Journal of Immunodeficiency & DisordersISSN: 2324-853X

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Research Article, J Immunodefic Disor Vol: 2 Issue: 1

Effect of Different Hydatid Cyst Molecules on Hela and Vero Cell Lines Growth In vitro

Nasir Aref1, Hedayatollah Shirzad2, Morteza Yousefi1 and Hossein Yousofi Darani3*
1Student Research Committee, Shahrekod University of Medical Sciencs, Shahrekord, Iran
2Department of Immunology, Shahrekod University of Medical Sciencs, Shahrekord, Iran
3Department of Parasitology, Faculty of medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Corresponding author : Hossein Yousofi Darani
Infectious and Tropical Diseases Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
Tel: 0098-3117922489
E-mail: [email protected]
Received: May 16, 2013 Accepted: August 05, 2013 Published: August 09, 2013
Citation: Aref N, Shirzad H, Yousefi M, Darani HY (2012) Effect of Different Hydatid Cyst Molecules on Hela and Vero Cell Lines Growth In vitro. J Immunodefic Disor 2:1. doi:10.4172/2324-853X.1000105

Abstract

Mast Cells as Reservoirs for HIV Latency

 

Hydatid cyst is the larval stage of the tapeworm, Echinococcus granulosus, a parasite responsible for hydatid disease or hydatidosis in human and livestock. This disease considered as one of the most important cosmopolitan zoonotic infection with different mammalian hosts being involved in the life cycle. It has been shown that in a large retrospective study of patients with cancer, the prevalence of hydatid cyst was significantly lower than in normal subjects. Antitumor activity of some other parasites have also been demonstrated.

Keywords: Hydatid cyst molecules; Cancer; Hella cells

Keywords

Hydatid cyst molecules; Cancer; Hella cells

Introduction

Hydatid cyst is the larval stage of the tapeworm, Echinococcus granulosus, a parasite responsible for hydatid disease or hydatidosis in human and livestock. This disease considered as one of the most important cosmopolitan zoonotic infection with different mammalian hosts being involved in the life cycle. It has been shown that in a large retrospective study of patients with cancer, the prevalence of hydatid cyst was significantly lower than in normal subjects [1]. Antitumor activity of some other parasites have also been demonstrated. For example anticancer activity of parasites such as Trypanosoma cruzi [2-8] Toxoplasma gondii [9-15], Toxocarac canis [16], Acantamoeba castellani [17,18] and Plasmodium yoelii [19] have been shown in experimental animals. In vitro investigations also revealed that some parasites such as Trypanosma cruzi, hydatid cyst protoscileces, and Toxoplama gondii show anticancer activities [7,16,20,21]. We previously showed that alive hydatid cyst protoscolices inhibited the proliferation ofWEHI-164 and BHK cells and have the capacity to induce cell death in WEHI-164 cells in vitro [21]. In this work the effect of different hydatid cyst molecules including crude protoscolices molecules, excretory secretory molecules of protoscolices, Laminated & germilal layers molecules and hydatid fluid moleculeson Hella and Vero cell lines growth in vitro have been investigated.

Materials and Methods

In this experimental study, Echinococcus granulosus hydatid cysts were collected from sheep or cattle from a slaughter house in Isfahan, Iran. Hydatid fluid were aspirated, collected and examined for presence of protoscolices. The fluids were then centrifuged at 2000×g, for 2 min, and the supernatant was concentrated and kept at -20 as hydatid fluid molecules. The sediment which was compact protoscolices washed with isotonic saline, sonicated and kept at -20 as crude protoscolice molecules. Also culture medium was added to compact alive protoscolices, and following 48 hours incubation centrifuged at 2000×g, for 2 min and the supernatant used as excretory secretory molecules of protoscilices (ES molecules). Laminated & germinal layers were removed from the cyst, homogenized and then sonicated and kept at -20 as Laminated & germilal layers molecules.
Culturing of tumor cells
Two cell lines including Hella and Vero cells were purchased from the Pasture Institute (Tehran, Iran). Cells were cultured as we performed before [21]. In each experiment, two culture flasks containing 10 mL culture medium with 100000 freshly prepared and viable Hella or Vero cells were used. The first flasks were treated with 500 μl of different molecules containing 5-10mg protein, while the second flasks were treated with 500 μl isotonic saline as control sample. All flasks were incubated in CO2 incubator for 48h and then cell counting was determined for each flask after 48 h. Cell counting was performed using a Neubauer’s chamber. Each experiment was performed in triplicate. In this study to evaluate the effect of different molecules on cancer cells, two criteria including increasing the number of dead cells or decreasing the numbers of alive cells were used. Trypan blue staining was used to discriminate between dead and alive cells. Jonckheere-TerpstraTest used for statistical analysis of the data.

Results

When crude extract of protoscolices was added to Hela cells a significant difference in number of dead cells between case and control groups was achieved (P=0.01). However the difference for alive cells was not significant (Figure 1). When excretory secretory molecules of protoscolices was used a significant difference was observed for both alive (P=0.014) and dead cells (P=0.02) (Figure 2).With the hydatid cyst fluid molecules the difference for alive cells was significant (P=0.031) and for dead cells was not significant (Figure 3). Finally when crude mixture of homogenized laminated & germinal layers was added a significant difference (P=0.01) was observed for dead cells. However the difference in alive cells was not significant (Figure 4). When different molecules incubated with Vera cells no significant effects on growth of those cells was detected.
Figure 1: Mean of alive or dead cells in flasks of Hella cells treated with hydatid cyst protoscolices crude antigens (case groups) or isotonic saline (control group).
Figure 2: Mean of alive or dead cells in flasks of Hella cells treated with Excretory secretory antigen of hydatid cyst protoscolices (case groups) or isotonic saline (control group).
Figure 3: Mean of alive or dead cells in flasks of Hella cells treated with hydatid cyst fluid (case groups) or isotonic saline (control group).
Figure 4: Mean of alive or dead cells in flasks of Hella cells treated with hydatid cyst laminated & germinal layers antigen (case groups) or isotonic saline (control group).

Discussion

Results of this investigation revealed that in cell cultures treated with crude protoscolices molecules, protoscolices ES molecules or Laminated & germinal layers molecules the number of dead Hela cells increased in comparison with control cell culture. Also in cell cultures treated with protoscolices ES molecules or hydatid fluid molecules the number of alive Hela cells decreased in comparison with control cell culture. However none of the molecules increased Vera dead cells or increased Vera alive cells.
The results about Hela cells are consistent with our previous finding that Protoscolices of hydatid cyst induced cell death in WEHI-164 Fibrosarcoma cells and also inhibited the proliferation of baby hamster kidney fibroblasts in culture medium [21]. The effects of parasite antigens on inhibition of cancer cells have also been shown in other investigations. Atayde et al. showed that a Trypanosoma cruzi surface molecule gp82 recombinant protein induced cell death in melanoma cells [22]. In other investigation it has been shown that some parasitic antigens have stimulatory or inhibitory effects on certain cell lines. However some other antigen showed no effect on proliferation or death of cells in culture medium.
Rigano et al. investigated the effect of hydatid cyst fluid or antigen B on Human Dendritic Cell differentiation. They showed that Antigen B and hydatid cyst fluid upregulated CD86 expression and downmodulated CD1a expression [23]. Kanan and Chain explored the effect of hydatid cyst fluid on differentiationof human monocyte to dendritic cell. Their results showed that hydatid cyst fluid modulated differentiation of dendritic cells. The presence of hydatid cyst fluid also increased CD14 expression and decreased expression of CD1a [24]. Nono et al. showed that Excretory/Secretory-Products of Echinococcus multilocularis Larvae Induce apoptosis in dendritic cells in vitro [25]. Results of these investigations are in agreement with our finding indicating that hydatid cyst molecules are able to effect cells growth in culture medium.
Apart from Echinococcus antigens, effect of some other parasite antigens on different cell lines has also been investigated. Huby et al. showed that ES products of the intestinal nematode, Nematodirus battus, decreased the number of epithelial cells in culture medium. Inversely ES products of two other parasites stimulated cell growth [26]. In another study Huby et al. investigated effects of the excretory/ secretory products of Trichostrongylus colubriformis on the growth of different cell lines. These products increased cell numbers of three epithelial intestinal cells. In contrast, the products inhibited the proliferation of epithelial ovarian cells and fibroblasts. Finally no effect was detected on the cell growth of hepatocytes [27]. Semnani et al. showed that Brugia malayi microfilariae induce cell death indendritic cells [28]. These investigation indicate that in addition to hydatid cyst molecules, other parasite molecules also effect cells growth in vitro.
Results of our investigation showed that hydatid cyst molecules affected tumor cell growth either by increasing dead cells or by decreasing alive cells. From these results along with other findings about the effect of parasite antigens on cell growth it can be inferred that parasite antigens interfere with different cell lines growth in vitro. However the mechanisms that may be involved remain concealed and further work is recommended to discover these mechanisms.

Acknowledgement

This work was supported by a grant from Shahrekord University of Medical Sciences, Shahrekord, Iran.

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