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CELLULAR AND MOLECULAR
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan (Y.-L.H., C.-C.L.); Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan (P.-L.K.); and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada (L.-T.L.)
Received October 3, 2004; accepted December 22, 2004.
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). This pathology is currently controlled by surgery and radiotherapy and is frequently supported by adjuvant chemoor hormonotherapies (Bange et al., 2001). However, breast cancer is highly resistant to chemotherapy, and there is still no effective cure for patients with advanced stages of the disease, especially in cases of hormone-independent cancer (Bange et al., 2001; Chopin et al., 2004). Effective chemopreventive treatment for breast cancer would have an important impact on breast cancer morbidity and mortality. A series of triterpenes are widely distributed in composite plants, and their biological activities have attracted a great deal of attention. Many triterpenoids have shown promising effects when applied as antineoplastic agents (Haridas et al., 2004; Saleem et al., 2004). Asiatic acid, a plant-derived triterpenoid compound, was extracted from the tropical medicinal plant Centella asiatica (Coldren et al., 2003). It has been found to prevent UVA-mediated photoaging, to inhibit 
-amyloid-induced neurotoxicity, and to possess antiulcer and antihepatofibric activities (Jew et al., 2000; Lee et al., 2000; Soo Lee et al., 2003; Dong et al., 2004). It also has been reported to exhibit a cytotoxic effect against Hep G2 cells by Ca2+ release and p53 up-regulation (Lee et al., 2002). However, the anticancer effect and mechanism of asiatic acid in human breast cancer remains unknown.
Eukaryotic cell cycle progression involves the sequential activation of cyclin-dependent kinases, whose activation is dependent upon their association with cyclins. The complex formed by the association of Cdc2 and cyclinB1 plays a major role at entry into mitosis (Sancar et al., 2004). The phosphorylation of Tyr15 of Cdc2 suppresses activity of Cdc2/cyclinB1 kinase complex. Dephosphorylation of Tyr15 of Cdc2 is catalyzed by Cdc25C phosphatases, and this reaction is believed to be the rate-limiting step for entry into mitosis (De Souza et al., 2000). Cell cycle progression is also regulated by the relative balance between the cellular concentrations of cyclin-dependent kinase inhibitors, such as members of the cyclin-dependent kinase-interacting protein/cyclin-dependent kinase inhibitory protein and inhibitor of cyclin-dependent kinase families, and that of cyclin-CDK complexes. The Cip/Kip family, including p21/WAF1 and p27/KIP, bind to cyclin-CDK complexes and prevent kinase activation, subsequently blocking the progression of the cell cycle at the G0/G1 or G2/M phases (Sancar et al., 2004).
The mitogen-activated protein kinases (MAPKs), a family of serine/threonine kinases, are mediators of intracellular signals in response to various stimuli. c-Jun NH2-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) 1/2 are the three main members of three different MAPK pathways that can be activated by growth factors, DNA damage, cytokines, oxidant stresses, UV light, anticancer drugs, and osmotic shock (Johnson and Lapadat, 2002; Olson and Hallahan, 2004). All three MAPK pathways can be differentially activated, and their involvement in apoptosis is highly context and model-dependent. JNK and p38 are activated by cellular stress and both have been associated with apoptosis (Huh et al., 2004; Xiao et al., 2004). However, there have also been reports indicating that the JNK is required for interleukin-3-mediated cell survival and that p38 is associated with the development of chemoresistance by activating nuclear factor-
B (Hendrickx et al., 2003; Yu et al., 2004). In contrast, the activation of the ERK1/2 pathway is generally considered to be a survival signal induced by mitogenic stimuli or growth factors against apoptotic signals (Johnson and Lapadat, 2002; Park et al., 2003; Olson and Hallahan, 2004). However, some of the literature contradicts ERK1/2's essential role in several types of chemotherapeutic or preventive agent-induced apoptosis, such as taxol, resveratrol, and quercetin (Bacus et al., 2001; She et al., 2001; Nguyen et al., 2004). This may be due to the cell type and cell content specificity of apoptosis inducers and their subsequent signaling transduction pathways.
In this study, we determined the cell growth inhibition activity of asiatic acid and examined its effect on cell cycle distribution and apoptosis in the two human breast cancer cell lines, MCF-7 and MDA-MB-231. Furthermore, to establish asiatic acid's anticancer mechanism, we assayed the levels of cell cycle control and apoptosis-related molecules, which are strongly associated with the signal transduction pathway of apoptosis and affect the chemosensitivity of tumor cells to anticancer agents.
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-actin, cyclinB1, cyclinA, Cdc2, Cdc25C, p21/WAF1, Bax, Bak, Bcl-2, phospho-Bcl-2, and Bcl-XL were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibodies to JNK, p38, ERK, phospho-JNK, phospho-p38, phospho-ERK, phospho-Cdc2, and phospho-Cdc25C; cytochrome c; and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 were obtained from Cell Signaling Technology Inc. (Beverly, MA). Cell Culture. Breast cancer cell lines MCF-7 (ATCC HTB-22) and MDA-MD-231(ATCC HTB-26) were obtained from the American Type Cell Culture Collection (Manassas, VA). MCF-7 cells were cultured in Dulbecco's modified Eagle's medium with nonessential amino acids, 0.1 mM sodium pyruvate, 10 µg/ml insulin, and 10% FCS. The MDA-MB-231 cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS and 1% penicillin-streptomycin solution (Invitrogen).
Cell Proliferation Assay. Inhibition of cell proliferation by asiatic acid was measured by sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) assay. Briefly, cells were plated in 96-well culture plates (1 x 104 cells/well). After 24-h incubation, the cells were treated with asiatic acid (0, 2.5, 5, 10, and 20 µM) for 48 h. Fifty microliters of XTT test solution, which was prepared by mixing 5 ml of XTT-labeling reagent with 100 µl of electron coupling reagent, was then added to each well. After 4-h incubation, absorbance was measured on an ELISA reader (Multiskan EX; Labsystem, Helsinki, Finland) at a test wavelength of 492 nm and a reference wavelength of 690 nm. Data were calculated as the percentage of inhibition by the following formula: inhibition % = [100 - (ODt/ODs) x 100]. ODt and ODs indicated the optical density of the test substances and the solvent control, respectively. The concentration of 50% cellular cytotoxicity of cancer cells (IC50) of test substances was calculated based on 48-h absorbance values.
Cell Cycle Analysis. To determine cell cycle distribution analysis, 5 x 105 cells were plated in 60-mm dishes and treated with asiatic acid (0, 5, and 10 µM) for 12 h. After treatment, the cells were collected by trypsinization, fixed in 70% ethanol, washed in phosphate-buffered saline, resuspended in 1 ml of phosphate-buffered saline containing 1 mg/ml RNase and 50 µg/ml propidium iodide, incubated in the dark for 30 min at room temperature, and analyzed by EPICS flow cytometer. The data were analyzed using Multicycle software (Phoenix Flow Systems, San Diego, CA).
Assaying the Levels of p53, p21/WAF1, Fas/APO-1, and Fas Ligand (mFasL and sFasL). p53 pan ELISA, WAF1 ELISA, Fas/APO-1 ELISA, and Fas Ligand ELISA kits were used to detect p53, p21/WAF1, Fas/APO-1 receptor, and FasL. Briefly, cells were treated with 0, 5, and 10 µM asiatic acid for the indicated times. The samples of cell lysate were placed in 96-well (1 x 106/well) microtiter plates coated with monoclonal detective antibodies and were incubated for 1 h (Fas/APO-1), 2 h (p53 or p21/WAF1), or 3 h (Fas ligand) at room temperature. It was necessary to determine the soluble Fas ligand in cell culture supernatant by using Fas Ligand ELISA kit. After removing unbound material by washing with washing buffer (50 mM Tris, 200 mM NaCl, and 0.2% Tween 20), horseradish peroxidase-conjugated streptavidin was added to bind to the antibodies. Horseradish peroxidase catalyzed the conversion of a chromogenic substrate (tetramethylbenzidine) to a colored solution, with color intensity proportional to the amount of protein present in the sample. The absorbance of each well was measured at 450 nm, and concentrations of p53, p21/WAF1, Fas/APO-1, and FasL were determined by interpolating from standard curves obtained with known concentrations of standard proteins.
Assay for Caspase-8 and -9 Activities. The assay is based on the ability of the active enzyme to cleave the chromophore from the enzyme substrate: Ac-IETD-pNA (Ac-Ile-Glu-Thr-Asp-pNA) for caspase-8 and LEHD-pNA (Ac-Leu-Glu-His-Asp-pNA) for caspase-9. Cell lysates were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM HEPES, 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, and 0.1% CHAPS, pH 7.4) for 2 h at 37°C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percentage of change of activity compared with the untreated control.
Immunoprecipitation/Immunoblot and ERK1/2 and p38 MAPK Kinase Activity Assays. Cells were treated with 10 µM asiatic acid in the absence or presence of MAPK inhibitors for specified time intervals. Mitochondrial and cytoplasmic fractions were separated using cytochrome c releasing apoptosis assay kit (BioVision Inc., Mountain View, CA). For immunoblotting, the cells were lysed on ice for 40 min in a solution containing 50 mM Tris, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM Na3VO4, 2 mM EGTA, 12 mM -glycerol phosphate, 10 mM NaF, 16 µg/ml benzamidine hydrochloride, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was centrifuged at 14,000g for 15 min, and the supernatant fraction was collected for immunoblotting. Equivalent amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis (10-12%) and transferred to polyvinylidene difluoride membranes. After blocking for 1 h in 5% nonfat dry milk in Tris-buffered saline, the membrane was incubated with the desired primary antibody for 1 to 16 h. The membrane was then treated with appropriate peroxidase-conjugated secondary antibody, and the immunoreactive proteins were detected using an enhanced chemiluminescence kit (Amersham Biosciences Inc., Piscataway, NJ) according to the manufacturer's instructions.
For association of p21/WAF1 with Cdc2, cell lysates (300 µg) were incubated with 10 µl of anti-Cdc2 for 1 h at 4°C. Immunocomplexes were resolved by 7.5% SDS-polyacrylamide gel electrophoresis. Association of p21/WAF1 with Cdc2 was detected by incubating the blots with anti-p21/WAF1 and anti-Cdc2 antibodies as described above. The ERK1/2 and p38 MAPK activities were determined using kits from Cell Signaling Technology Inc. according to the manufacturer's instructions.
siRNA Knockdown of p38 and ERK Expression. Breast cancer cell monolayers were transfected with SMARTpool p38/SPAK or MAPK1(ERK2) siRNA duplexes or nonspecific control siRNA duplexes (Upstate Biotechnology, Lake Placid, NY) by using LipofectAMINE 2000 (Invitrogen). At these times, immunoblot analysis showed that expression of p38 and ERK1/2 remained low but detectable, and expression of -actin was unaffected by siRNA treatment.
Statistical Analysis. Data were expressed as means ± S.D. Statistical comparisons of the results were made using analysis of variance. Significant differences (p < 0.05) between the means of control and asiatic acid-treated cells were analyzed by Dunnett's test.
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To examine the mechanism responsible for asiatic acid-mediated cell growth inhibition, cell cycle distribution was evaluated using flow cytometric analysis. The results showed that treating cells with asiatic acid caused a significant inhibition of cell cycle progression in both MCF-7 and MDA-MB-231 cells at 12 h (Fig. 1B), resulting in a clear increase of the percentage of cells in the S-G2/M phase compared with the control.
We next assessed the effect of asiatic acid on the induction of apoptosis in MCF-7 and MDA-MB-231 cells by DNA fragmentation assay. The results showed that asiatic acid treatment results in the formation of DNA fragments in both MCF-7 and MDA-MB-231 cells, as determined by agarose gel electrophoresis at 48 h (Fig. 1C). Additionally, a quantitative evaluation was then sought using ELISA to detect histone-associated oligonucleosome DNA fragments. Compared with vehicle-treated cells, 10 µM asiatic acid induced a 4.5- and 7.7-fold increase of oligonucleosome in MCF-7 cells, whereas oligonucleosome only increased 3.5- and 6.2-fold in MDA-MB-231 cells at 24 and 48 h, respectively (Fig. 1, D and E).
Asiatic Acid Increases p21/WAF1 Expression through a p53-Independent Pathway. Because our studies showed that asiatic acid treatment of breast cancer cells results in S-G2/M phase cell cycle arrest, we examined the effect of asiatic acid on cell cycle-regulatory molecules, including p21/WAF1, cyclinB1, cyclinA, Cdc25C, and Cdc2. Previous reports have indicated that MCF-7 cells have a normal tumor suppression gene, p53, whereas in MDA-MB-231 cells the major protein of the p53 gene has mutated and is accompanied by the absence of p53 function (Amellem et al., 1990; Negrini et al., 1994). As shown in Fig. 2A, asiatic acid failed to affect the expression of p53 at any of the examined time points in MCF-7 cells, but it increased the expression of p21/WAF1 in both MCF-7 and MDA-MB-231 cells (Fig. 2, B and C).
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Asiatic acid treatment of the cells resulted in a time-dependent decrease in the protein expression of cyclinB1, cyclinA, and Cdc25C as well as Cdc2 in both MCF-7 and MDA-MB-231 cells (Fig. 2D). In addition, exposure of cells to asiatic acid for 3 h resulted in an increase in the levels of inactive phospho-Cdc2 (Tyr 15) and phospho-Cdc25C (Ser 216). Results from time-dependent studies have indicated that decreasing functional Cdc25C by increasing phosphorylation was followed by an increase in phospho-Cdc2 (Fig. 2D). In addition, the association of p21/WAF1 and Cdc2 increased in a time-dependent manner in asiatic acid-treated MCF-7 and MDA-MB-231 cells, as detected by immunoprecipitation assay (Fig. 2E). We suggest that Cdc2 action was inhibited by a decrease in Cdc25C expression and an increase in the association of p21/WAF1 with Cdc2.
Fas/Fas Ligand Is Not Involved in Asiatic Acid-Mediated Apoptosis. To establish the sequence of events occurring during asiatic acid-induced apoptosis, we measured some of the molecular activity of the death receptor apoptotic pathway, including Fas/APO-1 receptor and its two ligands, mFas ligand and sFas ligand. However, treatment of either of these two cell lines with 5 or 10 µM asiatic acid failed to affect the levels of these proteins at any of the examined time points, including Fas/APO-1, mFas ligand, and sFas ligand (data not shown). In addition, asiatic acid also failed to affect the activation of caspase-8 in both MCF-7 and MDA-MB-231 cells (data not shown).
Asiatic Acid Induces the Execution of Apoptosis through Activation of the Mitochondrial Pathway. To investigate the mitochondrial apoptotic events involved in asiatic acid-induced apoptosis, we first analyzed the changes in the levels of proapoptotic protein Bax and antiapoptotic proteins Bcl-2 and Bcl-XL. Western blot analysis showed that treatment of MCF-7 and MDA-MB-231 cells with asiatic acid increased Bax protein levels (Fig. 3A). In contrast, asiatic acid decreased Bcl-2 and Bcl-XL levels, which led to an increase in the proapoptotic/antiapoptotic Bcl-2 ratio (Fig. 3A). In addition, asiatic acid also increased phosphorylation of Bcl-2 in both MCF-7 and MDA-MB-231 cells. However, asiatic acid failed to affect the Bak levels in either MCF-7 or MDA-MB-231 cells at any of the examined points in time.
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Cytosolic extracts were prepared under conditions to preserve the mitochondria, and cytosolic cytochrome c protein levels were measured by immunoblotting analysis. Figure 3B shows that the cytosolic fraction from untreated MCF-7 and MDA-MB-231 cells contained no detectable amounts of cytochrome c, whereas it did become detectable after 12 h of 10 µM asiatic acid treatment in both MCF-7 and MDA-MB-231 cells.
Hallmarks of the apoptotic process include the activation of cysteine proteases, which represent both initiators and executors of cell death. Upstream caspase-9 activities increased significantly as shown by the observation that treatment with asiatic acid increased caspase-9 activity in both MCF-7 and MDA-MB-231 cells. This is consistent with the release of cytochrome c into the cytosol (Fig. 3, C and D). Furthermore, when cells were pretreated with the specific caspase-9 inhibitor LEHD-CHO before asiatic acid treatment, the apoptosis induction effect of asiatic acid decreased in both MCF-7 and MDA-MB-231 cells (Fig. 3E).
Asiatic Acid Induces the Activation of ERK and p38 but Not JNK Pathway. We assessed the level of MAPKs (JNK, p38, and ERK1/2) in asiatic acid-treated MCF-7 and MDA-MB-231 cells. As shown in Fig. 4A, we did not observe the levels and activation (phosphorylation) of JNK in the two breast cancer cell lines exposed to 10 µM asiatic acid for 1, 3, 6, and 12 h. Instead, we found that exposure of either line of breast cancer cells to 10 µM asiatic acid resulted in a rapid and sustained activation of p38 and ERK1/2 (Fig. 4A). Activation (phosphorylation) of p38 and ERK1/2 was evident as early as 1 h after asiatic acid treatment and persisted for the duration of the experiment. On the other hand, the expression of p38 and ERK1/2 (unphosphorylated form) was not altered by asiatic acid treatment. Asiatic acid-mediated activation of p38 and ERK1/2 was additionally confirmed by determining phosphorylation of one of its substrates (ATF-2 and Elk-1 for p38 and ERK1/2, respectively). As shown in Fig. 4B, in comparison with the control, the Ser383 phosphorylation of Elk-1 increased after a 1-h exposure of MCF-7 and MDA-MB-231 cells to 10 µM asiatic acid. Phosphorylation of Elk-1 increased relative to the control at all four points in time (Fig. 4B). Similarly, phosphorylation of ATF-2 at Thr 71 increased in both asiatic acid-treated MCF-7 and MDA-MB-231 cells at 1, 3, 6, and 12 h, in contrast to the control.
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Decrease of Asiatic Acid-Induced Cell Cycle Arrest and Apoptosis by ERK1/2 and p38 Chemical Inhibitors. To experimentally verify the possible role of p38 and ERK1/2 in asiatic acid-induced apoptosis, MCF-7 and MDA-MB-231 cells were pretreated for 1 h with specific inhibitor for MEK1 (an upstream kinase in ERK1/2 signaling pathway), U0126, or a potent specific inhibitor for p38, SB203580. Subsequently, the inhibitor-treated cells were exposed to asiatic acid, and then the cell cycle distribution and apoptosis were determined. As shown in Fig. 5A, the asiatic acid-mediated p38 and ERK activation was effectively inhibited by 20 µM SB203580 and 20 µM U0126, respectively. Flow cytometric analysis of MCF-7 and MDA-MB-231 cells exposed to asiatic acid for 12 h showed the effect of 20 µM SB203580 or 20 µM U0126 on S-G2/M progression. Pretreatment of SB203580 for 1 h reduced the S-G2/M populations, both of which were accumulated by the asiatic acid treatment (Fig. 5B). Despite its ability to reduce the sub-G0 population, U0126 had no significant effect on the S-G2/M population (Fig. 5B).
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Figure 5C shows the effect of MEK and p38 inhibitor on asiatic acid-induced apoptosis in both MCF-7 and MDA-MB-231 cells. In comparison with control cells, the percentage of apoptotic cells was significantly higher in cultures exposed to 10 µM asiatic acid, but this effect was blocked by MEK inhibitor U0126 at 24 and 48 h (Fig. 5C). In contrast, cotreatment of cells with the p38 inhibitor SB203580 and asiatic acid resulted in a delay in the initiation of apoptosis. DNA fragmentation was not evident at 24 h but started to become significant only after 48 h of treatment (Fig. 5D).
The Role of p38 in Asiatic Acid-Mediated S-G2/M Arrest. Previous studies have indicated that regulation of Cdc25C phosphorylation and accumulation of the inactive phosphorylated Cdc2 by p38 is a critical event for initiating the G2/M checkpoint. As shown in Fig. 6A, pretreatment of cells with SB203580 decreased the levels of phospho-Cdc2 and blocked the degradation of Cdc2 observed after asiatic acid exposure. Similarly, SB203580 also abolished Cdc 25C degradation by increasing phospho-Cdc25 in both asiatic acid-treated MCF-7 and MDA-MB-231 cells. In contrast, U0126 failed to affect the asiatic acid-mediated changes in Cdc2 and Cdc25C expression and their phosphorylation in either MCF-7 or MDA-MB-231 cells. These data strongly suggest that the p38 MAPK pathway might plays an important role in asiatic acid-induced S-G2/M arrest.
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The Role of p38 and ERK1/2 in Mitochondrial Apoptotic Pathways in Asiatic Acid-Treated Breast Cancer Cells. We further investigated the mechanism that accounts for the actions of p38 and ERK1/2 in asiatic acidinduced apoptosis in breast cancer cells. We tested the involvement of the mitochondrial apoptotic pathways by examining the effect of p38 and ERK inhibitors on Bax, Bcl-2, and phospho-Bcl-2 expression, and caspase-9 activation. As shown in Fig. 7A, cotreatment of MCF-7 and MDA-MB-231 cells with asiatic acid and U0126 completely blocked asiatic acid-mediated Bax up-regulation, Bcl-2 down-regulation and phosphorylation. In contrast, these effects were not affected when cells were cotreated with asiatic acid and SB203580. Furthermore, the activation of caspase-9 induced by asiatic acid was completely attenuated by pretreatment with U0126 (Fig. 7B). Similarly, cotreatment of cells with asiatic acid and SB203580 failed to decrease the activity of caspase-9. These results suggest that activation of ERK1/2 plays an upstream role in asiatic acid-mediated mitochondrial apoptotic pathways.
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Genetic Inhibition of p38 and ERK Blocks Asiatic Acid-Mediated Cell Cycle Arrest and Apoptosis. Although SB203580 and U0126 are relatively selective inhibitors of p38 and ERK1/2, reports have suggested these compounds also interfere with G2 checkpoint-related proteins (Lali et al., 2000). We therefore also used genetic inhibition to specifically inhibit p38 and ERK to assess the consequences of p38 and ERK inhibition on asiatic acid-mediated cell cycle arrest and apoptosis. To do so, MCF-7 and MDA-MB-231 cells were transfected with a pool of siRNAs targeting p38 or ERK2, after which the cells were exposed to 10 µM asiatic acid for a specific time. As shown in Fig. 8, A and B, in comparison with oligonucleotide transfected control cells, transfection of cells with p38 and ERK2 siRNA reduced basal amounts of p38 and ERK1/2 and blocked the asiatic acid-induced activation of p38 and ERK1/2. Selective genetic inhibition of p38 not only blocked asiatic acid-induced S-G2/M phase arrest but also abrogated phosphorylation of Cdc2 and Cdc25C as well as the degradation of two proteins (Fig. 8, C and D). On other hand, specific knockdown ERK1/2 expression by ERK2 siRNA also inhibited asiatic acid-mediated apoptosis (Fig. 8E). In addition, asiatic acid-induced phosphorylation of Bcl-2, up-regulation of Bax, and activation of caspase-9 were significantly prevented by specific siRNA inhibition of ERK1/2 in both MCF-7 and MDA-MB-231 cells (Fig. 8, F and G). These consequences of p38 and ERK1/2 inhibition by genetic inhibition on asiatic acid-mediated S-G2/M arrest and apoptosis induction coincide with chemical inhibitors, indicating that p38 and ERK1/2 may play important roles in molecular regulation.
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). In this study, we first show that asiatic acid inhibits the growth of two breast cancer cell lines, MCF-7 and MDA-MB-231. Both MCF-7 and MDA-MB-231 cells treated with asiatic acid accumulated in the S-G2/M phase of the cell cycle and underwent apoptosis in a dose- and time-dependent manner. In our study, we have found that asiatic acid decreases the expression of cyclinB1, cyclinA, Cdc25C, and Cdc2, whereas it increases the amount of p21/WAF1 and phosphorylation of Cdc2, as well as phospho-Cdc25C. The association of p21/WAF1 and Cdc2 also increased in asiatic acid-treated MCF-7 and MDA-MB-231 cells. Therefore, we suggest that asiatic acid may prove to be a valuable tool for inhibition of Cdc2/cyclinB1 and Cdc2/cyclinA complex in breast cancers for the following reasons: 1) the down-regulation of asiatic acid on cyclinB1 and cyclinA expression; 2) the induction of p21/WAF1 by asiatic acid in a p53-independent manner, which may subsequently inhibit the function of Cdc2 by forming Cdc2/p21/WAF complex; and 3) the increase of phosphoCdc25C followed by an increase in inactivated phospho-Cdc2, suggesting that increased phospho-Cdc25C levels may also decrease functioning phosphatase for dephosphorylating and activating Cdc2.
Two major distinct apoptotic pathways have been described for mammalian cells. One involves caspase-8, which is recruited by the adapter molecule Fas/APO-1-associated death domain protein to death receptor upon Fas ligand binding (Hengartner, 2000). We did not observe any alteration of either Fas/APO-1 or Fas ligand (mFas ligand and sFas ligand) expression or caspase-8 activation in asiatic acid-treated MCF-7 and MDA-MB-231 cells. On the other hand, asiatic acid treatment resulted in a significant increase of Bax expression and a decrease in the amount of Bcl-2 and Bcl-XL, suggesting that changes in the ratio of proapoptotic and antiapoptotic Bcl-2 family proteins might contribute to the apoptosis-promotion activity of asiatic acid. In addition, elevation of phospho-Bcl-2 by asiatic acid treatment further helps to reduce its ability to bind with Bax, leading to an enhanced susceptibility of the cells to apoptosis (Hu et al., 1998). These regulatory effects of asiatic acid on the Bcl-2 family are correlated with the release of cytochrome c from the mitochondria into the cytoplasm and the activation of caspase-9. The importance of this pathway was further confirmed by the protection from programmed cell death that is conferred by caspase-9 inhibition.
Recent studies have shown that MAPKs signaling pathways regulate the eukaryotic cell cycle. p38 kinase has been demonstrated as essential for sustained G2 arrest induced by 
irradiation, decitabine, vanadate, and genistein (Wang et al., 2000; Frey and Singletary, 2003; Lavelle et al., 2003; Zhang et al., 2003). Reduced activity of Cdc25C and a subsequent increase in Cdc2 phosphorylation lead to cell cycle arrest at the G2/M phase (Bulavin et al., 2001). In this study, we found that activation of p38 was involved in the accumulation of inactive phospho-Cdc2, which may be due to the decrease of Cdc25 activation by phosphorylation, leading to subsequent G2 arrest. These effects, however, were abolished in MCF-7 and MDA-MB-231 cells that were cotreated with asiatic acid and SB203580. In addition, selectively known down p38 expression by p38 siRNA-based inhibition approach also decreased the effects of asiatic acid on the regulation of Cdc2 and Cdc25C and cell cycle arrest. Moreover, p38 inhibition by SB203580 also delayed apoptosis until 48 h, suggesting that the S-G2/M arrested cells were sensitive to apoptosis induced by asiatic acid treatment
In general, the JNKs and p38 kinase pathways are associated with increased apoptosis, whereas the ERK1/2 pathway is shown to suppress apoptosis (Johnson and Lapadat, 2002; Olson and Hallahan, 2004; Sah et al., 2004). Surprisingly, our results show that the activation of ERK1/2 is involved in asiatic acid-mediated apoptosis. This difference may be due to dissimilar species of cell types or to different extracellular stimuli. Previous studies have documented the involvement of ERK1/2 in induction of apoptosis by RRR-
-tocopheryl succinate, quercetin, resveratrol, and taxol (Bacus et al., 2001; She et al., 2001; Yu et al., 2001; Nguyen et al., 2004). Furthermore, several downstream targets of ERK1/2, including Fas/APO-1 receptor and p53 have been discovered to be involved in ERK1/2-triggered apoptosis (Goillot et al., 1997; Ko et al., 2004). However, these two proapoptotic factors were not the downstream target of ERK1/2 in our study, because their expression did not increase expression when ERK1/2 was activated by asiatic acid treatment. In contrast, ERK1/2 activation is involved in the events of asiatic acid-mediated mitochondrial apoptotic pathway, which is completely inhibited by using chemical inhibitor U0126 and ERK siRNA-based inhibition, including Bax augmentation, Bcl-2 reduction, Bcl-2 phosphorylation, and caspase-9 activation. However, since activating ERK1/2 to commit suicide is not a universal feature of mammalian cells, the actual mechanism by which asiatic acid converts the outcome of ERK1/2 signaling from mitogenesis to apoptosis requires further investigation.
In conclusion, the present study demonstrates that 1) the breast cancer cell lines MCF-7 and MDA-MB-231 are highly sensitive to growth inhibition by asiatic acid; 2) reduced survival of either of breast cancer cell lines after exposure to asiatic acid is associated with S-G2/M phase cell cycle arrest and apoptosis induction; 3) asiatic acid can inhibit cell cycle progression at the S-G2/M phase by increasing p21/Cdc2 interaction and decreasing the expression of Cdc2, Cdc25C, cyclinB1, and cyclinA; and 4) asiatic acid-induced cell growth inhibition in the MCF-7 and MDA-MB-231 cell lines is mediated by activation of p38 and ERK1/2 kinases, but not JNKs, p53 or Fas/Fas, ligand pathway. Finally, 5) it has also been demonstrated that the p38 pathway may operate in cell cycle arrest induced by asiatic acid and that the ERK1/2 cascade of events plays a role in the apoptosis, but not in the cell cycle regulation, of these cells. These data provide a basic mechanism for the chemopreventive properties of asiatic acid in breast cancer cells. Future in vivo studies using animal models and human patients would will ascertain whether this proapoptotic effect of asiatic acid might contribute its overall chemopreventive effects in the fight against breast cancer and possibly have future therapeutic applications.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; MEK, mitogen-activated protein kinase kinase; XTT, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate; FasL, Fas ligand; mFasL, membrane-bound Fas ligand; sFasL, soluble Fas ligand; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; siRNA, small interfering RNA.
Address correspondence to: Professor Chun-Ching Lin, Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Rd., Kaohsiung 807, Taiwan, Republic of China. E-mail: aalin{at}ms24.hinet.net
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