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) significantly 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.
3.2. 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 specific changes in cell morphology such as cell shrinkage, membrane blebbing, nuclear condensation and internucleosomal DNA fragmentation1. 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.
3.3. 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 assay26 was conducted by flow 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 significantly 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 significantly inhibit cell proliferation by inducing apoptosis of colon cancer HT-29 cells.
Table 1
Effects of IOP60b on apoptosis and cell cycle of HT-29 cells
Group (mg/mL) | Apoptotic cells (%) | | Cell cycle distribution (%) |
Live | Early apoptosis | Late apoptosis | G0/G1 | G2/M | S |
0(control) | 92.42 ± 0.23f | 0.40 ± 0.03a | 0.35 ± 0.03a | | 73.54 ± 0.40a | 8.00 ± 0.00c | 18.46 ± 0.40e |
0.625 | 80.16 ± 0.47e | 3.55 ± 0.21b | 10.55 ± 0.01b | | 76.42 ± 0.5b | 8.00 ± 0.00c | 15.58 ± 0.5d |
1.25 | 73.33 ± 0.63d | 4.34 ± 0.67b | 18.05 ± 0.02c | | 79.86 ± 0.82c | 8.00 ± 0.00c | 12.14 ± 0.82c |
2.5 | 67.17 ± 0.50c | 5.32 ± 0.22c | 23.11 ± 0.18d | | 83.61 ± 0.6d | 6.27 ± 0.62b | 10.12 ± 0.2b |
5 | 54.75 ± 1.60b | 6.89 ± 0.83d | 28.60 ± 0.16e | | 85.01 ± 0.6d | 5.46 ± 0.39b | 9.53 ± 0.82b |
10 | 51.50 ± 0.44a | 7.37 ± 0.70d | 28.81 ± 0.11e | | 90.03 ± 0.40e | 3.31 ± 0.63a | 6.66 ± 0.40a |
The data are average values of triplicate ± standard deviation. a−f Mean values with different letters are significantly different (P < 0.05) |
3.4. 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 radiation27. In the present study, flow 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 al28. 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 proliferation29.
3.5. Effects of IOP60b on Bcl-2, Bax, and Caspase-3 expressions in HT-29 colon cancer cells
Increasing evidences have identified 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 significantly 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-apoptotic31, and the Bax/Bcl-2 ratio was often used as an index of apoptosis32. 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 fragmentation4 and its activation marks the irreversible stage of apoptosis 33. Results in Fig. 4 showed that caspase-3 activity increased significantly at all time-points (P < 0.05)༌which proved that caspase-3 also played an important role in the process of HT-29 colon cancer cells apoptosis induced by IOP60b. This conclusion was consistent with the point mentioned by Cheng-Chih Tsaia et.al.34 who investigated apoptosis effects of I. obliquus extract on HCT-116 cell line.
3.6. Effects of IOP60b on Bcl-2, Bax, and Caspase-3 expressions in HT-29 cells
To further determine the role that the Bcl-2 families and caspase-3 played in IOP60b mediated apoptosis, western blot analysis was used to detect their effects on the protein levels of Bcl-2, Bax and a caspase-3. The expression of β-action was used as a loading control. As shown in Fig. 5, similar to the RT-PCR results, there was a significant decrease in the expression levels of Bcl-2 protein (P < 0.05), while significant increase in Bax and Caspase-3 in the IOP60b-treated groups compared to the negative control (P < 0.05). Besides, these trends were more pronounced as the treatment time increased from 0 h, to 72 h which indicated that IOP60b induced cell apoptosis of HT-29 cells in a time-dependent manner.
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 significant 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.