Full Paper. Jian Huang 1,2, Lijun Wu 2, Shin-ichi Tashiro 3, Satoshi Onodera 3, and Takashi Ikejima 1, *

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1 J Pharmacol Sci 102, (2006) Journal of Pharmacological Sciences 2006 The Japanese Pharmacological Society Full Paper Fibroblast Growth Factor-2 Suppresses Oridonin-Induced L929 Apoptosis Through Extracellular Signal-Regulated Kinase-Dependent and Phosphatidylinositol 3-Kinase-Independent Pathway Jian Huang 1,2, Lijun Wu 2, Shin-ichi Tashiro 3, Satoshi Onodera 3, and Takashi Ikejima 1, * 1 China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, , China 2 Department of Phytochemistry, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, , China 3 Department of Clinical and Biomedical Science, Showa Pharmaceutical University, Tokyo , Japan Received April 13, 2006; Accepted September 25, 2006 Abstract. Oridonin, isolated from Rabdosia rubescences, has been reported to exert cytotoxic effects on L929 cells. In this study, we investigated the mechanisms of FGF-2 protection of L929 cells from oridonin-induced apoptosis. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signal did not mediate this effect because the PI3K inhibitor wortmannin failed to reverse this protection and PKB activation was not observed in this process. In contrast, the extracellular signal-regulated kinase (ERK) was responsible for this rescue because its inhibition abolished the protective effect of fibroblast growth factor (FGF)-2. ERK had dual regulatory functions: mediating cell apoptosis or preventing cells from initiating the apoptotic response by phosphorylation or promoting expression of Bcl-2 in dependence of different stimuli. In L929 cells treated with oridonin alone, the activated ERK decreased the ratio of Bcl-2/Bax by mediating the phosphorylation of Bcl-2, resulting in apoptosis; the Ras inhibitor manumycin A and Raf inhibitor GW5074 failed to inhibit this apoptosis, indicating that there is a signal other than Ras/Raf pathway activated ERK. However, in the presence of FGF-2, Bcl-2 phosphorylation was blocked, and the Ras/Raf/ERK signal pathway was activated and protected against the oridonininduced apoptosis by the alternative function of promoting of Bcl-2 expression. Keywords: oridonin, fibroblast growth factor (FGF)-2, apoptosis, extracellular signal-regulated kinase (ERK), Bcl-2 Introduction Oridonin (Fig. 1), a diterpenoid isolated from the plant Rabdosia rubeseens, has been reported to have various pharmacological and physiological effects such as scavenging active oxygen free radicals, anti-inflammation, anti-bacteria, and anti-tumor effects (1 4). Previous reports have demonstrated that oridonin exhibits remarkable inhibitory effects on breast carcinoma, nonsmall cell lung cancers, acute promyelocytic leukemia, and glioblastoma multiforme (5, 6). Our previous study has also showed that oridonin has cytotoxic effects on *Corresponding author. ikejimat@vip.sina.com Published online in J-STAGE doi: /jphs.FPJ06004X various cancers such as human melanoma A375-S2, human cervical carcinoma HeLa, human breast adenocarcinoma MCF-7, and murine fibrosarcoma L929 (7). Apoptosis, or programmed cell death, is an important process in biological systems, including normal cell turnover, immune system, embryonic development, metamorphosis, and endocrine-dependent tissue atrophy. Apoptosis occurs when a cell initiates a series of biochemical and morphological events that result in a decrease in cell volume, dilatation of the endoplasmic reticulum, condensation and fragmentation of nuclear chromatin, and formation of membrane-bound apoptotic bodies (8 11). Apoptosis can be triggered in multiple ways; and it is well known that many protein such as cysteine-dependent aspartate-specific proteases (caspase) family, Bcl-2 family, mitogen-activated protein kinase 305

2 306 J Huang et al (MAPK) family, p53, and phosphoinositide 3-kinase (PI3K) signal transduction pathways play important roles in regulating apoptotic process (12 16). Fibroblast growth factor-2 (FGF-2) is a multifunctional cytokine involved in many biological processes including proliferation, migration, neoangiogenesis, and cell survival (17 19). Serum and growth factor withdrawal induces apoptosis in several cell lines, indicating that growth factors have a protective effect against apoptosis (20). FGF-2 is known to bind and activate specific tyrosine kinase receptors (FGFRs), which couple to multiple intracellular signal pathways (21, 22). The activation of tyrosine kinase receptors by other growth factors participates in cell survival through downstream signal cascades such as the mitogenactivated ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (PKB) pathway (23 26). Upstream of MEK/ERK are Ras and Raf, which are predominantly activated by FGF-2 (27). These signals promote cell survival through several mechanisms including the regulation of Bcl-2 family members (28 31). Bcl-2 family members are characterized by containing at least one of four Bcl-2 homology domains (BH1 BH4). Some of these proteins such as Bax, Bad, and Bak function to promote apoptosis, whereas others like Bcl-2, Bcl-X L, and Mcl-1 inhibit apoptosis (32). The balance between the two types of Bcl-2 family members has been reported to partially control cell fate and the ratio of Bcl-2/Bax alters through two different mechanisms, transcriptional regulation or phosphorylation of Bcl-2 family members by the ERK pathway (33, 34). In the present study, we demonstrated that FGF-2 prevented the L929 cell apoptosis induced by oridonin through an ERK-dependent pathway and a PI3K/PKBindependent signal pathway. ERK not only mediated cell apoptosis but also protected cells against apoptotic response relying on the difference of stimulus. In L929 cells treated with oridonin alone, a signal different from the Ras/Raf pathway activated ERK, and the activated ERK mediated cell death by promoting the phosphorylation of Bcl-2. When the L929 cells were stimulated with FGF-2, the Ras/Raf/ERK signal pathway was activated and rescued from the apoptosis induced by oridonin by an alternative effect of up-regulation of Bcl-2 expression. Materials and Methods Reagents Oridonin (Lot: ) was obtained from the Beijing Institute of Biological Products (Beijing, China). The chemical structure (Fig. 1A) of oridonin Fig. 1. Chemical structure (A) and HPLC detection (B) of oridonin. was assigned by comparing the chemical and spectral data ( 1 H-NMR, 13 C-NMR) with those reported in the literature (35). The purity of the oridonin was measured by HPLC (column: 4.6 mm 250 mm, type: CAPCELL PAK C18 ACR; Shiseido, Tokyo; solvent phase: methanol:h 2 O 55:45) and determined to be 97.4% (Fig. 1B). Oridonin was resolved in dimethyl sulfoxide (DMSO) to make a stock solution. The DMSO concentration was kept below 0.05% throughout the cell culture period and did not exert any detectable effect on cell growth or cell death. FGF-2 was from Gibco (Grand Island, NY, USA). The Ras inhibitor manumycin A, Raf inhibitor GW5074, ERK inhibitor PD98059, p38 inhibitor SB203580, and c-jun N-terminal kinase (JNK) inhibitor SP were obtained from Calbiochem (La jolla, CA, USA). The PI3K family inhibitor wortmannin, Hoechst 33258, and Giemsa staining solution were purchased from Sigma Chemical (St. Louis, MO, USA). Fetal calf serum

3 FGF-2 Protects Against Apoptosis 307 (FCS) was from the Dalian Biological Reagent Factory (Dalian, China). TACS TM 2 TDT-DAB In Situ Apoptosis Detection Kit was obtained from Trevigen (Gaithersburg, MD, USA). Cell culture The murine fibrosarcoma cells L929 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FCS and 0.03% L-glutamine (Gibco) and maintained at 37 C with 5% CO 2 in a humidified atmosphere. Cytotoxity assay L929 cells were incubated in 96-well plates (NUNC, Roskilde, Denmark) at a seeding density of cells per well. The cells were pretreated with wortmannin, PD98059, SB203580, SP600125, manumycin A, or GW5074 at the given concentrations for 1 h, and FGF-2 for 15 min, and then incubated with oridonin for different time periods. Four hours before the end of incubation, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) solution (5.0 mg/l) was added to each well. The resulting crystals were dissolved in 100 µl DMSO. Absorbance was measured with an ELISA reader (TECAN SPECTRA; Tecan, Wetzlar, Germany). The cytotoxic effect was expressed as the relative percentage of cell growth inhibition as calculated below: Cell growth inhibition (%) = [(control absorbance experimental absorbance) / (control absorbance blank absorbance)] 100 Observation of morphological changes L929 cells were seeded into culture plates and cultured overnight. The control L929 group was treated with the RPMI 1640 culture medium. Oridonin was added to the cells and cultured for 12 h. For measurement of chromatin condensation, the cells were fixed with Carnoy solution (ethanol:chloroform:acetic acid 6:3:1) at room temperature for 10 min, and then stained with Giemsa solution at room temperature for 10 min. Morphology of L929 cell nuclei was observed under a light microscope (Olympus, Tokyo). The L929 cells were cultured as above and treated with oridonin for 24 h. Apoptotic nuclear morphology was also assessed using Hoechst Cells were fixed with 3.7% paraformaldehyde at room temperature for 30 min, and then they were washed and stained with 167 µm Hoechst at 37 C for 30 min. The cells were washed and resuspended in phosphatebuffered saline (PBS) for morphologic observation by fluorescence microscopy (Leica, Wetzlar, Germany). Terminal deoxynucleotidyl transferase-mediated Dutp nick end-labeling (TUNEL) assay The TUNEL assay was used for detection of DNA strand breaks. The detection was carried out according to the instructions of the TACS TM 2 TDT-DAB In Situ Apoptosis Detection Kit. Briefly, the cells were rinsed once with PBS and fixed in 3.7% buffered formaldehyde at room temperature for 10 min. The fixed cells were pretreated with 10% H 2 O 2, and end-labeling was performed with TdT labeling reaction mix at 37 C for 1 h. Nuclei exhibiting DNA fragmentation were visualized by incubation in 3',3-diamino benzidine (DAB) for 7 min. Lastly, the cells were counter-stained with methyl green and observed under a light microscopy. The nuclei of apoptotic cells were stained dark brown and TUNELpositive L929 cells were determined as a relative percentage by randomly counting 100 cells. Western blot analysis L929 cells were cultured for different time periods. Both adherent and floating cells were collected, and then Western-blot analysis was performed as follows. Briefly, the cell pellets were resuspended in lysis buffer consisting of 50 mm Hepes (ph 7.4), 1% Triton-X 100, 2 mm sodium orthovanada, 100 mm sodium fluoride, 1 mm edetic acid, 1 mm PMSF, 10 mg/ L aprotinin (Sigma), and 10 mg/l leupeptin (Sigma) and lysed at 4 C for 60 min. After 13,000 g centrifugation for 15 min, the protein content of the supernatant was determined by a protein assay reagent (Bio-Rad, Hercules, CA, USA). The protein lysates were separated by electrophoresis in 12% SDS polyacrylamide gel and blotted onto a nitrocellulose membrane. Each membrane was blocked with 5% skim milk and then incubated with the indicated primary antibodies against PKB, phospho- PKB, ERK, phospho-erk (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2, phospho-bcl-2, and Bax (Oncogene, Boston, MA, USA). After that, the membrane was incubated with secondary antibodies, goat anti-rabbit, and goat anti-mouse IgG conjugated with peroxidase (HRP) (Santa Cruz Biotechnology) and visualized by using DAB as the HRP substrate. Statistical analysis of the data All results were confirmed in at least three separate experiments. The data are expressed as means ± S.D. Statistical comparisons were made by Student s t-test, P<0.05 was considered significant. Results Oridonin induces apoptotic cell death in L929 cells Oridonin induced L929 cell death in a concentration-

4 308 J Huang et al and time-dependent manner. Oridonin at µm exerted a potent cytotoxic effect on L929 cells, and treatment of L929 cells with 30 µm oridonin for 12 h resulted in approximately 66% cell death (Fig. 2A). To characterize the oridonin-induced L929 cell death, we examined the morphologic changes by Giemsa and Hoechst staining. When L929 cells were cultured with 30 µm oridonin for 12 h, apoptotic morphologic changes were observed as compared with the medium control group. Oridonin-treated L929 cells underwent contraction and showed nuclear fragmentation (Fig. 2: B and C). By 12 h, apoptotic morphologic changes were further confirmed by Hoechst staining of cell nuclei. In the control group, nuclei of L929 cells were round and homogeneously stained, but the 30 µm oridonin-treated cells showed marked granular apoptotic bodies (Fig. 2: D and E). FGF-2 blocks oridonin-induced L929 apoptosis Murine L929 cell line expresses FGFR, and activation of this receptor and its downstream signals occurs at 1 5ng/ ml (20). Therefore, we tested whether FGF-2 could prevent oridonin-induced L929 cell death at the concentration of 1 10 ng/ml. FGF-2 significantly reduced the cell death in the L929 cells incubated with 30 µm oridonin for 12 h (Fig. 3). A 5 ng/ml dose of FGF-2 was effective at inhibiting oridonin-mediated cell death. This protective effect of FGF-2 was not simply due to an increased proliferation of L929 cells because FGF-2 in the absence of oridonin did not measurably increase the cell number. To further determine that FGF-2 blocked oridonininduced apoptosis, TUNEL assay was carried out. When L929 cells were treated with oridonin alone, the TUNEL-positive cell ratio was about 37.2 ± 1.2%. However, in the presence of FGF-2 at 5 or 10 ng/ml, the ratio of apoptotic cell increased to 18.5 ± 2.1% and 16.9 ± 2.7%, respectively (Table 1). The results suggested that FGF-2 played the protective role against oridonin-induced apoptosis, and 5 ng/ml FGF-2 had almost the maximum protective effect against the cell death in L929 cells. PI3K/PKB signals do not mediate the protection of FGF-2 against oridonin-induced L929 cell death To investigate the effect of PI3K on the ability of FGF-2 to prevent oridonin-induced apoptosis in L929 cells, the PI3K inhibitor wortmannin was applied (Fig. 4A). When L929 cells were cultured with oridonin and FGF-2 for 12 h, 200 nm wortmannin did not prevent FGF-2 rescue of oridonin killing. It has been reported that PKB lies downstream of PI3K in the signal transduction pathway to regulate cell growth (36). Fig. 2. Oridonin induces apoptotic cell death. The cells were treated with various doses of oridonin for 6, 12, or 24 h (A). Cell growth inhibition rate was measured by MTT assay. n = 3, Mean ± S.D. The cells were treated with medium or 30 µm oridonin for 12 h, and cellular morphologic changes were observed by Giemsa (B, C; 200 magnification) or Hoechst (D, E; 200 magnification) staining. Western blot analysis was carried out to detect the expression of PKB and phosphorylated PKB (Fig. 4B). After treatment of L929 cells with oridonin and FGF-2

5 FGF-2 Protects Against Apoptosis 309 Fig. 3. FGF-2 prevents L929 cells from oridonin-induced cell death. After pretreatment with 1, 5, or 10 ng/ml FGF-2, the cells were cultured with 30 µm oridonin for 12 h. Cell growth rate was measured by MTT assay. n = 3, mean ± S.D. *P<0.05, **P<0.01 vs oridonin group. Table 1. Quantitative analysis of TUNEL-positive L929 cells FGF-2 (ng/ml) Apoptotic cells (%) TUNEL ± ± ± 2.1** ± 2.7** L929 cells were treated with 30 µm oridonin in the absence or presence of FGF-2 of different concentrations for 12 h. The TUNEL assay was used for detection of apoptotic cells. The results were representative of three independent experiments. All data were expressed as means ± S.D. processed by Student s t-test and considered statistically significant at **P<0.01 vs oridonin group. for 12 h, the expression level of PKB and phosphorylated PKB was similar to that of the oridonin group, and the PI3K inhibitor wortmannin did not change the PKB and phospho-pkb expression. All these results suggested that the PI3K/PKB signal pathway did not mediate FGF-2-induced resistance to oridonin in L929 cells. The ERK signal pathway is involved in the FGF-2 protection against oridonin-induced apoptosis in L929 cells ERK is one of the MAPK family members, and the other two are JNK and p38. In our study, specific inhibitors for p38 (SB203580), JNK (SP600125), and ERK (PD98059) were applied to evaluate the function of MAPK in FGF-2 protection of oridonin-induced L929 Fig. 4. PI3K/PKB signal pathway is not involved in the FGF-2 suppression of oridonin-induced apoptosis. The cells were pretreated with 5 ng/ml FGF-2 (F) for 15 min or 200 nm wortmannin (W or) for 1 h, and then, they were cultured with 30 µm oridonin (Or) for 12 h (A). Cell growth inhibition rate was measured by MTT assay. n = 3, mean ± S.D. The cells were treated as in A, and cell lysates were separated by 12% SDS-PAGE electrophoresis, and the expression of PKB and phosphorylated PKB proteins was detected by Western blot analysis (B). apoptosis. After incubation of L929 cells with oridonin or oridonin and FGF-2 for 12 h, 5 µm SB and 5 µm SP neither affected oridonin cytotoxity nor FGF-2 rescue of it (Fig. 5: A and B). In case of PD98058, when L929 cells were treated with oridonin alone, 5 µm PD98059 inhibited the cell death; and in contrast, in the presence of FGF-2, PD98059 not only failed to inhibit the cell death, but also effectively reversed the FGF-2 rescue of oridonin-induced apoptosis (Fig. 5C). To further confirm this result, expression of ERK and phosphorylated ERK protein was examined by Western blot analysis. As shown in Fig. 5D, FGF-2 promoted ERK phosphorylation, compared with the medium group, in the presence or absence of oridonin. Moreover, the ERK inhibitor PD98059 partially reduced ERK phosphorylation. The ERK pathway is predominantly activated through a Ras-dependent mechanism and is required for cell proliferation and survival. To further confirm that the Ras/Raf/ERK cascade signal transduction pathway is involved in FGF-2 protection against oridonin-induced apoptosis, the Ras inhibitor manumycin A and Raf inhibitor GW5074 were applied. In the presence of oridonin alone, neither 10 µm manumycin A nor 5 nm GW5074 blocked oridonin cytotoxity. Consistent with

6 310 J Huang et al Fig. 5. ERK mediates the anti-apoptotic effects of FGF-2. The cells were pretreated with 5 ng/ml FGF-2 (F) for 15 min or 5 µm SB (S B) (A), 5 µm SP (S P) (B), or 5 µm PD98059 (P D) (C) for 1 h; and then, they were cultured with 30 µm oridonin (Or) for 12 h. Cell growth inhibition rate was measured by MTT assay. n = 3, mean ± S.D. **P<0.01. The cells were treated as in C, and cell lysates were separated by 12% SDS-PAGE electrophoresis, and the expression of ERK and phosphorylated ERK proteins was detected by Western blot analysis (D). our previous results, when the cells were added with FGF-2, 10 µm manumycin A and 5 nm GW5074 effectively reversed the FGF-2 protective property (Fig. 6A). Western blot analysis was also performed to examine the effect of manumycin A and GW 5074 on the expression of phosphorylated ERK. As shown in Fig. 6B, phosphorylated ERK expression triggered by oridonin alone was not influenced by manumycin A and GW5074, however, in the presence of FGF-2, both manumycin A and GW5074 inhibited ERK phosphorylation. These results suggested that the Ras/Raf/ERK cascade signal pathway was activated and played the protective role in mediating the FGF-2 suppression of oridonin-induced apoptosis. The increased ratio of Bcl-2/Bax expression is required for FGF-2-mediated rescue in L929 cells The ERK signal pathway is reported to regulate Bcl-2 expression or its phosphorylation to control the cell fate. Therefore, we also examined the expression of Bcl-2 family protein Bcl-2 and Bax in L929 cells treated with oridonin in the absence or presence of FGF-2 by Western-blot analysis (Fig. 7). The results showed that treatment of L929 cells with 30 µm oridonin for 12 h decreased the ratio of Bcl-2/Bax by promoting Bcl-2 phosphorylation and Bax expression. The ERK inhibitor PD98059 inhibited Bcl-2 phosphorylation, but did not change Bax expression. In the presence of FGF-2, the expression of Bcl-2 is up-regulated and the expression of Bax is down-regulated, compared with the oridonin group, and phosphorylated Bcl-2 was also blocked. When the L929 cells cultured with oridonin and FGF-2 were added with PD98059, PD98059 reduced Bcl-2 expression, but did not change Bax expression (Fig. 7). These results suggested that in the presence of FGF-2, the ERK signal pathway was activated and protected the oridonin-induced apoptosis by up-regulation of Bcl-2 expression. Discussion The FGF-2 has been shown to prevent cell death induced by several chemotherapeutic agents including paclitaxel, doxorubicin and 5-flurouracil in human prostate cancer cells and rat tumors (19). However, the underlying mechanism has not been clearly elucidated. Polypeptide growth factors are known to mediate the cell survival through the PI3K/PKB signal pathway

7 FGF-2 Protects Against Apoptosis 311 Fig. 6. Ras and Raf participate in FGF-2 rescue of oridonin-induced apoptosis. The cells were pretreated with 5 ng/ml FGF-2 (F) for 15 min or 10 µm manumycin A (M A) or 5 nm GW 5074 (G W) for 1 h, and then, they were cultured with 30 µm oridonin (Or) for 12 h. Cell growth inhibition rate was measured by MTT assay. n = 3, mean ± S.D. **P<0.01. The cells were treated as in A, and cell lysates were separated by 12% SDS-PAGE electrophoresis, and the expression of ERK and phosphorylated ERK proteins was detected by Western blot analysis (B). Fig. 7. Western-blot analysis of Bax, Bcl-2, and phosphor-bcl-2 protein expression in L929 cells treated with oridonin in the absence or presence of FGF-2. The cells were pretreated with 5 ng/ml FGF-2 (F) for 15 min or 5 µm PD98059 (P D) for 1 h, and then, they were cultured with 30 µm oridonin for 12 h. Cell lysates were separated by 12% SDS-PAGE electrophoresis, and the expression of Bax, Bcl-2, and phosphorylated Bcl-2 proteins was detected by Western blot analysis. (23 25). This effect partly involves the phosphorylation of the pro-apoptotic protein Bad either directly by PKB or through the intermediate activation of Pak. Phosphorylated Bad is sequestered in the cytoplasm, preventing it from exerting its pro-apoptotic effect on mitochondria (37 39). Recent work has also demonstrated that the expression of Mcl-1 and Bcl-2 could be regulated by the PI3K/PKB pathway (40). In this study, however, FGF-2-mediated rescue of oridonin-induced apoptosis was independent of these signals because the PI3K inhibitor wortmannin failed to inhibit this rescue, and PKB was not activated by FGF-2. Therefore, the PI3K/PKB signal pathway was not involved in this FGF-2 rescue of apoptosis induced by oridonin. The function of ERK is controversial in the regulation of apoptosis. In a sense, ERK signal provides a protective pathway by which some growth factors prevent apoptosis and this promotion of cell survival is through several mechanisms including the regulation of antiapoptotic Bcl-2 family members (33). Recent work has also demonstrated that ERK promotes apoptosis in response to certain chemotherapeutic agents, and this effect is partially due to the increased phosphorylation of Bcl-2 (34). In this study, inhibition of ERK with PD98058 inhibited oridonin-induced apoptosis, indicating that ERK mediated this process. However, in the presence of FGF-2, PD98059 not only failed to inhibit the cell death, but also effectively reversed the FGF-2 rescue of oridonin-induced apoptosis. The inhibition of upstream proteins Ras and Raf also reversed this rescue, but did not influence oridonin killing. All those results indicated that ERK was activated by a signal different from the Ras/Raf pathway, and the activated ERK could mediate oridonin-induced apoptosis. However, when L929 cells were stimulated with FGF-2, and then given access to oridonin, the Ras/Raf/ERK cascade signal pathway was activated and played the protective role against the cell death, instead. We also examined the effect of ERK on the expression of Bcl-2. The result showed that ERK mediated the phosphorylation of Bcl-2 in oridonin-treated L929 cells, resulting in the cell death, and PD98059 inhibited the phosphorylation of Bcl-2. However, in the presence of FGF-2, phosphorylation of Bcl-2 was inhibited, and the expression of Bcl-2 was significantly increased, compared with the oridonin group, and inhibition of ERK down-regulated the expression of Bcl-2. This effect is consistent with the previous report that the ERK signal pathway was implicated in either mediating cell apoptosis or protection from various apoptotic signals though two different mechanisms: the phosphorylation or transcriptional up-regulation of Bcl-2 family member (33, 34). Taken together, all these results demonstrated that in the oridonin-induced apoptosis, the phosphorylation of Bcl-2 was responsible for the cytotoxity of oridonin via activated ERK kinase, and that the increased expression of Bcl-2 is required for FGF-2-mediated rescue in L929 cells by means of the

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