Rhein (98%) was purchased from Nanjing Qingze Medicine Ltd. (Nanjing, Jiangsu, China). Lysine was purchased from Beijing Solarbio Science and Technology Co. (Beijing, China). RHL was created in our department (Patent No. 200810089025.8). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich (Shanghai, China). The remaining chemicals were of standard analytical grade.

Human cervical carcinoma HeLa cell line was obtained from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, China. The HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Chemical Co., St. Louis, MO, USA), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

The cell proliferation assay was examined with the MTT method, according to the manufacturer’s instructions. Cells were seeded in 96-well plates (Costar, Cambridge, MA, USA) with 2,500 cells/well. Following overnight incubation, triplicate wells were treated with various concentrations of RHL for 48 h. Then, 20 μl MTT solutions (5 mg/ml in PBS) were added to each well and incubated for 4 h at 37°C. The MTT formazan was dissolved in 150 μl DMSO and absorbance was measured using a Microplate Reader (Multiskan MK3; Thermo Labsystem, USA) at a wavelength of 570 nm.

Cells were collected and resuspended in 200 μl binding buffer. Then, 10 μl FITC-labeled enhanced Annexin V (Baosai Biotechnology Ltd., Beijing, China) and 100 ng PI were added. Following incubation in the dark (15 min at room temperature or 30 min at 4°C), the samples were diluted with 300 μl binding buffer. Flow cytometry was carried out using a FACScan instrument (Becton-Dickinson) and the data were processed using WinMDI/PC-software.

Cells were harvested and washed with PBS solution. The whole cellular extracts were prepared by incubating cells on ice in lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1% Nonidet P-40, 0.1% SDS, protease inhibitors (1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin and 5 μg/ml pepstatin) and phosphatase inhibitors (20 mM β-glycerophosphate, 50 mM NaF and 1 mM Na₃VO₄). The cell lysates were cleared by centrifugation at 12,000 × g for 12 min. Protein concentrations were determined using the Bradford assay. Equal amounts of lysate (40 μg) were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked in TBST containing 5% non-fat skim milk at room temperature for 2 h and probed with primary antibodies overnight at 4°C. Membranes were then blotted with an appropriate horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Proteins were visualized using enhanced chemiluminescence Western blot detection reagents (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

The growth inhibitory effect of RHL on HeLa cells was examined using MTT assay. Cells were cultured for 48 h, with or without different concentrations of RHL. A decreased cell proliferation following treatment with various concentrations of RHL is shown (Fig. 1A). The IC₅₀ value of RHL for the HeLa cells was ~80 μM. The cells were cultured with RHL (50, 75, 100 and 125 μM) for the indicated times and analyzed with the MTT assay (Fig. 1B). HeLa cells treated with the indicated dose of RHL showed decreased cell proliferation in a time-dependent manner.

FITC-Annexin V/PI staining showed that RHL at 50 μM induced early apoptosis in HeLa cells (Fig. 2A). The ratio of early apoptosis was significantly enhanced when cells were incubated with 75 or 100 μM RHL for 48 h. To determine whether RHL-induced apoptosis is mediated by the activation of caspase and/or PARP, HeLa cells were treated with RHL at various concentrations for 48 h, RHL-induced caspase-3/7 and PARP cleavage in a dose-dependent manner (Fig. 2B). Taken together, these results strongly suggest that RHL triggers caspase-dependent apoptosis in HeLa cells.

MAPKs are a family of proteins that transduce signals from the cell membrane to the nucleus in response to a wide range of stimuli and regulate vital biological functions, including gene expression, mitosis, differentiation, apoptosis, proliferation and motility (12,13). Three major groups of MAPKs exist: JNK, p38 MAPK and ERK.

To investigate the molecular mechanisms underlying growth inhibition, the effect of RHL on the activities of MAPKs in HeLa cells was examined. As shown in Fig. 3, Western blot analysis revealed that RHL treatment increased the phosphorylation of JNK, p38 MAPK and ERK1/2 in a dose- and time-dependent manner in HeLa cells. These results suggest that RHL inhibits the growth of cervical cancer cells by modulating the activation of various MAPKs.

To further examine the role of various MAPKs in the inhibition of cervical cancer cell growth, pharmacological inhibitors of MAPKs (the JNK inhibitor SP600125, the p38 MAPK inhibitor SB203580 and the MEK inhibitor U0126) were used in combination with RHL.

The specificity of each inhibitor for various kinases on RHL-treated cells was assessed. Inhibitors were added and incubated for 1 h, followed by RHL treatment (75 μM) for 48 h. As shown in Fig. 4A, the specificity of each inhibitor was confirmed. Moreover, the cleavage of caspase-3/7 and PARP triggered by RHL was blocked by SP600125 and SB203580, whereas the cleavage of caspase-3/7 and PARP triggered by RHL was enhanced by U0126. As shown in Fig. 4B, in the absence of RHL, the inhibitors of JNK, p38 MAPK and MEK alone did not significantly alter proliferation in the HeLa cells. When the HeLa cells were treated with RHL in the presence of three inhibitors, respectively, the situation was altered. MTT results showed that inhibition of the p38 MAPK activity with SB203580 and inhibition of the JNK activity with SP600125 rescued RHL-mediated cell growth inhibition in HeLa cells, whereas the inhibition of ERK activity with U0126 enhanced RHL-induced growth inhibition. These results suggest that JNK and p38 MAPK play a partial role in mediating HeLa cell apoptosis triggered by RHL.