The Apoptotic Effect of Polysaccharides with Specic Molecular Weight Extracted from Inonotus Obliquus on HT-29 Colon Cancer Cells

Background: Recently, much attention has been paid to natural products owing to their effective anticancer effects with relatively low toxicity, especially Inonotus obliquus (I. obliquus). Results: Polysaccharides with different molecular weights were ltered from water extract of I. obliquus using grading membrane ltration method. IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa) was found to have the highest yield and the highest inhibition ratio of HT-29 cancer cells, therefore it was chosen to evaluate the apoptotic effect of HT-29 cancer cells. After treated with different concentrations of IOP60b, morphological changes including cell shrinkage and nuclear condensation and DNA fragmentation in HT29 cancer cells were observed, and cells in early apoptosis and late apoptosis signicantly (p<0.05) increased in a dose-dependent manner by arresting cell cycle at G0/G1 phase. To further explore the underlying mechanism, RT-PCR and Western blotting were applied. Results indicated that both Bcl-2 family and Caspase family were involved in the process of apoptosis regulation and IOP60b induced cellular apoptosis via upregulation of Bax/Bcl-2 ratio and activation of Caspase-3. Conclusion: These data suggested that IOP60b might be the potential candidate for the clinical prevention and treatment of colorectal cancer.


Introduction
Colorectal cancer is one of the most common global gastrointestinal malignant tumors, which has caused great concern worldwide particularly in western and developed nations due to its great threat to human being health 1 . In fact, the incidence of colon cancer is closely related to the personal lifestyle. As far as we know, with the enhancement of the living standard and the change of the dietetic habit of people, the morbidity and mortality of colon cancer have been on the rise during the past few decades 2 .
At present, surgical resection, radiotherapy and chemotherapy are the dominant methods applied for colon cancer treatment. However, there still exist some drug resistance and severe side effects including hair loss, bleeding, diarrhea and immunosuppression 3 , which signi cantly limits their application. Thus, natural product offers a potential alternative approach for tumor therapy owing to their effective anticancer effects with relatively low toxicity 4 .
Inonotus obliquus (I. obliquus), a white rot fungus, has been widely used as a folk remedy in Russia since the 16th century 5 for its non-toxic effects in treating gastrointestinal cancers and digestive system diseases 6 . Polysaccharides are considered to be the main bioactive constituents of I. obliquus 7 , and have triggered much attention in recent years due to their biological activities, including anti-cancer 8 , anti-in ammatory 9 , anti-oxidation 10 , hypolycemic effect 11 , and immuno-stimulating 12 . Moreover, it has been reported that polysaccharides from I. obliquus have cytotoxicity on multiple tumor cells, such as human stomach carcinoma 13,14 , human hepatoma 14,15 , human lung carcinoma 14,16,17 , kidney adenocarcinoma 14 , human breast adenocarcinoma 14 , human ovary adenocarcinoma 14 , human endometrial epithelial cells 14 , murine melanoma cells 14,16,18 , human T lymphadenoma jurkat cells 15 et al..
Whereas it has been mentioned that the extraction of I. obliquus by water and ethanol could directly inhibit the proliferation of colon cancer cells which was mainly composed of polysaccharides and proteins, among which polysaccharides accounted for the most proportion 7,19,20 . A preliminary deduction is that polysaccharides from I. obliquus play a major role in its anticancer effects on colon carcinoma. Few studies were regarding the effect of polysaccharides with speci c molecular weight extracted from I. obliquus on colon cancer. In addition, studies on the mechanism of anti-colon cancer of I. obliquus showed that the aqueous extract of I. obliquus induced apoptosis of colon cancer cells by down-regulating Bcl-2 protein expression and up-regulating Bax and Caspase-3 protein expression 21 .
While Lee et al. 22 found that the ethanol extract of I. obliquus inhibited colon cancer by arresting the cell cycle G1phase. The mechanism of polysaccharides from I. obliquus on HT-29 colon cancer is still unclear. Therefore, the current study was carried out to further explore the anticancer activities of polysaccharides with speci c molecular weight isolated from I. obliquus and the related mechanism.

Materials and reagents
The fruiting bodies of I. obliquus were collected from Changbai Mountain, Jilin, China. Human colon carcinoma cells HT-29 were obtained from Cancer Hospital Chinese Academy of Medical Sciences, Beijing, China. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidi ed atmosphere with 5% CO 2 . All reagents used for cell culture were from Hyclone (USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan). Tissue DNA Kit was purchased from Solarbio (Beijing, China). Annexin V-FITC Apoptosis Detection Kit was purchased from Njjcbio (Nanjing, China). Antibodies to Bcl-2, Bax, Caspase-3 and β-actin were obtained from Wanleibio (Shenyang, China).

Extraction, puri cation and fraction of polysaccharides
The crude water-soluble polysaccharides (CIOP) were extracted from I. obliquus samples with distilled water at 60 °C according to the method outlined by Xu et al 23 . After removal of the protein by using the Sevag reagent, the CIOP solution was mixed with 95% ethanol to reach a nal concentration of 60% ethanol (v/v), and then the mixture was stored at 4 °C overnight. After centrifugation, dialysis and freeze drying, the precipitate was termed IOP60. 10 mg/mL IOP60 solution was prepared, then passed through a 0.22 µm lter, and assembled with ultra ltration centrifuge tubes (Merck Millipore Amicon™). After centrifugation and freeze drying, the fractions were collected and named as IOP60a (3 kDa ≤ molecular weight ≤ 10 kDa), IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa), IOP60c (30 kDa ≤ molecular weight ≤ 50 kDa), IOP60d (50 kDa ≤ molecular weight ≤ 100 kDa), and IOP60e (molecular weight ≥ 100 kD), respectively. The yield was calculated based on the weight of each fraction to the weight of the total IOP60.

Cell viability analysis
HT-29 cells were seeded for 12 h in 96-well plates (2 × 10 6 cells/mL), and then treated with 1.25 mg/mL polysaccharide fraction (IOP60a, IOP60b, IOP60c, IOP60d, IOP60e) for 24 h, respectively. CCK-8 assay was used to detect the cell viability according to the manufacturer's protocol 24 . All determinations were done in six duplicates. Inhibition ratio of tumor cell proliferation was calculated according to the formula below: Where As, Ac and Ab were the absorbance of treated cells, untreated cells and control group, respectively.

DNA fragmentation analysis
IOP60b-treated (48 h) HT-29 cells were washed and resuspended in 1 mL PBS. Cell total DNA was extracted according to the manufacturer's instructions. The extracted DNA samples were run on the 1.2% agarose gel in 1 × TAE buffer. After that, the gel was visualized with a UV light by Gel Imaging System, and photographed.

Cell apoptosis and Cell cycle analysis by ow cytometry
The percentage of speci c apoptotic cells was determined by ow cytometry according to the manufacturer's procedure. Brie y, HT-29 cells were plated in a 6-well plate (1 × 10 5 cells/mL) and treated with different concentrations of IOP60b (0.625, 1.25, 2.5, 5, 10 mg/mL) for 48 h. The cells were collected and washed twice with cold PBS, and then resuspended in 500 µL binding buffer containing 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) for 10 min in the dark, nally analyzed using Millipore guava easy Cyte ™ ow cytometer (Millipore, USA) and Guavasoft 3.1.1 software.
Similarly, HT-29 cells were rst plated in a 6-well plate (1 × 10 5 cells/mL) and treated with different concentrations of IOP60b (0.625, 1.25, 2.5, 5, 10 mg/mL) for 48 h and then used for cell cycle analysis. Cells were centrifuged at 12000 × g for 5 min and then xed with 70% (v/v) ethanol and stored at 4 °C overnight. Cells precipitate were washed with PBS and treated with 100 µL RNase, incubating for 30 min at 37 °C. After centrifugation, cells were incubated with PI at 4 °C for 30 min in the dark. Cell cycle distribution was detected by ow cytometry and analyzed using ModFit LT 5.0 software.

Real-time quantitative RT-PCR analysis
HT-29 cells (5 × 10 5 cells/mL) were seeded in 6-well plates and treated with 5 mg/mL IOP60b for 0 h, 24 h, 48 h, 72 h, respectively. Total RNA was extracted using total RNA extraction kit (GeneMark ™ ) and then reversed to cDNA by using the All-in-One ™ First-Strand cDNA Synthesis Kit (GeneCopoeia, USA) according to the manufacturer's protocol. Real-time PCR was performed with 2 × Es Taq MasterMix (Dye) (CWBIO, Beijing, China). The following primers (Sangon Biotech, Shanghai, China) were used for ampli cation: Total proteins were extracted by using a Total Protein Extraction Kit (Wanleibio, China), and the protein concentration was determined by using a BCA Protein Assay Kit (Wanleibio, China) according to the manufacturer's protocol. Equal amounts of proteins were boiled for 5 min and separated by SDS-PAGE, then transferred onto a PVDF membrane. Blocking of non-speci c binding was achieved by placing the membrane in a dilute solution of 5% skim milk powder in tris-buffered saline (TBS) for 1 h. After blocking, a dilute solution of primary antibody (Bcl-2, Bax, Caspase-3 and β-actin) at a concentration of 1:500 was incubated with the membrane overnight at 4 °C under gentle agitation. The primary antibody was then diluted with TBST wash buffers. After rinsing the membrane to remove unbound primary antibody, the membrane was incubated with a dilute solution of secondary antibody (HRP-labeled goat anti-rabbit Ig G) at a concentration of 1:5000 for 45 min at 37 °C. The membrane was washed with TBST buffers again.
The Electrochemilu-minescence (ECL) reagent was used to measure the chemiluminescence intensity. Finally, the band intensities were quantitated using Gel-Pro-Analyzer software.

Statistical analysis
All the measurements and analyses were carried out in triplicate. The experimental results were presented as means of three determinations ± SD (standard deviation). Origin (version 8.5) and SPSS (version 16.0) with Tukey's multiple comparisons were used for the statistical and graphical evaluation. The statistical signi cance of mean differences was based on p-Values of < 0.05.

Results And Discussion
3.1. Yields and inhibition ratios of polysaccharides with different molecular weights Results in Fig. 1 showed that the yield of IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa) signi cantly higher than that of any other polysaccharide fraction (P < 0.05). As for the inhibition ratio of HT-29 colon cancer cells, IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa) was the highest, reaching 61.9%, which was followed by IOP60a (3 kDa ≤ molecular weight ≤ 10 kDa), while IOP60e with the highest molecular weight(≥ 100 kDa)was the lowest (28.3%). As was reported, polysaccharides with lower molecular weight generally had higher biological activity 14,25 , which was consistent with our results, that probably because polysaccharides with lower molecular weight were more likely to pass free radicals. Therefore, IOP60b was chosen to explore its effects on apoptosis of HT-29 colon cancer cells and the underlying mechanism.

IOP60b induced morphological changes and DNA fragmentation
Optical microscopy was used to observe morphological changes in HT-29 cells after treated with IOP60b (0, 0.625, 1.25, 2.5, 5 and 10 mg/mL) for 48 h. It can be seen from Fig. 2 that cells in the control group adhered to the wall and grew vigorously. The nuclei and cell bodies were large, and they were fusiform or polygonal. The cytoplasm was uniform and transparent with high transmittance. After 48 hours of IOP60b treatment, the number of adherent cells decreased and the cell contents increased which reduced the transmittance, the cells shrunk or even shattered, the nuclei were concentrated, and the cell volume became smaller, rounded and deformed. In addition, as the concentration of IOP60b increased, the changes in cell morphology became more pronounced.
Agarose gel electrophoresis was used to detect the nuclear DNA from HT-29 cells. As can be seen in Fig. 3, the untreated cells represented by Lane A showed normal chromosomal DNA with no DNA ladder, while DNA isolated from HT-29 cells treated with different concentrations of IOP60b for 48 h (Lane B-F) was all degraded into giant DNA fragments, and the DNA ladder phenomenon became more apparent as the increasing concentrations of IOP60b. As we all know, apoptosis is an extremely important process to maintain cellular homeostasis, accompanying with speci c changes in cell morphology such as cell shrinkage, membrane blebbing, nuclear condensation and internucleosomal DNA fragmentation 1 . Therefore, combined the cell morphological changes with nuclear DNA fragmentation, it is speculated that IOP60b may induce colon cancer HT-29 cell death through apoptotic pathway.

IOP60b induced apoptosis in HT-29 cells
To evaluate whether the IOP60b treatment (0, 0.625, 1.25, 2.5, 5 and 10 mg/mL) for 48 h in HT-29 cells was associated with apoptosis, annexin V-FITC/PI apoptosis assay 26 was conducted by ow cytometry. Results in Table 1 demonstrated that as the concentration of IOP60b increased from 0.625 mg/mL to 10 mg/mL, the percentages of cells in early apoptosis compared with the negative control group increased by 3.15%-6.97%, and the percentage of cells in late apoptosis signi cantly increased ranging from 10.20-28.46% (P < 0.05). In addition, the total number of apoptotic cells in HT-29 treated with 0.625, 1.25, 2.5, 5 and 10 mg / mL IOP60b increased by 13.35%, 21.64%, 27.68%, 34.74% and 35.43%, respectively. It was apparent that IOP60b induced cells in early apoptosis and late apoptosis increased in a dose-dependent manner. Thus, we can conclude that IOP60b can signi cantly inhibit cell proliferation by inducing apoptosis of colon cancer HT-29 cells.

IOP60b triggers cell cycle arrest in HT-29 cells
The cell-division cycle is a vital process which consists of four distinct phases: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis or meiosis). Deregulation of the cell cycle is the most common abnormality in human cancer. The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation 27 . In the present study, ow cytometry was used to investigate the cell cycle distribution treated with different concentrations (0.625 1.25 2.5 5 and 10 mg/mL) of IOP60b for 48 h. In the presence of IOP60b, we observed a dose-dependent increase in the percentage of cells in G0/G1 phase accompanied by a corresponding reduction in the percentages of cells in S and G2/M phases (Table 1). These data suggest that IOP60b induced G0/G1 phase arrest in HT-29 cells, which was in agreement with the results of Youn et al 28 . The mechanism by which polysaccharides from I. obliquu inhibits cell proliferation and arrests cell cycle can be explained as follows: On the one hand, polysaccharides can inhibit ribosome synthesis in G0/G1 phase, thereby inhibiting cell protein synthesis and reducing mitosis, leading to cell proliferation. On the other hand, the polysaccharides interferes with the synthesis of DNA by inhibiting the synthesis of RNA and protein, thereby arresting the cells in the G0/G1 phase and ultimately inhibiting cell proliferation 29 .

Effects of IOP60b on Bcl-2, Bax, and Caspase-3 expressions in HT-29 colon cancer cells
Increasing evidences have identi ed natural product might control cancer via the direct or indirect modulation of apoptosis-related genes 30 . RT-PCR was used to investigate the expression of caspase-3, Bax, Bcl-2, and Bad at different time points. As shown in Fig. 4, after 0 h, 24 h, 48 h and 72 h treatment with 5 mg/mL IOP60b, the expression level of Bcl-2 gene in the drug-exposed groups was signi cantly decreased, whereas the level of Bax gene was dramatically increased in a time-dependent manner compared to the negative control (P < 0.05). As we all know, Bcl-2 plays a role of anti-apoptosis in the Bcl-2 family, while Bax play a role of pro-apoptotic 31 , and the Bax/Bcl-2 ratio was often used as an index of apoptosis 32 . Therefore, we deduced that IOP60b promote HT-29 colon cancer cells apoptosis by regulating Bax/Bcl-2 ratio. Moreover, we examined whether caspase-3 activity would change with increasing concentrations of IOP60b. As was reported,caspases-3 protease, can target structural substrates and induce cancer cell breakdown and DNA fragmentation 4 and its activation marks the irreversible stage of apoptosis 33 . Results in Fig. 4 showed that caspase-3 activity increased signi cantly at all time-points (P < 0.05) which proved that caspase-3 also played an important role in the process of An article by Lee et al. 21 mentioned that aqueous extracts from the fruiting bodies of I.obliquus could inhibit HT-29 colorectal cancer cells, and the anti-apoptotic protein Bcl-2 was found to be inhibited and the pro-apoptotic protein Bax was promoted, at the same time, the levels of procaspase-3 showed a decreasing trend implying that procaspase-3 underwent cleavage and activation into caspase-3, which in turn affected apoptosis in HT-29 cells. Youn et al. 35,36 employed Western blotting to analyze the protein expression of procaspase-3 in HepG2 liver cancer cells which were treated with aqueous extracts of I. obliquus, results showed that as the concentration of I. obliquus increases, there was a signi cant decrease in procaspase-3. It was explained that extracts from I. obliquus fruiting bodies could aid in procaspase-3 activation into caspase-3. Nomura et al. 37 also found that inotodiol, a lanostane triterpenoid from I. obliquus can inhibit P388 leukemia cells through caspase-3 activation.
In this case, our results are in agreement with these previous studies wherein extracts of I. obliquus have been reported to exhibit anticancer effects via upregulating Bax, Caspase-3 activity and downregulating Bcl-2 activity 19,20 .That was to say, polysaccharides from I. obliquus induce apoptosis in HT-29 cells through the mitochondrial pathway.

Conclusions
In the present study, polysaccharides with different molecular weights were successfully puri ed from IOP60 with ultra ltration centrifuge tubes. IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa) was selected to verify the effects and mechanisms of apoptosis because it had the highest yield and inhibition ratio of HT-29 colon cancer cells. It was observed that IOP60b induced morphological changes and DNA ladder phenomenon of HT-29 colon cancer cells. The results of ow cytometry indicate that IOP60b induced cells in early apoptosis and late apoptosis increased in a dose-dependent manner and signi cant G0/G1 phase arrest in HT-29 cells. Moreover, according to the RT-PCR and western blot results, it could be preliminarily inferred that, IOP60b induced cellular apoptosis of HT-29 cells through the mitochondrialmediated apoptosis pathway in which the expression of Bax and Caspase-3 were upregulated and the expression of Bcl-2 was downregulated. These data suggested that polysaccharides with speci c molecular weight (10 kDa ≤ molecular weight ≤ 30 kDa) could signi cantly inhibit the growth of HT-29 cells and induce apoptosis. IOP60b might be the potential candidate for the clinical prevention and treatment of colorectal cancer, while the underlying mechanism still needs further study.