Fresh, ripe fruits of high quality flowers of Chrysanthemum indicum were procured from Xi’an traditional medicine group (Shaanxi, China) in March 2006 and the characteristics were consistent with that described in the Pharmacopeia of the People’s Republic of China. Moreover, Chrysanthemum indicum was also authenticated by Professor Wang Jun-Xian, a taxonomist in the Department of Pharmacy in Xi’an Jiongtong University. The plant materials were air dried at room temperature and then powdered. The dried and powdered fruit of Chrysanthemum indicum (500 g) were extracted with 95% ethanol (EtOH) twice under refluxed temperature. After evaporation of organic solvent under reduced pressure, the resultant Chrysanthemum indicum EtOH extract (CIE) was concentrated under reduced pressure to give 66.5 g (13.3%) EtOH extract. The dry extract was stored in a refrigerator at -20°C until use in the experiments. CIE was dissolved in phosphate buffered solution (PBS) and diluted in cultured medium before use, and the control group was made up of medium, PBS and the cells.

A human MHCC97H HCC cell line was purchased from the Liver Cancer Institute of Fudan University (Shang Hai, China). A human ECV304 cell line was obtained from the Cell Bank of Academia Sinica (Shang Hai, China). MHCC97H cells and ECV304 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) in a humidified incubator containing 5% CO₂ in air at 37°C before use and subcultured with 0.25% trypsin-0.02% EDTA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine 123 (Rh123), annexin V and propidium iodide (PI) were purchased from Sigma Corporation (Sigma, St.Louis, MO, USA). DMEM, FBS and trypsin were obtained from GibcoBRL, Grand Island, NY, USA. Anti-caspase-3, anti-caspase-9, anti-cytochrome C, and anti-β-actin were purchased from Santa Cruz Company. Anti-CDK4 and P21 were obtained from eBioscience Corporation.

Hepatocytes were isolated by the collagenase perfusion method[9] from 10-wk-old male Sprague-Dawley rats anesthetized with intraperitoneal administration of ketamine. The viability of the isolated hepatocytes was over 90% as determined by 0.2% trypan blue exclusion. The cells were plated in 35 mm plastic dishes at a density of 3 × 10⁵ cells/mL in 2 mL of Williams’ Medium E supplemented with 10% FBS, and were cultured in a humidified atmosphere of 5% CO₂ and 95% air at 37°C overnight. After overnight incubation, the culture medium was changed to fresh medium, and cultures were incubated with varying concentrations of CIE 400, 800 and 1200 μg/mL for 24 h. According to a previous report[25], the cells were then trypan blue stained and a hemocytometer was used to determine the total cell count and viable cell number. Viability of cells were determined as follows: viability (%) = viable cell number/total cell count × 100%.

ECV304 cells were seeded in a 96-well plate (5 × 10⁴ cells/well). After 24 h seeding, cells were treated with CIE (400, 800 and 1200 μg/mL) for 24 h, in 3 parallel wells each, with untreated cells serving as a control, then the MTT assay as described by Xiao et al[26] was performed. At 24 h, 20 μL MTT solution (5 mg/mL) was added to each well and incubated for a further 4 h. The medium was removed and 200 μL DMSO was added to each well. Absorbance (A) at 570 nm was measured using a microculture reader. The percentage of viable cells was calculated as follows: (A of experimental group/A of control group) × 100%.

MHCC97H cells were seeded into 96-well plates at a density of 5 × 10⁴/well and were then incubated with different concentrations of CIE (400, 800 and 1200 μg/mL) for 24, 48 and 72 h, then the MTT assay was performed. At 24, 48 and 72 h, 20 μL MTT solution (5 mg/mL) was added to each well, and the cells were further incubated at 37°C for 4 h. The MTT assay and calculation of the percentage of viable cells were the same as described above.

In accordance with the study of Chen et al[27], the apoptotic rates were analyzed by flow cytometry using an annexin V-FITC/PI kit. Staining was performed according to the manufacturer’s instructions, and flow cytometry was conducted on a FACS Caliber (Becton Dickinson, Mountain View, NJ, USA). Cells that were annexin V (-) and PI (-) were considered viable cells. Cells that were annexin V (+) and PI (-) were considered early apoptotic cells. Cells that were annexin V (+) and PI (+) were considered late apoptotic cells.

Cell cycle analyses were carried out by the method of Vinodhkumar et al[28]. Briefly, cells were incubated in culture media alone or culture media containing 400-1200 μg/mL of CIE, at 37°C for 48 h. Cells were harvested in cold PBS, fixed in 70% EtOH, and stored at 4°C. Fixed cells were washed with PBS once and suspended in 1 mL of PI staining reagent 50 mg/mL containing 100 μg/mL Rnase, and were then incubated in the dark for 30 min. The distribution of the cell cycle was measured by a Becton Dickinson FACS analysis system and quantitation of cell cycle distribution was carried out using Multicycle Software.

Loss of MMPΔψ_m was assessed by flow cytometry, using a fluorescent indicator Rh123, as described by Tang et al[29] and Li et al[30]. Briefly, cells were treated with different concentrations of CIE. Then, Rh123 working solution was added to the culture at a final concentration of 2 μg/mL and then incubated in the dark at 37°C for 30 min. Cells were then washed with PBS, and fluorescence of Rh123 was detected immediately using a FACS Caliber, at an excitation wavelength of 488 nm and emission wavelength of 525 nm.

Cancer cells (2.5 × 10⁷/well) were treated with different concentrations of CIE for 24 or 48 h. To extract cytoplasmic protein as by the method of Li et al[31], cells were collected by centrifugation at 200 r/min for 10 min at 4°C. The cells were washed twice with ice-cold PBS, followed by centrifugation at 200 r/min for 5 min. The cell pellet was then suspended in ice-cold cell extraction buffer for 30 min on ice. In addition, as described by Hsu et al[32], cells were then lysed in a sample buffer, followed by sonication and denaturation. Protein concentrations were measured using DC Protein Assay (Bio-Rad, Hercules, CA) and equal amounts of protein (50 μg) were subjected to SDS-PAGE on 12% gel. The proteins were then electrophoretically transferred to nitrocellulose membranes and processed for immunoblotting. Membranes were first blocked with 5% non-fat dry milk overnight at 37°C and immunolabeled using primary antibodies. Goat anti-rabbit horseradish peroxidase-conjugated antibodies (Cell Signaling Technology, Beverly, MA) were used as secondary antibodies and detected with enhanced chemiluminescence (Amersham, USA). Equal loading of each lane was evaluated by immunoblotting using the same membranes with β-actin antibodies after detachment of previous primary antibodies. The band density for the target protein in each sample was measured with image analysis software (Gene Genus, Gene Company) and normalized to β-actin expression.

All data were expressed as mean ± SE. Statistical analysis was performed with analysis of variance (ANOVA) using the statistical software SPSS 11.0. P-values < 0.05 were regarded as statistically significantly.

In order to compare the effects of CIE on rat hepatocytes and human ECV304 cells, the numbers of viable cells were measured. As shown in Figure 1A, CIE did not decrease the number of viable rat hepatocytes, used as a normal cell model. To confirm the activity of CIE in human cells, we measured the number of viable cells treated with varying concentrations of CIE in human endothelial cells (ECV304). CIE did not reduce the number of viable ECV304 cells at any dose (Figure 1B). Therefore, the effect of CIE in inhibiting proliferation of MHCC97H cells and its mechanism were examined in subsequent experiments.

When MHCC97H cells were incubated with 400-1200 μg/mL CIE for 24, 48, 72 h, as shown in Figure 2, there was a significant dose-dependent reduction in cell viability. The IC₅₀ value at 24 h was 1009 ± 130 μg. When 1200 μg/mL CIE was incubated with cancer cells for 72 h, viable cells amounted to only 25% of control. These findings indicated that CIE significantly decreased proliferation of MHCC97H cells in a dose- and time-dependent manner. Hence, the proliferation inhibitory effect of CIE on MHCC97H and its mechanisms were tested in the following experiments.

MHCC97H cells were incubated with different CIE concentrations (400, 800 and 1200 μg/mL) for 24 h and were analyzed by flow cytometry. Pretreatment of MHCC97H cells with various concentrations of CIE induced significant apoptosis (Figure 3A). The numbers of early and late apoptotic cells were significantly increased compared with the control group (Figure 3B). The proportion of early and late apoptotic cells in the 1200 μg/mL treatment group was more than 10 times higher than in the drug-free cells.

After incubation with CIE at different concentrations (400, 800, 1200 μg/mL), the cells were examined by phase contrast microscopy for evidence of morphological apoptosis induced by CIE (Figure 4). The control cells showed a typical polygonal and intact appearance (control), whereas the CIE-treated cells displayed morphological changes with preapoptotic characteristics, such as cellular shrinkage (400, 800 μg/mL), rounding (800 μg/mL), and poor adherence (1200 μg/mL), as well as round floating shapes (1200 μg/mL).

To explore whether CIE-induced apoptosis involved the MMPΔψ_m, we used a fluorescent indicator, Rh123 to detect the MMPΔψ_m when MHCC97H cells were treated with 400-1200 μg/mL of CIE for 24 h. As shown in Figure 5A and B, after exposure to different CIE doses, cells exhibited much lower Rh123 staining (236.7 ± 9.3, 170.7 ± 13.9, 105 ± 10.5) than controls (275 ± 14.5: P < 0.05 or P < 0.01), indicating that CIE can significantly decrease MMPΔψ_m associated with cancer cell apoptosis.

To determine whether apoptosis induced by CIE was a mitochondrial-dependent caspase pathway, we further tested whether cytochrome C could be released from the mitochondria into the cytoplasm. As shown in Figure 6A, although there was no detectable cytochrome C in the cytosolic fraction of continuously growing MHCC97H cells, the level of cytochrome C released from the mitochondria increased dose-dependently in the presence of CIE concentrations ranging from 400 to 1200 μg/mL. In accordance with mitochondrial cytochrome C release into the cytoplasm, caspase-9 protein expression was increasingly detected. Accordingly, caspase-3 protein expression was also increased dose-dependently on exposure to CIE (Figure 6A and B). Taken together, these findings suggest that CIE exerted a significant apoptotic effect on MHCC97H cells in a concentration-dependent manner through the mitochondrial pathway, and was accompanied by a decrease in MMPΔψ_m, release of cytochrome C, and activation of caspase-9 and caspase-3.

The cell cycle of cancer cells was also determined by flow cytometry. MHCC97H cells treated with CIE 400, 800, 1200 μg/mL for 48 h showed an accumulation of cells in the S phase of the cell cycle. In contrast, the population of cells in G0-G1and G2/M phases was significantly decreased, especially at 400 μg/mL CIE (Figure 7A-C). In addition, as shown in Figure 7A, cancer cells incubated with higher doses of CIE for 48 h also showed a sub-G1 peak indicating apoptosis. These observations suggest that a small number of cancer cells escape from the S phase and undergo apoptosis, particularly at the 1200 μg/mL CIE concentration. Therefore, with the dose-dependent increase in cancer cell apoptosis, the proportion of cells arrested in the S phase by CIE decreased as the CIE concentration increased from 400 to 1200 μg/mL.

To determine the mechanism by which CIE arrested the cell cycle in the S phase, Western blotting was used to determine the expression levels of cell cycle-regulating proteins including P21 and CDK4. P21 protein expression was markedly higher than that of the control group at all CIE doses tested. In contrast, CDK4 levels were significantly lower than that of the control group, as shown in Figure 8A and B. The results suggested that CIE could arrest the cell cycle via upregulation of P21 and downregulation of CDK4.

Ethnopharmacology used in folk medicine continues to be an important source of discovery and development of novel therapeutic agents in cancer. The flowers of Chrysanthemum indicum, a Compositae plant, is common in ethnopharmacology, and has long had wide spread use in the treatment of hypertension, colitis, pneumonia and carbuncles by traditional Chinese practitioners. Recently, much attention has been devoted to the anticancer activity of Chrysanthemum indicum, especially in hepatocellular carcinoma (HCC). However, the underlying mechanisms of the pharmacological effect of the plant extract in cancer therapy have been largely undetermined.

Induction of apoptosis and arrest of the cell cycle by plant extracts has become a principal mechanism by which anticancer therapy is effective. Apoptosis triggered by the activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. Permeabilization of the outside mitochondrial membrane plays a vital role in cell apoptosis, during which loss of the mitochondrial membrane potential and release of cytochrome C into the cytosol, followed by caspase-9-dependent activation of caspase-3 occurs, resulting in apoptosis. Dysregulation of the cell cycle mechanism has also been shown to perform an important function in growth of various cancer cells, including HCC. The S phase is associated with DNA synthesis and plays a crucial role in cell cycle progression. One of the CDKIs, P21, can influence key functions in the control of the cell cycle by negatively regulating CDK4 activity, and plays an important role in regulation of the S phase of the cell cycle.

So far, there has been no evidence found to show that the mitochondrial pathway is involved in induction of apoptosis and cell cycle arrest by Chrysanthemum indicum extract (CIE) in human HCC cells. Therefore, the present study examined the anticancer activities of CIE and related mechanisms in MHCC97H cell lines. The data showed that CIE could induce apoptosis and arrest the cell cycle of MHCC97H cells. CIE exerted a significant apoptotic effect on MHCC97H cells through the mitochondrial-dependent caspase-3 pathway, and arrested the cell cycle in the S phase in cancer cells by upregulation of P21 and downregulation of CDK4.

This study suggests that Chrysanthemum indicum could be a promising plant with potential in the novel treatment of human cancer, particularly HCC.

The manuscript written by Li ZF et al describes that Chrysanthemum indicum extract can induce apoptosis and cell cycle arrest in a hepatoma cell line. Many patients with HCC still die each year, and novel therapeutic strategies are needed. The data are encouraging and promising.