Extended Abstract

Introduction
Colorectal cancer is the third most common cancer in men and the second most common in women. Currently, treatment options for colorectal cancer depend on various factors, such as tumor location, stage of disease, and overall patient health. Treatment may encompass surgery, the use of multiple drugs, such as fluoropyrimidines, irinotecan, oxaliplatin, and capecitabine, radiation therapy, chemotherapy, or a combination of them. On the one hand, patients with colorectal cancer face complications related to these treatment options. Hence, the need for more influential treatment methods is well felt.
The use of parasites or parasite antigens is one of the newest therapeutic approaches for cancer, although it has not yet been used or approved in human trials. In studies conducted on the carbohydrate antigens of hydatid cysts and the cross-reactivity of sera from breast cancer patients and healthy individuals with these antigenic fractions, carbohydrate antigens of hydatid cysts were found in all stages of the cyst and their frequency was higher in the hyaline layer. The simultaneous presence of hydatid cysts and hepatocellular carcinoma is rare, and Echinococcus granulosus seems to have protective effects against this cancer. Among the species of the Taeniidae family, which are common parasites worldwide, the genus Echinococcus is of great importance in terms of health and economy, particularly in human and animal communities. Hydatid cysts, usually caused by Echinococcus granulosus, consist of a single cavity with a thick wall. This unilocular cyst consists of two layers: Germinal layer and laminated layer. In fertile cysts, protoscolices are located on the inner or germinal membrane of the cyst. The hyaline layer has a dense and voluminous structure that is only observed in the genus Echinococcus and is highly enriched in carbohydrate compounds. Hydatid cyst antigens have anti-melanoma activities, and these effects are related to the immune system’s response to parasitic antigens. The anti-tumor activity of hydatid cyst fluid against breast cancer has also been attributed to increased immune responses to parasitic antigens. Therefore, various methods can be used, including the isolation and synthesis of compounds and molecules of parasites such as Toxoplasma gondii and Echinococcus granulosus to identify and produce compounds that contribute to eliciting the immune system against cancers. Hence, this study aimed to determine the cytotoxic effects of purified carbohydrates from the hyaline layer, fluid, and protoscolices of hydatid cysts on the human colorectal cancer cell line (LS174T).
Methods
This descriptive-analytical study was conducted on two sheep and two cow livers infected with hydatid cysts and the human colorectal cancer cell line (LS174T).
Two sheep and two cow livers infected with unilocular fertile hydatid cysts caused by Echinococcus granulosus in the terminal stages of the disease and containing cyst fluid were collected from a slaughterhouse. The cyst surfaces were sterilized with 70% alcohol, and the cyst contents were collected using 20 cc syringes.
The collected cyst fluid was examined under a light microscope for the presence of protoscolices. After observing the presence of protoscolices, the cyst contents were centrifuged at 4000 rpm for 15 minutes. The supernatant, as the hydatid cyst fluid, was carefully removed and stored in Falcon tubes. The resulting precipitate, which consisted of protoscolices separated from the cyst fluid, was stored in Falcon tubes until use. Subsequently, the contents of the Falcon tubes containing the cyst fluid were poured into sterile Petri dishes and concentrated and dried for 24 hours to obtain crude cyst fluid antigens, which were stored at -20°C for subsequent study stages. The precipitate of compressed protoscolices at the bottom of the Falcon tubes was sonicated using ultrasonic waves. Then, they were incubated at 37°C for 48 hours. After 48 hours, the liquid in the Petri dishes was dried, and the Petri dishes were removed under sterile conditions and transferred to a laminar flow hood. Then, the dried liquid in the Petri dishes was scraped with a sharp object and poured as a powder into small microtubes, and was sonicated using a Bandelin electronic (Germany) sonicator for 2 pulses of 15 seconds each. These sonicated protoscolices, as crude protoscolice antigens, were also stored at -20°C in the freezer for further studies. Moreover, a portion of the collected cyst wall was cut into smaller pieces, washed several times in a mixture of phosphate buffered saline (PBS), penicillin (300 IU), and streptomycin (0.3 mg/ml), and frozen and thawed several times for 24 hours to help remove the germinal layer. The hyaline layer was then separated and, after homogenization, was sonicated for 5 pulses of 15 seconds and 10 pulses of 15 seconds for sheep and cow hyaline wall antigens, respectively, ultimately turning into slurry, which was stored as a crude hyaline cyst wall antigen at -20°C in the freezer for future studies.
A chloroform-methanol 20 extraction method was employed to isolate glycoproteins and glycolipids from the antigens isolated from the three components of the cysts: Hyaline layer, fluid, and crude protoscolices.
A β-elimination method at 37°C was employed to isolate carbohydrates from the glycoprotein and glycolipid components of the antigens extracted from different parts of the cyst.
Bovine and ovine glycolipid and glycoprotein antigen samples were transferred to activated dialysis bags.
A phenol-sulfuric acid method was employed to confirm the presence of carbohydrates in each of the antigen solutions isolated from the cyst.
The cytotoxicity of carbohydrates purified from antigens isolated from hydatid cysts on colorectal cancer cells was evaluated using the methyl tetrazolium (MTT) assay. The MTT assay was employed to assess the cytotoxicity of twelve bovine and ovine glycolipid and glycoprotein antigens on colorectal cancer cells.
In order to further investigate and screen the antigens, the cytotoxicity of carbohydrates purified from the five antigens with the highest cytotoxicity against the LS174T cell line was re-evaluated using the MTT assay.
Results
Carbohydrates purified from glycoproteins isolated from sheep hydatid cyst fluid, glycolipids isolated from sheep hydatid cyst protoscolices, glycolipids isolated from bovine hydatid cyst hyaline layer, glycoproteins isolated from bovine hydatid cyst hyaline layer, and glycoproteins isolated from bovine hydatid cyst protoscolices demonstrated the greatest inhibitory effects on the growth of LS174T cells. In order to further screen, the inhibitory effects of carbohydrates purified from these five antigens on the growth of LS174T cancer cells were re-evaluated using the MTT assay, and the carbohydrates purified from the five selected antigens significantly inhibited cell growth. Carbohydrates purified from glycoproteins extracted from sheep hydatid cyst fluid, glycolipids extracted from sheep hydatid cyst protoscolices, and glycolipids extracted from bovine hydatid cyst hyaline layer exhibited the greatest inhibitory effects on the growth of LS174T cancer cells (P<0.05).
Conclusion
According to the results, carbohydrates purified from antigens isolated from the cyst significantly inhibited the growth of colorectal cancer cells. Carbohydrates purified from five antigens, including glycoproteins from sheep cyst fluid, glycoproteins from bovine cyst hyaline layer, glycolipids from bovine cyst hyaline layer, glycoproteins from bovine cyst protoscolices, and glycolipids from sheep cyst protoscolices, appeared to have the greatest inhibitory effects on cell growth. Treatment of cells with carbohydrates purified from glycoproteins extracted from sheep hydatid cyst fluid, glycolipids from sheep hydatid cyst protoscolices, and glycolipids from bovine hydatid cyst hyaline layer gave rise to the greatest inhibitory effects on cancer cell growth.
Ethical Statement
This study was approved by the Research Ethics Committees of Shahid Chamran University of Ahvaz (EE/1401.2.24.226665/scu.ac.ir).
Funding
This article has been extracted from the Ph.D. dissertation of Zahra Jafari in Parasitology from the Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz.
Conflicts of Interest
No conflict of interest.
Acknowledgment
The authors would like to thank the Research Vice-Chancellor of Shahid Chamran University of Ahvaz for the financial support.

Key message: Carbohydrates isolated from glycoproteins and glycolipids of hyaline layer and protoscolices of the hydatid cyst fluid exhibited significant anticancer effects on the LS174T colorectal cancer cell line.