Introduction

Blastocystis hominis is one of the most common intestinal protozoan parasites of humans. It is also known to show diverse morphologies and reproductive processes (Govind et al. 2002). The extreme debate regarding its role in the pathogenicity had led to the recent findings on both phenotypic and genotypic characteristics of asymptomatic and symptomatic human-derived B. hominis isolates (Tan et al. 2008). Recently, the elevation of oxidative damage in rats inoculated with human B. hominis has also been demonstrated (Chandramathi et al. 2009). We have also observed that the elevation of urinary hyaluronidase in these rats is suggestive of invasive activity. Furthermore, elevated levels of proinflammatory interleukin 6 (IL-6) and IL-8 cytokines in the serum of these rats have further confirmed the pathogenic potential of B. hominis (personal communication).

Generally, when a host’s immune system is triggered by an infection of parasites, a massive production of ROS or oxidative burst is activated by macrophages that are coupled with the inflammatory system (Rosen et al. 1995). Macrophage or phagocyte activation causes the release of reactive species, which leads to lipid peroxidation, protein damage, and DNA strand breaks (Ohshima and Bartsch 1994), which has enabled studies to correlate the inflammation by parasitic infection with cancer (Fitzpatrick 2001). Hence, it is imperative for us to investigate the cellular responses and immunomodulation caused by B. hominis infection especially in malignancy as it is one of the most common parasites seen in any stool survey.

To date, there have been limited studies done on the cytopathic effect or cellular cytokine responses of Blastocystis sp. infection (Walderich et al. 1998; Long et al. 2001; Puthia et al. 2008). These studies investigated mainly on two types of cytokines pertinent to inflammatory responses. The present study investigates the effect of solubilized antigen from B. hominis on the cell viability together with gene expressions of cytokine, transcriptional factor, and apoptotic mediators in both peripheral blood mononuclear cells (PBMCs, which represent the immune cells) as well as colorectal cancer cells in an attempt to assess if they influence the growth of cancer cells.

Materials and methods

Isolation of peripheral blood mononuclear cells

Human blood sample from a healthy volunteer (approximately 12 ml) was collected in sterile ethylenediaminetetraacetic acid (EDTA) tubes. PBMCs were isolated using Histopaque®-1077 (Sigma-Aldrich, USA) according to the density gradient centrifugation method (Boyum 1974). The collected PBMCs were washed with phosphate-buffered saline (PBS) and resuspended in growth media of Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin–streptomycin, and 2.5 μg/ml fungizone prior to the introduction of antigen.

Cultivation and collection of HCT116

Human colorectal carcinoma cell line, HCT116, was purchased from the American Type Culture Center and maintained in 25-cm3 culture flask containing 5-ml growth medium of RPMI 1640 supplemented with 5% FBS, 2 mM l-glutamine, 100 U/ml penicillin–streptomycin, and 2.5 μg/ml fungizone and incubated in an incubator set with 100% humidity, atmosphere containing 5% CO2, and a temperature of 37°C. During the harvesting process, the cells were liberated from the substratum of the culture flask using 0.25% trypsin–EDTA. The detached cells were washed with PBS resuspended in growth medium before introducing with antigen isolated from B. hominis.

Axenization of B. hominis and isolation of solubilized antigen

B. hominis isolate was obtained from a human subject. Cysts were isolated using the Ficoll-Paque technique according to Zaman and Khan (1994). Harvested cysts were washed in sterile saline and cultured in Jones medium supplemented with 10% heat-inactivated horse serum and incubated at 37°C (Suresh and Smith 2004). After 2 days, the isolate was checked under a microscope to confirm the presence of B. hominis (vacuolar form), and the axenization procedure was commenced according to the method previously established and further modified in our laboratory (Lanuza et al. 1997). Axenic B. hominis (vacuolar forms) were harvested using Ficoll-Paque according to the density gradient centrifugation method. The harvested organisms were resuspended in basal Jones medium (without supplementation) and lysed by sonication method. The homogenate was next incubated overnight at 4°C. After incubation, the homogenate was centrifuged at 13,000×g for 15 min. The supernatant, which contains the solubilized antigen from B. hominis (Blasto-Ag), was filter-sterilized, and the protein concentration was determined by Bradford assay.

Introduction of solubilized antigen (Blasto-Ag) to PBMC and HCT116

Freshly isolated PBMCs and harvested HCT116 cells (5 × 104 and 1 × 103 cells per well, respectively) in 100-μl growth medium were seeded into 96-well plates. Cell concentrations were determined by preliminary test whereby the proliferation percentage exhibited by 5 × 104 cells per well of PBMC was equivalent to 1 × 103 cells per well of HCT116. After overnight incubation at 37°C in a CO2 incubator containing 5% CO2, Blasto-Ag with concentrations ranging from 0.001 to 20 μg/ml was added to each well containing cells and was further incubated for 48 h. Then, proliferation percentage was measured using MTT assay as described previously (Mosmann 1983). From here, concentration of Blasto-Ag known for optimal proliferation for both types of cells was determined. This concentration was used to investigate the gene expression levels of cytokines, DNA transcription factor, and apoptosis stimulating genes in both PBMCs and HCT116 cells using real-time reverse transcription polymerase chain reaction (RT–PCR) technique.

Real-time RT–PCR

PBMC and HCT116 cells (5 × 105 and 1 × 105 cells per milliliter, respectively) were seeded into separate culture flasks containing 5-ml growth medium. Blasto-Ag with a final concentration of 1 µg/ml (concentration known to give optimal proliferation from preliminary tests) was introduced into each flask. Cells introduced with PBS were used as controls. After 48 h of incubation, the total RNA was isolated from the PBMC and HCT116 cells using Invitrogen Macro-to-Midi Total RNA Purification System (CA, USA). Purified RNA was next used to synthesize complementary DNA (cDNA) by the PCR method using High-Capacity RNA-to-cDNA Kit (Applied Biosystems, USA). Real-time PCR analysis was carried out using inventoried primers (TaqMan® Gene Expression Assays, Applied Biosystems). Genes studied were IL-6, IL-8, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), nuclear factor kappa light-chain enhancer of activated B cells (NF-κB), protein 53 (p53), and cathepsin B (CTSB) as depicted in Table 1. Real-time RT–PCR was performed on StepOnePlus™ Real-Time PCR System (Applied Biosystems). The PCR reaction was prepared according to the protocol generated by StepOne™ Software v2.0. The quantification approach used in this study was comparative CT (threshold cycle) method or also known as 2−ΔΔCt method (Livak and Schmittgen 2001).

Table 1 Gene expressions of IL-6, IL-8, TNF-α, IFN-γ, NF-κB, p53, and CTSB in both PBMC and HCT116
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Statistical analysis

For statistical analyses of real-time RT–PCR gene expressions, the ΔCT value of the treated sample was compared against nontreated sample (control) using Student’s t test. A P value of 0.05 was considered as the minimum threshold of significance.

Results and discussion

Previously, reports have shown that blastocystosis is coupled with intestinal disorders (Barahona Rondon et al. 2003; El-Shazly et al. 2005). Following this, researchers have developed an in vitro model using B. hominis culture filtrates to investigate its ability in triggering inflammatory cytokine responses and transcription factors in human colonic epithelial cells (Long et al. 2001; Puthia et al. 2008). These studies have concluded that the protozoan induces and regulates the immune responses of intestinal epithelial cells, thus suggesting that Blastocystis infection causes different pathophysiological events. However, the effects of B. hominis on various gene expressions in PBMCs and HCT116 cells have not been investigated. Past studies have reported that PBMCs can display various gene expression patterns for certain diseases including autoimmune diseases and cancer (Maas et al. 2002; Burczynski et al. 2005). The HCT116 cell line has also been broadly used for gene expression studies in colorectal cancer (Wang et al. 2006).

In the present study, increased cell proliferation observed in PBMCs stimulated with Blasto-Ag (Fig. 1) leads to a speculation that antigen stimulation causes immune cells to propagate in order to combat the infection. To date, this is the first study demonstrating the cytopathic effect of Blasto-Ag on PBMCs. Whereas increased cell proliferations seen in antigen-stimulated HCT116 cells (Fig. 1) suggest that infection by Blastocystis may facilitate the growth of colon cancer cells, in contrast, Walderich et al. (1998) and Long et al. (2001) have reported that Blastocystis culture filtrates did not show any effect on the survival rate of adenocarcinoma HT29 cell cultures. This suggests that the effect of excretory–secretory antigen of B. hominis may differ from its solubilized antigen and therefore needs further investigations.

Fig. 1
figure 1

Proliferation of PBMC and HCT116 cells introduced with various concentrations of Blasto-Ag (three independent experiments). PHA, mitogen (8 μg/ml) was used as positive control. Values are given in mean ± SD. *P < 0.05 and **P < 0.01 are the comparisons done against the lowest concentration (0.001 μg/ml). Values are normalized against sample blank where Blasto-Ag was substituted with sterile Jones medium (without any supplements)

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In the current study, Blasto-Ag stimulation has caused various patterns of cytokine and apoptotic gene expressions in both PBMC and HCT116 (Table 1). Generally, Th1 cytokines (e.g., IFN-γ and TNF-α) are known to increase the cellular immune responses while Th2 cytokines (e.g., proinflammatory IL-6 and IL-8) are known for humoral immune responses, which are usually triggered by extracellular microbes (Romagnani 1996). Past studies done on PBMCs and colon cells have reported that upregulation of proinflammatory IL-6 and IL-8 cytokines is induced by activation of NF-κB (Landi et al. 2003; Rhodes and Campbell 2002). NF-κB is a protein complex that controls the transcription of DNA, and it is correlated with cell proliferation and tumorigenesis (Jo et al. 2000).

In our study, the downregulation of IFN-γ and TNF-α together with the upregulation of IL-6, IL-8, as well as NF-κB gene expressions was seen in the PBMCs stimulated with 1 μg/ml of Blasto-Ag (Table 1). These observations suggest that, as an example of extracellular allergen, the Blasto-Ag has stimulated the humoral immune responses in PBMCs, which may lead to inflammatory reactions and propagation of the cells to combat with the infection. Besides, the upregulation of proapoptotic genes namely p53 and CTSB in PBMCs indicates that the infection may also induce apoptosis or programmed cell death in these cells. Collectively, these results may imply that Blastocystis infection has the tendency to create resistance against protective responses of immune cells by inducing the cells to die. Such activity can facilitate the survival and propagation of this enteric protozoan in humans.

We have also observed a significant downregulation of IFN-γ and upregulation of IL-6 and NF-κB gene expressions in HCT116 cells. These results imply that Blasto-Ag may play a role in weakening the cellular immune activation of HCT116 cells while promoting the growth of colon cancer cells. A previous study has reported that IFN-γ expression was decreased in cervical carcinoma biopsies when compared with normal cervical tissues (Pao et al. 1995). Apart from being involved in inflammation processes, IL-6 has also been recognized as a tumor-promoting growth factor (Spaeth et al. 2009) including colorectal tumor (Becker et al. 2005). Thus, the noticeable upregulation of IL-6 in HCT116 as seen in our study may also indicate that Blasto-Ag has the ability to enhance colon cancer progression. In contrast to PBMCs, proapoptotic genes namely p53 and CTSB were significantly downregulated in HCT116 cells, suggesting that Blastocystis infection possesses the ability to inhibit the apoptotic effect of colon cancer cells that could induce the growth of an existing tumor.

In conclusion, the cell proliferative effects and gene expressions of the present study have successfully exhibited results, which favor the fact that antigen from B. hominis, at a certain concentration, could facilitate the growth of an existing tumor or cancer cells while having the ability to downregulate immune cell responses. Thus, there is a vital need to screen tumor-harboring or colorectal cancer patients for B. hominis infection especially on those who are not undergoing chemotherapy. Our findings could also be used as point of reference to further investigate the immunomodulation of Blastocystis infection in normal as well as in cancer patients.