|Anti-proliferative and Pro-apoptotic Effect of Smilax glabra Roxb. Extract on Hepatoma Cell Lines
Fei Saa, Jian-Li Gaoa, Kwok-Pui Fungb,c, Ying Zhenga**, Simon Ming-Yuen Lee a,c*, Yi-Tao Wang a
Affiliation and address:
a Institute of Chinese Medicine Sciences, University of Macau, Av. Padre Tomás Pereira S.J., Taipa, Macao, China
b Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China
c Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China
*Corresponding author. Dr. Simon Ming Yuen Lee, Tel.: +853 397 4695; fax: +853 841 358; E-mail addresses: firstname.lastname@example.org
**Corresponding author. Dr. Ying Zheng, Tel.: +853 397 4687; fax: +853 841 358; E-mail addresses: email@example.com
Running title: Anti-proliferative effect of Smilax glabra Roxb. Extract
KEY WORDS: Smilax glabra Roxb., apoptosis, HepG2, Hep3B, caspase, mitochondrial, cytochrome c, p38, ERK, JNK
CBA, cytometric bead array; ∆Ψm, change of mitochondrial transmembrane potential; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; NMR, nuclear magnetic resonance; MAPK, mitogen-activated protein kinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; MS, mass spectrometry; IC50, 50% of inhibitory concentration; JNK, c-Jun N-terminal kinase; PARP, poly (ADP-ribose) polymerase, UV, ultraviolet
Smilax glabra Roxb. (SGR) is the root of a traditional Chinese herb, referred to as tu fu ling in Chinese medicine. It is an inexpensive traditional Chinese medicine commonly used for treatment of liver diseases, and a few studies have indicated that SGR has anti-hepatocarcinogeneic and anti-cancer growth activities. In the current study, raw SGR plant was extracted with Accelerate Solvent Extractor, and the molecular mechanism by which S. glabra Roxb. extract (SGRE) has an anti-proliferative effect on the human hepatoma cell lines, HepG2 and Hep3B, was determined. We showed that SGRE inhibited HepG2 and Hep3B cell growth by causing cell cycle arrest at either S phase or S/G2 transition and induced apoptosis, as evidenced by a DNA fragmentation assay. SGRE induced apoptosis by alternation of mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly ADP-ribose polymerase. The SGRE-mediated mitochondria-caspase dependent apoptotic pathway also involved activation of p38, JNK, and ERK mitogen-activated protein kinase signaling. Isometric compounds of astilbin (flavonoids) and smilagenin (saponin) have been identified as the main chemical constituents in SGRE by HPLC-MS/MS. These results have identified, for the first time, the biological activity of SGRE in HepG2 and Hep3B cells and should lead to further development of SGR for liver disease therapy.
Keywords: Smilax glabra Roxb., liver cancer, apoptosis
Hepatocellular carcinoma is the fifth most commonly diagnosed cancer, with more than 1 million deaths reported annually worldwide . Apoptosis has been characterized as a fundamental cellular activity that maintains the physiological balance of organisms. It also plays a critical role as a protective mechanism against carcinogenesis by eliminating damaged or abnormally excessive cells induced by various carcinogens . Emerging evidence has demonstrated that the anti-cancer activities of certain chemotherapeutic agents are involved in the induction of apoptosis, which is regarded as the preferred way to manage cancer [2, 3]. In order to develop an effective means for the prevention and treatment of hepatocellular carcinoma and related liver diseases, we have isolated and identified several chemical extracts and pure compounds from Chinese medicine with anti-hepatoma and anti-liver disease effects [4-7].
SGR, a member of the Smilacaceae family and a rhizome of the Liliaceae plant, is referred to as tu fu ling in Chinese medicine. It is a crude drug used in many traditional prescriptions in Asia. It is inexpensive with a low toxicity to organs, but may be highly advantageous in its long-term use for chronic inflammatory diseases such as rheumatoid arthritis and hepatitis . Traditional Chinese medicinal uses of SGR have included the dissipation of heat, resolution of toxins, elimination of moisture, and promotion of mobility to the joints. It is commonly used clinically to prevent leptospirosis, and to treat syphilis, acute bacterial dysentery, acute and chronic nephritis, mercury poisoning, and rheumatoid arthritis . Moreover, it has been used in the preparation of traditional medications administered to cancer patients in Thailand . Previous investigators have demonstrated that SGR can inhibit cell proliferation of the squamous cell carcinoma cell line, JTC226, and the inhibitory rate was approximately 90% . Moreover, the saponins of SGR can inhibit the growth of EAC, S180, and H22 cells . Recent in vivo investigations have demonstrated that extract prepared from SGR, N. sativa seeds, and H. indicus roots, as used in Sri Lanka, can offer significant protection against diethylnitrosamine-induced hepatocarcinogenic changes in rats [13, 14]. Furthermore, the effect of this preparation on HepG2 cells, a human hepatoma cell line, has been investigated in vitro, and the extract possesses direct cytotoxic activity that contributes to the anti-hepatocarcinogenic effects . These studies indicate that SGR has potential protective effects against hepatocarcinogenesis. In this study, we isolated chemical extracts from SGR (SGRE) and attempted to determine the molecular mechanism of action of the inhibitory effect in human hepatoma HepG2 and Hep3B cells.
Materials and Methods
Chemicals and cell culture
SGR was purchased from the Zhuhai Medicine Company (Zhuhai, China). HPLC grade-methanol, -ethanol, and -acetonitrile were purchased from Merck (Darmstadt, Germany). Deionized Milli-Q water was used in all experiments (Millipore, Mill-Q & Rios Systems, Bedford, MA).
The human hepatoma cell lines, HepG2 and Hep3B (containing hepatitis B virus genome), were purchased from the American Type Culture Collection (ATCC, VA). Cell culture medium was purchased from Invitrogen (Guangzhou, China). Hep3B cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 µg/mL of streptomycin, and 100 unit/mL of penicillin in 75 cm2 tissue culture flasks in a humidified incubator at 37 ˚C with 5% CO2. HepG2 cells were cultured in RPMI medium supplemented with 10% (v/v) FBS, 100 µg/mL of streptomycin, and 100 unit/mL of penicillin in 75 cm2 tissue culture flasks in a humidified incubator at 37 ˚C with 5% CO2.
Preparation of plant extracts
Two hundred grams of SGR were prepared from a methanol extract with Accelerate Solvent Extractor (ASE200, Dionex, CA) at 1500 psi and 60 ˚C. The resulting extract was concentrated by rotary evaporation, dissolved in Milli-Q water, and then filtered with a 0.45 μm Millipore filter unit. The same volume of petroleum ether was added to the extract for double phase extraction for 12 h. The petroleum ether layer was discarded, and 5 times volume of 99% alcohol (EtOH) was added. After 2 h, the sediment was discarded and the final extract (i.e., SGRE) was freeze-dried to a powder form, 6.442 g in weight. The freeze-dried extract was used in both chemical analysis and pharmacological studies. The extract was dissolved in PBS containing 1% DMSO to give stock solutions of 100 mg/mL.
UV-VIS spectral methods for qualitative and quantitative analyses of SGRE
The methanol solution of the SGRE was scanned; the UV spectrum exhibited an absorption maxima at 280 nm (band II) and 300-330 nm (band I); characteristic absorption bands of a dihydroflavonol skeleton. To investigate the contents of the total flavonoids in the extract, AlCl3 colorimetry [16, 17] was used and rutin (Sigma, MO) was used as a reference chemical standard. As a result, the total flavonoid content in the extract was found to be 39.37 mg/g.
HPLC instrumentation and conditions
An Agilent 1100 series LC/MSD VL trap system (Agilent Technologies, Palo Alto, CA) was used for sample analysis. An Agilent ZORBAX Eclipse XDB-C18 (150 mm × 4.6 mm, I.D.; 3.5 μm, particle size) column was used (Agilent). The separation was achieved using a gradient elution with Milli-Q water (a) and acetonitrile (b) as follows: 83–82% (a) for 0-10 min, 82–78% (a) for 10-15 min, 78–75% (a) for 15-16 min, 75–55% (a) for 16-35 min, 55–25% (a) for 35-45 min, 25–0% (a) for 45-50 min, and 100–100% (b) for the final 10 min. The flow rate was 0.6 mL/min, the column temperature was set at 35 ˚C, and the detection wavelength was 330 nm.
An Agilent trap mass spectrometer was interfaced to the HPLC system with an electrospray ionization (ESI) source. The sample was detected in both the positive and negative ion modes, and scanned from m/z 150-1000. The capillary voltage was set at -4 kV, the gas used for drying and spraying was nitrogen, the nebulizer pressure was 50 psi, the flow rate of dry gas was 11 L/min, the dry gas temperature was 350 ˚C, the collision energy was set at 1.5 V, and the compound stability and trap drive level were set at 80%. The HPLC-MS data were acquired using the program, Data Analysis for LC/MSD Trap, version 3.2 (Build 121), which corresponded with LC/MSD Trap software 5.2 (Build 382).
Cell viability assay
Cells were seeded in 96-well microplates (2 104 cells/well in 100 µl of medium). SGRE was added to the cells in serial concentrations and incubated for 24, 48, and 72 h. Medium was discarded before 30 l of tetrazolium dye (MTT) solution (5 mg/mL in PBS) was added to each well and incubated for an additional 4 h. DMSO (100 l) was added to dissolve the formed formazan crystals. The plate was then read in a microplate reader (1420 Multilabel counter victor3, Perkin Elmer, MA) at 570 nm. MTT solution with DMSO (without cells and medium) was used as a blank control.
Cell cycle analysis
Cells were seeded in 6-well plates and incubated with 0.6 mg/mL of SGRE in a humidified incubator (37 ˚C with 5% CO2) for 24, 48, and 72 h, respectively. The adherent cells were washed with PBS, and then 300 µl of trypsin was incubated with cells for 5 min at room temperature to collect the cells. After centrifugation at 350 g for 5 min at 4 ˚C, the cell pellet was obtained. The cell pellet was then resuspended with 1 mL of cold 70% EtOH at 4 ˚C for 12 h. The cell pellet was collected again by centrifugation at 350 g for 5 min at 4 ˚C. Finally, 1 mL of propidium iodide (PI) stain solution (20 µg/mL of PI and 8 µg/mL of DNase-free RNase) was added to the samples, and the samples were then analyzed by flow cytometry (BD FACS CantoTM, Franklin Lakes, NJ). The results were analyzed with Mod Fit LT 3.0 software.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
The TUNEL assay was performed according to the manufacturer’s instructions (Apo-BrdUTM TUNEL Assay Kit, Molecular Probes, Leiden, Netherlands). Cells were fixed with 1% paraformaldehyde (PFA) in PBS on ice for 15 min. For a further fixation step, 70% EtOH was added and cells were kept on ice for 10 min. After 3 h of labeling at 37 ˚C with the DNA-labeling solution, cells were incubated with Alexa Fluor 488 conjugated anti-BrdU antibodies for 30 min at room temperature. Cells were analyzed by flow cytometry and were mounted on slides. The morphology of the cells was observed with a fluorescent microscope (Axiovert 200, Carl Zeiss, Thornwood, NY) mounted with a camera (Carl Zeiss AxioCam HRc, Carl Zeiss).
Analysis of mitochondrial membrane potential
Mitochondrial injury was assessed by JC-1 dye, a Mitochondrial Potential Sensors (Molecular Probes). Red fluorescence of the J-aggregate form of JC-1 indicates intact mitochondria, whereas green fluorescence shows a monomeric form of JC-1 that is due to the breakdown of the mitochondrial membrane potential. Cells were seeded in 6-well plates for 6 h. The medium of each well was discarded and treated with 1 mL of medium (5 mg/mL JC-1) for 15 min at 37 ˚C and 5% CO2 in the dark, then washed twice in PBS and serial concentrations of SGRE, and re-incubated in a humidified incubator (37 ˚C with 5% CO2 ) for 24 h. The cells were collected and centrifuged, the cell pellet re-suspended in 1 mL of medium, measured by flow cytometry and mounted on slides. The morphology of the cells was observed under a fluorescent microscope.
Cytochrome c labeling
Cytochrome c-release was assessed by a SelectFX Alexa Fluor 488 Cytochrome C Apoptosis Detection Kit (Molecular Probes). Cells were seeded in 24-well plates and treated with SGRE for 8 h. The media was discarded and the cells were washed with warm PBS, fixed with fresh 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 and incubated in a blocking buffer (10% heat-inactivated normal goat serum) for 30 min at room temperature. The cells were then incubated for 1 h with 1 µg/mL primary antibody (anti-cytochrome c mouse IgG, Molecular Probes) at room temperature. Green fluorescence was observed with a fluorescent microscope.
Active caspase-3 protein and poly ADP-ribose polymerase (PARP) levels
The BD™ CBA Human Apoptosis Kit (BD, NJ) was used to quantify the active caspase-3 and PARP protein levels. Cytometric Bead Array (CBA) employs a particle with a discrete fluorescence intensity to detect a soluble analyte. This kit provides 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 a 6-well plate and incubated with desired concentrations of SGRE in a humidified incubator (37 ˚C with 5% CO2) for 24 h. Cells were harvested and washed with PBS. The cells of each sample were counted to 1.0 106 and 50 µl of cell lysis buffer was added to each sample for 30 min on ice and vortexed at 10 min intervals. Cellular debris was pelleted by centrifugation at 12,500 rpm for 10 min. The protein concentrations of all samples were measured with a 2-D Quant Kit (Amersham Biosciences, Piscataway, NJ). Each sample was normalized in a final concentration of 0.2 µg/µl. Thirteen standard curves (standards ranging from 0 to 6000 unit/mL) were obtained from one set of calibrators. For each sample and the standard mixture of lysate standard (caspase-3 and PARP beads), 50 µl of a sample or a standard of beads was added to the mixture of 50 µl of 2 mixed capture beads, incubated for 1 h, and then mixed with 50 mL of PE detector beads for an additional 1 h. After that, samples were washed before data acquisition by flow cytometry. The results were analyzed by FCAP Array, version 1.0.
Expression of phospho-ERK1/2, -JNK1/2, and -p38
Expression of ERK1/2, JNK1/2, and p38 were measured by a Cell Signaling Master Buffer Kit (BD). Three types of beads in a CBA provided a capture surface for phospho-ERK1/2, -JNK1/2, and -p38 proteins. Cells were seeded in a 6-well plate and incubated with the desired concentrations of SGRE in a humidified incubator (37 ˚C with 5% CO2) for the specific times, as indicated. Cells were harvested and washed with PBS. Cells of each sample were counted to 1.0 106 and 50 µl of cell-denaturation buffer was added to each sample and immediately placed in a boiling water bath for 5 min. Cellular debris was pelleted by centrifugation at 10,000 g for 5 min. The protein concentrations of all samples were measured by a 2-D Quant Kit (Amersham Biosciences). Each sample was normalized to a final concentration of 0.2 µg/µl. Standard curve (standards ranging from 0 to 1000 pg/mL) were obtained from one set of calibrators. For each sample and the standard mixture of lysate standard (phospho-ERK1/2, -JNK1/2, and -p38 beads), 50 µl of sample or standard beads was added to the mixture of 50 mL of three mixed capture beads, and 50 µl of PE detector beads were incubated for 4 h. After that, samples were washed before data acquisition with flow cytometry. The results were analyzed by FCAP Array, version 1.0.
The data are expressed as the mean ± SD from at least three independent experiments. Differences between groups were analyzed using a Student’s t-test.
The typical HPLC chromatogram and total ion chromatogram (TIC) of SGRE are shown in Fig. 1A and B. The MS/MS spectra of the main peaks in the chromatogram are shown in Fig. 1a-d. Compounds 1, 2, and 3, indicated in Fig. 1A and B, had the same molecular weight of approximately 450 m/z (Fig. 1 a and b) and were predicted to be isomeric compounds of Astilbin [18, 19] (Fig. 1C) which structure was classified as dihydroflavonol. In addition, compound 4 was predicted to be a glycoside of a steroid, namely smilagenin (MW = 416.6), and the chemical structure of its’ aglycone was shown in Fig.1D.
Cell viability assay
The growth of the HepG2 and Hep3B cells in the presence of various concentrations of SGRE was examined. Under the experimental conditions (24 and 48 h), SGRE exhibited a marked growth inhibitory effect on both HepG2 and Hep3B cells. The IC50 for HepG2 and Hep3B cells ranged from approximately 1.8 mg/mL (24 h) to 1.1 mg/mL (48 h) and from 2.6 mg/mL (24 h) to 0.8 mg/mL (48 h), respectively (Fig. 2). Both inhibitory curves of SGRE exposure to HepG2 and Hep3B cells were similar, suggesting a parallel effect of cell death.
Cell cycle analysis
The effect of different concentrations of SGRE on cell-cycle progression of HepG2 and Hep3B cells was studied after 24, 48 and 72 h of drug exposure. Treatment of cells with 0.6 mg/mL SGRE led to profound changes in the cell cycle profiles after incubation of up to 72 h (Fig. 3A and B). SGRE treatment resulted in a time-dependent significant accumulation of cells in S phase with concomitant losses from G1 phase (Fig. 3A and B). These results could be explained by the hypothesis in which S-phase accumulation is suggested to be caused by a decrease in the progression through the cell cycle or an inhibition of S/G2 phase transition. These results suggested an anti-proliferative effect of SGRE on cells and possible induction of cell cycle arrest at the either S phase or S/G2 transition.
Apoptosis of SGRE treated Hep3B cells was further examined with the TUNEL assay at 24, 48, and 72 h of treatment. The nuclei of the treated cells demonstrated nuclear shrinkage and condensed chromatin, which was consistent with the morphological hallmark of an apoptotic nucleus (Fig. 4A). A significant number of cells containing DNA strand breaks were found after treatment with 1.0 mg/mL of SGRE for 48 h. The green fluorescence intensity indicated that the quantity of apoptotic cells of the 1.0 mg/mL treatment group was approximately 10 times higher than the drug-free cells. After treatment for 72 h, the green fluorescence intensity was approximately 20 times higher than that of the control cells (Fig. 4B). This result revealed a time-dependent increase in the quantity of apoptotic Hep3B cells.
Some chemotherapeutic drugs induce apoptosis via mitochondrial pathways by altering the mitochondrial transmembrane potential, ∆Ψm. We used a JC-1 probe to detect the ∆Ψm after cells were treated with 0.5 mg/mL of SGRE for 6 h. Mitochondria with normal Ψm concentrates JC-1 into aggregates (red/orange fluorescence), while in depolarized mitochondria, JC-1 forms monomers (green fluorescence). As compared to untreated (control) cells, the red/orange fluorescence decreased by 89% in HepG2 cells (Fig. 5) and by 47% in Hep3B cells (data not shown) after exposure to SGRE under the same condition.
Cytochrome c labeling
Cytochrome c released from the mitochondria to the cytosol is implicated in mitochondria dependent apoptosis. Cytochrome c staining in the cytosol of both HepG2 and Hep3B cells showed markedly stronger levels of green fluorescence than that of control cells after 6 h of treatment with 1.0 mg/mL SGRE (Fig. 6). SGRE treated cells showed obvious punctate green fluorescence staining or appeared to have green fluorescence accumulated in large aggregates compared to control cells (Fig. 6).
Expression of active caspase-3 protein and PARP levels
In drug-induced cell death via apoptosis, 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. Active caspase-3 protein and PARP cleavage protein levels were measured by the BD™ CBA Human Apoptosis Kit. All the samples were counted to 1.0 106 cells and normalized to a final protein concentration of 0.2 µg/µl. The results were analyzed by FCAP Array, version 1.0, and are shown in Fig. 7. Our results demonstrated that there was a significant dose-dependent increase in protein levels of cleaved PARP and active caspase-3 in both HepG2 and Hep3B cells (Fig. 7). Both results indicated that SGRE induced apoptosis via the caspase-dependent pathway.
Cell signaling cascades of mitogen-activated protein kinases (MAPKs)
In order to evaluate if SGRE induced the HepG2 cell death through activation of the cell signaling cascades of mitogen-activated protein kinases (MAPKs), we assessed the kinetics of p38 MAPK, ERK1/2 and JNK phosphorylation simultaneously by BD™ CBA Cell Signaling Flex Set system. All samples were counted to 1.0 106 cells and the protein concentrations were normalized to 0.2 µg/µl. The results were analyzed by FCAP Array, version 1.0, and are shown in Fig. 8. Results showed that the expression levels of phosphorylated forms of p38, JNK1/2, and ERK1/2 increased significantly and dose-dependently in SGRE-induced HepG2 and Hep3B cell death.
The underlying mechanism of the pharmacological action of SGR in cancer therapy is still largely unclear. In the present study, SGRE has been identified to inhibit HepG2 and Hep3B cell growth by inducing apoptosis, as evidenced by activation of the depolarization of the mitochondrial transmembrane potential, mitochondrial cytochrome-c release, caspase-3 and PARP cleavage, S/G2 cell cycle arrest, and finally, DNA fragmentation.
Cell cycle progression was arrested in S/G2 phase in SGRE treated hepatoma cells. The results indicated that there was a modulation of events at the S and G2 checkpoints, which may provide an opportunity to enhance SGRE-induced cytotoxicity in hepatoma cells. The results indicated that SGRE can be used to sensitize malignant cells to drugs that destabilize DNA during replication, such as methotrexate (MTX), which is a cell cycle-specific (S phase) chemotherapeutic agent that is currently used to treat human osteosarcoma .
It has been reported that the induction of cell detachment is a prerequisite for the activation of caspase-3 in an apoptosis execution process [21, 22]. The quantity of active caspase-3 protein (Fig. 7A and B) increased after SGRE treatment. Also, our results showed that caspase-3 activity was dramatically increased after treatment with 1.0 mg/mL of SGRE for 24 h; the activity was 2.8 times higher in HepG2 cells than drug-free (control) cells, and 4.5 times higher in Hep3B cells, as measured by the EnzChek Caspase-3 Assay Kit (data not shown). We demonstrated here that the caspase-3 protein is critically involved in SGRE–inducing apoptosis. PARP is a 116-kDa nuclear protein involved in DNA repair, and a well-characterized substrate for caspase-3. Activated caspase-3 cleaves PARP, generating 89- and 24-kDa inactive fragments . Fig. 7C and D indicated that the amount of cleaved PARP increased depending on the SGRE concentration and the quantity of active caspase-3. These results suggested that the caspase-3 protein is critically involved in SGRE-induced apoptosis. Taken together, a mitochondrial-dependent caspase-3 pathway may be involved in SGRE-induced apoptosis of hepatoma cells.
The MAPK superfamily consists of three serine/threonine kinase cascades . ERKs respond to growth factors or other external mitogenic signals by promoting cell proliferation and opposing cell death signals. p38 and JNK are typically described as stress-activated kinases that promote programmed cell death , and it is now widely accepted as a simplified scheme that p38 and JNK mediate apoptotic signals. We thus wondered about the involvement of these pathways in the SGRE-induced apoptosis of hepatoma cells. Phospho-p38 and -JNK1/2 were activated dose dependently after SGRE treatment (Fig. 8 C-F). Our results support the hypothesis that p38 and JNK1/2 are activated in the process of SGRE -induced apoptosis in human hepatoma cells (HepG2 and Hep3B).
ERK promotes growth, differentiation and proliferation, and its activation alone does not fully reflect the complex biology of cancer cells, especially in clinical material . Wang et al.  showed that after treatment, apoptosis was linked to ERK activation in the HeLa cervical cancer cell line, whereas its resistant variants, Hela-R1 and Hela-R2, established by a continuous exposure to increasing concentrations of cisplatin, showed a reduced activation of ERK . However, the pattern of ERK activation seems to be complex, as it was reported to be biphasic in some kinetic studies [29-31]. Therefore, substantial evidence suggests that the activation of the ERK pathway increased the cell death threshold in an unknown way. The expression of phospho-ERK1/2 was up-regulated by SGRE-induced apoptosis in our experiment. Interestingly, in spite of a similar trend of up-regulated protein expression levels of phospho-ERK1/2, -JNK1/2, and -p38 in both cell lines, the activation profile of these three kinases was shown to be different in response to SGRE.
In this study, SGRE, a standardized chemical extract, has been analyzed by UV spectrophotometry and HPLC-MS/MS. Flavonoids (mainly isometric compounds of astilbin) and saponins (e.g. smilagenin) have been identified as the main chemical constituents. Recent investigations have shown that the flavonoids of SGR are the major active components which have immunomodulating effects . Previous reports have shown that astilbin, isolated from SGR, possesses anti-inflammatory and pain relief activities . In addition, the Smilax steroids, sarsasapogenin and smilagenin, have been reported to be active components in the treatment of senile dementia, cognitive dysfunction, and Alzheimer's disease. They improve memory by elevating the low muscarinic acetylcholine receptor density in brains of memory-deficient rat models . In addition, the saponins of SGR have been reported to facilitate the body's absorption of other drugs and phytochemicals. In short, our study suggested that the SGRE containing mainly flavonoids and saponins induced apoptosis in HepG2 and Hep3B cells through activation of the mitochondrial and caspase-3-pathways, which involved activation of p38, ERK1/2 and JNK1/2 MAPK signaling cascades.
We thank Sandy Lao, Leon Lai, Chi Weng Leong, Emilia Conceição Leong, Hio Wa Lam, and Xiao Yu for their participation in the preliminary experiments. This study was supported by grants from the Research Committee, University of Macau, Macao SAR (Ref. No.: RG054/05-06S and RG058/05-06S) and grants from the Science and Technology Development Fund, Macao SAR ((Ref. No.: 078/2005/A2 and 012/2006/A).
 Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ, Cancer statistics. CA Cancer J. Clin. 55: 10-30, 2005
 Schuchmann M, Galle PR, Sensitizing to apoptosis-sharpening the medical sword. J. Hepatol. 40: 335-336, 2004
 Ma Y, Hendershot LM, The role of the unfolded protein response in tumour development: friend or foe? Nat. Rev. Cancer. 4: 966–977, 2004
 Lam WY, Leung KT, Law PT, Lee SM, Chan HL, Fung KP, Ooi VE, Waye MM, Antiviral effect of phyllanthus nanus ethanolic extract against hepatitis B virus (HBV) by expression microarray analysis. J. Cell. Biochem. 97: 795-812, 2006
 Lee SM, Li ML, Tse YC, Leung SC, Lee MM, Tsui SK, Fung KP, Lee CY, Waye MM, Paeoniae Radix, a Chinese herbal extract, inhibit hepatoma cells growth by inducing apoptosis in a p53 independent pathway. Life Sci. 71: 2267-77, 2002
 Xiao Y, Yang FQ, Li SP, Gao JL, Hu G, Lao SC, Leong C E, Fung KP, Wang YT, Lee SM , Furanodiene induces G2/M cell cycle arrest and apoptosis through MAPK signaling and mitochondria-caspase pathway in human hepatocellular carcinoma cells. Cancer Biol. Ther. 2007 (In Press)
 Tang PM, Chan JY, Au SW, Kong SK, Tsui SK, Waye MM, Mak TC, Fong WP, Fung KP, Pheophorbide A, an active compound isolated from scutellaria barbata, possesses photodynamic activities by inducing apoptosis in human hepatocellular carcinoma. Cancer Biol. Ther. 5: 1111-1116, 2006
 Jiang J, Xu Q, Immunomodulatory activity of the aqueous extract from rhizome of Smilax glabra in the later phase of adjuvant-induced arthritis in rats. J. Ethnopharmacol. 85: 53–59, 2003
 Pharmacopoeia Commission of PRC (Editors), Pharmacopoeia of the People’s Republic of China, Vol. I. Chemical Industry Press, Beijing, PR China: 14-15, 2005
 Itharat A, Houghton PJ, Eno-Amooquayec E, Burke PJ, Sampsonb JH, Ramanb A, In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer. J. Ethnopharmacol. 90: 33–38, 2004
 LI GX, Pharmacology Toxicity and Clinical Uses of Chinese Medicine, Tianjing Technological Translation Publishing Inc., Tianjing, China: 42, 1992
 You GQ, Xu LH, Lin JN, Zhang M, The anti-tumor research of total saponins in Smilax glabra. J. The Pharmacology and Clinical Chinese Medicine. 17: 14-15, 2001
 Iddamaldeniya SS, Wickramasinghe N, Thabrew I, Ratnatunge N, Thammitiyagodage MG, Protection against diethylnitrosoamine-induced hepatocarcinogenesis by an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra: a preliminary study. J. Carcinog. 2: 1-6, 2003
 Iddamaldeniya SS, Thabrew MI, Wickramasinghe SM, Ratnatunge N, Thammitiyagodage MG, A long-term investigation of the anti-hepatocarcinogenic potential of an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra. J. Carcinog. 9: 5-11 2006
 Thabrew MI, Mitry RR., Morsy MA, Hughesb RD, Cytotoxic effects of a decoction of Nigella sativa, Hemidesmus indicus and Smilax glabra on human hepatoma HepG2 cells. Life Sci. 77: 1319–1330, 2005
 Gardner PT, McPhail DB, Crozier A, Duthie GC, Electron spin resonance (ESR) spectroscopic assessment of the contribution of quercetin and other flavonols to the antioxidant capacity of red wines. J. Agric. Food Chem. 79: 1011-1014, 1999
 Galvez M, Martin CC, Houghton PJ, Ayuso MJ, Antioxidant activity of methanol extracts obtained from Plantago species. J. Agric. Food Chem. 53: 1927-1933, 2005
 Yuan JZ, Don DQ, Chen YJ, Li W, Kazuo K, Tamotsu N, Yao XS, Study on dihydroflavonol glycosides from Rhizome of Smilax glabra. J. China J. Chin. Mater. Med. 29: 867-870, 2004
 Chen T, Li JX, Cai Y, Xu Q, A flavonol glycoside from Smilax glabra. J. Chin. Chem. Lett. 13: 537 – 538, 2002
 Alleva R, Benassi MS, Pazzaglia L, Tomasetti M, Gellert N, Borghi B, Neuzil J, Piccib P, α-Tocopheryl succinate alters cell cycle distribution sensitizing human osteosarcoma cells to methotrexate-induced apoptosis. Cancer Letts. 232: 226–235, 2006
 Rytomaa M, Martins LM, Downward J, Involvement of FADD and caspase-8 signaling in detachment-induced apoptosis. Curr. Biol. 9: 1043–1046, 1999
 Puthalakath H, Villunger A, O’Reilly LA, Beaumont JG, Coultas L, Cheney RE, Huang DCS, Strasser A, Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis. Science. 293: 1829– 1832, 2001
 Amano H, Yamagiwa M, Akao T, Mizuki E, Ohba M, Sakai H, A novel 29-kDa crystal protein from Bacillus thuringiensis induces caspase activation and cell death of jurkat T Cells. Biosci. Biotechnol. Biochem. 69: 2063-2072, 2005
 Chang L, Karin M, Mammalian MAP kinase signaling cascades. Nature. 410: 37–40, 2001
 Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME, Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 270: 1326–1331, 1995
 Davidson B, Konstantinovsky S, Kleinberg L, Nguyen MTP, Bassarova A, Kvalheim G, Nesland JM, Reich R, The mitogen-activated protein kinases (MAPK) p38 and JNK are markers of tumor progression in breast carcinoma. Gynecol. Oncol. 20: 1-9, 2006
 Chu G, Chang E, Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA. Proc. Natl. Acad. Sci. USA. 87: 3324– 3327, 1990
 Wang X, Martindale JL, Holbrook NJ, Requirement for ERK activation in cisplatin-induced apoptosis. J. Biol. Chem. 275: 39433–39445, 2000
 Hayakawa J, Ohmichi M, Kurachi H, Ikegami H, Kimura A, Matsuoka T, Jikihara H, Mercola D, Murata Y, Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell line. J. Biol. Chem. 274: 31648–31654, 1999
 Mansouri A, Ridgway LD, Korapati AL, Zhang Q, Tian L, Wang Y, Siddik ZH, Mills GB, Claret FX, Sustained activation of JNK/p38 MAPK pathways in response to cisplatin leads to Fas ligand induction and cell death in ovarian carcinoma cells. J. Biol. Chem. 278: 19245–19256, 2003
 Schweyer S, Soruri A, Meschter O, Heintze A, Zschunke F, Miosge N, Thelen P, Schlott T, Radzun HJ, Fayyazi A, Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation. Br. J. Cancer. 91: 589– 598, 2004
 Chang BJ, The research of function as anti-inflammation, pain release and drain off water in Smilax glabra and astilbin. J. The Pharmacology and Clinical of Chinese Medicine. 20: 11-12, 2004
 Hu Y, Xia Z, Sun Q, Orsi A, Rees D, A new approach to the pharmacological regulation of memory: Sarsasapogenin improves memory by elevating the low muscarinic acetylcholine receptor density in brains of memory-deficit rat models. Brain Res. 1060: 26-39, 2005
Fig.1. HPLC chromatogram, total ion chromatogram, and mass/mass spectra of the SGRE. (A) The HPLC chromatogram of SGRE at 330 nm. (B) The total ion chromatogram of the SGRE in the negative mode (most of the major peaks were marked in the chromatograms); (a) the mass spectra of compounds 1, 2, and 3 in the positive mode; (b) the mass spectra of compounds 1, 2, and 3 in the negative mode; (c) the mass spectrum of compound 4 in the positive mode; and (d) the mass spectrum of compound 4 in the negative mode. (C) Predicted chemical structure of compounds 1, 2 and 3, belong to isomeric compounds of astilbin. (D) Predicted chemical structure of the aglycone of compound 4 (smilagenin).
Fig.2. HepG2 and Hep3B cells were treated with drug-free medium or medium containing different concentrations of SGRE for 24 or 48 h. Cell growth was determined by MTT assay and was directly proportional to the absorbance at a wavelength of 570 nm. Error bars represent the mean ± SEM (n=16).
Fig.3. Effect of SGRE on the cell-cycle distribution of HepG2 and Hep3B cells. (A) Flow cytometric analysis of PI-stained HepG2 and Hep3B cells treated with 0.6 mg/mL SGRE for 24, 48, and 72 h. The x-axis represents fluorescent intensity on a logarithmic scale, whereas the y-axis represents the number of events. (B) The results were analyzed by Mod Fit LT 3.0. Columns, mean of three independent plates; bars, SD; the results were reproducible in three additional independent experiments. *, P < 0.05; $, P< 0.01; #, P< 0.001; P value compared with the control group (0 h).
Fig.4. (A) Morphological observation of TUNEL stained Hep3B cells by fluorescence microscopy. Hep3B cells were treated with medium alone (Control) or 1 mg/mL of SGRE for 24, 48, and 72 h. Photographs were taken at a magnification of 20 x. (B) The remaining cells were analyzed by flow cytometry. The x-axis indicates green fluorescence intensity on a logarithmic scale; the y-axis indicates the number of events.
Fig.5. Analysis of mitochondrial transmembrane potential in HepG2 cells. HepG2 cells were stained with JC-1 and treated with 0.5 mg/mL SGR for 6 h. The cells were photographed at a magnification of 20 x under bright field (A-B), red fluorescence (C-D) and green fluorescence (E-F).The remaining cells were analyzed by flow cytometry and revealed a loss of intact mitochondria transmembrane potential in treated (H) vs. untreated (G) cells. Representative density plots of green vs. red fluorescence are shown. Red fluorescence: intact mitochondrial potential; green fluorescence: breakdown of mitochondrial transmembrane potential.
Fig.6. Representative microscopic photos of cytochrome c immunostaining in HepG2 and Hep3B cells treated with 1.0 mg/mL SGRE. Cytochrome c immunofluorescence was observed with Oregon Green. HepG2 or Hep3B cells (B and D, respectively) exhibited higher cytochrome c immunostaining in the cytosol than the control cells (A and C). Similar results were obtained from three separate experiments (original magnification, 20 x).
Fig.7. Protein expression levels of active caspase-3 and cleaved PARP in SGRE-induced apoptosis. Both HepG2 and Hep3B cells were treated with medium alone (control) or different concentrations of SGRE for 24 h. Cells of each sample were counted to 1.0 106 and all the samples were normalized to a final protein concentration of 0.2 µg/µl. The results were analyzed by FCAP Array, version 1.0. Active caspase-3 expressions in HepG2 and Hep3B cells are shown (A and B, respectively). Cleaved PARP expression in HepG2 and Hep3B cells are presented (C and D, respectively). The x-axis indicates the concentration of SGRE. The y-axis indicates unit of proteins per mL. Columns, mean of three experiments; bars, SEM; *, P < 0.05; $, P< 0.01; #, P< 0.001; P value compared with a control group.
Fig.8. Protein expression levels of phospho-ERK1/2, -JNK1/2, and -p38 of SGRE-induced apoptosis. Both HepG2 and Hep3B cells were treated with medium alone (control) or different concentrations of SGRE for 24 h. All the samples were normalized to a final protein concentration of 0.2 µg/µl. The results were analyzed by FCAP Array, version 1.0. The phospho-ERK1/2, -JNK1/2, and -p38 expression in HepG2 and Hep3B cells are shown A- F, respectively. Columns, mean of three experiments; bars, SEM; *, P < 0.05; $, P< 0.01; #, P< 0.001; P value compared with the control group.