Printer Friendly

A novel polysaccharide, isolated from Angelica sinensis (Oliv.) Diels induces the apoptosis of cervical cancer HeLa cells through an intrinsic apoptotic pathway.

ABSTRACT

A novel polysaccharide isolated from Angelica sinensis, named APS-1d showed cytotoxic activity towards several cancer cell lines in vitro. However, the precise antitumor mechanisms of this compound are unknown. In this study, we investigated the pro-apoptotic effects of APS-1d in human cervical cancer HeLa cells both in vitro and in vivo, and further elucidated the mechanisms of this action. Inhibition of HeLa cell proliferation was determined by MTT assay and the therapeutic efficacy of APS-1d was evaluated by human cancer xenografts in nude mice. Cell apoptosis was examined with flow cytometry and TUNEL assay. The mechanism of action of APS-1d was investigated by Western blot analysis. APS-1d decreased HeLa cell proliferation in a concentration- and time-dependent manner in vitro. In addition, APS-1d significantly inhibited tumor growth in athymic nude mice. Characteristic manifestations of apoptosis including apoptotic morphological features and the sub- [G.sub.0]/[G.sub.1] peaks were observed when the cells were treated with APS-1d. Further analysis showed that APS-1d-induced apoptosis was associated with the regulation of Bcl-2 family protein expression, a decrease in the mitochondrial membrane potential, and an increase in the cytosolic cytochrome c levels. Sequentially, APS-1d increased the activities of caspase-9, -3, and poly (ADP-ribose) polymerase in a concentration-dependent manner, however, no obvious activation of Bid and caspase-8 was observed. Pretreatment with Z-LEHD-FMK, a specific inhibitor of caspase-9, significantly attenuated APS-1d-induced cell apoptosis, and activation of caspase-3. Taken together, our studies indicate that APS-1d is capable of inhibiting HeLa cell proliferation and inducing apoptosis in these cells which primarily involves the activation of the intrinsic mitochondrial pathway.

Keywords: Angelica sinensis Antitumor Apoptosis HeLa cell Polysaccharide

Introduction

Although the efficacy of chemotherapy for the majority of cancer types has improved over the last three decades, high toxic effects of chemotherapeutic drugs causing a severe reduction in quality of life are still formidable problems in clinical medicine (Rein and Kurbacher, 2001). Therefore, it is important to develop novel potent, but low toxic anti-cancer reagents, including natural products.

Since the discovery that Letinan, a polysaccharide from Lentinus edodes (Berk.) Sing inhibited mouse sarcoma 180 and displayed very low toxicity compared with chemical antitumor drugs (Chihara et al., 1969), a number of polysaccharides with antitumor activity have been reported, such as Panax ginseng, Coriolus versicolor and Agaricus blazei (Kobayashi et al., 2005; Nakazato et al., 1994; Shin et al., 2002). The root of Angelica sinensis (Oliv.) (Chinese Danggui), a well-known Chinese herbal medicine, has been used historically in gynecology for thousands of years (Sarker and Nahar, 2004). In the last few years, polysaccharides as one of the main compounds in Angelica sinensis have also attracted much attention (Cao et al., 2006a). Previous work has shown that polysaccharides inhibited tumor growth mainly through stimulation of humoral and cell-mediated immunity, so they were regarded as biological response modifiers (Zaidman et al., 2005). We and other investigators also revealed that the crude polysaccharide from Angelica sinensis possessed antitumor effects in mice transplanted with sarcoma 180, leukemia L1210 and Ehrlich ascitic cancer via activation of the host immune response (Shang et al., 2003).

Our group recently isolated several kinds of polysaccharides from Angelica sinensis, and studied their effects on cancer cells. Interestingly, a novel polysaccharide, named APS-1d having a backbone composed of (1,4)-[alpha]-D-glucopyranosyl (Glcp) residues, and branches composed of (1,6)-[alpha]-D-Glcp residues with a terminal [beta]-L-arabofuranose (Araf) residue exhibited significant anti-tumor effects in vitro, especially in human cervical cancer HeLa cells (Cao et al., 2006b). However, the precise molecular and cellular mechanisms remain unclear. An increasing number of reports have confirmed that polysaccharides or their complexes could have cytotoxic effects on various tumor cell lines in vitro, but are less toxic to normal cells. Moreover, it is reported that some kinds of polysaccharides with a backbone mainly composed of Glcp residues, such as Cladonia furcata polysaccharide and Maitake mushroom polysaccharide, could induce apoptosis in cancer cells (Fullerton et al., 2000; Lin et al., 2001). Therefore, we examined whether APS-1d has a similar apoptotic effect on HeLa cells.

Apoptosis is an energy-dependent type of programmed cell death. In general terms, apoptotic pathways can be sub-divided into two categories, the extrinsic pathway and the intrinsic pathway (Wajant, 2002). The extrinsic pathway is initiated by ligands engagement of cell surface receptors (Fas, TNF receptor, and TRAIL receptor) with their respective ligands (FasL, TNF, and TRAIL) to activate membrane-proximal caspases (caspase-8 and - 10) (Mehmet, 2000). The intrinsic pathway requires disruption of the mitochondrial membrane and the release of mitochondrial proteins, such as cytochrome c (Cyt c). Once Cyt c is in the cytosol, Cyt c together with Apaf-1 activates caspase-9, and the latter then activates caspase-3 (Desagher and Martinou, 2000).

Therefore, we conducted this study to elucidate the cellular mechanism of APS-1d on HeLa cells both in vivo and in vitro. Using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and flow cytometric analysis, APS-1d was found to induce apoptosis and also tended to induce S phase arrest in HeLa cells. In our research, APS-1d-induced apoptosis was accompanied by the alteration of expression of the Bcl-2 family members, disruption of mitochondrial potential, release of Cyt c from mitochondria and activation of capase-9 and downstream caspase-3. These results might be helpful in understanding the antitumor mechanism of polysaccharides, and to develop a novel antitumor agent.

Materials and methods

Cell lines and reagents

Human cancer cell line HeLa (cervical cancer), obtained from Xi'an Cell Engineering Center (Xi'an, China), was cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G, and 100 [micro]g/ml streptomycin in a humidified 5% [CO.sub.2] atmosphere at 37 [degrees]C.

APS-1d was prepared in our laboratory as described previously (Cao et al., 2006b). Briefly, the powdered roots of Angelica sinensis (Oliv.) Diels (5.5 kg) were defatted with alcohol and then decocted 3 times with 4 volumes of water. The aqueous extract was concentrated and treated with 3 volumes of ethanol for precipitation. The gel-like precipitate was suspended in water and dialyzed against distilled water (exclusion limit 3.5 kDa). The nondialyzable portion was frozen at -20 [degrees]C, then thawed and centrifuged to remove insoluble materials. After the freeze-thaw process was repeated 6 times, the supernatant was lyophilized and the brown product (APS-0) was obtained. APS-0 was dissolved in distilled water, and loaded onto a DEAE-sephadex A-25 column. The column was eluted with distilled water followed by 0.3 M and 0.5 M NaCl, respectively. The water-eluted fraction (APS-1) was further fractionated on a column of Sephadex G-100, eluted with 0.1 M NaC1 and separated into four fractions. The forth fraction, which molecular weight was lower than the other fractions, was pooled and applied onto the column of Sephadex G-100 once more. The relevant fraction was concentrated, dialysed and lyophilized to get white powder (APS-1d, 2.1 g).

The molecular weight of APS-1d was determined to be 5.1 kDa by high-performance gel-permeation chromatography. The percentage of total sugar was determined to be 91.5% by phenol-sulfuric acid method. The optical rotation was [alpha] 20D+45.9(c 0.2, [H.sub.2]O). The component sugars of APS-1d, determined by GC, were glucose and arabinose with a molar ratio of 13.8:1. Using methylation analysis, partial acid hydrolysis, FT-IR, 1D and 2D NMR (H/H-COSY, HSQC and HMBC) experiments, the structure of APS-1d was elucidated to be a backbone composed of 1,4-[alpha]-D-glucopyranosyl residues, with branches attached to 0-6 of some residues. The branches were composed of 1,6-[alpha]-D-glucopyranosyl residues, and terminated with [beta]-L-arabinofuranose residues. Moreover, the percentage of nitrogen content in APS-1d was determined to be 0.12% using a Vario EL III Element analyzer (Elementar, Germany). [.sup.1.H]-NMR spectrum showed no signs of protein or lipopolysaccharide.

Cell proliferation assay

Cell proliferation was analyzed by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, as previously described (Mosmann, 1983). Briefly, exponentially growing cells in 96-well plates were treated with different concentrations (0.3-300 [micro]g/ml) of APS-1d in complete medium or the medium alone. MTT (5 mg/ml, Sigma-Aldrich, MO, USA) 20 [micro]l was added 24, 48 or 72 h later. After the plates were incubated at 37 [degrees]C for 4 h, the supernatant was aspirated, and 150 [micro]l dimethyl sulfoxide (DMSO) was added to each well.

Absorbance was measured at 570 nm by a 96-well microplate reader (Bio-Rad, Tokyo, Japan). The percentage of surviving cells was calculated as follows: the ratio of cell survival (%)=(mean absorbency in test wells)/(mean absorbency in control wells) x 100.

Cell cycle analysis and apoptosis assay

HeLa cells were seeded in six-well plates (5 x [10.sup.5] cells/well) and allowed to grow for one day before being exposed to APS-1d (0, 3, 30 or 300 [micro]g/ml). Cells were collected after 72 h. For cell cycle analysis, the cells were collected by trypsinization, fixed in 70% ethanol, washed in phosphate buffered saline (PBS), resuspended in 1 ml of PBS containing 1 mg/ml RNase and 50 [micro]g/ml propidium iodide (PI), and then incubated for 30 min in the dark at room temperature. DNA content of the cells was measured by an Elite-ESP flow cytometer (Beckman Coulter, Miami, FL, USA), and the population of each phase was calculated using the Elite Multicycle software (Phoenix Flow Systems, San Diego, CA, USA). For the apoptosis assay, the cells were washed twice with PBS, and then stained with annexin V-FITC and PI, which were included in the Apoptosis Detection Kit (Biovision Research Products, Mountain View, CA, USA), for 30 min in the dark before being analyzed by flow cytometry. Experiments were conducted three times, and the results are reported as the mean of the three experiments.

Assay of antitumor activity in vivo

Female athymic BALB/c nude mice (6 weeks of age) were purchased from Shanghai National Center for Laboratory Animals (Shanghai, China) and maintained in pathogen-free conditions. Exponentially growing HeLa cells suspended in PBS were injected subcutaneously into the left flanks of nude mice (1 x [10.sub.7] cells in 200 [micro]1). When the size of established tumors reached about 100 [mm.sub.3] (around one week after tumor cells were inoculated), 24 mice were randomized into three groups (n=8) and received a daily intraperitoneal injection of 0.9% NaCl (control group), 50 mg/kg APS-1d or 100 mg/kg APS-1d in a 0.2-ml volume. Mice were weighed regularly. Tumor size was measured with a caliper and tumor volume (TV) was calculated by the formula, TV =(L+W)/2 x (L x W) x 0.5236, where L is the maximum diameter of the tumor and W is the minimum diameter. At day 28 after the inoculation of 0.9% NaCl or APS-ld, all the animals were sacrificed with an overdose of sodium pentobarbital. The tumors were removed carefully, fixed in 10% neutral formalin in PBS, and then embedded in paraffin for TUNEL detection. The animal experiments were performed in accordance with the 'Guidelines for Animal Experimentation' of the Fourth Military Medical University.

TUNEL assay

TUNEL assay was conducted by using the Dead-End Colorimetric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer's instruction. The slides were counterstained with hematoxylin. Omission of the terminal deoxynucleotidyl transferase enzyme during processing was used as a negative control. Apoptotic cell densities were expressed as the mean number of the five random microscopic high power fields (HPF, 400 x ).

Analysis of mitochondrial potential ([delta][psi]m)

HeLa cells were seeded in six-well plates (5 x [10.sub.5] cells/well) and allowed to grow for one day before being exposed to APS-1d (0, 3, 30 or 300 [micro]g/ml). After 72 h, the cells were harvested, washed twice with PBS, and exposed to the [delta][[psi].sub.m]-specific stain rhodamine 123(10 [micro]g/ml. Sigma-Aldrich) for 30 min at 37 J[degrees]C. The cells were then washed twice with PBS, resuspended in 0.5 ml of PBS, and analyzed by flow cytometry with an excitation wavelength of 505 nm and emission wavelength of 534 nm for changes in [delta][[psi].sub.m]. The mean values of fluorescence intensity were calculated. The experiments were repeated three times.

Preparation of cytosolic protein fractions

After treatment with the given concentrations of APS-1d for 72 h, cells were collected. Cytosolic protein fractions were prepared as described previously (Park et al., 2003). The protein in the cytosolic fraction was measured by the Bradford assay using bovine serum albumin as the standard and Cyt c was detected by Western blot analysis using anti-Cyt c antibody.

Western blot analysis

Cells were washed in PBS and lysed in 100 [micro]l of buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 [micro]g/ml pepstain, and 10 [micro]g/ml leupetin. After 20 min, extracts were centrifuged at 12,000 rpm for 10 min at 4 [degrees]C and supernatants were stored at -80 [degrees]C until use. The total protein was determined using the Bradford assay. 50 [micro]g protein lysine was separated by 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After being blocked in the 5% non-fat milk solution in washing buffer containing 10 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, and 0.05% Tween-20 for 2 h, membranes were incubated overnight at 4 [degrees]C with different primary antibodies: Bcl-2, Bax, Bak, Bcl-[X.sub.L] (Santa Cruz); Bid, caspase-9, caspase-8, and caspase-3 (Cell Signaling Technology, Danvers, MA, USA); poly (ADP-ribose) polymerase (PARP) (BD Pharmingen, San Jose, CA, USA); and [beta]-actin (Sigma-Aldrich). Blots were washed with washing buffer and incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. After washing, signals were detected using the Super Signal West Pico Trial Kit (Pierce, Rockford, IL, USA).

Inhibitors of caspases

40 [micro]M of caspase-8 inhibitor Z-IETD-FMK or caspase-9 inhibitor Z-LEHD-FMK (Calbiochem, CA, USA) were incubated with HeLa cells for 2 h at 37 [degrees]C prior to the addition of APS-1 d.

Statistical analysis

Data are expressed as means [+ or -] SD. Statistical significance (p< 0.05) for each variable was estimated by Student's t-test or ANOVA followed by Bonferroni's post analysis.

Results

APS-1d inhibited the growth of cervical cancer HeLa cells in vitro

The cytotoxic effects of APS-1d on HeLa cells were assessed using the MTT assay. As shown in Fig. 1, APS-1d inhibited the growth of HeLa cells in a time- and concentration-dependent manner and exhibited significant inhibition at concentrations of 30, 100 and 300 [micro]g/ml after APS-1d treatment for 24, 48, and 72 h (p < 0.05). At the highest concentration of 300 [micro]g/ml, the inhibition ratio was 36.0% after 72 h.

[FIGURE 1 OMITTED]

APS-1d induced apoptosis of HeLa cells in vitro

To confirm induction of cell apoptosis by APS-1d, annexin V-FITC/PI assay based on flow cytometry was used. As shown in Fig. 2A and B, the majority of the normal cells appeared in the annexin V-negative section, and APS-1d-treated HeLa cells increased the annexin V-positive cell population to 9.1 [+ or -] 1.6%.

The change in DNA content during progression of the cell cycle was also evaluated by flow cytometry. A representative example depicting the effect of APS-1d treatment for 72 h on cell cycle phase distribution is shown in Fig. 2C. In the cells undergoing apoptosis, a special DNA peak (usually called the sub-[G.sub.0]/[G.sub.1] peak or apoptotic peak) appeared. This peak is thought to be one of the characteristics of apoptosis. As shown in Fig. 2D, treatment with 30, 100 or 300 [micro]g/ml of APS-1d for 72 h resulted in the accumulation of cells in the S phase (29.2 [+ or -] 6.0, 33.9 [+ or -]1.7 or 33.7 [+ or -]9.7%, respectively), compared with that in the control (22.7[+ or -]1.0%). In addition, the sub-[G.sub.0]/[G.sub.1]] apoptotic fraction was markedly increased when APS-1d was applied. Taken together, these results demonstrated that APS-1d suppressed HeLa cell proliferation by induction of apoptosis.

[FIGURE 2 OMITTED]

APS-1d inhibited tumor growth and promoted HeLa cells apoptosis in vivo

Athymic nude mice xenografted with HeLa cells were divided into control, 50 mg/kg or 100 mg/kg of APS-1d-treated groups. Tumor growth was monitored daily after tumor cell inoculation. Tumors in the control group grew rapidly, reaching an average volume of 2507.4 [+ or -] 584.4 [mm.sub.3] on day 28 after inoculation of HeLa cells. In contrast, mice administered APS-1d intraperitoneally (50 or 100 mg/kg) showed significantly reduced growth of HeLa cells and the tumor volume remained at an average of 1597.5 [+ or -] 269.8 or 957.3 [+ or -] 348.0 [mm.sup.3] (Fig. 3A and B, p < 0.05). No sign of toxicity, as judged by monitoring of body weights (Fig. 3C) and lungs, livers and kidneys, was observed in APS-1d-treated mice.

To further confirm APS-1d-induced apoptosis in vivo, TUNEL assay was performed. As shown in Fig. 3D, the application of APS-1d increased the apoptotic staining (dark-brown) in a dose-dependent manner (5.4[+ or -]3.6 cells/HPF in control, 12.2 [+ or -]4.0 cells/HPF in 50 mg/kg APS-1d group, and 26.2 [+ or -] 9.9 cells/HPF in 100 mg/kg APS-1d group, respectively). These data suggested that APS-1d attenuated tumor growth in vivo via induction of tumor cell apoptosis.

[FIGURE 3 OMITTED]

APS-1d increased the levels of pro-apoptotic proteins Bax and Bak and decreased anti-apoptotic proteins Bcl-2 and Bcl-[X.sub.L] levels

The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. In order to understand the mechanism of APS-1d-induced apoptosis, this study examined the expression of pro-apoptotic (Bax, Bak and Bid) and anti-apoptotic (Bcl-2 and Bcl-[X.sub.L]) proteins. Western blotting revealed that Bax and Bak proteins increased dramatically in the 30 and 300 [micro]g/ml APS-1d-treated groups, compared with control cells (Fig. 4). In contrast, Bcl-2 and Bcl-[X.sub.L] were down-regulated by APS-1d in a dose-dependent manner. Surprisingly, no significant change in Bid expression was observed in cells treated with APS-1d compared with control cells. These results suggest that the alteration in expression of Bcl-2 family proteins may contribute to HeLa cell apoptosis induced by APS-1d.

[FIGURE 4 OMITTED]

APS-1d treatment regulated the intrinsic apoptotic pathway

Taking into account the previous data which showed that there was no significant change in Bid expression in cells treated with APS-1d, we presumed that it was unlikely that APS-1d-induced apoptosis involved the extrinsic apoptotic pathway. Therefore, we further investigated the effects of APS-1d on activation of caspase-8, a molecular marker associated with the extrinsic apoptotic pathway. Western blots revealed no significant change in the cleavage of caspase-8 in APS-1d-treated cells compared with control cells (Fig. 5C). These results suggest that the extrinsic apoptotic pathway is not involved in APS-1d-induced apoptosis in HeLa cells.

[FIGURE 5 OMITTED]

Down-regulation of Bcl-2 may cause Cyt c release from the mitochondria into the cytoplasm, and subsequent activation of death effectors, such as caspase-3. We, therefore, examined whether treatment with APS-1d regulates the intrinsic apoptotic pathway by affecting the mitochondria. We first determined the mitochondrial potential in HeLa cells treated with APS-1d. Decreases in mitochondrial potential were observed in the treatment groups compared to control cells (Fig. 5A). The relative decrease of mitochondrial potential in 30 and 300 [micro]g/ml APS-1d-treated cells were 10.5% and 29.2%, respectively.

We next investigated the involvement of the mitochondrial-mediated intrinsic apoptotic pathway by assessing the release of Cyt c from the mitochondria into the cytoplasm, and the cleavage of the caspase cascade. We extracted the cytosolic section from the cells treated with APS-1d (0, 30 or 300 [micro]g/ml) and examined Cyt c by Western blotting. As shown in Fig. 5B, a significant increase in Cyt c release was observed when HeLa cells were treated with APS-1d, and this release was found to be concentration-dependent. In addition, total cell lysates were analyzed to detect the expression of capase-9, caspase-3, and PARP by Western blotting. As shown in Fig. 5C, cleaved/activated caspase-9 (37 and 35 kDa), caspase-3 (19 and 17 kDa), and PARP (85 kDa) were detected in the APS-1d-treated cells, and when the APS-1d concentration increased, the cleaved capase-9, caspase-3, and PARP expression was significantly augmented. These results indicate that treatment with APS-1d resulted in activation of the intrinsic mitochondrial apoptotic pathway in HeLa cells.

Effect of caspase inhibitors on APS-1d-induced apoptosis

Caspases are broadly grouped into initiator or effector caspases according to the roles they play in the apoptosis inducing system. The initiator caspases including caspase-1, -8, -9, typically caspase-8 and caspase-9, are activated by two alternative pathways (Salvesen and Dixit, 1997). The first involves cell death receptor-mediated apoptosis through caspase-8, while the second involves mitochondria-mediated apoptosis through caspase-9. To further demonstrate the involvement of caspase activation in the apoptotic effect, the caspase-8 specific inhibitor Z-IETD-FMK and caspase-9 specific inhibitor Z-LEHD-FMK were used to evaluate the possible role of caspases in APS-1d-induced apoptosis. We pre-incubated cells with selective caspase inhibitors before exposure to APS-1d, and then examined the percentage of apoptotic cells using flow cytometry. As indicated in Fig. 6A, pre-incubation with Z-LEHD-FMK [caspase-9 inhibitor) significantly attenuated the APS-1d-induced increase in the number of apoptotic cells. However, pre-incubation with Z-IETD-FMK (caspase-8 inhibitor) did not inhibit APS-1d-induced apoptosis.

[FIGURE 6 OMITTED]

Furthermore, an important role for caspase-9 in APS-1d-induced apoptosis was also confirmed by the observation that Z-LEHD-FMK efficiently attenuated the cleavage of caspase-3 induced by APS-1d in HeLa cells (Fig. 6B). The caspase-8 inhibitor did not affect the activation of caspase-3.

These results indicated that APS-1d can induce apoptosis in HeLa cells via the intrinsic (mitochondrial) apoptotic pathway.

Discussion

Polysaccharides are important components of plants, fungi, yeasts, algae and lichens, and have attracted more and more attention in the biochemical and medical areas due to their immunomodulatory and antitumor effects (Ooi and Liu, 2000). Recently, some polysaccharides exhibiting cytotoxic activity on human cancer cells were reported, and some of these polysaccharides showed significant apoptosis-inducing activities. However, most of the polysaccharides inducing apoptosis in cancer cells were isolated from mushrooms or fungi rather than from plants.

APS-1d is a novel polysaccharide isolated from Angelica sinensis and can significantly suppress the proliferation of several types of human cancer cells in vitro. The present study further assessed its apoptosis-inducing effect on human cervical cancer cells. This is the first report of a polysaccharide from Angelica sinensis inducing apoptosis in cancer cells both in vivo and in vitro, and suggests a potential therapeutic role of polysaccharides isolated from plants in the treatment of cervical cancer.

Tumor development and growth are considered a result of the high proliferative capacity of tumor cells. Apoptosis and its related signaling pathways have a profound effect on the progression of cancer (Lowe and Lin, 2000). In the present study, we assessed the capacity of APS-1d to suppress tumor cell proliferation and investigated whether this effect might occur by inducing apoptosis in HeLa cells. Cell proliferation assays showed that APS-1d inhibited HeLa cell growth in a concentration-dependent manner. To further test the antitumor effect of APS-1d, athymic nude mice xenografted with HeLa cells were used. After treatment with APS-1d for 3 weeks, tumor volumes were significantly reduced. Thus, we demonstrated that APS-1d inhibited HeLa cell proliferation not only in vitro but also in vivo. Moreover, we used several methods to ensure the accuracy of the apoptosis results in this study. A special apoptotic peak appeared when APS-1d-treated HeLa cells were analyzed by flow cytometry. The results of the TUNEL assay further confirmed that APS-1d induced apoptosis in mice xenografted with HeLa cells. Taken together, these results suggest that APS-ld can induce HeLa cell apoptosis. Furthermore, cell cycle analysis showed that cell cycle progression of APS-ld-treated cells was blocked in S phase, which was similar to the effect of other polysaccharides from plants, such as Lycium barbarum (Zhang et al., 2005).

Most anticancer agents either directly induce DNA damage or indirectly induce secondary stress-responsive signaling pathways to trigger apoptosis by activation of the intrinsic apoptotic pathway, and some can simultaneously activate the extrinsic receptor pathway, increasing evidence suggests that Bcl-2 family proteins play central roles in cell apoptosis (Burlacu, 2003). Our data demonstrated that APS-ld treatment can reduce Bcl-2 and Bcl-[X.sub.L] expression, and increase Bax and Bak expression in HeLa cells. It is reported that overexpression of the anti-apoptotic molecules such as Bcl-2 or Bcl-[X.sub.L] blocks Cyt c release in response to a variety of apoptotic stimuli. In contrast the pro-apoptotic molecules such as Bax and Bid promote Cyt c release from the mitochondria (Liu et al., 1996). Interaction of Cyt c with a cytosolic apoptosis protease activating factor, Apaf-1, induces recruitment of procaspase 9 into a high-molecular-weight complex, termed the apoptosome, which gives rise to activated caspase-9 and -3. Subsequently, active caspase-3 mediates inhibition of caspase-activated DNase (ICAD) and PARP processes to account for DNA fragmentation as well as other morphological and biochemical changes during apoptosis (Hao et al,. 2005; Zou et al., 1999). Here, we tried to elucidate the signaling mechanisms that underlie APS-1d-induced apoptosis. Our results from Western blotting analysis showed that APS-1d induced the release of Cyt c from the mitochondria into the cytoplasm and the cleavage of caspase-9, caspase-3, and PARP. In addition, the caspase-9 inhibitor blocked the cleavage of caspase-3, and the formation of apoptotic cells, suggesting that the mitochondrial pathway mediates APS-1d-induced apoptosis in HeLa cells.

The structure of the polysaccharide is strongly related to its antitumor activity. Previous research has mainly focused on the structure of the [beta]-glucan and suggested that the (l,3)-[beta]-glucan with the (l,6)-[beta]-glucan branches was important for the antitumor activity by increasing immune-competent cell activity (Kodama et al., 2002; Wasser, 2002). However, there are some antitumor polysaccharides with different chemical structures, such as [alpha]-glucan (Kiho et al., 1989). To our knowledge, there is no report of a polysaccharide with a structure similar to APS-1d displaying an antitumor effect and having apoptosis-inducing activity. Thus, our research will facilitate the understanding of the structural basis of the polysaccharides with antitumor effects and their antitumor mechanisms. However, as a novel polysaccharide, whether APS-ld is effective against other cancer types will be worthy of future investigation.

In conclusion, these studies demonstrate that APS-1d has an inhibitory action on the growth of human cervical HeLa cells both in vitro and in vivo, which is related to the induction of S phase arrest and the promotion of apoptosis. APS-1d induces apoptosis by regulating Bcl-2 family protein expression and further activating the intrinsic mitochondrial apoptotic pathway. Our findings provide a novel understanding of the antitumor mechanism of this polysaccharide from Angelica sinensis and might be helpful in developing a new antitumor agent.

Acknowledgements

This work was supported by National Natural Science Foundation of China (30701081).

References

Burlacu, A., 2003. Regulation of apoptosis by Bcl-2 family proteins. J. Cell. Mol. Med. 7, 249-257.

Cao, W., Li. X.Q., Liu, L., Wang, M., Fan, H.T., Li, C, Lv, Z., Wang, X., Mei, Q., 2006a. Structural analysis of water-soluble glucans from the root of Angelica sinensis (Oliv.) Diels. Carbohydr. Res. 341, 1870-1877.

Cao, W., Li, X.Q., Liu, L., Yang, T.H., Li, C., Fan, H.T., Jia. M., Lv, Z.G., Mei, Q.B., 2006b. Structure of an anti-tumor polysaccharide from Angelica sinensis: (Oliv.) Diels. Carbohydr. Polym. 66, 149-159.

Chihara, G., Maeda, Y., Hamuro, J., Sasaki, T., Fukuoka, F., 1969. Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) sing. Nature 222, 687-688.

Desagher, S., Martinou, J.C., 2000. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10, 369-377.

Fullerton, S.A., Samadi, A.A., Tortorelis, D.G., Choudhury, M.S., Mallouh. C., Tazaki, H., Konno, S., 2000. Induction of apoptosis in human prostatic cancer cells with beta-glucan (Maitake mushroom polysaccharide). Mol. Urol. 4, 7-13.

Hao, Z., Duncan, G.S., Chang, C.C., Elia, A., Fang, M., Wakeham, A., Okada, H., Calzascia, T., Jang, Y., You-Ten, A., Yeh, W.C., Ohashi, P., Wang, X., Mak, T.W., 2005. Specific ablation of the apoptotic functions of cytochrome C reveals a differential requirement for cytochrome C and Apaf-1 in apoptosis. Cell 121, 579-591.

Kiho, T., Yoshida, I., Nagai, K., Ukai, S., Hara, C., 1989. (1-3)-alpha-D-glucan from an alkaline extract of Agrocybe cylindracea, and antitumor activity of its O-(carboxymethyl)ated derivatives. Carbohydr. Res. 189, 273-279.

Kobayashi, H., Yoshida, R., Kanada, Y., Fukuda, Y., Yagyu, T., Inagaki, K., Kondo, T., Kurita, N., Suzuki, M., Kanayama, N., Terao, T., 2005. Suppressing effects of daily oral supplementation of beta-glucan extracted from Agaricus blazei Murill on spontaneous and peritoneal disseminated metastasis in mouse model. J. Cancer Res. Clin. Oncol. 131, 527-538.

Kodama, N., Komuta, K., Nanba. H., 2002. Can maitake MD-fraction aid cancer patients? Altern. Med. Rev. 7, 236-239.

Lin, X., Cai, Y.J., Li, Z.X., Liu, Z.L., Yin, S.F., Zhao, J.C., 2001. Cladonia furcata polysaccharide induced apoptosis in human leukemia K562 cells. Acta Pharmacol. Sin. 22, 716-720.

Liu, X., Kim, C.N., Yang, J., Jemmerson, R., Wang, X., 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147-157.

Lowe, S.W., Lin, A.W., 2000. Apoptosis in cancer. Carcinogenesis 21, 485-495.

Mehmet, H., 2000. Caspases find a new place to hide. Nature 403, 29-30.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.

Nakazato, H., Koike, A., Saji, S., Ogawa, N., Sakamoto, J., 1994. Efficacy of immunochemotherapy as adjuvant treatment after curative resection of gastric cancer. Study Group of Immunochemotherapy with PSK for Gastric Cancer. Lancet 343, 1122-1126.

Ooi, V.E., Liu, F., 2000. Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr. Med. Chem. 7, 715-729.

Park, M.T., Choi, J.A., Kim, M.J., Um, H.D., Bae, S., Kang, C.M., Cho, C.K., Kang, S., Chung, H.Y., Lee, Y.S., Lee, S.J., 2003. Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells. J. Biol. Chem. 278, 50624-50634.

Rein, D.T., Kurbacher, C.M., 2001. The role of chemotherapy in invasive cancer of the cervix uteri: current standards and future prospects. Anticancer Drugs 12, 787-795.

Salvesen, G.S., Dixit, V.M., 1997. Caspases: intracellular signaling by proteolysis. Cell 91, 443-446.

Sarker, S.D., Nahar, L., 2004. Natural medicine: the genus Angelica. Curr. Med. Chem. 11, 1479-1500.

Shang, P., Qian, A.R., Yanxg, T.H., Jia, M., Mei, Q.B., Cho, C.H., Zhao, W.M., Chen, Z.N., 2003. Experimental study of anti-tumor effects of polysaccharides from Angelica sinensis. World J. Gastroenterol. 9, 1963-1967.

Shin, J.Y., Song. J.Y., Yun, Y.S., Yang, H.O., Rhee, D.K., Pyo, S., 2002. Immunostimulating effects of acidic polysaccharides extract of Panax ginseng on macrophage function. Immunopharmacol. Immunotoxicol. 24, 469-482.

Wajant, H., 2002. The Fas signaling pathway: more than a paradigm. Science 296, 1635-1636.

Wasser, S.P., 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60, 258-274.

Zaidman, B.Z., Yassin, M., Mahajna, J., Wasser, S.P., 2005. Medicinal mushroom modulators of molecular targets as cancer therapeutics. Appl. Microbiol. Biotechnol. 67, 453-468.

Zhang, M., Chen, H., Huang, J., Li, Z., Zhu. C., Zhang, S., 2005. Effect of lycium barbarum polysaccharide on human hepatoma QCY7703 cells: inhibition of proliferation and induction of apoptosis. Life Sciences 76, 2115-2124.

Zou, H., Li, Y., Liu, X., Wang, X., 1999. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11549-11556.

W. Cao (a), (1), X.-Q. Li (a), (1), X. Wang (b), (1), H.-T. Fan (a), X.-N. Zhang (a), Y. Hou (a), S.-B. Liu (a), Q.-B. Mei (a), *

(a) Department of Pharmacology, School of Pharmacy, the Fourth Military Medical University, 169 West Changle Road. Xi'an, Shaanxi 710032, China

(b) Department of Pathophysiology, School of Basic Medicine, Xi'an Medical University, Xi'an, Shaanxi 710021, China

* Corresponding author. Tel./fax: +86 29 8477 4552.

E-mail address: qbmmei@gmail.com (Q.-B. Mei).

(1) These authors contributed equally to this paper.

0944-7113/$ - see front matter [C] 2009 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2009.12.014
COPYRIGHT 2010 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2010 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Cao, W.; Li, X.-Q.; Wang, X.; Fan, H.-T.; Zhang, X.-N.; Hou, Y.; Liu, S.-B.; Mei, Q.-B.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jul 1, 2010
Words:5447
Previous Article:Selective induction of apoptosis in glioma tumour cells by a Gynostemma pentaphyllum extract.
Next Article:Effects of triterpenes from Ganoderma lucidum on protein expression profile of HeLa cells.
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters