CWO was purchased from Zhejiang RuiAn Pharmaceutical Company (Lot No. 011001). CWO (0.1 mL) was diluted in 10 mL methanol. The solution thus obtained was filtered through a 0.45 &mgr;m Econofilter (Agilent Technologies, Palo Alto, CA, USA) and injected into Agilent Series 1100 (Agilent Technologies, USA) liquid chromatograph, equipped with a vacuum degasser, a quaternary pump, an autosampler, and a diode array detection (DAD) system, and analyzed under the conditions described in a previous report[7]. In brief, A Zorbax ODS column (250 mm × 4.6 mm I.D., 5 &mgr;m) with a Zorbax ODS C18 guard column (20 mm × 3.9 mm I.D., 5 &mgr;m) was used. Solvents that constituted the mobile phase were A (water) and B (acetonitrile). The elution conditions applied were: 0-15 min, linear gradient 30%-47% B; 15-30 min, isocratic 47% B; 30-40 min, linear gradient 47%-60% B; 40-50 min, linear gradient 60%-90% B; 50-60 min, linear gradient 90%-100% B; and finally, washing the column with 100% B for 10 min before reconditioning the column for 15 min with 30% B. The flow-rate was 1 mL/min and the injection volume was 10 &mgr;L. The column operated at 25°C. The analytes were monitored with DAD at 214 nm and 256 nm. In addition, methanol solutions, containing known concentrations of standards including curcumenone, curcumenol, neocurdione, curdione, isocurcumenol, furanodienone, curcumol, germacrone, curzerene, furanodiene, and β-elemene were prepared and subjected to the LC-DAD system for comparison. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), JC-1 dye, caspase-3 assay kit, and 488 cytochrome C apoptosis detection kit were purchased from Molecular Probes. Human apoptosis kits were purchased from BD Bioscience.

Cell culture and drug treatment: The human hepatoma cell line HepG2 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Life Technologies Inc., Gaithersburg, MD), 100 &mgr;g/mL streptomycin, and 100 U/mL penicillin in 75 cm² tissue culture flasks in a humidified atmosphere at 37°C with 5% CO₂. CWO was dissolved in 1 mL DMSO to make a 1 mmol/L stock solution and diluted to the concentrations as needed. The final volume of drug solution added to medium was 1%. Control samples contained 1% DMSO.

Growth inhibitory assay: Cells were seeded in 96-well microplates (1 × 10⁵ cells/well in 100 &mgr;L medium). CWO was added to the cultures in serial concentrations and cultures were incubated for 48 h. Medium was discarded and 30 &mgr;L tetrazolium dye (MTT) solution (5 mg/mL in PBS) were added to each well. Plates were incubated for additional 4 h. DMSO (10 &mgr;L) was added to dissolve the formed formazan crystals. The plate was read in a microplate reader at 570 nm. MTT solution with DMSO (without cells and medium) acted as blank while the DMSO (1%)-treated cells served as control of 100% survival.

Agarose gel electrophoresis for analysis of DNA fragmentation: HepG2 cells were treated with the drug for 72 h while the DMSO (1%) containing medium treated cells served as control. Adherent cells (2 × 10⁶ /mL) were harvested, washed once with 400 &mgr;L PBS, and taken up in 400 &mgr;L lysis buffer (containing 200 mmol/L Tris-HCl (pH 8.3), 100 mmol/L EDTA, and 1% SDS). Twenty &mgr;L of 10 mg/mL proteinase K were added for protein digestion, and the tubes were incubated in a 37°C water bath overnight. The samples were allowed to cool down to room temperature before 300 &mgr;L saturated NaCl solution were added. After centrifugation for 15 min at 9000 r/min, supernatants were collected. DNA fibers were obtained by adding 1 mL cold absolute ethanol (EtOH) and a centrifugation for 20 min at 4°C at 16 000 r/min. DNA fibers were washed once with 500 &mgr;L of -20°C 70% EtOH, and the DNA pellet was dried in a 70°C oven. Fifteen &mgr;L TE buffer (10 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA) containing 0.2 mg/mL RNase A were added. After an incubation at 37°C for 90 min, 10 &mgr;L of each sample were loaded on a 1.5% TBE agarose gel to visualize DNA.

Cell cycle analysis: Cells were seeded in 6 well plates and treated with CWO at various concentrations for 48 and 72 h. DMSO (1%)-treated cells served as control. After treatment, media were discarded. The adherent cells were washed with PBS, and 300 &mgr;L trypsin were added for 5 min at room temperature to detach the cells. After centrifugation at 350 g at 4°C for 5 min, the cell pellet was resuspended with 1 mL cold 70% EtOH at 4°C for 12 h and centrifuged again for 5 min at 4°C at 350 g. Finally, 1 mL propidium iodide (PI) staining solution (20 &mgr;g/mL PI, 8 &mgr;g/mL DNase free RNase) was added to the samples. The samples were analyzed by flow cytometry (FCM) (BD FACS Canto^TM). The results were analyzed by Mod Fit LT 3.0 software.

Measurement of mitochondrial transmembrane potential (ΔΨm):ΔΨm was assessed by JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyan-ine iodide), Mitochondrial Potential Sensors (Molecular Probes, Leiden, Netherlands). Red fluorescence J-aggregate form of JC-1 indicates intact mitochondria, whereas green fluorescence shows monomeric form of JC-1 due to breakdown of the mitochondrial membrane potential. Cells were seeded in 6-well plates and then incubated with the desired concentrations of CWO for 48 h. The medium of each well was discarded, cells were treated with 1 mL medium containing 5 mg/mL JC-1 for 15 min at 37°C and 5% CO₂ in the dark, washed twice in PBS, resuspended in 1 mL medium and measured by FCM.

Immunostaining of cytochrome C: Cytochrome C release was assessed by SelectFX Alexa Fluor 488 cytochrome C apoptosis detection kit (Molecular Probes, Leiden, Netherlands). Cells were seeded in 24-well plates, and treated with various amounts of CWO in an humidified atmosphere (37°C in 5% CO₂) for 24 h while the DMSO (1%)-treated cells served as control. Media were discarded and the cells were washed with warm PBS, fixed with freshly prepared 4% formaldehyde in PBS for 15 min at 37°C, and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. The cells were washed, incubated in a blocking buffer (10% heat-inactivated normal goat serum (NGS) for 30 min at room temperature, and finally for 1 h with 1 &mgr;g/mL primary antibody (anti-cytochrome C, mouse IgG) at room temperature. Green fluorescence was observed by fluorescence microscopy.

Caspase-3 enzymatic activity assay: Caspase-3 enzymatic activity was determined by measuring the cleavage of Ac-DEVD-R110 according to the protocol with the caspase-3 assay kit supplied by Molecular Probes. Cells were treated with various amounts of CWO in an humidified atmosphere (37°C in 5% CO₂) for 24 h while DMSO (1%)-treated cells served as control. Cells were harvested at a concentration of a minimum of 1 × 10⁶/mL, pellets were collected, appropriate cell lysis buffer was added, and the samples were incubated on ice for 30 min. The samples were then centrifuged and supernatants were collected and transferred to microplates. Cell lysis buffer was used as a no-enzyme control to determine the background fluorescence of the substrate. At the same time, 1 &mgr;L of 1 mmol/L Ac-DEVD-CHO inhibitor was added to selected samples. One &mgr;L of DMSO was added to no-inhibitor samples to serve as control and incubated for 10 min simultaneously. Finally, 0.05 mmol/L Z-DEVD-R110 substrate was added and samples were incubated for 30 min prior to the fluorescence measurement.

Measurement of cleaved PARP and active caspase-3 protein levels: The BD™ CBA human apoptosis kit (BD, Franklin Lakes, USA) was applied to quantify the active caspase-3 and Poly-ADP-ribose polymerase (PARP) protein levels; cytometric Bead Array (CBA) employs a particle with a discrete fluorescence intensity to detect a soluble analyte. This kit provided two types of bead populations with distinct fluorescence intensities that have been coated with capture antibodies specific for cleaved caspase-3 and PARP. Cells were seeded in 6-well plates and incubated with various concentrations of CWO in an humidified atmosphere (37°C with 5% CO₂) for 48 h. 1.0 × 10⁶ cells per sample were counted, harvested, and washed with PBS. Fifty &mgr;L of cell lysis buffer was added to each sample for 30 min on ice and samples were vortexed at 10-min intervals. Pellet cellular debris was removed by centrifugation at 12 500 r/min for 10 min. Protein concentrations were measured by 2-D Quant Kit (Amersham Biosciences, Piscataway, USA). Each sample was normalized in a final concentration of 0.2 &mgr;g/&mgr;L. Thirteen standard curves (standard ranging from 0 to 6000 U/mL) were obtained from one set of calibrators. For each sample and the standard mixture of lysate standard (caspase-3 and PARP beads), 50 mL of sample or standard of beads were added to the mixture of 50 mL of 2 mixed capture beads incubated for 1 h, and mixed 50 mL of PE detector bead for another 1 h. After that, samples were washed before data acquisition with a FCM. The results were analyzed by FCAP Array V1.0.

The data are expressed as mean ± SD from at least 3 independent experiments. Differences between groups were analyzed using a Student’s t-tests.

CWO treatment inhibited the growth of HepG2 cells in a dose-dependent manner, and the IC50 of CWO was approximately 70 &mgr;g/mL (Figure 1). We then examined whether CWO inhibited HepG cell growth through inducing cell death and apoptosis. HepG2 cell were treated with different concentrations of CWO for 72 h. Figure 2 shows that DNA fragmentation was evidently observed at a concentration of 70 &mgr;g/mL after 72 h of treatment.

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Figure 1 HepG2 cells were treated with the indicated concentrations of CWO for 48 h. Cell growth was determined by the MTT assay and was directly proportional to the absorbance at a wavelength of 570 nm. Data are expressed as mean ± SD from three independent experiments.

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Figure 2 Agarose gel of electrophoresis of genomic DNA obtained from HepG2 cells treated with different concentrations of CWO for 72 h. DNA fragmentation with a ladder pattern is a characteristic for apoptosis.

The effect of different concentrations of CWO on cell-cycle progression was studied after 48 and 72 h of drug exposure. CWO treatment resulted in the accumulation of cells in S/G₂ phase with concomitant losses from G₀/G₁ phase (Figure 3). Since substantial proportions of cells were dead in groups treated with 50 &mgr;g/mL and 70 &mgr;g/mL CWO, only cultures treated with 35 &mgr;g/mL were used for the analysis as shown in Figure 4.

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Figure 3 Effect of CWO on HepG2 cell cycle progression. Flow cytometric analysis of propidium iodide-stained HepG2 cells treated with 35 &mgr;g/mL CWO for 48 h (A) and 72 h (B). The results of HepG2 cells treated with CWO were analyzed by Mod Fit LT 3.0. Data expressed as mean ± SD from three independent experiments. ^bP < 0.01, ^dP < 0.001 vs control.

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Figure 4 Analysis of change of ΔΨm in HepG2 cells. HepG2 treated with 17.5, 35 , 50, and 70 &mgr;g/mL for 48 h, were stained with JC-1 probe. The cells were analyzed by FCM. Red and green fluorescence were measured by FL2 and FL1 channel, respectively. Red fluorescence indicates intact mitochondrial potential while green fluorescence indicates breakdown of mitochondrial potential. The ratio of intensity of FL2 to FL1 indicates the change of ΔΨm. Data expressed as mean ± SD from four independent experiments. ^bP < 0.001 vs control.

Some chemotherapeutic drugs induced apoptosis via mitochondrial pathways by altering ΔΨm. To monitor the ΔΨm, we used JC-1 probe to determine the ΔΨm in cells that were treated with CWO for 48 h at different concentrations. Mitochondria with normal ΔΨm concentrate JC-1 into aggregates (red/orange fluorescence), while in depolarized mitochondria, JC-1 forms monomers (green fluorescence). As compared to non-treated HepG2 cells, green fluorescence increased while red/orange fluorescence decreased after CWO exposure. Figure 4 indicates an only minor shift from red/orange to green fluorescence in groups treated with 17.5 &mgr;g/mL and 35 &mgr;g/mL CWO, while 50 &mgr;g/mL and 70 &mgr;g/mL CWO led to significant changes in ΔΨm.

Cytochrome C release from mitochondria to the cytosol is implicated in mitochondria dependent apoptosis[16]. Cytochrome C staining in the cytosol of HepG2 cells showed markedly stronger green fluorescence than in control cells in a dose-dependent fashion (Figure 5). CWO treated cells showed obvious punctuate green fluorescence staining or appeared to have green fluorescence accumulated in large aggregates compared to the control.

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Figure 5 Cytochrome C release into the cytosol in CWO treated HepG2 cells for 12 h. Cytochrome C immunofluorescence was observed with fluorescent microscope. A: Control; B: 17.5 &mgr;g/mL; C: 35 &mgr;g/mL; D: 70 mg/mL; E: 120 mg/mL. Fine punctate/granular stainings for cytochrome C are observed. Cytochrome C release also increases the global cytosolic fluorescent signal. Similar results were obtained for 3 independent experiments (× 20).

Many studies previously have demonstrated that programmed cell death is associated with the activation of caspase as key elements involved in the sequence of events that lead to cell death[17]. Caspase-3 particularly, is essential for propagation of the apoptotic signal after exposure to many DNA-damaging agents and anticancer drugs. We examined caspase-3 activity after cells were treated with 17.5-70 &mgr;g/mL CWO for 24 h. The result clearly demonstrated that caspase-3 activity increased in a dose-dependent manner (Figure 6). Caspase-3 activities were 2 and 5.5 times higher in 35 &mgr;g/mL and 70 &mgr;g/mL CWO treated cultures, respectively, when compared to the control. When the cellular samples were incubated with specific Ac-DEVD-CHO inhibitors simultaneously, caspase-3 activity was blocked.

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Figure 6 The fluorescence was increased in a dose-dependant manner after 24 h treatment with CWO. Caspase-3 cleaves substrate Ac-DEVD-R110 to emit green fluorescence. Higher fluorescent intensity indicates higher caspase-3 enzymatic activity. Ac-DEVE-CHO inhibitor can inhibit caspase-3 enzymatic activity. Data are expressed as mean ± SD from three independent experiments. ^bP < 0.001, ^dP < 0.01 vs control.

Drug-induced cell death via apoptosis pathway, signaling can generally be divided into receptor- and mitochondrial-mediated pathways. These pathways converge at several downstream points including the mitochondria, caspase activation, and substrate cleavage[18]. Figure 7 showed that there was significant increase in protein levels of cleaved PARP and active caspase-3 in a dose-dependent manner as determined by BD™ CBA Human Apoptosis Kit. HepG2 cells treated with 17.5, 35, and 70 &mgr;g/mL CWO were 6, 4, and 7 times higher in caspase-3 protein level than control group (Figure 7A). The level of cleavage of PARP had a 2-fold increase in HepG2 cells treated with 70 &mgr;g/mL CWO compared to the control (Figure 7B).

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Figure 7 Protein expression levels of active caspase-3 and cleaved PARP in CWO-induced apoptosis. HepG2 cells were treated with medium alone (control) or different concentration of CWO for 48 h. Cells of each sample were counted to 1.0 × 10⁶ and all the samples were normalized to final protein concentration in 0.2 &mgr;g/&mgr;L. It was detected with BD^TM CBA Human Apoptosis Kit (BD, Franklin Lakes, USA) according to manufacturer instruction. The results were analyzed by FCAP Array V1.0. Active caspase-3 protein level in HepG2 was shown in (A) and the cleaved PARP protein level in HepG2 was shown in (B). The x-axis indicated the concentration of CWO while the y-axis indicated amount of proteins (unit per mL). Concentration of active caspase-3 and cleaved PARP in test samples were determined using the standard curve. Data expressed as mean ± SD from three independent experiments. ^bP < 0.01, ^dP < 0.001 vs control.

Ezhu has a long history on treating liver diseases and protecting liver functions. Chinese Pharmacopoeia indicated that the rhizomes of three species including Curcuma phaeocaulis, C. kwangsiensis and C. wenyujin are used as Ezhu. The essential oil of Ezhu has been reported to possess various biological roles such as antimicrobial, anti-inflammatory, and anti-tumor activity. Some sesquiterpene compounds isolated from essential oil of Curcuma wenyujin (CWO) has been identified to have hepatoprotective effects. The total effects of the complex interactions of different compounds in extracts isolated from CWO are not well characterized.

Hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer with more than 1 million deaths reported annually worldwide. Many sesquiterpenes are identified to possess protective effects against carcinogenesis or tumor growth. For example, artemisinin, a sesquiterpene lactones, killed human oral cancer cells through apoptosis and may be useful as an alternative treatment for oral cancer. In order to develop effective means for prevention and treatment of HCC and related liver diseases, extensive research is being carried out to isolate and identify chemical extracts and pure compounds from Chinese medicine with hepatoprotective, anti-hepatoma, anti-multidrug resistant hepatoma, and anti-viral effects.

This is the first study to report the biological activity and mechanism of action of CWO on HCC cells.

CWO induce apoptosis in HepG2 cells through activation of mitochondrial and caspase-3-pathway and the result suggests the potential value of development of Ezhu on treatment of liver diseases and further study on anti-apoptotic effect of CWO may lead to identification of new lead compounds and novel drug targets for treatment of liver cancer and diseases.

This is an interesting study showing that CWO inhibits growth and induces apoptosis in human HepG2 hepatoma cells. These findings are of interest.