to suspend the pellets, the suspensions were centrifuged at 12,000 × g for 5 min at 4°C. Discarding the supernatant, the pellets were collected, and stored into 4°C for the next experiments.

Preparation of sirNA and transfection
CyclinA2-siRNA (siCyclinA2) and nonspecific siRNA (simock) were synthesized from Shanghai Gene Pharma Technology Co. Ltd (Shanghai, China). siCyclinA2 sequence is as follows: sense 5ʹ-CAGGACCAGGAGAAUAU CATT-3ʹ; antisense 5ʹ-UGAUAUUCUCCUGGUCCUGTT-3ʹ. Simock: sense 5ʹ-UUCUC CGAACGUGUCACGUTT-3ʹ, antisense 5ʹ-ACGUGACACGUUCGGAGATT-3ʹ. The siRNAs and Lipofectamine™2000 with Opti-MEM culture medium were mixed gently, then incubated at room temperature for 20 min. The complex was added to each cell wells. The culture medium was exchanged with fresh medium after 6 h, and the cells were collected for flow cytometry analysis after 24 h.

Annexin V-PI apoptosis rate analysis
The cells were treated with Ary at 1.25, 2.5, 5, 10, and 20 μg/mL for 24 h, collected in tube (1 × 10 6 cells/ mL), and added 195 μL Annexin V-FITC binding buffer and 5 μL Annexin V-FITC. The cell suspensions were incubated at room temperature in dark for 10 min, and centrifuged. The cell pellets were collected, and added 190 μL Annexin V-FITC binding buffer and 10 μL PI staining solution. After being mixed gently, the cell suspensions were placed in the dark drawer. After being filtrated with 300 mesh nylon net, the cells were immediately detected with flow cytometry.

Immunofluorescence staining
Immunofluorescence staining was performed as previously described [44]. Briefly, the treated cells were fixed with 4% paraformaldehyde for 15 min, and then added 0.1% Triton X-100 incubation for 5 min. The cells were blocked with 3% bovine serum albumin (BSA), and followed by incubation with mouse antibody against ERK1/2 or Phos-ERK1/2 at 4°C over nigh. The horseradish peroxidase-linked anti-mouse antibodies were used to stain ERK1/2 or phos-ERK1/2. DAPI was use to stain cell nuclear. The cells were observed under Olympus Confocal laser scanning microscope. The cell images were treated using Meta Morph software.

Flow cytometric analysis
The treated cells were harvested and fixed with cold 70% ethanol. RNA enzyme at 50 μg/mL was added to the cells, and incubated in 37°C for 30 min. The cells were stained with propidium iodide and analyzed its cell cycle by flow cytometry.

statistical analysis
All experiments were repeated at least three times and the data were presented as the mean ± SD. Differences between data groups were assessed of Student's t-test or analysis of variance (ANOVA) or independent sample t test (SPSS16.0). P values less than 0.05 was considered statistical significance.