Ganoderma Lucidum Polysaccharides Target a Fas / Caspase Dependent Pathway to Induce Apoptosis in Human Colon Cancer Cells

Colon cancer, which caused by cellular pathology in the appendix, colon, or in the rectum, is the third most common cancer in the world. Clinically, chemotherapy and radiation are typically used before or after surgery in colorectal treatment. Unfortunately, even with modern intensive medical therapy, a high ratio of recidivations and formation of metastases lead to a low cure rate and scarcely improve long-term survival rate (Luigi et al., 2009). Furthermore, the toxicity produced by chemotherapy inflicts on immune system of cancer patients in the course of treatment. Currently, compounds from herbaceous plants are recognized as pharmacological agents to inhibit cancer growth and lessen the side effects of radiation and chemotherapy (Chen et al., 2009; Chen et al., 2010). GLP, isolated from Ganoderma lucidum, have been used in East Asian countries to remedy conditions like aging, oxidation, radiation and hypertension for thousands of years (Chen et al., 2009; Pillai et al., 2010; Li et al., 2011). Previous studies have showed that GLP can mediate cytotoxicity in number of cancer cell lines including lung cancer, prostate cancer and breast cancer. Further studies showed that


Ganoderma Lucidum Polysaccharides Target a Fas/Caspase Dependent Pathway to Induce Apoptosis in Human Colon Cancer Cells
Zengenni Liang, Yu-Tong Guo, You-Jin Yi, Ren-Cai Wang*, Qiu-Long Hu, Xing-Yao Xiong* GLP programmed cell death through endothelial growth factor overexpression and tumor angiogenesis inhibition in highly metastatic mouse melanoma B16F10 cells (Sun et al., 2011), through endotoxin-induced intercellular cell adhesion molecule-1 (ICAM-1) upregulation in the neointima in mice (Liu et al., 2010), through cell differentiation and p53 activation in human monocytic leukemia THP-1 cells (Hsu et al., 2011), through activation of mitochondrial signaling pathway in MCF-7 breast cancer cells (Shang et al., 2011).GLP can also boost anticancer immunity by changing lymphocyte phenotype in H22 hepatoma mice (Zhou et al., 2009), by enhancing CD56 + NK-cell cytotoxicity in cord blood (Chien et al., 2004), by promoting the proliferation of B cells in mice (Jiang et al., 2003).Although GLP have been demonstrably effective in fighting cancer by reducing cell growth and boosting cell apoptosis, the anti-cancer effects of GLP have not been reported in human colon cancer cell line  In this study, we isolated and purified polysaccharides from Ganoderma lucidum (Leyss.et Fr.) Karst.Thereafter, HCT-116 cell line was used as a test model to measure the hypothesis that GLP (>10 kDa) affected anticancer effects by MTT assay, migration assay and morphologic observation.We also examined the roles of lactate dehydrogenase (LDH), cellular intracellular Ca 2+ , Fas and caspase cascades in GLP-induced apoptosis in HCT-116 cells.

Preparation of GLP
Slices of Ganoderma lucidum (Leyss.et Fr.) Karst.were provided by State Key Laboratory of Sub-health Intervention Technology, State Administration of Traditional Chinese Medicine.GLP were isolated and purified using a procedure as described (Guo, 2012).Crude polysaccharides were gained through derosination, removal of oligosaccharides, hot water extraction, deproteinization, decolorization, concentration under vacuum, and freeze-dried.GLP solution (10 mg/ mL in water) was ultrafiltered through 10, 30, and 50 kDa molecular weight cut off membranes (MWCO, Millipore, US), respectively, in an Amicon 8200 stirred cell (Millipore Corporation Bedford, MA).The filtrate (molecular weight of polysaccharides >10 kDa) was freeze-dried, dissolved in high glucose DMEM with 10% FBS to get a stock solution of 10 mg/mL, passed through 0.22 μm filter and stored at 4°C which was prepared for subsequent analysis.

Cell Culture
Colon cancer HCT-116 cell line was obtained from Institute of Basic Medical Sciences (Beijing, China).The cells was planted in DMEM medium with high glucose supplemented with 10% FBS in 25 cm 2 tissue culture flasks under a humidified atmosphere of 5% CO2 at 37°C.Frozen cells were thawed and passaged 3 times, which were subsequently used for this trial.

Cell viability assay
HCT-116 cells (3×10 4 cells/well) were seeded onto 96-well plates.After a 12 h recovery period, cells were incubated in fresh medium without or with various concentrations of GLP (0.313,0.625,1.25,2.5 and 5 mg/ mL) for 24, 48 and 72 h, respectively, then added 5 mg/ mL of MTT solution (20 μL/well) to each well for an additional 4 h.Subsequently removing the supernatant, water-insoluble formazan was dissolved in 150 μL DMSO to each well for 10 min.The absorbance at wavelength of 492 nm was recorded using a microplate reader (MK3, Thermo, USA).Cells with starch (0-10 mg/mL) treatment were used as control.The inhibitory rate was calculated using the following formula: Inhibitory rate (%) = (1mean A treatment /mean A control ) × 100%.

Wound-healing assay in vitro
A 24-well plate was spread by monolayer HCT-116 cells and scratched wounds by 10 μL pipette tips.Cells were washed twice with PBS to remove loose cells, and then exposed to 0.625, 1.25, 2.5, 5 and 10 mg/mL of GLP.Following incubation for 48 h, the wounds were photographed under an inverted microscope (BX60, Olympus, Japan).The number of migrant cells was analyzed with Image J software from three visual fields in each group.

Morphologic observation
To directly assess the effects of GLP on HCT-116 cells, GLP-induced morphological change was examined by two types of electron microscopes.Transmission electron microscope: GLP-treated cells and untreated cells were trypsinized and suspension-fixed in 1% glutaraldehyde for 2 h.After rinsing with phosphate buffered solution (PBS, pH7.4), cells were postfixed with 1% osmium tetroxide (OsO4) for another 2 h.Dehydration was carried out in an ascending grade of ethanol (50-100%, v/v).Samples were infiltrated with 50% quetol in ethanol for 1 h and additional 1 h incubation with 100% quetol for 6 h, followed by polymerized at 60°C.After 39 h, all groups were cut into ultra-thin sections, stained with 4% uranyl acetate for 10 min.Photographs were taken under a transmission electron microscope (JEM-1230, Tokyo, Japan).Scanning electron microscopy: After GLP inoculation, cells were processed and analyzed as previously described (Wahab et al., 2009).Cells were fixed in 1% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in a graded of ethanol (50-100%, v/v), critical-point dried, affixed, gold-sputtered and finally viewed by a scanning electron microscope (JSM-6380LV, Tokyo, Japan).

Lactate Dehydrogenase (LDH) Release Assay
The apoptotic effect of GLP was examined by measurement of LDH release (Nakagawa et al., 2005).1.5×10 5 cells per well were plated in 24-well cell culture plate, followed by overnight incubation.Supernatants were collected and added in a black 96-well culture plates (200 μL per well) after cells were exposed to increasing concentrations of GLP (1.25, 2.5 and 5 mg/mL) for 48 h.LDH release was measured using LDH cytotoxicity assay kit (Beyotime, China) as per the manufacturer's instructions.Absorbance at a wavelength of 450 nm in each well was determined using an enzyme linked immunoassay instrument.

Detection of Intracellular Ca 2+ level
GLP-treated cells were rinsed twice with PBS, resuspended in Hanks' buffer (pH 7.4) containing 0.2% bovine serum albumin (BSA), and treated with 10 μmol/L of fura-2 acetoxymethyl ester (Fura-2 AM) at 37℃ for 30 min in the dark.Cells were then harvested, washed three times and resuspended in hanks' buffer.The fluorescence were monitored by recording in the ratio mode excitation wavelength at 340/380 nm and 510 nm emission wavelength using a varioskan flash (Thermo, USA).Maximal fluorescence (Fmax) was obtained in the presence of 10% Triton X-100 (final concentration 0.1%) and followed by minimum fluorescence (Fmin) with 5 mmol/L of EDTA.Intracellular Ca 2+ was calculated from the fluo-2 fluoresce intensity using the equation: where Kd of fluo-2 for Ca 2+ is 224 nM (Grynkiewicz et al., 1985;Hirst et al., 1999).

Statistical Analysis
All experiments were performed in triplicate and the data were expressed as mean ± S.D. Statistical analyse was performed using the statistical software SPSS 18.0, with highly significance at P< 0.01.

Isolation and structural analysis
The yield of crude polysaccharides was about 3.08%.

GLP dose-and time-dependently inhibit HCT-116 cell growth
Primary, different doses of GLP (0-5 mg/mL) and various cultivation times were applied in order to detect the cytotoxicity of GLP in HCT-116 cells.As shown in Figure 1A, GLP inhibited the viability of HCT-116 cells in a dose-and time-dependent manner (P<0.01).The rate of cell proliferation were markedly decreased in human colon cancer cells treated with doses above 2.5 mg/mL after 72 h (P<0.01),whereas there was no significant influences at dose of 0.313 mg/mL.The inhibitory concentration of 50% (IC50) for 24, 48 and 72 h were 9.25, 5.72 and 3.69 mg/ mL, respectively.The MTT assay also showed that starch incubation (0-5 mg/mL) promoted weak proliferation of HCT-116 cells (data not shown).

GLP delayed HCT-116 cell migration
To determine whether GLP impact the migratory capacity of HCT-116 cells, scratch wound assays were performed.As shown in Figure 1B, GLP concentration dependently inhibited the migratory capacity of human colon cancer contrast with control group.By 48 h incubation with 0.625, 1.25, 2.5, 5 and 10 mg/mL GLP, the number of migratory cells decreased by 22.7%, 29.1%, 39.9%, 63.1% and 84.4%, respectively (P<0.01).

Scanning electron microscopy
To investigate whether the observed cell viability inhibition was caused by apoptosis, the surface ultrastructure of GLP-treated cells was examined by SEM electron microscopy.Untreated cells showed uniform cellular distribution and numerous microvilli on their surface.In GLP-treated cells, 2.5 and 5 mg/mL of GLP were significant changes in cell morphology that corresponded to a typical cellular surface morphology of apoptosis, including cell membrane blebbing, cellular density and microvilli reduction, apoptotic body formation (as shown in white arrow) (Figure 2).

Transmission electron microscopy
Induction of apoptosis was also inspected using transmission electron microscopy.Control cells had a well regular cell shape, many microvilli protruding from their surfaces, homogeneous chromatin distribution (Figure 3A), rough endoplasmic reticula and healthy-looking mitochondrion (arrow) (Figure 3B).On the contrary, 5 mg/mL GLP lead to irregular cell shape, a decreased microvillar density, mitochondria swelling, chromatin concentration and marginalization (arrow) (Figure 3C), cytoplasm vacuolization and apoptotic bodies formation (arrow) (Figure 3D).Treatment with 10 mg/mL GLP induced collapse of plasma membrane, resulting in cell decomposition and death (Figure 3E).

GLP promoted LDH release and intracellular [Ca 2+ ]
The aim was to determine whether GLP-induced apoptosis was involved in these crucial events in HCT-116 cells.LDH activity of culture medium had a dosedependent increase (71.66 ± 11.47, 97.65 ± 5.87 and 144.13 ± 11.00 versus 49.77 ± 2.15 in control group, P<0.01) at the concentrations of GLP (1.25, 2.5 and 5 mg/mL) (Figure 4A).The intracellular Ca 2+ levels, in contrast to control, were also significantly promoted after incubation with GLP for 24 h (P<0.01)(Figure 4B).

GLP activated caspase-8 and altered abundance of apoptosis-associated protein
We next investigated the possible mechanism of GLPinduced apoptosis by Western blot experiments.As shown in Figure 5A, 5 mg/mL of GLP significantly enhanced caspase-8 activity compared to control (P<0.01).The result suggests that the activation of caspase-dependent apoptosis signaling pathways might be involved in GLPinduced apoptosis in human colon cancer cells.In western blot assay, we found that the levels of caspase-3 and Fas proteins were up-regulated in HCT-116 cells cultured in the solution with 5 mg/mL GLP at 12 h-48 h.We also found a direct correlation between the levels of caspase-3 and Fas proteins and incubation period of GLP (Figure 5B).

Discussion
Due to serious side effects of chemotherapy in cancer patients, we require new therapeutic strategies to be found.Polysaccharides from herb plants especially mushroom have been proven to be effective anti-tumor compounds with no toxicity (Shi et al., 2013;Giavasis, 2014;Ning et al., 2014).It has been reported that GLP exert strong anti-tumor effect when the molecular weight was larger than 1×10 4 (Liu et al., 2010).The reasons may be that polysaccharides with molecules weight in the range have not only a good dissolubility in water but also a relative complex structure (Dai et al., 2013).GLP have been found to possess anticancer activity without toxicity, but the molecular mechanisms involved remain unresolved.We previously identified that GLP and 5-fluorouracil (5-FU) contributed synergistically to inhibit growth and induce apoptosis against human colon cancer cells in vitro.The effect was associated with dose-response relationship of GLP exposure (Liang et al., 2012).
In our current study, high molecular weight GLP larger than 10 kDa was obtained by ultrafiltration and observed its anti-cancer effects on human colon cancer HCT-116 cells.The data showed a range of doses between 0.625 and 5 mg/mL GLP had a robust dose and time-dependent inhibitory effects on proliferation in HCT-116 cells (Figure 1).Considering the effect of osmotic pressure produced by GLP (0.313-5 mg/mL), the polysaccharide starch was applied to control the efficacy of GLP suppression in A. GLP activated caspase-8.1×10 6 HCT-116 cells per well were incubated with GLP at 0, 2.5, 5 mg/mL for 24 h.Caspase-8 activity was measured by colorimetric assay as described.*P<0.01,compared to control group.All data were expressed as the mean ± SD. (n=3 HCT-116 cells.The results also showed that starch (0.313 -10 mg/mL) dose-dependently promoted cell growth, suggesting the osmotic pressure in the cell culture with these concentrations of GLP did not markedly induce cell death.Cell migration is a critical process for sustained maintenance and development of every type of cancer cells (Condeelis and Pollard, 2006).Especially, malignant cancer cells put into use their own migratory capacity to invade adjacent healthy tissues and the vasculature, then ultimately to inflict devastating organizer destruction (Yamaguchi and Condeelis, 2007).Thus, limitation of this process is a novel therapeutic strategy for controlling tumor growth and deterioration.In the present study, we reported that GLP mediated dose-dependent inhibitory effects on cell migration of human colon cancer HCT-116 cells (P<0.01).The data showed that GLP may cause antitumor effects by suppression cell growth and migration in HCT-116 cells.
Cell apoptosis triggered by most of cancer chemopreventive agents and anticancer drugs is an essential process in prevention of tumor promotion (Núñez et al., 2010).It is commonly measured by the increase in release of LDH, TUNEL staining, intracellular Ca 2+ concentration, cytoplasm condensation, DNA and nuclear fragmentation (Liu et al., 2006).Under SEM and TEM observation, vacuoles, apoptotic bodies, chromatin margination and cell degradation appeared in GLP -incubated cells (Figure 2 and Figure 3).As further proof of the apoptotic effects of GLP, LDH release and intracellular Ca 2+ concentration were also determined.The permeabilization of the plasma membrane is a key signature for apoptotic cells, which can be quantified in tissue culture settings by measuring the release of the intracellular enzyme LDH (Sun et al., 2013).As an important messengers and a key regulator, Ca 2+ can control apoptosis in response to a variety of pathological conditions from external and internal environment though a wide range of Ca 2+ sensitive factors that are compartmentalized in various intracellular organelles including the mitochondria, ER and cytoplasm.GLP has been reported to promote calcium levels in the brain of D-gal-treated mice (Li et al., 2011).In this study, GLP also induced apoptosis by promoting the release of LDH (Figure 4A) and increased the level of intracellular Ca 2+ (Figure 4B).These results clearly indicated that GLP significantly induced apoptosis in HCT-116 cells.
Next, we investigated the possible mechanisms of GLP-mediated apoptosis.Intrinsic (mitochondrial) and extrinsic (death receptor) apoptotic pathways are two major ways to stimulate cell apoptosis.In extrinsic pathway, ligand-bound death receptors such as Fas and TRAIL receptors have been well characterized to be a crucial regulator, which mediate activation of caspase cascades in some types of cancer cells (Sayers, 2011).It is demonstrated that caspase-3/8 activation is physically associated with the signaling complex during Fas-induced apoptosis (Mandal et al., 2005).In addition to the caspasedependent pathway, cell apoptosis also has been reported to be stimulated via caspase-independent pathway (Liu and Chang, 2011).In this study, GLP activated caspase-8 (Figure 5A) and up-regulated caspase-3 (Figure 5B), indicating that caspase-dependent pathway is related to GLP-induce apoptosis.It also increased the level of Fas protein (Figure 5B).Additionally, the addition of GLP treatment is attributed to enhanced active caspase-8 signaling and levels of Fas or caspase-3.These findings implied that GLP-induced apoptosis might activate Fasmediated caspase-dependent pathway in HCT-116 cells.Interestingly, we found cytosolic Ca 2+ was released during the cell apoptosis induced by GLP, which is believed to be a key activation signal in mitochondrial signaling pathway.Therefore, whether mitochondrial pathway participates in GLP-stimulated apoptosis remains need further investigation.
In conclusion, the present study offers a novel insight into the cytotoxicity and apoptosis of GLP in human colon cancer HCT-116 cells, suggesting that these findings provide potential perspectives for further research on pharmacology of GLP as a possible candidate for the cancer prevention or treatment of colon cancer.

Figure 1 .
Figure 1.GLP Suppressed Cell Growth and Migratory.A. Growth-inhibiting effects of GLP on HCT-116 cells.Cells were treated with 0-5 mg/mL GLP for 24, 48 and 72 h, respectively, and then determined by MTT assay.Experiment was repeated three times and similar results obtained.B. GLP delayed motility of HCT-116 cells.After wounding with pipette tips, cells were treated with 0-10 mg/mL GLP for 48 h.The number of migratory cells in the wound edge migration was quantitated by ImageJ software.*P<0.01,represents significant difference compared with the untreated control.Data are presented as mean ± SD (n=4)