Magnolol Suppresses Proliferation of Cultured Human
Colon and Liver Cancer Cells by Inhibiting DNA
Synthesis and Activating Apoptosis
Shyr-Yi Lin,1,2Jean-Dean Liu,2Hui-Chiu Chang,3Shauh-Der Yeh,1Chien-Huang Lin,4 and Wen-Sen Lee1,5*
1Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
2Department of Internal Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan 3Department of Physiology, Kaohsiung Medical University, Kaohsiung, Taiwan
4Graduate Institute of Biomedical Technology, Taipei Medical University, Taipei, Taiwan 5Department of Physiology, School of Medicine, Taipei Medical University, Taipei, Taiwan
Abstract Magnolol, a hydroxylated biphenyl compound isolated from the Chinese herb Hou p'u of Magnolia officinalis, has been reported to have anti-cancer activity. In the present study, magnolol at very low concentrations of 3±10 mM inhibited DNA synthesis and decreased cell number in cultured human cancer cells (COLO-205 and Hep-G2) in a dose-dependent manner, but not in human untransformed cells such as keratinocytes, ®broblasts, and human umbilical vein endothelial cells (HUVEC). Magnolol was not cytotoxic at these concentrations and this indicates that it may have an inhibitory effect on cell proliferation in the subcultured cancer cell lines. [3H]thymidine incorporation and
¯ow cytometry analyses revealed that magnolol treatment decreased DNA synthesis and arrested the cells at the G0/G1 phase of the cell cycle. Moreover, the magnolol-induced cell cycle arrest occurred when the cyclin-CDK system was inhibited, just as p21 protein expression was augmented. When magnolol concentration was increased to 100 mM, apoptosis was observed in COLO-205 and Hep-G2 cells, but not in cultured human ®broblasts and HUVEC. COLO-205 cells implanted subcutaneously in nude mice formed solid tumors; subsequent daily i.p. injections of magnolol led to profound regression of these tumors of upto 85%. In these tumors, an increase in the expression of p21 protein level and the occurrence of apoptosis were observed. These ®ndings demonstrate for the ®rst time that magnolol can inhibit the proliferation of tumor cells in vitro and in vivo. J. Cell. Biochem. 84: 532±544, 2002. ß2001 Wiley-Liss, Inc.
Key words: p21; apoptosis; cell cycle; G0/G1 arrest
The occurrence of gastrointestinal cancers has increased strikingly during the last decade. For instance, colorectal cancer is the second leading cause of cancer mortality in Western societies and one of the most common malig-nancies worldwide [Chinery et al., 1997]. Liver cancer is one of the most prevalent and deadly cancers in the world, causing almost one million deaths annually [Akriviadis et al., 1998]. Al-though intensive research during the past few
years has led to considerable improvement in the treatment and diagnosis at an early stage, for the patient at this time, the prognosis is still not good. Therefore, experimental and clinical investigators continue to search for new ther-apeutic strategies. One approach, as pursued in this study, seeks to identify medicinal agents capable of retarding the cell cycle and/or activating the cellular apoptotic response in the cancerous cells.
Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, is a widely used Chinese herbal medicine known as Hou p'u. Traditionally, it has been employed to treat such diverse ailments as headache, fever, etc. [Fujita et al., 1973]. Studies using puri®ed magnolol have shown that it can function to scavenge hydroxyl radicals [Haraguchi et al.,
ß2001 Wiley-Liss, Inc. DOI 10.1002/jcb.10059
Grant sponsor: Foundations of Jin Lung Yen and Sagittar-ius Life Science Corp.
*Correspondence to: Wen-Sen Lee, Graduate Institute of Medical Sciences and Department of Physiology, School of Medicine, Taipei Medical University, Taipei 110, Taiwan. E-mail: [email protected]
1997], inhibit neutrophil adhesion [Shen et al., 1998], suppress the in¯ammatory response [Wang et al., 1992], inhibit platelet aggregation [Ko et al., 1989], and may also exhibit certain antimicrobial actions [Chang et al., 1998]. Of particular interest for the present study is the magnolol-induced suppression of a cutaneous papilloma, which was induced in mice treated with dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) [Konoshima et al., 1991]. Our study was done to investigate any anti-cancer effects of magnolol on human hepatoma and colon carcinoma cells.
MATERIALS AND METHODS Cell Culture
Four human malignant cell lines (COLO-205, HT-29, Hep-G2, and Hep-3B) and three human primary cells (keratinocytes, ®broblasts, and HUVEC) were used in this study. COLO-205 (CCL-222; American Type Culture Collection) originated from a poorly differentiated human colon adenocarcinoma. Hep-G2 (HB-8065; American Type Culture Collection) was derived from a human hepatoma. HT-29 (HTB-38; American Type Culture Collection) originated from a human colon adenocarcinoma. Hep-3B (HB-8064; American Type Culture Collection) originated from a hepatocellular carcinoma. HUVEC was harvested from the human umbi-lical vein by enzymatic dissociation as pre-viously described [Jaffe et al., 1973]. Human gingival ®broblasts were harvested by enzyma-tic dissociation. The cells were grown in RPMI 1640 (for COLO-205 and HT-29), Eagle's mini-mal essential medium, MEM, (for Hep-G2 and human ®broblasts), or M199 (for HUVEC) sup-plemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 mg/ml), and 0.3 mg/ml glutamine in a humidi®ed incubator (378C, 5% CO2). Human keratino-cytes were grown in keratinocyte serum free medium (KSFM) supplemented with bovine pituitary extract and epidermal growth factor (EGF). Magnolol (Pharmaceutical Industry, Technology and Development Center, Taiwan) was added at the indicated doses in 0.1% DMSO. For control specimens, the same volume of the 0.1% DMSO without magnolol was added.
[3H]Thymidine Incorporation
As previously described [Jain et al., 1996; Lee et al., 1997], COLO-205 or Hep-G2 at a density
of 1 104 cells/cm3 were applied to 24-well plates in growth medium (RPMI 1640 or MEM plus 10% FCS). After the cells had grown to 70± 80% con¯uence, they were rendered quiescent by incubation for 24 h in RPMI 1640 containing 0.04% FCS. Magnolol in 0.1% DMSO or 0.1% DMSO without magnolol (control) in RPMI supplemented with 2% FCS for COLO-205 or MEM with 10% FCS for Hep-G2 was added to the cells at various concentrations and the cultures were allowed to incubate for 21 or 6 h, respectively. During the last 3 h of the incubation with or without magnolol, [3 H]thy-midine was added at 1 mCi/ml (1mCi 37 kBq). Incorporated [3H]thymidine was extracted in 0.2 N NaOH and measured in a liquid scintilla-tion counter.
Cell Counting
As a measurement of cell proliferation, the cells at a density of 1 104cells/cm3were seeded onto 24-well plates and grown in RPMI 1640 (for COLO-205) supplemented with 2% FCS or MEM (for Hep-G2) supplemented with 10% FCS. Media with and without magnolol were changed daily until cell counting. At various times of incubation, cultures were treated with trypsin-EDTA and the released cells were counted in a Coulter apparatus.
Analysis of DNA Fragmentation
Apoptotic cell death may be documented by various methods, including identi®cation of internucleosomal DNA fragmentation by lad-dering on DNA gel electrophoresis. Analysis of DNA fragmentation was performed as pre-viously described [Ho et al., 1996]. Brie¯y, the magnolol and control-treated cells were seeded on 100-mm dishes. The DNA was extracted twice with equal volumes of phenol and once with chloroform-isoamyl alcohol (24:1 v:v), then precipitated with 0.1 volume of sodium acetate, pH 4.8, and 2.5 volumes of ethanol at ÿ208C overnight, and ®nally centrifuged at 13,000g for 1 h. Genomic DNA was quantitated, and equal amounts of DNA sample in each lane were electrophoresed in a 2% agarose gel. The DNA was visualized by ethidium bromide staining. Genomic DNA isolated from 10 mM terbina®ne-treated HT-29 cells showing DNA fragmenta-tion was included in this experiment to serve as a positive control.
Trypan Blue Exclusion Assay
Cell viability was estimated by Trypan blue exclusion assay as previously described [Ho et al., 1997].
Flow Cytometry
As previously described [Lai et al., 1996], the cells were seeded onto 100-mm dishes and grown in RPMI 1640 supplemented with 10% FCS. After the cells had grown to subcon¯uence, they were rendered quiescent and challenged with 2% FCS. Then, after release using trypsin-EDTA, they were harvested at various times, washed twice with PBS/0.1% dextrose, and ®xed in 70% ethanol at 48C. Nuclear DNA was stained with a reagent containing propidium iodine (50 mg/ml) and DNase-free RNase (2 U/ ml) and measured using a ¯uorescence-acti-vated cell sorter (FACS). The proportion of nuclei in each phase of the cell cycle was determined using established CellFIT (Becton Dickinson, San Jose, CA) DNA analysis soft-ware.
Protein Extraction and Western Blot Analysis The frozen tumor was pulverized in liquid N2, and then mixed with lysis buffer (Tris-HCl 0.5M, pH 6.8, SDS 0.4%). For cell cultures, the cells were seeded onto 150-mm dishes. After the cells had grown to subcon¯uence, they were rendered quiescent. The cells were released from quiescence with culture medium supple-mented with 2% FCS (for COLO-205) or 10% FCS (for Hep-G2). Magnolol in 0.1% DMSO or 0.1% DMSO without magnolol was added to the cells at various concentrations and the mixture was allowed to incubate for 18 h. The cells were washed with phosphate buffered saline (PBS) and then lysed in lysis buffer (Tris HCl 0.5 M, pH 6.8; SDS 0.4%). Western blot analysis was performed as previously described [Lee et al., 1998]. The protein samples (50 mg/ lane) were loaded onto 10% sodium dod-ecyl sulfate±polyacrylamide gel electrophoresis (SDS±PAGE). The gel electrophoresis was performed at constant 150 V for 3±4 h at 16± 188C. After protein separation, each gel was transferred onto an Immobilon-P membrane. Immunodetection was carried out by probing with proper dilutions of speci®c antibodies at room temperature for 2 h. Anti-p21, anti-p27, anti-cyclin D1 and D3, anti-CDK2 and CDK4, and anti-PCNA monoclonal antibodies
(Trans-duction, San Diego, CA) and anti-G3PDH monoclonal antibody (Biogenesis, Kingston, NH) were used at a concentration of 1:1,000 dilution. Anti-cyclin A and E polyclonal anti-bodies (Transduction, San Diego, CA) were used at a concentration of 1:250 dilution. The secondary antibodies, alkaline phosphatase-coupled anti-mouse or anti-rabbit antibody (Jackson, Westgrove PA), was incubated at room temperature for 1 h at a concentration of 1:5,000 or 1:1,000 dilution, respectively. The speci®c protein complexes were identi®ed using NBT/BCIP (nitro-blue-tetrazolium chloride/ bromo-chloro-3-indolyl-phosphate)(Kirkegaard Perry Laboratory, Gaithersburg, MD). In each experiment, membranes were also probed with anti-G3PDH antibody to correct for differences in protein loading.
Immunoprecipitation
Immunoprecipitation was performed as pre-viously described [Ho et al., 1996]. Brie¯y, CDK2 was immunoprecipitated from 200 mg of protein by using anti-CDK2 antibody (2 mg) and protein A agarose beads (20 ml). The precipitates were washed ®ve times with lysis buffer and once with PBS. The pellet was then resuspend in sample buffer (50 mM Tris, pH 6.8; 100 mM Bromophenol blue, and 10% glycerol) and incu-bated at 908C for 10 min before electrophoresis to release the proteins from the beads.
CDK2 Kinase Assay
As previously described [Wu et al., 1996], CDK2 immunoprecipitates from magnolol-treated and control COLO-205 cells were washed three times with lysis buffer and once with kinase assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, and 1 mM DTT). Phosphor-ylation of histone H1 was measured by incubat-ing the beads with 40 ml of ``hot'' kinase solution [0.25 ml (2.5 mg) histone H1, 0.5 ml [g-32P] ATP, 0.5 ml 0.1 mM ATP, and 38.75 ml kinase buffer] at 378C for 30 min. The reaction was stopped by boiling the sample in SDS sample buffer for 5 min, and the products were analyzed by 12% SDS±PAGE. The gel was dried and visualized by autoradiography.
Antisense Oligonucleotide
The oligonucleotides were designed as pre-viously described (Evdokiou et al., 1999). The antisense oligonucleotide (p21AS) was based on the p21 coding sequence, which is
complementary to the region of the initiation codon (50-TCCCCAGCCGGTTCTGACAT-30)
and the scramble p21 (p21S) as control (50
-ACCTGTGCTCCGACACGTCT-30). These
oli-gonucleotides were purchased from Sigma-Genosys, Inc. Phosphorothioate oligodeoxynu-cleotides were added to the cells at a ®nal concentration of 2 nM at 1 h before the cell was challenged with 2% FCS.
Measurement of Cytosolic-Free Ca2 Concentrations
As previously described [Grynkiewicz et al., 1985], the cells were seeded on coverslips and grown in RPMI 1640 medium supplemented with 10% FCS. After the cells had grown to 60± 70% con¯uence, the medium was changed to RPMI supplemented with 1% FCS for 24 h. The medium was then replaced with a fresh RPMI supplemented with 1% FCS containing 5 mM fura-2/AM (Molecular Probes, Eugene, Oregon), and then incubated for 45 min at 378C. After washing with Phocal buffer (HEPES 10 mM; NaCl 125 mM; KCl 5 mM; glucose 10 mM; MgCl2 2 mM; NaH2PO40.5 mM; NaHCO35 mM; CaCl2 1.8 mM) for three times, the coverslip was then put into a quartz cuvette containing 1.8 ml of Phocal buffer with a magnetic stir bar. Fluor-escence activity was monitored with a ¯uo-rescence spectrophotometer (Hitachi F-4500, Tokyo, Japan) at 510 nm with excitation at 340 and 380 nm. [Ca2]
i was calibrated from the ¯uorescence intensity using the following equation: [Ca2]
i Kd Q [(RÿRmin)]/(RmaxÿR)], where R represents the ¯uorescence intensity ratio Fl1/Fl2, in which l1 (340 nm) and l2 (380 nm) are the ¯uorescence detection wave-lengths for the ion-bound and ion-free indicator, respectively. The values of Fmax, Fmin, Rmax, and Rmin were obtained at the end of each experi-ment by the sequential addition of 10 mM ionomycin (BIOMOL Research Laboratories, Plymouth Meeting, PA) and 50 mM ethylene glycol-bis(b-aminoethyl ether) N,N,N0,N0
-tetra-acetic acid (EGTA). Q was the ratio of Fmin to Fmaxat l2 (380 nm). The dissociation constant (Kd) was taken as 240 nM.
Tumor Growth Assay
The cells were implanted by injection of a tumor suspension (3 106cells in 0.1 ml RPMI 1640) subcutaneously in the ¯anks of male nude mice (BALB/c-nu, National Animal Center, Taiwan). Each mouse was implanted with one
bolus of tumor cells on day 7. Dosing with magnolol was begun 7 days after implantation (day 0), when the tumors reached 70 mm3in volume. Either the vehicle or magnolol at a dose of 5 mg/50 g, which had been used in various in vivo studies [Watanabe et al., 1983; Hsieh et al., 1998], were given i.p. 5 times/week for 2 weeks. Magnolol was formulated by dissolving it in 5.5% Cremophore RH-40 (BASF, Germany). Tumor volume in mm3 was calculated by the formula: volume 1/2 W2 L, where W width and L length of the tumor [Osborne et al., 1987]. Mice were weighed and sacri®ced at day 14, and their general health was monitored during the assay.
TUNEL Staining
TdT-mediated dUTP-biotin nick end labeling was used to detect DNA breaks in apoptotic cells in situ [Tsai et al., 1996]. The tissue sections were rinsed three times with phosphate-buf-fered saline, incubated in avidin (25 mg/ml in PBS and 0.4% Triton-X 100) for 30 min at room temperature to block endogenous biotin, rinsed three times with PBS, and then incubated in 3% H2O2 at room temperature for 10 min. After another three rinses with PBS, the cells were rinsed with TdT buffer and incubated in 50 ml of TdT buffer (10 U of TdT and 0.5 nmol of biotinylated dUTP) at 378C for 60 min. Biotiny-lated dUTP incorporated into DNA breaks in the nuclei was detected by an avidin-biotin complex method (DAB/nickel chromogen).
Statistics
All data were expressed as the mean value SEM. Comparisons were subjected to ANOVA followed by Fisher's least signi®cant difference test. Signi®cance was accepted at P < 0.05.
RESULTS
Inhibition of Cell Proliferation in Magnolol-Treated Cells
In the experiments of Figure 1a,b, COLO-205 and Hep-G2 cells were cultured for 5 days with or without magnolol (3ÿ10 mM), and then the cells were harvested and counted. These data show that minute concentrations of magnolol reduced cell counts by 35±50% in both colon and liver cancer cells. The data of Figure 1c show that treatment with 10 mM magnolol for 6 days had no effect on cell growth in culture of un-transformed human keratinocytes, ®broblasts,
and HUVEC. To con®rm further that the results of our studies of cell growth inhibition in COLO-205 and Hep-G2 were not due to cell death caused by magnolol treatment, we conducted viability assays by treating the cells with magnolol for 5 days at the maximal dose (10 mM) used in the studies of cell growth inhibition. Trypan blue exclusion assays indi-cated that there was no signi®cant difference in cell viability between vehicle-treated and magnolol-treated cells (Fig. 1d). As illustr-ated in Figure 1e,f, this inhibitory effect of magnolol on growth was also observed in other transformed cultured cells, such as hepa-toma (Hep-3B) and colon cancer (HT-29) cell lines.
Arrest of Cell Cycle in G0/G1
In order to examine further the actions of magnolol on the cell cycle, the cells were switched to media with 0.04% FCS to render them quiescent and to synchronize their cell cycle activities. Then they were returned to
media with 2% FCS and, at various times there-after, they were treated with [3H]thymidine. Figure 2a shows a reduction of the thymidine incorporation into COLO-205 during the S-phase of the cell cycle. Figure 2b shows that magnolol inhibited [3H]thymidine incorpora-tion into COLO-205 cells in a dose-dependent manner to as low as 50% of control values, when magnolol concentration was only 10 mM. Similar inhibition was also observed in Hep-G2 (data not shown). Figure 2c shows a representative ¯uorescence-activated cell sorter (FACS) ana-lysis of DNA content after 18 h release from quiescence at the time of the thymidine incor-poration peak by incubation in culture media supplemented with 2% FCS and DMSO or magnolol. The data reveal that magnolol in-duced a signi®cant accumulation of cells in the G1-phase of the cell cycle, suggesting that the observed growth inhibitory effect of magnolol was due to an arrest of DNA replication, thereby, inhibiting further progress in the cell cycle.
Fig. 1. Effect of magnolol on cell number in human malignant and normal cells. Dose-dependent inhibition of cell growth was observed in COLO-205 (a) and Hep-G2 (b) cells treated with magnolol. Media with and without magnolol were changed daily until cell counting. Cells were allowed to grow for 5 days after DMSO or magnolol treatment. c: Treatment with magnolol at 10 mM concentration for 6 days does not affect growth of untransformed human keratinocytes, ®broblasts, and HUVEC. d: Treatment of COLO-205 and Hep-G2 cells with 10 mM
magnolol for 5 days does not cause cell death. This inhibitory effect of magnolol on growth was also observed in HT-29 (e) and Hep-3B (f). Six (COLO-205, keratinocytes, and ®broblasts) or three (HUVEC, Hep-G2, HT-29 and Hep-3B) samples were analyzed in each experiment, and values represent the mean SEM. Comparisons were subjected to ANOVA followed by Fisher's least signi®cant difference test. Signi®cance was accepted at P < 0.05. *Magnolol-treated group different from control group.
Alterations in Cell Cycle Activity
Observation of intracellular events asso-ciated with the progression of cell cycle activity have suggested that coordinated successive activation of certain CDKs occurs late in the G1-phase and is instrumental in the transition from the G1- to the S-phase [Hunter and Pines, 1994; Morgan, 1995]. This CDK activation is in turn modulated by association with a number of regulatory subunits called cyclins, and with a group of CDK-inhibitory proteins designated CKIs [Sherr and Roberts, 1995]. Among these CKIs are two known as p21 and p27. Cyclins
have been identi®ed as cyclins A, D1, D3, and E. The most common cyclin-dependent-kinases are designated CDK2 and CDK4. In our study of this complex of regulatory factors, we have extracted proteins from COLO-205 and Hep-G2 human cancer cells in culture. The extracted
Fig. 2. Effect of magnolol on cell cycle in human malignant cells. a: [3H]Thymidine incorporation after cells were released from quiescence by incubation in culture media supplemented with 2% FCS and 0.1% DMSO (open dot) or 10 mM magnolol (solid dot). b: Dose-dependent inhibition of [3H]thymidine incorporation in COLO-205 by magnolol. Cultures were incubated with magnolol for 21 h, and [3H]thymidine was added only for the last 3 h of this incubation. c: FACS analysis of DNA content after 18 h release from quiescence by incubation in culture media supplemented with 2% FCS and 0.1% DMSO (C18) or 50 mM magnolol in 0.1% DMSO (M18). Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle determined using established CellFIT DNA analysis software is shown in the right upper corner.
Fig. 3. Effect of magnolol on p21 protein levels, p21-CDK2 association, and CDK2 kinase activity. a: The time course of p21 change of COLO-205 in response to magnolol (50 mM) treatment. b: Magnolol dose-dependently increases p21 level in COLO-205 and Hep-G2, but not in HUVEC. Proteins were extracted from cultured cells and probed with proper dilutions of speci®c antibodies. c: Dose-dependent upregulation of p21-CDK2 association in COLO-205 by magnolol. p21-CDK2 was immunoprecipitated by anti-CDK2 antibody, and p21-CDK2 association was detected by anti-p21 antibody. d: Dose-dependent inhibition of CDK2 kinase activity by magnolol. e: Antisense p21 oligonucleotide abolished the magnolol-mediated increase of p21 protein level and decrease of thymidine incorporation. Antisense or scramble p21 was added to COLO-205 at a ®nal concentration of 2 nM at 1 h before the cell was challenged with 2% FCS and magnolol treatment. Cultures were incubated with magnolol for 21 h and [3 H]thy-midine was added only for the last 3 h of this incubation. Values are mean SE (n 6). Comparisons were subjected to Student's t-test. Signi®cance was accepted at P < 0.05. *M or M S-treated group different from DMSO-S-treated group. D, 0.1% DMSO; M, 10 mM magnolol. S, scramble p21 oligonucleotide; AS, antisense p21 oligonucleotide.
proteins were analyzed by gel electrophoresis as described in Materials and Methods.
As illustrated in Figure 3a, treatment of COLO-205 with magnolol resulted in an increase in p21, but not p27 (Fig. 4); the increase was maximal by 18 h after treatment. This upregulation was in a dose-dependent manner (Fig. 3b). Similar results were observed using Hep-G2 cells (Fig. 3b). In contrast, at a dose as high as 50 mM, magnolol did not alter the p21 in untransformed HUVEC (Fig. 3b). In accord with the established notion that p21 is a CDK inhibitor, we found in magnolol-treated cells that the formation of the p21-CDK2 complex was increased (Fig. 3c) and the assayable CDK2 kinase activity was decreased (Fig. 3d). In contrast, the formation of the p21-CDK4 com-plex was not changed (data not shown). We concluded that such decreased CDK2 activity could account (in part at least) for the impair-ment in the transition from G1- to S-phases. Finally, to show that in the magnolol-treated cells, increased p21 expression correlated with decreased DNA synthesis activity, the experi-ment illustrated in Figure 3e was carried out. Thus, in the sample labeled M for magnolol-treated, the p21 electrophoretogram band was increased in intensity, while 3H-thymidine incorporation was reduced by about 30%. Sam-ple M AS was treated with a p21 antisense
oligonucleotide (AS), which blocked the expres-sion of p21. Consequently, in this sample, p21 level did not increase and the magnolol addition to sample M AS failed to induce the decreased 3H-thymidine incorporation, which was evident in the M (magnolol added) sample.
In order to determine whether the changes of the levels of cyclins and CDK2 were also invol-ved in the magnolol-induced reduction of CDK2 kinase activity, we examined cyclin and CDK protein levels in response to magnolol treat-ment. Magnolol at concentrations of 10±30 mM did not induce any signi®cant changes of the levels of cyclin A, D1, D3 and E, CDK2, CDK4, p27, and proliferating cell nuclear antigen (PCNA). However, at a concentration of 50 mM, cyclin A and E as well as CDK2 were down-regulated (Fig. 4).
Elevation of the Cytosolic-Free Ca2Is Involved in the Magnolol-Mediated Increase
of p21 and Decrease of Thymidine Incorporation in COLO-205
Previous study in rat neutrophils has shown that magnolol induced an increase in cytosolic-free Ca2 via inositol trisphosphate pathway [Wang and Chen, 1998]. Accordingly, we further investigated the possible involvement of cyto-solic-free calcium in the magnolol-mediated increase of p21 in COLO-205. The intracellular fura-2/AM ¯uorescence activity (an indicator of cytosolic-free calcium concentration) in COLO-205 cells treated with or without magnolol was examined. Figure 5a showed an initial rapid spike with sustained high [Ca2]
i curve in the treated COLO-205. The magnolol-induced elevation of [Ca2]
ioccurred in a rapid and dose-dependent manner. Pretreatment of COLO-205 cells with 1,2-bis(2-aminophenoxy)-ethane-N,N,NõÂ,NõÂ-tetraacetic acid acetoxyme-thyl ester (BAPTA/AM, an intracellular chela-tor) reversed the magnolol-induced suppression of thymidine incorporation (Fig. 5b), suggesting that the magnolol-induced elevation of [Ca2]
i was involved in the magnolol-induced antipro-liferation. Moreover, pretreatment of COLO-205 with 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U73122, phospholipase C inhibitor) at a dose of 1 mM, which inhibits the hydrolysis of phos-phatidylinositol biphosphate (PIP2) to inositol triphosphate (IP3) and blocked magnolol-induced increase of [Ca2]
i (data not shown), abolished the magnolol-induced p21 protein
Fig. 4. Effect of magnolol on cyclin, and CDK protein levels. Protein was extracted from the cultured COLO-205 at 18 h after magnolol treatment and probed with proper dilutions of speci®c antibodies. Magnolol at a range of concentrations (10±30 mM) did not induce any signi®cant changes of the levels of cyclin A, D1, D3 and E, CDK2, CDK4, p27, and PCNA. However, treatment of COLO-205 with magnolol at a concentration of 50 mM for 18 h did induce downregulation of cyclin A and E as well as CDK2, but no change in cyclin D1 and D3, CDK4, p27, and PCNA. Membrane was probed with anti-G3PDH antibody to verify equivalent loading.
elevation and inhibition of thymidine incorpora-tion (Fig. 5c). These data suggested that mag-nolol induced an IP3-dependent elevation of free cytosolic Ca2, which in turn, caused an increase of p21 protein level and an inhibition of DNA synthesis in COLO-205. Since, it has been demonstrated that elevation of intracellular calcium can activate protein kinase C (PKC) [Oancea and Meyer, 1998], we further exam-ined whether PKC pathway was involved in the magnolol-induced p21 expression in COLO-205. Pretreatment with PKC inhibitor, staurosporine, dose-dependently reversed mag-nolol-induced p21 protein expression (Fig. 5d), suggesting that the PKC pathway was involved in the magnolol-induced p21 protein expression in COLO-205 cells.
Magnolol-Induced Inhibition of Cell Proliferation Is Reversible
We also examined the reversibility of the magnolol-induced inhibition of cell prolifera-tion. Treatment of the COLO-205 (Fig. 6a) and Hep-G2 (Fig. 6b) cells with magnolol (10 mM) for 6 days induced a 55% reduction of cell number as compared with the cells treated with 0.1% DMSO. However, treatment of the cells with magnolol (10 mM) for 3 days followed by 0.1% DMSO without magnolol for an additional 3 days induced only 25% inhibition as compared with the cells treated with 0.1% DMSO for 6 days. These results suggest that the magnolol-induced cell growth inhibition was largely reversible.
Fig. 5. Elevation of cytosolic-free calcium was involved in the magnolol-induced p21 induction and antiproliferation in COLO-205. a: Dose-dependent elevation of [Ca2]
iin COLO-205 by magnolol (arrow) in the fura-2/AM loaded COLO-COLO-205 cells. Vehicle treatment (0 mM magnolol) did not cause any elevation of [Ca2]
i. b: Intracellular Ca2chelator, BAPTA/AM, abolished the magnolol-induced decrease of thymidine incor-poration. U73122 and BAPTA/AM were added to COLO-205 at a ®nal concentration of 1 mM at 30 min before the cells were challenged with 2% FCS and magnolol treatment.
*Magnolol-treated group different from control (DMSO) group. M, 10 mM magnolol. c: Phospholipase C inhibitor, U73122, abolished the magnolol-mediated increase of p21 protein level and decrease of thymidine incorporation. d: Staurosporine abolished the magnolol-mediated increase of p21 protein level. Staurosporine was added to COLO-205 at 30 min before the cell was challenged with 2% FCS and magnolol treatment. P21 and G3PDH were detected after 18 h of incubation. DMSO, 0.1% DMSO; M, 50 mM magnolol; S50, 50 nM staurosporine; S100,100 nM staurosporine.
Magnolol Effect on DNA Fragmentation The particular evidence for the occurrence of apoptosis includes the fragmentation of DNA as illustrated in Figure 7. The cells were grown in RPMI 1640 (for COLO-205) or MEM (for Hep-G2) supplemented with 10% FCS and then treated with 100 mM magnolol for various times. Genomic DNAs extracted from magnolol-treatedCOLO-205(Fig.7a)andHep-G2(Fig.7b) were examined by gel electrophoresis and found to display the DNA ladder patterns characteristic of cells undergoing apoptosis. As shown in Figure 7c,d, control HUVEC and human ®broblasts did not show this DNA ladder pattern.
Magnolol Causes Tumor Regression In Vivo Given the inhibition by magnolol of the growth of transformed colon cells in culture, we next determined whether administration of magnolol could affect the growth of tumors derived from human colon cancer cells in an in vivo setting. A reduction in tumor volume be-tween mice given magnolol vs. those given vehicle (Cremophore RH-40) was detectable by day 3, three days after initiation of magnolol treatment. This difference became progres-sively more conspicuous, with the average tumor volume of magnolol-treated mice at 15% that of vehicle-treated mice at 14 days of treatment (Fig. 8a±c). Since, retardation of the cell cycle and/or activation of the cellular apoptotic response are two major mechanisms preventing tumor growth, we examined the
magnolol effect on the cell cycle and apoptosis occurrence of the solid tumor derived from the implanted COLO-205. The particular evidence for the occurrence of apoptosis in the tumor isolated from the magnolol-treated animal includes the fragmentation of DNA as illu-strated in Figure 8d. The content of p21, a CDK-inhibitory protein, was increased in the tumor isolated from the magnolol-treated mouse (Fig. 8e), suggesting that the inhibition of the progression of cell cycle activity was involved in the magnolol-induced tumor regres-sion. As illustrated in Table I, intraperitoneal injection of magnolol at a dose of 100 mg/kg, which has been used in various in vivo studies [Watanabe et al., 1983; Hsieh et al., 1998], did not affect the body weight and was not cytotoxic for the vital organs.
DISCUSSION
The present study was undertaken to exam-ine the anti-cancer effect of magnolol on human cancer cells. Our in vitro studies demonstrated that magnolol at concentrations of 3±10 mM inhibited DNA synthesis and the growth rate of COLO-205 and Hep-G2 cells in a dose-depen-dent manner, but not in human untransformed cells such as keratinocytes, ®broblasts, and HUVEC. These results were not due to necrotic cell death and indicated that there was an inhi-bitory effect of magnolol on the mechanisms for cell division in the subcultured cancer cells. Fur-thermore, when magnolol concentration was increased to 100 mM, apoptosis was observed in
Fig. 6. Reversibility of the magnolol-induced inhibition of cell proliferation. Magnolol-induced inhibition of COLO-205 (a) and Hep-G2 (b) cell proliferation was reversed by removal of magnolol. Open columns, the cells were treated with 0.1% DMSO for 6 days; solid columns, the cells were treated with 10 mM magnolol for 6 days; striped columns, the cells were treated with 10 mM magnolol for 3 days and then the medium
was replaced with 0.1% DMSO without magnolol for an additional 3 days. Six samples were analyzed in each experiment. *6-d magnolol-treated group different from control (6-d DMSO) group and 3-d magnolol 3-d DMSO-treated group. #3-d magnolol 3-d DMSO group different from 6-d magnolol-treated group and control group.
COLO-205 and Hep-G2 cells, but not in cultured human ®broblasts and HUVEC. In vivo studies show that i.p. administration of magnolol caused a striking and substantial regression of the COLO-205 tumor mass. To our knowledge, this is the ®rst demonstration that magnolol inhibits the growth of colon cancer cells both in vitro and in vivo.
This inhibitory effect of magnolol on growth does not appear to be limited to the COLO-205 and Hep-G2 cells, as similar inhibition has also been observed in other transformed cultured cells, such as Hep-3B and HT-29 (Fig. 1e,f). Importantly, at the doses (3±10 mM) used in our in vitro studies, magnolol was not cytotoxic for the cultured untransformed human cells (such
Fig. 7. Magnolol induces DNA fragmentation in human malignant cells. A typical DNA ladder pattern associated with apoptosis was seen in COLO-205 (a) and Hep-G2 (b) cells treated with 100 mM magnolol for at least 36 h. In contrast, a typical DNA ladder pattern was not observed in human ®broblasts (c) or HUVEC (d) treated with 100 mM magnolol for
up to 48 h. In these experiments, the cells were grown in RPMI 1640 (for COLO-205), MEM (for Hep-G2 and ®broblasts), or M199 (for HUVEC) supplemented with 10% FCS. Genomic DNA isolated from 10 mM terbina®ne-treated HT-29 cells were included in this experiment to serve as a positive control.
as keratinocytes, ®broblasts, and HUVEC), nor did the magnolol have any effect on cell pro-liferation in these cultures. Moreover, magnolol at a concentration of 100 mM caused the
occur-rence of apopotsis in COLO-205 and Hep-G2, but not in cultured human ®broblasts and HUVEC. In the animal study, intraperitoneal injection of magnolol at a dose of 100 mg/kg,
Fig. 8. Magnolol reduces the growth rate of tumors in nude mice. Gross appearance of subcutaneous tumors after treatment with DMSO (a) or magnolol (b) for 14 days. Insets show the isolated tumors. c: Average tumor volume of DMSO-treated (open circles, n 5) vs. magnolol-treated (®lled circles, n 4) nude mice. Mice were injected subcutaneously with COLO-205 cells at day ÿ7. At day 0, mice were injected intraperitoneally with DMSO or magnolol (100 mg/kg) 5 times/week for 2 weeks. d: Induction of apoptosis by magnolol in COLO-205 tumor as
shown by DNA fragmentation using electrophoresis of genomic DNA (left panel) and DNA breaks stained in situ by the TdT-mediated dUTP-biotin nick end labeling methods (right panel). Arrows indicate representative apoptotic cells. e: Western blot analysis of p21 protein levels in COLO-205 tumor. COLO-205 tumors were harvested for protein extraction at 14 days after DMSO or magnolol treatment. P21 (indicated by arrow) protein levels were quanti®ed, and p21 signals were corrected against those for G3PDH to equalize loading sample size.
which has been used in various in vivo studies (Watanabe et al., 1983; Hsieh et al., 1998), was not cytotoxic for the vital organs (Table I). Thus, this magnolol extracted from magnolia tree bark seems to possess a selective suppressive action on several malignant cell types.
By thymidine incorporation and ¯ow cyto-metry analyses, we demonstrate that magnolol at low concentrations (3±10 mM) induced retar-dation and reduction of COLO-205 and Hep-G2 cells entrance into the S-phase. Cell cycle progression is regulated by successive, coordi-nated activation of CDKs [Hunter and Pines, 1994; Morgan, 1995], whose activity is con-trolled by their association with a series of regulatory subunits called cyclins and a group of CDK-inhibitory proteins designated CKIs [Sherr and Roberts, 1995]. Among these CKIs are two known as p21 and p27. Cyclin A-CDK2 and cyclin E-CDK2 complexes form late in the G1 phase as cells prepare to synthesize DNA [Lees, 1995], and formation of the cyclin E com-plex is a rate-limiting step in the G1/S transi-tion [Sherr, 1993]. By gel electrophoresis and TUNEL staining analyses, we also demonstrate that magnolol in a higher concentration (100 mM) caused apoptosis in COLO-205 and Hep-G2 cells. Apoptosis is a cell suicide mechanism that requires specialized cellular machinery. The central component of this machinery is a pro-teolytic system involving caspases, a highly conserved family of cysteine proteinases with speci®c substrates [Thornberry and Lazebnik, 1998]. Observation of an increased expression of p21 protein (Fig. 7d) and the occurrence of apoptosis (Fig. 7e) in the solid tumor isolated from the magnolol-treated mouse suggests that both cell cycle inhibition and apoptotic cell death contribute to the magnolol-induced tumor
regression. These ®ndings might explain why magnolol can induce a profound regression (85± 90% inhibition) of the solid tumor derived from subcutaneous implantation of COLO-205 cells, while there is only 35±50% growth inhibition in cultured malignant cell lines.
Although the primary mode of action of mag-nolol remained obscure, our results suggested that cell membrane associated inositol trispho-sphate signaling pathway was involved in the magnolol-induced elevation of [Ca2]
i and increase of p21 protein level. Binding of calcium to PKC single C2- or diacylglycerol to tandem C1-domains has been demonstrated to release the pseudosubstrate inhibitory domain, which in turn, activates the PKC isoforms [Newton, 1995; Quest, 1996]. PKC activation has been associated with p21 expression and growth inhibition of A431 [Toyoda et al., 1998] and IEC-18 [Frey et al., 1997] cells. Our results indi-cated that PKC pathway was also involved in the magnolol-induced p21 induction in COLO-205 cells.
The experimental ®ndings presented in this paper introduce our basic observations that this herbal ``magnolol'' can speci®cally inhibit can-cer cell proliferation and induce apoptosis in cancer cells both in cell cultures and also in subcutaneous sites in mice. Although animal studies of magnolol-mediated anti-tumoral action are still ongoing, the ®ndings from the present in vitro and in vivo studies in magnolol anti-cancer effect suggest the potential appli-cations of magnolol in the treatment of human cancer.
ACKNOWLEDGMENTS
We thank Professor Winton Tong (University of Pittsburgh, PA) and Professor Ling-Ru Lee (University of California-Berkeley, CA) for critical reviews of the paper, Dr. Chien-Huang Lin (Taipei Medical University, Taipei, Taiwan) for valuable discussion, and Dr. How Tseng (Taipei Medical University, Taipei, Taiwan) for editorial assistance.
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