Introduction

Blastocystis is an anaerobic microorganism which is commonly found in the human stool sample (Windsor et al. 2002). It is known to cause many nonspecific symptoms such as diarrhea, abdominal pain, and flatulence (Suresh et al. 2009) which may be observed in immunocompromised and immunocompetent hosts (Doyle et al. 1990; Cirioni et al. 1999). High rates of Blastocystis infection are found in developed countries (Wong et al. 2008). To date, nine different subtypes of Blastocystis have been reported in human based on genomic studies (Yoshikawa et al. 1998, 2000, 2004). The pathogenesis of Blastocystis remains as a controversial issue, as it cannot be clearly assigned to certain genotype. Recently, the solubilized antigen of Blastocystis was shown to facilitate the in vitro proliferation of human colorectal carcinoma cells (HCT116) (Chandramathi et al. 2010). It was also observed to cause oxidative damage in rats inoculated with human-derived Blastocystis isolate (Chandramathi et al. 2009). However, these studies were limited to only one single isolate. Therefore, the present study attempts to evaluate the effect of solubilized antigen isolated from five different subtypes of Blastocystis on colon cancer cells, HCT116 proliferation. In addition to that, we also compared the gene expression of cytokines, nuclear transcription factors, and apoptotic genes (Table 1) in colon cancer cell line in the presence of Blastocystis (Figs. 1, 2, 3, and 4).

Table 1 Inventoried primers used in quantitative real-time RT-PCR analysis (TaqMan® Gene Expression Assays, Applied Biosystem)
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Fig. 1
figure 1

Gene expression profiles of Th2 cytokines in HCT116 cells upon exposure to 1 μg/ml of different subtypes of Blastocystis. Values are presented in fold difference observed in comparison with respective control (untreated samples) and normalized against endogenous gene (human β-actin). Error bars represent the relative quantity ± SD (n = 3). A positive fold difference refers to upregulation and vice versa. The significant difference between ΔC of different subtypes is represented by *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA (SPSS version 17). All the studied genes used inventoried primers sourced from Homo sapiens using probes labeled with 6-carboxyfluorescein (FAM) dye and minor groove binder (MGB) quencher (Applied Biosystems)

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Fig. 2
figure 2

Gene expression profiles of Th1 cytokines in HCT116 cells upon exposure to 1 μg/ml of different subtypes of Blastocystis. Values are presented in fold difference observed in comparison with respective control (untreated samples) and normalized against endogenous gene (human β-actin). Error bars represent the relative quantity ± SD (n = 3). A positive fold difference refers to upregulation and vice versa. The significant difference between ΔC of different subtypes is represented by *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA (SPSS version 17). All the studied genes used inventoried primers sourced from H. sapiens using probes labeled with FAM dye and MGB quencher (Applied Biosystems)

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Fig. 3
figure 3

Gene expression profiles of gene transcription factor in HCT116 cells upon exposure to 1 μg/ml of different subtypes of Blastocystis. Values are presented in fold difference observed in comparison with respective control (untreated samples) and normalized against endogenous gene (human β-actin). Error bars represent the relative quantity ± SD (n = 3). A positive fold difference refers to upregulation and vice versa. The significant difference between ΔC of different subtypes is represented by *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA (SPSS version 17). All the studied genes used inventoried primers sourced from H. sapiens using probes labeled with FAM dye and MGB quencher (Applied Biosystems)

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Fig. 4
figure 4

Gene expression profiles of apoptotic genes in HCT116 cells upon exposure to 1 μg/ml of different subtypes of Blastocystis. Values are presented in fold difference observed in comparison with respective control (untreated samples) and normalized against endogenous gene (human β-actin). Error bars represent the relative quantity ± SD (n = 3). A positive fold difference refers to upregulation and vice versa. The significant difference between ΔC of different subtypes is represented by *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA (SPSS version 17). All the studied genes used inventoried primers sourced from H. sapiens using probes labeled with FAM dye and MGB quencher (Applied Biosystems)

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Methods

Preparation of solubilized antigen from Blastocystis

The axenic Blastocystis was collected using the Ficoll-Paque density gradient centrifugation method as described previously by Chandramathi et al. (2010) and Chan et al. (2012). Sonication was carried out at a frequency of 60 Hz and 0.5 amplitude for 10 cycles. The lysate was observed under a microscope to ensure that a complete lysis had taken place. The sonicated samples were left overnight at 4 °C and were centrifuged at 60,000×g for 15 min. The supernatants were filter sterilized, and the protein concentrations were determined using the Bradford assay (Bio-Rad, USA).

Human colorectal carcinoma cell line, HCT116

Human colon cancer cell line HCT116 was obtained from the American Type Culture Center and grown at 37 °C in a humidified atmosphere containing 5 % CO2 in 5 ml RPMI 1640 growth medium supplemented with 5 % (v/v) fetal bovine serum, l-glutamine (2 mM), penicillin–streptomycin (100 U/ml), and Fungizone (2.5 μg/ml) in a 25-cm3 culture flask. The cells were detached by trypsinization. The detached cells were washed once with PBS and then resuspended in the RPMI 1640 medium prior to the introduction of antigen.

Proliferation assay

HCT116 cells were harvested and seeded into 96-well plates (1 × 103 cells per well) and grown in a 100-μl growth medium. The cells were incubated overnight at 37 °C in a CO2 incubator containing 5 % CO2 before introducing the antigen with concentrations ranging from 0.01 to 10 μg/ml. A preliminary test was conducted to determine the optimum concentrations of antigen to be added into each well. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed after incubating cells for 48 h to determine cell proliferation in vitro (Mosmann 1983). This assay measures the capacity of mitochondrial succinate dehydrogenase enzymes in living cells which reduces MTT to purple-colored formazan. The intensity of the color was measured spectrophotometrically at 595 nm.

Real-time polymerase chain reaction analysis

Optimum concentration (1 μg/ml) of Blastocystis antigen (which gave a maximum proliferation of HCT116 cells) was introduced into a culture flask containing 5 ml medium and 1 × 105 cells. Jones' medium was introduced instead of antigen for control experiments. Total RNA was extracted from pelleted HCT116 cells via the Ambion RNAqueous®-4PCR Kit (Ambion, CA, USA) according to the manufacturer's instructions. Complementary DNA was synthesized from total RNA samples extracted from HCT116 cells using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Real-time PCR was performed with the Applied Biosystems StepOne™ System. The threshold cycle (C T) value is defined as the cycle number at which the fluorescence crosses a fixed threshold above the baseline. For a relative quantification, fold changes were measured using the ΔΔC T method. The C T value of each sample was measured and compared to endogenous gene, β-actin. Relative transcripts were determined by the formula 2-∆∆C T.

Statistical methods

The level of significance between the various subtype-induced cell proliferations was determined using one-way ANOVA. MTT tests were performed in triplicate. In all cases, three independent experiments were carried out. Results are presented as the mean value ± standard error of the mean. The level of gene expression difference between treated and non-treated (control) samples were assessed using one-way ANOVA. A p value of 0.05 was considered as the minimum threshold of significance. All statistical analyses were performed using the SPSS Software version 17.0.

Results

The proliferation of colon cancer cells was initially assessed after 24 h of incubation with solubilized antigen. The optimum concentration of the antigen for the proliferation of colon cancer cells was at 1.0 μg/ml. Table 2 shows the proliferations of cancer cells in the presence of various Blastocystis subtypes. Out of the five different Blastocystis subtypes tested, subtype 3 showed a statistically significant proliferation of HCT116 (Table 2). Blastocystis antigen caused the upregulation of Th2 and Th1 cytokines (Figs. 1 and 2) and downregulation of IFN-γ and p53 in HCT116 cells (Figs. 1 and 4) as determined by real-time polymerase chain reaction (RT-PCR) analysis. It is noteworthy that subtype 3 Blastocystis antigen caused a significantly higher stimulation of cathepsin B (CTSB) and TGF-β gene expression (Figs. 1 and 4).

Table 2 Proliferation of HCT116 upon exposure to 1.0 μg/ml Blastocystis antigen
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Discussion

Study has already been conducted previously to correlate Blastocystis and HCT116, but the Blastocystis isolate used was devoid of any molecular characterization (Chandramathi et al. 2010). In another study, the pathogenic potential of symptomatic Blastocystis was reported to be higher compared to asymptomatic isolate when introduced to colorectal cancer cells (Chan et al. 2012). It is important to assess if different subtypes can influence cancer cells proliferation, especially when there have been conflicting reports on the subtypes of Blastocystis and its pathogenicity role (Kaneda et al. 2001; Tan et al. 2008). Therefore, the present study exploits the fact that Blastocystis stimulates proliferation of cancer cells and is being used in this study to assess if subtype variation of Blastocystis can influence its pathogenic potential towards cancer cells. Colon epithelial cells which are found at the surface of gastrointestinal tract forms a mechanical barrier and play an important role in gut mucosal immune response and defense mechanism against the invading pathogens. In this study, we hypothesized that in Blastocystis-infected colon cancer patients, the expression of many immunological components by these cells may probably contribute to pathophysiological changes in the host. In a previous study, the inflammatory cytokine responses were observed to be modulated in human epithelial cells in the presence of Blastocystis (Long et al. 2001). It was also speculated that Blastocystis has the ability to downregulate the host immune response at the beginning stage of colorectal cancer to improve its survival (Long et al. 2001). In this study, RT-PCR work has revealed that colon cancer cells express significantly higher levels of IL-6 and IL-8 in the presence of Blastocystis antigen (Fig. 1), providing a possible explanation for the observed proliferation. Therefore, IL-6 and IL-8 probably can be associated with cellular immune response activation towards Blastocystis antigen. Similar results were observed in HCT116 before incubation with a solubilized antigen of symptomatic Blastocystis isolate (Chan et al. 2012). However, the mean value of IL-8 expression is considerably high for subtypes 2 and 3 (Fig. 1). We speculate that the release of inflammatory cytokines such as IL-6 and IL-8 together with reactive oxygen species contribute to pathogenesis of carcinogenesis. Previously, IL-6 has been associated with proliferation of colon carcinoma in a number of studies (Galizia et al. 2002; Chung and Chang 2003; Becker et al. 2005). Of interest is the high expression of CTSB detected in response to subtype 3 Blastocystis antigen (Fig. 4). Cathepsin B has been widely associated with carcinogenesis and highly expressed in colorectal cancer (CRC) patients (Herszenyi et al. 2008). It has also been shown to enhance the motility of cancer cells and has tumor-promoting ability via upregulation of TGF-β (Elliott and Blobe 2005). RT-PCR analysis also showed downregulation of IFN-γ and p53 expression which was more significant in subtype 3-treated HCT116 cells (Figs. 1 and 4). This indicates Blastocystis infection, probably reducing the apoptosis in colon cancer cells. p53 is known to play a vital role in CRC and is known as a tumor suppressor gene, and its dysregulation has been widely accepted as a carcinogenesis proponent. Furthermore, elevated p53 levels have been reported in stage III colorectal tumors (Adrover et al. 1999).

Conclusion

There has been far no information on whether different subtypes of Blastocystis can influence proliferation of colon cancer cells. Therefore, this study basically provides a better understanding of how proliferation of HCT116 can be possibly affected by the solubilized antigen isolated from different subtypes of Blastocystis in vitro. The proliferation analysis and gene expression findings in the present study implicate a possible pathogenic role for subtype 3 Blastocystis. The data obtained also suggest the importance of screening Blastocystis in patients with an early diagnosis of CRC which may help to prevent the worsening of this condition.