Induction of apoptosis in HCT-116 colon cancer cells by polysaccharide of Larimichthys crocea swim bladder

Larimichthys crocea swim bladder is a traditional food and medicine widely used in China. The in vitro anticancer effects of polysaccharide of L. crocea swim bladder (PLCSB) in HCT-116 human colon cancer cells was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. At concentrations ranging between 0 and 800 μg/ml PLCSB, cancer cell viability was decreased by PLCSB in a concentration-dependent manner. In particular, 400 μg/ml PLCSB significantly (P<0.05) induced apoptosis, which was demonstrated by 4,6-diamidino-2-phenylindole staining and flow cytometry analysis. To elucidate the mechanisms underlying the anticancer effect of PLCSB in HCT-116 cancer cells, the expression of apoptosis and metastasis-associated genes was analyzed by reverse transcription-polymerase chain reaction and western blot analysis. A total of 400 μg/ml PLCSB significantly induced apoptosis in HCT-116 cells (P<0.05) via the upregulation Bax, p53, p21, apoptotic protease activating factor 1, caspase-3, -8, and -9, as well as Fas and the downregulation of B-cell lymphoma 2 (Bcl-2), Bcl-extra large and Fas ligand (L). The results of this study demonstrated that PLCSB exhibits an anticancer effect on HCT-116 colon cancer cells, in vitro.


Introduction
The swim bladder is important organ in Osteichthyes that is used to maintain balance and contains ~10% polysaccharide. Larimichthys crocea is used as drug in traditional Chinese medicine (TCM) and it has been reported that L. crocea swim bladder may remove free radicals and protect against cancer (1). Polysaccharides are an important material for producing drugs, and the polysaccharide obtained from Phellinus linteus and Pleurotus ostreatus have been shown to exhibit an anticancer effect in colon cancer cells in vitro (2,3).
Apoptosis induction in cancer cells is characterized by changes in cell morphology, which include cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation (4). Apoptosis presents a critical defense mechanism against cancer, which leads to the death of potentially harmful cells. Dysregulated apoptotic processes have been implicated in numerous diseases; these lead to the inhibition of cell death and the progression of diseases, including cancer (5). Identifying the critical events involved with carcinogenesis may present an opportunity to prevent cancer development using TCM that triggers apoptosis, particulary with the extraction of natural substances. However, TCM may augment disease progression. In addition to the effects on protein expression and function, an increasing number of studies have reported that a large number of components of TCM may exert effects on the human genome, via the direct or indirect modulation of gene expression (6).
Western blot analysis. Total cell lysates were obtained using extraction buffer as previously described (12

Results
Inhibitory effects of PLCSB on cancer cell growth. The inhibitory effects of PLCSB on HCT-116 cell growth were analyzed by MTT assay. HaCaT keratinocyte cells were evaluated and the growth inhibitory rates were not associated with the concentration of PLCSB. When the cells were treated with 0-400 µg/ml PLCSB, no significant difference in the growth inhibitory rates were identified, and the rates were all <10%. When the cells were treated with >400 µg/ml PLCSB, the growth inhibitory rate increased and, following treatment with 800 µg/ml PLCSB, the rate was 100% (Fig. 1A). At concentrations ranging between 0 and 400 µg/ml, cell viability was decreased by the PLCSB in a concentration-dependent manner. At the concentration of 600 µg/ml, the survival rate of the cells treated with PLCSB reached 0% (Fig. 1B). No inhibitory or toxic effects on the growth of normal human cells were observed at concentrations of 0-400 µg/ml PLCSB; however, an inhibitory effect on growth was exhibited in HCT-116 cancer cells. These results indicated that PLCSB only exerts an effect on cancer cells. Consequently, concentrations of 100, 200 and 400 µg/ml PLCSB were selected for subsequent experiments.

Induction of apoptosis by PLCSB.
To determine a possible mechanism underlying the growth inhibitory activity of PLCSB in HCT-116 cancer cells, the induction of apoptosis was analyzed. The extent of chromatin condensation was    following incubation with 100, 200 or 400 µg/ml PLCSB for 48 h. The expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 and Bcl-xL exhibited significant changes (P<0.05) following treatment with PLCSB (Fig. 3). These results indicate that treatment with PLCSB leads to apoptosis induction in HCT-116 cells via a Bax-, Bcl-2-and Bcl-xL-dependent pathway.
p53 and p21 gene expression. As shown in Fig. 4, PLCSB significantly increased the level of p53 and p21 mRNA expression (P<0.05). These changes in p53 and p21 expression as a result of PLCSB treatment may lead to the induction of apoptosis in HCT-116 cells. These results showed that PLCSB exhibits significant anticancer activity via the induction of apoptosis.
Apaf-1 gene expression. The mRNA and protein expression of Apaf-1 was increased by treatment with PLCSB, with the greatest Apaf-1 expression observed in the 400-µg/ml PLCSB-treated cells (8.85-and 6.45-fold that of the mRNA and protein expression in the untreated cancer cells, respectively) (Fig. 5). The Apaf-1 mRNA and protein expression in 200-µg/ml PLCSB-treated cells was 5.57-and 3.90-fold that of the control cells. The 100-µg/ml PLCSB-treated cells also showed higher Apaf-1 mRNA and protein expression than the control cells (3.59-and 1.94-fold, respectively), but this was lower than that of cells treated with 200 and 400 µg/ml PLCSB.
Caspase gene expression. The mRNA expression levels of caspase-3, -8 and -9 were extremely low in untreated control  HCT-116 cells; however, the levels significantly increased following treatment with 400 µg/ml PLCSB (P<0.05). Following PLCSB treatment, the mRNA expression of caspase-3, -8 and -9 were gradually increased in a dose-dependent manner (Fig. 6). Furthermore, the induction of apoptosis by PLCSB was associated with the upregulation of caspase-3, -8 and -9 mRNA and protein expression.
Fas and FasL gene expression. This study further determined whether the apoptosis-inducing actions of PLCSB were associated with inhibition of Fas and FasL gene expression. As shown in Fig. 7, PLCSB demonstrated induction activity of apoptosis in HCT-116 cells, as indicated by increased mRNA and protein expression of Fas along with decreased FasL expression when compared with untreated cancer cells (P<0.05).

Discussion
The swim bladder has been historically used as a folk medicine.
Recently, swim bladder has been shown to alleviate various inflammatory conditions, and it may also augment the function of platelets, capillary vessels and clotting factors (11).
Polysaccharides are the main component of swim bladder; however, few studies have investigated the polysaccharides of the swim bladder, and these studies showed that polysaccharides of the swim bladder posses anti-inflammatory effects (12,13). To the best of our knowledge, the present study was the first to investigate the anticancer effect of apoptosis induction by PLCSB in vitro. The results demonstrated that the PLCSB exhibited a marked apoptosis-inducing effect against the HCT-116 colon cancer cells. Apoptosis induction in cancer cells is a potentially promising approach for cancer therapy (14). In the present study, PLCSB decreased the growth of HCT-116 cells via the induction of apoptosis. Apoptotic cells with degraded DNA exhibit hypodiploid DNA content and are presented as sub-G1 peaks on DNA histograms, which are used to count the percentage of apoptotic cells (15). The formation of apoptotic bodies was observed, in addition to increased sub-G1 DNA (apoptotic cells) accumulation in cells treated with PLCSB.
Apoptosis is a critical cellular event and, thus, eludicating its mechanisms of action may present potential for improvements in tumor diagnosis and therapy (16). In normal cells, the anti-apoptotic protein Bcl-2 is expressed on the outer mitochondrial membrane surface (17). The apoptosis regulator BAX promotes apoptosis by binding to and antagonizing the Bcl-2 protein (18). Bcl-xL is a member of the Bcl-2 protein family and it is hypothesized that the relative amount of pro-and anti-survival Bcl-2 family of proteins determines whether a cell undergoes apoptosis (19). The Bax, Bcl-2 and Bcl-xL genes are predominantly expressed during apoptosis and, thus, the effects of these genes on apoptotic activity were determined.
p53, which is a tumor supressor, upregulates expression of the Bax protein, which has been found to be involved in p53-mediated apoptosis (20). p53 is a transcription factor, which regulates the Bax downstream target gene when activated in response to stress (21). The expression of the p21 gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli (22).
The Apaf-1 gene encodes a cytoplasmic protein, which presents one of the most important factors in the apoptosis regulatory network. The Apaf-1 protein binds and cleaves caspase-9 preproprotein, releasing the mature, activated caspase-9, which stimulates a subsequent caspase cascade that causes the cancer cell to undergo apoptosis (23). Caspase-8 initiates disassembly in response to signals from extracellular apoptosis-inducing ligands (24). Caspases present a proteolytic network within the cell in which upstream initiator caspases are activated early in the apoptotic process (caspase-9), leading to the activation of downstream caspases (caspase-3). Caspase-3 amplifies caspase-9 and caspase-9 initiation signals to induce nuclear disassembly (25).
Fas is a death domain-containing member of the tumor necrosis factor receptor superfamily, which is associated with apoptosis of cancer cells. The FasL-Fas system has been investigated with respect to its death-inducing function. The Fas receptor exerts an apoptotic signal by binding to FasL, which is expressed on the surface of other cells (26). FasL signals via trimerization of FasR, which spans the membrane of the target cell. This trimerization usually leads to apoptosis (27).
In the death receptor pathway, a signaling cascade leads to the activation (using an adaptor, Fas-associated protein with death domain) of a caspase cascade involving caspase-8 and -3. BH3-only proteins are firstly upregulated or activated by cytotoxic injury during the mitochondrial pathway, and subsequently activate and oligomerize Bax, which leads the oligomerized Bcl-2 family members, Bax/Bak, to induce the release of cytochrome c from the mitochondria into the cytosol. Consequently, cytochrome c and Apaf-1 form a complex, the apoptosome, which activates caspase-9 and subsequently caspase-3 (28). In agreement with the results of the present study, the induction of apoptosis in drug or functional material-treated cancer cells has been reported to increase Bax, p53, p21, Apaf-1, caspase-3,-8,-9 and Fas gene expression, and decrease Bcl-2, Bcl-xL and FasL gene expression (9,(29)(30)(31)(32).
In the present study, PLCSB induced a high level of apoptotic activity in HCT-116 colon cancer cells, in vitro. PLCSB, particularly at high concentrations, induced apoptosis, which was demonstrated by DAPI staining, flow cytometry analysis, and changes in the mRNA and protein expression of apoptosis-related genes. PLCSB may be used as health product or medicine for cancer prevention and treatment in the future.